United Arab Emirates University

Scholarworks@UAEU
Theses Electronic Theses and Dissertations

12-2014

Effects of Antihistamines on the Function of

Human Alpha-7 Nicotinic Acetylcholine
Receptors.
Seyedeh Soha Khanian

Follow this and additional works at: http://scholarworks.uaeu.ac.ae/all_theses

Part of the Pharmacology Commons

Recommended Citation
Khanian, Seyedeh Soha, "Effects of Antihistamines on the Function of Human Alpha-7 Nicotinic Acetylcholine Receptors." (2014).
Theses. Paper 159.

This Thesis is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarworks@UAEU. It has been accepted for
inclusion in Theses by an authorized administrator of Scholarworks@UAEU. For more information, please contact fadl.musa@uaeu.ac.ae.
 u1..a..J I ii..u I a II G I La!J I Cisul b

lnlEU Lnited Arab

... � J "
Emirates University
.

United Arab Emirates University

College of Medicine and Health Sciences

Department of Phanllacology and Therapeutics

EFFECTS OF ANTIHISTAMINES ON THE FUNCTION OF HUMAN

ALPHA-7 NICOTINIC ACETYLCHOLINE RECEPTORS

Seyedeh Soba Kllanian

This thesis is submitted in partial fulfillment of the requirements for the degree of
Master of Medical Sciences (Pharmacology and Toxicology)

Under the Supervision of Dr. Bassem Shaban Sadek

December 2014
 ii

Declaration of Original Work

L eyed h oha Khanian, the undersigned, a graduate tudent at the United Arab

Em irates U n i versity ( U E ), and the author of thi thesis entitled " Eff

antihistamine. on the Junction oj hllman a7-nicotinic ace tyLcholine receptors",

hereb , 01 m n l y declare that this the is is an original research work that has been

done and prepared b Ine under the upervision of Dr. Bassem Shaban Sadek and co-

supervision of Professor M urat Oz, in the Col lege of Medicine & H ealth Sciences at

EU. Thi work ha not been previously formed as the basis for the award of any

academ ic d gree, diploma or a imi lar title at this or any other tmiversity. The

materi als borrowed from other sources and i nc l uded in my thesis have been properly

cited or acknowledged.

tudent ' s Signature ----;7---:�,..,���'-----.- Date 16·2.·Z.0fC

 iii

Copyright © 20 1 4 Seyedeh Soha Khani an

A l l Rights Reserved
The i E amining Committee:

1) Supervi or: Dr. Bassem Shaban Sadek

Title: Assi tant Profe sor

D partment: D partment : Phannacology and Therapeutics

In titution : Col l e

ignature . . . . . .
2) Member: Professor Murat Oz

Title: Profe or

Department: Phannacology and Therapeutic

In titution: Col lege of Medicine and Health Sciences

i gnaturc \ ..,AA,c �. . � . . . . . . . . . . . . . .. . . . Date D.1Z<.: 15�h/t�14

3) Member: Dr. Sandeep Subramanya

Title: As i stant P rofessor

Department: Physiology

ft
Institution: co ege of Medicine an (? � a Sciences

S igna�� g . . . . . . . . . . . . . . . . . . . . Date . ! :f . i R .R " Jit . . .

4) External Examiner: Professor Maria Beatrice Passani

Title: Professor

Department: eurosci ence, Psychology, Drug Design and Chi l d Care

.
Institution: Uni e

Signature . .. . . . U . .. . . . . . .
. . . . . . . . .
.. . . .. . .
. . . . . .... ... . Date . . ).?: ...� � . .� l �t.
. . . . . . . . . . .
 v

Th is Master The is is accepted by:

Dean of the Col lege of Medic i ne and H ealth Science : Professor Templeton

ignalure: �� Date tG 2 (.0-

1:;-

Dean of the Col l ege of Graduate Studies:

S ignatur<:;. � Date: \ b \ \
2.. 20 IS"

Copy �of L
 vi

Ab tract

The effe ts of hi tam ine H I receptor ( H I R) antagonists (antihistam i n es),

namel), promethazine ( PMZ), orphenadrine (ORP , chl orpheniran1 ine (CLP ),

pyri lamine ( PYR), di phenhydram i ne ( D P H ) , cetrizine ( CTZ), and triprol i dine (TRP)

\ \ ere tested on the function of the c l oned U7 ubunit of the h uman n icotinic

acety lcholine receptor (urnAChR ) expressed in Xenopu oocytes using the two­

electrode voltage-c lamp techni que. Antihistami nes i nllibited the urnAChR i n

theorder of PYR > CLP > T R P > P M Z > O R P 2: DPH 2: CTZ. Among

antih istam ine , PYR howed highest re ersi ble inhi bition of acetylcho l i ne ( 1 00 �lM)­

induced currents with I C50 val ue of 6.2 f..L M . In contrast, the standard H 2- H4R

antagonists, cimetid i ne (CMT),ranitidine ( RNT) , r u zatidine (NZT) , c lobenpropit

(CLB), ciproxifan (CPR), pito l i sant ( P I T), andJNJ-7777 1 20 (JNJ) when tested at

h igh concentration (1 0 �lM ) howed negl igible i nh ibition ofurnAChRs. Further

experiments using PYR, the strongest inhibitor of u7-nAChR, indicated thatPYR

i nhibition was not dependent on the membrane potential and could not be reversed

by increasing acetylcho l i ne concentrations suggesting a non-competitive i nhibition

of n icotinic receptors. In l ine with functional experi ments, docking studies i ndicated

that PYR can potential ly bind al losteri cal l y with the U7 transmembrane domain . Our

results indicate that first generation H I R antagonists i nh i bit the function of human

urnAChR, with varying potencies, and emphasize the importance of urnAChR for

future harmacologicalltoxicological profil ing of these ant i histami nes.

Keywords: nicotin ic receptor; A ntih istamine ; Xen opus oocyte; Pyrilam ine
 vii

Title and Abstract (in Arabic)

�I

(ORP)-�Yw.J3I ,(PMZ) - -L:i... 3Y.

�.J . \ -: . - I I
-�� .J\..U;;.I
.
•

_( DPH)lJ:lAI� ,(PYR)lJ:lAi

wl.:J �1 01 �) � u\""'O�1 lY> ;i,ol';'i, IQlI �1.:lll\ _microelectrode voltage c lamp

.fi.i�i lJ:lAI �y-,-! ��I wl.:l�1 LB-! lY> _PYR>CLP>TRP>PMZ>ORP>DPH>CTZ

- .:lijl· - - .lilli l
� _ .J:!-' � -.- .J

\.ili 4..w:i ,- �� 11 �.Y' J:':""

-,,( -
y.:
-( ,:1\ w)lW... . 1.\ • .:l! (10 M ) '-
"� -- � , ..JI.blli ',
 viii

Wl.:,. WI 4...w:i ' :11 u)l. . . L.': . ,J .J':/1 �I'

.W....J1 J.=�i L.J� ':11 1.lJ.b...J ".w...r .. 11
. .J. . .' .:r--"' . .' . lY' I..?'"' J
4..1..w.J
�
.lJ _
_
.' _ _
 ix

Acknowled gment

I am grateful to my God for bestowing on me the opportuni ty to how my

grat itude to a l l those who deserve it. Yet, it is trul unfortunate that it i not possible

to mention here the name of the idea l s of my l i fe who have been the source of

in pirat ion for me!

M deepest thanks to m parents, though words woul d never show my

appre iution of their effo rts for m success throughout my l i fe. If it was not for their

prayer and encouragement, for sure I would have been unable to reach this stage.

They taught me not to be happy eating whi l st there's someone hungry around, of

wearing what the poor wi l l e e on m e and cannot wear, o f keeping a belonging for

mysel f whi l e around me there's someone wishing to have it. I'm thankful for a l l of

that . . . and much more.

My thanks to all those who taught me even a single word i n this world trying

to educate me, to make me fly from my ego to modesty, from sel fishness to

benevolence and from i gnorance to a\ areness. I'm sendi ng my gratitude to al l my

teachers ever since primary school , whose i n fl uence was the most on my persona l i ty

to those of my teachers i n this master program.

I feel i ndebted to Professor Abdu Adem (Chai rman, Department of

Pharmacology and Therapeutics, C o l l ege of Medicine and Health Sciences, UAE

U n iversity) and Professor San1 i r Attoub (Associate Professor, Department of

Pharmaco logy and Therapeutics, C o l l ege of Medicine and Health Sc iences, UAE

University) for their support i n my study and research.

 x

Dr. Ba m haban adek ( i tant Profes or, Department of Phamlucol ogy

and Th rapeutics, o l l ege of Medicine and Health ciences, AE Univers ity ) was

m academic upervisor who planned thi research . I thank him for offering me the

opportun i ty t work on this proj ect. I appreciate his patience, understandi ng, and

fol l o'w-up.

Furtherm re, I would l ike to thank Professor Murat Oz ( Department of

Pharmacology and Therapeutics, Col lege of Med i c i ne and Health Sciences, UAE

U n i ver ity), co-supervisor of this proj ect, i n whose lab I spent mo t o f my time

during the course of this master program . He did not hesitate i n offeri ng me his

guidance.

The last part of this proj ect which made use of computational molecular

doc k ing studies, was conducted with the hel p and i nstruction of Dr. Mohammad

Ghattas ( Assistant Professor, Col lege o f Phannacy, AI-A i n Universi ty of Science and

Technology), to whom I would l ike to send my thanks and regards. I am, also,

thankful to Dr. Khai ri M ustafa Salem ( Dean, College of Phamlacy, AI-Ain

University of Science and Technology) and a l l other Facul ty members thereafter for

al lowing me to work in their station.

Not forgetting the l aboratory team, I would l i ke to thank Mrs. Abrar Ashoor

who ski l l ed me with the main tech niques appl ied in this proj ect. Beside this, I

benefited from some of the i n formation and graphics present i n her thesis whi l e

comp i l i ng my thesis and preparing PowerPoint sl ides. I am, also, thankful t o Dr.

Seyd N urulain who hel ped me a lot in the preparation and i solation of the frog

oocytes for my experiments and supported me in the laboratory work .

 xi

La tly, I wish all my fam i l y member and friends to accept my gratitude,

thanking them [or being there for me. Yet, I feel unfair not to how my thank ful ness

to all heroe of m country who in pired me to land for the righteous and tackle the

inj u tice wherever it i i n the world. And la t but not the least, a l l thanks goes to God

- the Creator of the �hol e univers .

 xii

Ded ication

"I'm the harrOlt'ing moan oj broken-hearted OIphans 11'ho G>vaken from

extreme hunger at midnight, and an}' kind hand does not care s them. The y are

[{{raid (?fdarkness and /ol?e/ine s. There are no warm arms which give them shelter ",

Dr. Jlo taJa Chamran (Robin on, 2013)

1 'woliid like to dedicate thi work to Dr. A10stafa Chamran, the great

re earcher and scientist oj my country.

 xiii

Table of Contents

Title . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Dec larati n of Ori ginal Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i
opyright . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i
ignatur .................................................................................................................... iv
b tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
Title and b tract ( in Arabic) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
ckJ10\vl dgl11ent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i i
Table of ontent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi i i
L i t o f Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Li t of F igure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
I ntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ]
1.1 H i stor) of Histamine-Receptor-Blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1 .2 Histami ne Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1 .3 H i stami ne H I - Receptor and Antihistami nes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1 .-+ ChemicaL Functional and Toxicological pro fi l e o f antihistamines . . . . . . . . . . . 10
1 .5 Acetylchol ine and the cholinergic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1 .6 Acetylcho l i ne Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1 . 6. 1 icotinic acetylcholine receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
.

2. 1 Material s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2. 1 . 1 Female Xenopus laev i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2 . 1 .2 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2 . 1 .3 The Experimental rig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2 . 1 .4 . Other n1aterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2. 1 Preparation of sol utions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.2 I solation of oocytes from Xenopus laev i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2.3 Oocyte preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.4 Synthesi s of cRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2 .2 . 5 Inj ection o f cRNA i nto oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
 xiv

2.2.6 Drug preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.2.7 lectroph siological record i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.2.8 Dock ing studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6

2.2.9 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3 Re ult . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.1 Functional Expre ion of the Human a7nAChR i n Xenopus Oocytes . . . . . . . 48
3.2 The E ffect o f el ected HR B lockers on the Function of H uman a7 . . . . . . . . . . 49
3.3 The Role o fI ntracel l ular Calcium Levels in Pyri lami ne- I nhibition o f a7 . 54
3.4 The Vol tage-Dependence o f Pyri l am i ne Action on the u7nACh . . . . . . . . . . . . 5 5
3.5 tudie on the Binding S ite o f Pyri lamine on u7nACh Receptor . . . . . . .. . . . . . . 5 8
3.6 Docking tudie for P ri lam i ne at urnAChR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.7 I nhi bition of P Y R and its Competition with Ketam ine o n urnACh R . . . . . . . 61

3.8 P Y R E ffect o n Glyc i ne and 5-HT3 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
B i b l i ography . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
 xv

List of Tables

Table 1-1: Pattern of hi tam ine receptor di tri bution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Table 1 -2: tructural and functional clas i fi c ation of H ,R anti h i stam i nes . . . . . . . . . . . . . . . . 10
Table 1 -3: drenergic agonists and antag ni ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Table 2-] : Chemicals req uir d for experi ments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Table 2-2 : Calcium-free o l ution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Table 2-3: D96 solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Tabl e 2-4: Oocytes storag� solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Tabl e 3- 1 : The W AflG cores o[ the pyri lamine and ketamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
 xvi

List of Figures

Figure 1 -1: Dynamic of neuronal hi tami ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Figure 1 -2: Transcription and translation of histam ine receptors . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . 5
Figure 1 -3: Common tructmal Feature of c l assical 1 st generation antihistami nes. 14
Figure 1-4: chematic representation of a cho l inergic neuroeffector j unction . . . . . . . . . . 16
Figure 1 -5: Central cho l i nergic pathways regulate global functions . . . . . . . . . . . . . . . . . . . . . . . . . 1 8
Figure 2- 1 : Xenopu Leavi African Frog 31
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Figure 2-2 : Experi mental rig for electrophysiological recordi ngs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3

Figure 3 - 1 : Functional expression of the h a7-nACh receptor i n Xenopus oocytes. 49
Figure 3 - 2 : Chemical structures of H l -H4R antagoni sts used in the current study . . . 50
F igure 3 - 3 : Relati e potencies of H l R antagonists tested on human . . . . . . . . . . . . . . . . . . . . . . . . 5 1
Figure 3-4: Relative potencies of H2R-H4R antagonist tested on human . . . . . . . . . . . . . . . 5 1
Figur 3 - 5 : Effects of pyri l am i ne on a7 nAChR-mediated ion currents . . . . . . . . . . . . . . . . . . . . 52
Figure 3-6: Time and concentrati on-dependence of P YR inhibition of . . . . . . . . . . . . . . . . . . . . . 5 3
Figure 3 - 7 : PYR i nh ibits a7-nAChR i n a concentration-dependent manner . . . . . . . . . . . . 54
Figure 3 - 8 : Effect of P Y R on ACh-i nduced currents is i ndependent of . . . . . . . . . . . . . . . . . . . . 55
Figure 3-9: PYR i nh i bition of ACh-induced currents is i ndependent of vol tage . . . . . . 5 7
Figure 3 - 1 0 : Concentration-response curves forACh-induced c urrents . . . . . . . . . . . . . . . . . . . . 59
F igure 3 - 1 1 : The binding mode of the pyri lamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1
Figure 3 - 1 2 : Comparison of the effects of PYR and ketamine on a7-nAChR . . . . . . . . . . 62
F igure 3 - 1 3 : Com parison of the effect of P Y R on arnA C R, 5-HT3R, and GlyR . . . . . 63
 1

1 INT R O DUCTION

1. 1 H i tor}, of H i t a m i n e-Receptor-BJocker

1 Ii tam ine was known as a chemical before it presence in biological systems

was con fi mled. It i now almo t one hundred year since S i r H emy Dale & his

col league at th Wel lcome Laboratories isolated histam ine from the mould ergot

( Par on & Gan e l l i n , 2006). I n 1 907 W indaus and Vogt described the synthesis of 2-

(imidazole-4-) J) ethylamine; much later this compound received the name histami ne

(Greek hUo , · tissue ' ) due to relatively high concentrations found in tissues. I n 1 9 1 0

Dale and Laidl avv isolated the compound from animal tissues, but originally it was

thought to be there j ust as a metabol ite of h istidine, having no physiol ogical

sign i fi c ance of its own . Duri ng the 1 920s it became evident that histam ine evokes

several pharmacological activi ties, resembl ing very much the actions of the (at that

time) not wel l-defined ' H substance,' a more or l ess hypothetical compound thought

to be rel eased from i nj ured tissue; Eventua l l y it was found that H substance was a

complex m ixture of compounds, of which h i stamine was only one, playing a major

rol e i n anaphyl ax i s (Ti mmerman & van der Goot, 2009). The storage of histami ne i n

mast cel l s was establ ished b y Riley and West when they found that chemicals

capable of i nduc i ng h i stamine release also dismpted mast cells and this was

accompanied by a fall i n tissue h i stamine content ( R i ley & West, 1 952) Further, there

was a strong positive correlation between histami ne content and mast cel l

populations i n a variety of tissues. H owever, Ri ley and West were careful to point

out that some tissues contained too few mast cel l s to account for their histam i ne

content, and suggested that other cel l s may store histamine. Subsequently, it was

shown that the bl ood basophil, blood platelets i n some species and the
 2

enter chromaffin-like cell ( EeL) in the stomach were additional sources of

histam ine. In mammalian pecies hi tam ine is now known to be present in a l l ti ssues

in amount ranging from les than 1 to over 1 00 mg g l and, in general , the skin,

connective ti ue, l ung and much of the gastrointe tinal tract are rich In

hi stam ine( Par ons & Ganel l i n , 2006).

Depolarization o f brain s l i ces causes a ea-dependent release o f Histam i ne. In

the brai n : eer b Ilum has the lowest levels o f H i stamine whereas Hypothalamus has

the high st. The rol e of hi tam ine in anaphyl axis lead to an i ntensive search for

hi tam ine blocking agents; the fi rst so-cal led anti hi stam ines were found i n the l ate

1 93 0 , and in the 1 940 the anti hi tami nes were introduced as antiasthmatics and as

antiallergics i n genera l . Very many ant ih istam ines have been devel oped since. They

are al l of l i m ited use i n asthma, but are quite effective in treating other al lergic

re ponses, such as hay fever and skin rashes. For many years i t remained unclear

why certain e ffects of h i stam ine (e.g., stimulation of gastric acid secretion or

increa ed heart rate) were not blocked by highly act i ve anti histami nes. In the 1 960s

A h and S h i l d proposed that a second type of histamine receptor m ight exist in heart

and stomach tissues. Subsequently, in 1 974, B l ack et al. reported for the fi rst time on

selective blockers of the actions of histamine in both the heart and the stomach. The

new type of receptor was named H 2. the receptor discovered earl ier was designated

H I from then on. As with the antihistamines - now renamed H I blockers - several

sel ective H 2 blockers fol l owed the fi rst H 2 antagonist burimamide; c imetidine and

ranitidine, antigastric ulcer agents, have become blockbuster medi c i nes. I n the 1 980s,

it became clear that histam i ne not only has the characteristics of a local honnone, but

is a neurotransmitter as wel l . S hortly thereafter, Schwartz and group in Paris

identi fied a third c l ass of h i stam i ne receptors. These H3 receptors occur mainly as
 3

pre ynaptic receptor , functi n i ng b th a auto- and het roreceptors by regulating the

release of histam i ne (auto receptor) and other neurotransmitters (heteroreceptor).

Prett oon thereafter a large number of selecti e H3 agon ists and antagonists were

found: 0 far. everal antagoni t are undergoing c l inical testi ng for different central

nerYOLlS stem ( C )-related di ea es. During a search for new G-protein-coupled

re eptor (GPCR) genes, using the sequence of the f-b receptor gene as template, a

fourth t pe of histam ine receptor was found in the early 2000s. Th is H4 receptor i s

pre ent espec i a l l y i n peri pheral organs, o n immunoactive cel l s where it l i kely

mediate th pro i n flammatory prope11ies of histam ine. H i stam ine-induced

i n flammation is not blocked by H I , H2, or H3 antagoni ts. There is a low level of

homology between the H J , H], and H31H4 receptor proteins. The latter pair has a

rel atively h igh degree of homology, explai ning why i m i dazole-derived l i gands of the

H 3 receptor typical l y have a considerable affinity for the H.j receptor as wel l ; the

rever e is also true. Sel ective nonim idazol e H3 and H.j l i gands are avai l able, though

( ee Timmerman & van der Goot, 2009) .

 4

t·MIAA

HIS

Figure 1 - 1 : Dynam ics o f neuronal histamine

tep in the ynthe is. release and metabo l i sm o f hi tamine are shown : Ll -histidine
(Hi ) tran port i nto nen·e term i na l : 2. h i stamine (HA) synthesis by histidine
decarbox) l ase: 3. formation of hi tamine containing vesicles· 4, histam ine rel ease by
exocytosi : 5 , act ivation of po t-synaptic receptors: 6. feedback inl1ibition of
hi tamine s)11the is and release by H3autoreceptors: 7, histamine transport by
a troC)1es ( re-uptake by nerve tel111i nals ha not been found ): 8. metabol ism by
histamine- -meth;ltran ferase ( H MT): 9. oxidation of t-MH by monoami ne oxidase­
B . The cell ular loca l i zation of steps 7-9 remain poorly understood. /- MH, tele­
methyl histam i ne : t-M I AA. tele meth l i mi dazoleacetic acid (Basic Neurochemistry,
olecular and Cel l uar Aspects, 6th edition)

1 .2 H i s ta m i ne Recepto rs

The effects of h istam i ne are tri ggered through h i stam i ne receptors on

di fferent cells. To present date four subtypes of receptors (histamine HI receptor

( H,R ). h i stam i ne H2 receptor ( H2 R), h i stamine H3 receptor ( H3R), and histamine H4

receptor (H4R) have been described. A l l these receptors belong to the G-protein-

coupled receptor fam i l y and are heptahel ical transmembrane molecules transducing

the extracell ul ar si gnal using G-proteins and i ntrace l l ular second

 5

H,-DNA
Receptor

H,
H1--mRNA
� Transcription
Tr anslation

H2-mRNA
1 Transcnptlon
TranslatlOf'1
I

Intron 3

� Transcription

=��""II!'I==::JI
H 3 mRNA-fu ll length
�. CI
Translation

I ntron 3

H3 mRNA--80 amrno acids deleted

from 3ro lntracellular loop
2. I
Translation

H3 mRNA--80 am[no acids deleted

from 3rd lntracelluLar loop
+ 8 amino acids added 10 C terminus Translation
3. i I I

1 Tmn""'ption

H ... mRNA-fu l1 length

1. IC=================�
Translation •

2. Splice varian1s likely

Figure 1 -2: Transcription and translation of histam i ne receptors( H i l l & Baker. 2004

messenger systems akamura, l tadani . Hidaka, Ohta. & Tanaka. 2000; Oda.

Mori kawa, Saito. Masuho. & Matsumoto, 2000).They transduce extrac e l l ular signals

via Gq. Gs. G ilo and G i/o. respectively; at the H 2 receptor histami ne is a potent

stimulant of cAMP accumul ation whereas at the H3 receptor activation leads to

i nh ibition of cAMP production. The signal i ng mechanisms for the H-\ receptor appear

to involve i ncreases i n i ntracel l ul ar calcium l evels though sti l l under investigations.

In common with other GPC Rs, three recombinant histami ne receptors ( H,-H3Rs)

show constitutive activity, that is, spontaneous activity i n the absence of agonist and,

under these c ircumstances, the antagonists may be rec l assi fied as in erse agonists
 6

( Parsons & Ganel l i n . 2006). The affi nity of histam ine to di fferent HRs varies

signi ficantly. with K, alue ranging from 5- 1 0 nM for the H3R and H..t Rs2- 1 0 mM

for th HIR and H 2 Rs (Endo. 1 982)(Thurrnond et aI. , 2004 ) . H Rs form di mers and

even ol igomer \-"hich al low cooperation between HRs and other GPCRs. Spec i fic

a ti ation or block of HR shows that the di ffer in expression. signal transduction or

function (Hanuskova & Pie ko a. 20 1 3 ; Jutel, Akdis, & Akdis, 2009).

Tabl 1 - 1 : Pattern o f hi tamine receptors distributi on and main actions they

mediat (Hanuskova & P levkova, 20 1 3 ) .
Abbreviation : V - atri oventricular, CNS - central nervous system, GJ -
gastroi ntestinal , NK - natural k i l ler

Recept o r Location Respo nses

u btyp e

blood vessels. ensory nerves i ncrease vascular penneabi l i ty,

HI (smooth muscle bronchi GJ
tract, cardiac tissue
endothel i urn,CN S )
enhance release of histam ine and
vascular bed, nasal epith e l i um,
H2 nasal submucosal glands,
mucosa of stomach, CNS,
cardiac
tissue, uterus, smooth
m uscl e
vasodi l ation,
sti mulation of airway sensory
nerves,
eosi nophi I chemotaxi s,
smooth muscle contraction i n
bronchi and G 1 tract,
stimulation o f vagal nerves
i nducing reflex smooth
muscl e contraction in ai rways,
decrease A V node conduction
time,
arachidonic acid
derivati ves from platelets,
increase nitric oxide production
via iNOs
stimulate mucous glands i n
airways,
increase vascular permeabi l i ty,
direct chronotropic effect on
atri um and i notropi c action
on ventric l e,
rel axation of lower esophageal
 7

pre ynaptic n rves in the
peri pheral sympathetic
adrenergic
system, na al submucosal
glands, C (histami nergic
ner es), airways, GI tract
eosi nophi I s, mast cel ls,
H .j basophi l s, neutroph i l s, na al
turbi nates (nerves), lung, col on,
epicanthus, bone marrow,
spleen, l iver
sphincter timulatio n of
suppr ssor T cel ls.
decrease in neutroph i l and
basophi l chemotaxis and
activation,
prol iferation of lymphocytes,
activity of N K cells
suppression of norepi nephrine
release at
presynaptic nerve ending,
sti mulate nasal submucosal
gland secretion,
opposes bronchoconstriction and
gastric ac id secretion
chemotax is and chemoki nesis of
mast cel ls and
eosi nophi l s, enhancement of the
activity of other
chemoattractants (e.g.
chemoki nes) on eosi noph i l s,
up-regUlation of adhesion
molecules

1.3 H ista m i ne H I-Receptor a n d A n t i h is t a m i n es

A gene on c hromosome 3 encodes for the 4 8 7 am i no acid human H I R prote i n

w i t h a very l arge third i ntracel l ular loop ( 2 0 8 amino acids; through which i t mediates

i nteractions with the G qlll fan1 i ly of G protei ns) and a relatively short ( 1 7 am ino

ac ids) intracel l u l ar C-termi nal tai l . Sti mulation of the receptor-G q/ l l protein complex

results i n activation of the enzyme phosph o l i pase C � l eading to the formation of the

i ntracell ular second messengers i nositol 1 , 4, 5-triphosphate ( I P3) and diacy lglycero l .

I P3 is rel eased i nto the cytosol and then stimulates the release o f intracel l ular free

ci+ ions (which, for example, can result in smooth muscle contraction) whi l e

diacylglycerol can activate protei n k inase C and mediate responses such a s cell
 8

pro l i feration and the activation of the lrasncription of pro-i n fl ammator gene ( H i l l &

Baker, 2004)

Antih istam ine . the va t maj ority of wh ich exert their anti histaminic action by

acting as inver e agonists at the H,R receptor , are the econd most commonly used

cia of medication after antibiotics and have well -documented anti-al lergic and

anti-inflammatory effect ; HI R antih1 tamines are traditiona l l y classi fied i nto six

group ba ed on their pharmacological structures: ethanolam i nes eth y l ene diam ines,

alkylam i ne , p i perazine, piperidi nes, and phenothiazines. Yet, due to l ack of c l i nical

relevance, cunently HI Rantagon ists (antihistami n s) are c l assi fied as 'first

generation' edating antihistam ines) and 'second generation' (relati ely non-

edating). Fi rst-generation antihistam ine such as chl orphenamine (CLP),

cyproheptadine (CYP), hydroxyzine ( H YD), and promethazine (PMT) are highly

l i pophi l i c . ha e low molecular \ eights and are not recogrUzed by the P-glycoprotei n

efflux pump \vhich makes them penetrate read i l y i nto the blood brain barrier.

Additionally, they are non- elective in binding to the H I Rs because most of them

have weak antimuscari nic anticho l i nergic effects, some show al pha-adrenergic

blocking effects (As a phenothi azine HI -antih i stam i ne, promethazine exhibits

anticholinergic, local anesthetic, sedati ve, and weak a-adrenoceptor antagonistic

actions ( Meyler's ide Effects of Drugs : The I nternational Encyclopedia of Adverse

D rug Reactions and I nteractions, 2006a)), and others are inhibitors of both h i stamine

and 5-hydroxytryptam i ne activity (cyproheptadine).

These compounds are also used as sedati ves, sleeping aids, anti-emetics and

in the treatment of vestibular disorders; however, the wide variety of adverse effects

attributed to this c l ass such as antimuscarinic anticho l inergic effects (dry mouth,
 9

bl ulTed ision and d sfunctional urine oiding), gastrointestinal up et, jalmdice and

pancytopenia cannot be neglected . Moreover, some fi rst-generation H,R

anti histami nes are drugs of abu e leading to euphoria and hal l ucinations. Second­

generation H, R antihistam ines such as cetiri zine, fexofenadi ne, loratadine and

mizo l astine, each \\ i th di fferent tendencies to occupy central nervous system H,R

(Yarie from none for fexofenadine to 3 0% for cet i ri zine) ,are considerably less l i kely

than fi rst-g nerati n drug to cau e adver e effects; They penetrate poorly into the

, and have significantly Ie s affi nity for muscari nic cho l i nergic, al pha-

adrenergic and 5-hydroxytr ptami nergic receptors (Mahdy & Webster, 20 1 1 ) .

Rec ntl indicated, both sedating and non-sedat ing anti histami nes have the potential

to affect reaction time and ph siological tremor (Ka anagh, Grant, & Anoopkumar­

Dukie, 20 ] 2) most l i kel due to altered neurotransmission in chol inergic and

h i staminergic pathways.

Beari ng i n m i nd that anticho l i nergic effects o f H l antihistam i nes have been

c l arified only in muscarinic ( but not nicotinic) acetyl chol i ne receptors, the cUlTent

study i nvestigates effects of d i fferent histamine receptor antagonists on functions of

a7- nAChRs, which are subtype of Nicotinic Ach receptors. HI and m uscari nic

acet lchol i ne receptors al l being G-Protein coupled receptors ( GPCR) share a

sequence s i m i l arity of approximatel y 45% that faci l itates binding of antihistami nes to

M receptors leading to modulation of the chol inergic transm ission(Howarth,

1 999;Merk, 200 1 ). N icot i n i c acetyl choline receptors on the other hand, are members

of l i gand gated ion channels fami l y . The pharmacological behavior of the nAChR i n

three basic steps, starts with the neurotransmitter acetylcho l i ne (ACh) binding t o the

receptor, which is fol l owed by the openi ng of the i ntri nsical ly coupled ion channel

with subsequent ion fl ux activity . F i nal l y, the AChR in a process, where the ion
 10

channel becomes c l o ed I n the prolong d pre ence o f h. becomes desensitized

(Ki m e t a I . , 2006).

Variety of tudies ha et up n icotinic y terns as im portant pl ayers of

cogniti ve functi n . C n idering the effect of h i stam inergic drug on cognitive

perfo rmance, histamine s em to be an i nterest ing candidate for impo rtant

i nteraction \-,"ith nicoti nic y tern regard i ng cogn itive function ( Kholdebarin et aI . ,

2007). W h i l e therapy trategie for cogni tive i m pairments have traditi onal ly been

target ing the choli nergic neurotransmi sion, treatments with cho l inesterase inhi bitors

or mu cari n i c agonists have been genera l l y unproducti ve( B l andina, E foudebe. Cenn i,

M annaion i . & Passani , 2004 ). Nevertheless, this study was conducted to investigate

e ffect of hi tamine receptor antagonists on function of the c loned human nictotinic

acetylcho l i ne receptors i n xenopll leQ1'is frog oocytes expression system.

lA C h e m i cal, Fu n c t i o n a l a n d T o x i c o l ogica l p ro fi l e of a n t i h ista m i n es

Antihistamines act as competitive antagonists of histami ne at HI histamine

receptors, thus inhibiting HI receptor-mediated reactions, such as vasodi latation,

sneezing. and itchi ng. H istamine release from mast cel l s and basophi l s makes a

maj or contribution to the a l l ergic response, and ant i h i stami nes are widely used i n the

treatment of certain symptoms of al lergic di sease.

Table 1 -2 : Structural and functional c l assi fication of HJ R antih i stami nes.

 11

h e m ica l cia Fu n c t i o n a l cia

Fir t (o l d ) genera t i o n Seco n d (new)

genera t i o n

A l ky l a m i n e Chlorpheniramine Acrivastine
P i p e razi n e Hydroxyzine Cetirizine
P i p e ri d i n e K totifen loratadine
E t h a n o la m i n e Di phenhydram ine, -
Orphenadri ne
E t h y l e n ed i a m i n cs Pyri l amine (M pyram ine) -
P h e n o t h iazi n e Promethazine -

Oth e r Doxepin, Triprol idine Azelastine

Within a fev\' year of the discovery of the fi rst antihistam ine, phenbezamine

( ntegan), tlu'ee other antihi tami nes became avai lable and are sti l l in use today :

mep ram me ( pyri l am i ne ) maleate ( Bovet, Horc lois, & Wal thert, 1 944).

diphenhydram ine ( Lowe, M ac M i l l an, & Katser, 1 946), and tripelennamine

( Yonkman, Chess, M athieson, & Hansen, ) 946). Despite their pronounced adverse

effects these were the fi rst rea l l y useful drugs for the symptomatic rel ief of al lergic

disorders (Meyler's Side E ffects of Drugs : The I nternational Encyclopedia of

Adverse Drug Reactions and I nteractions, 2006b) . Duri ng the l ast 35 years the so-

cal led second-generation HI antihistam ines have emerged with greater potency,

longer durations o f action, and m i n i mal sedative effects. W ith a few exceptions,

antihistamines are rapidly and completely absorbed after oral administrati on: peak

p l asma concentrations are reached after 1 -4 hours and are highly vari able, owing to

d i fferences in tissue distribution and metabol ism ( Walsh et a I . , 200 1 ).

According to the I nternational Encyclopedia o f Adverse Drug Reactions and

I nteractions, fi rst-generation antihistanu nes, besides i nteracting with HI histam ine

receptors, also have affi ni ty for 5-HT receptors, al pha-adrenoceptors, and m uscarinic

receptors. Moreover, they reduce cyc l i c GMP concentrations, mcrease

 12

atrioventri cular nodal conduction, and inhibit acti ation of ai rway vagal afferent

nerve . They ea il eros the blo d-brain barrier, and their consequent v. ell­

documented edative and anticho l i nergic effects, together with hort hal f- l i ves,

greatl ) l i m i t their u e in th treatment of a l l ergic symptoms. evertheless, first

generation drugs are sti l l widely consumed mai nly in combination with other drugs

as v r the counter products. The incidence of adverse effects is very high with these

fi rst generation drugs perhaps up to 50%, espec i a l l y in telms of sedative and

anti mu cari nic e ffect . The marked anticho l i nergic effect of these compounds can

cau e dl) ne s of the oral and respi ratory mucousa; other anti muscarinic effects are

less common but nasal stuffi ness, blUlTing of vision, uri nary retention and

consti pation can all occur. A l though these adverse effects are rarel y serious and often

d i sappear with continued therapy, they o ften are so troublesome that medication must

be withdrav.n. Prolonged use of combination products conta i n i ng anti histami nic

decongestants have been reported to cause facial dyski nesias includ i ng

b1epharo pasm, swal lowing d i fficulties and dysarthria. I n 1 999 Chan-Tack reported

neuroleptic malignant syndrome among extrapyram idal symptoms of promethazine

antih i stam i n (Chan-Tack, 1 999) due to antidopaminergic e ffects.

q uesti onnai re showed that women taking anti histami nes and/or cold

fom1Ulations had a tone average 9 d B higher than those not taking such medication

(LEE, M ATTHE W S, M IL L S, DUBNO, & A D KINS, 1 998). Audiography showed

d i fferences in threshold of 6.4 and 1 2. 8 dB at 500 and 1 000 Hz respectively. The

medications i nvolved were primarily mec lozine for dizzi ness and terfenad i ne for

allergy.
 13

ervou system timulation is less frequent than C depression. But v hen

occur . it cau. e in omn ia, i rritabi l i ty and tremor; nightmare and hal lucinations. I n

ovelt i n t xication, these e ffects may b e rel ated t o antichlol inergic e ffects. Drugs that

hav e effect on the brain ( hypnotics. sedati ve , narcotic analgesics, neuroleptic drugs,

ale hoI, l i tll ium, anticon ul ants) wi l l interact with antihi stami nes, especi a l l y the

li rst-generLltion drug .

On the other hand, the 2nd generation antihi stami nes are rel atively free from

anticho l inergic. anti erotonergic and alpha-adrenergic activity and being rel atively

hydrophi l i c they cause marked ly less sedation ( Druce, 1 990; SIMONS, 1 989;

Trzeciako\-y k i & Levi, 1 98 3 ) . I n a double- bl ind, cross over study l evoceti rizne Smg

once-daily for 4 days wa compared with cetri zine 1 0 mg loratadine 1 0mg,

promethazine 3 0mg, and placebo in terms of CNS i nh i bitory effects in 20 healthy

volunteers(H i ndmarch, Jolmson, Meadows, K i rkpatrick, & Shamsi, 200 1 ) W ith the

exception of promethazine none of the drugs had disruptive or sedative effects on

obj ective measurements in a comprehensive battery of psychomotor and cognitive

tests.

There are several novel anti histam ines ( incl uding desl oratadi ne, fexofenadi ne,

levocabast i ne, and l evoceti ri zine) that are either metabo l i tes or enanti omers o f

existing drugs. These have been devel oped i n response t o widespread concerns about

the potential for cardiotox icity (Although it is widely bel i eved that cardiotoxicity of

antihistan1 i nes is l i m i ted to second-generation compounds, both hydroxyzine and

diphenhydram ine can block potassi um channels) and the i m pact of drug-drug

interactions associ ated with some earl ier second-generation HI receptor

 14

antagoni sts( yler' ide Effect of Drug : The Intemational Encycl opedia of

Adverse Drug Reaction and Interactions, 2006b).

Common t r u c t u ra l Fea t u re o f cia s i c a l fi r t genera t i o n a n t i h ist a m i n es :

_ ar matic ri ngs, connected to a central carbon, nitrogen, or oxygen.

pacer between central atom and the amine, usually 2-3 carbons in

length. (Can be l i near, ring, branched, saturated or unsaturated) .

The amine i ub tituted with small alkyl groups

Chiral ity at X and hav i ng the n ngs l D d i fferent pl anes lDcreases

potency of the drug.

* A l ky l am i ne drugs lack the ··spacer molecule" (which is usually a

nitrogen or oxygen) between the two aromatic ri ngs and at least one of the

rings has n itrogen i n c l uded in the aromatic system .

Figure 1 -3 : Common Structural Features of cl assi cal first generation antihistam ines.

The early antih i stami nes bore some structural resemblance to histam i ne and

l ike histami ne, contained an ethy l am i ne group; but, the structures o f the many

antihistam ines that are avai l able are unrelated; however, the traditional c l assi fication
 15

i acconl i ng t o the ch m ical tructure. In view of that, the fol lowing anti histamine

[rom di fferent groups \-vere used in our tudy.

1.5 Acety l c h o l i n e a n d t h e c h o l i n e rgic p a t hw ay

.. cet Icho l i ne (ACh ) i the neurotransm itter at a l l autonom ic gangl ia, al l

p tgangl ioni para ympatheti c nerves, postganglionic sympathet ic nerve

inneryating endocrine weat gland , the neuromuscular j unction, and neurons in the

central nervous ) stem. ACh is synthesized by the acety lation of choline and acetyl

coenzyme by chol ine acetyltransferase. Depolarization of chol inergic neurons

rel ea es Ch by exocytosi i nto the various synapses where it acts on muscari nic

( 1 1 -Ms) metabotropic receptor or nicotinic (Nm or Nn) ionotropic receptors. ACh is

i nactivated by acetylchol i ne esterase to acetic ac id and chol i ne . Fol lowing reuptake

i nto chol i nergic neurons, cho l i ne is reused for ACh synthesis", T.e. Westfal l,

Ency lopedia of eurosci ence. Moreover, ACh is a neurotransmitter widely

distributed in the central (and also peri pheral, autonom ic and enteric ) nervous

ystem. It is tared i n presynaptic vesicles, released intothe synaptic c l eft, and binds

to t\-vo dist i nct types of receptors : nicoti n i c Ach CnACh) receptors and m uscarin i c

A c h ( mA C h ) receptors. Neuronal nACh receptors are located in pre-and post­

synapti c membranes of neurons and also in extra synaptic locations throughout the

C S. I n the cho l inergi c neurons (they i n c l ude cel l s running from the c ranial nerve

n uc l ei of the brai n stem to the medu l lary tegmentum and pontomesencephal ic

tegmentum, continuing rostra l l y through the d iencephalon to the telencephalon

( W oo l f 1 99 1 )) ACh action i n t h e synaptic c l e ft is terminated b y AChE, a n enzyme

that hydrolyzes acetylchol i ne i nto cho l i ne and acetate. Conti nued ACh production

and release is suppo rted by a presynaptic h igh-affinitychol ine transporter (CHT)

 16

\\ hich recover almo t 5 0% o f the cho l ine derived from the eh hydrolys i ( Figure

1 -4 ).

:l.:tl"l

Figure 1 -4 : Schematic representati on of a chol i nergic neuroeffector j unction

( Westfal l . 2009)

In addition to cranial nerve n uc l e i . there are three major cholinergic subsystems i n

the brain:

1. chol i nergic neurons originating from varIOUS basal forebrain

nucl e i ( particularly, the nucleus basa l i sMeynert), from where t hey i lU1ervate

main l y the cortex ( e . g . neocortex. c ingulate cortex) and h ippocampus.

2. cho l inergic neurons originatingfrom brai nstem nuclei ( e . g. the

pedunculoponti ne and l aterodorsal tegmental nuc l e i ), from where they

provide widespread i nnervations to the thalamus and midbrain dopami nergic

 17

area and also d scendi ng inner ation t o the caudal pons and brain stem

( Dan i , J. A . , & Bertrand, 2007). The l aterodor al tegmental nucleus projects

mai nl to the ventral tegmental area; the pedunc ulopontine nucleus

i nner ates both the ventral tegmental area and the substantiani gra( Maskos,

2008 : Mena- egovia, J . , W i nn , P . , & Bolam, 2008; esack, S. R . , & Grace,

2 0 1 0).

3. Choli nergic subsystem arisi ng from a col lection of chol inergic

i nterneuron in the striatum, which provides very dense local innervation.

These interneurons constitute about 1 -3% of the stri atal neurons and interact

with the rich dopam i nergic i nnervation of the striatum ari sing from the

substantianigra pars compacta and the ventral tegmental area (Zhou, F. M . ,

W i l son, C . 1 . . & Dan i , 2002 ).

 18

Figure 1 -5 : Central chol i nergic pathways regulate global functions.

These pathways rely upon the cerebral cortex; such functions include attention,
arousaL motivation, memory and consc iousness .The basal forebrain contai ns two
group of cho l inergic neurons: 1 ) the medial septal group (medial septal nucleus and
vertical diagonal band : ms and vdb) that proj ect chol i nergic axons to the
hippocampus and parahi ppocampalgyrus and 2) the nucleus basa l i s group (nucleus
basa lis, substantiai nnomi nata and horizontal diagonal band: bas, si. hdb) that proj ect
chol inergic axons to al l parts of the neocortex, parts of l i mbic cortex and to the
amygdala. The choli nergic pontomesencepha lon neurons ( laterodorsal tegmental and
pedunculopontine tegmental n uc l e i : lelt and ppt) proj ect onto forebrain, thalamus
hypothalamus and basal forebrain. This fi gure is reprinted from Progress in
eurobiology, 37, Woo l f, N. J., Chol i nergic systems i n the mammal i an brain and
spinal cord p. 482 ( 1 99 1 )

Moreover, the cho l inergic systems h ave been the centre of attention when i t

comes t o di seases containing cognitive defic its such as A l zheimer Disease and seems

to play a pivotal rol e in l earning and memory . Cognitive i m pairments in AD pat ients
 19

are mai nl) characterized by the 10 of cho l i nergic neurons which are the most

affected in the disea e. In the central ner ous system ACh has a variety of effects as a

neur m dulat r involved i n synaptic plasticity and stabil ity. Acetylcho l i ne enhances

the amp l itude of naptic pot ntial fol lowing long-ternl potentiation ( LTP) in many

r gion of the brain but mainly in the neocortex, CA I , dentategyrus and piriform

cortex . ACh receptors (ACh Rs) decrease the conductance of vol tage-gated and Ca2+_

dependent curr nts (Conej ero-Goldberga Daviesa, & U l l oa 2008)An overarching

reocculTing theme has been that lesions of the cholinergic proj ection neurons of the

basal forebrain impact negati ely on acquisition learn i ng and blockade of m uscarinic

receptor i n the target structures produce simi l ar behavioral deficits(Robi nson, L . ,

P l att. B . , & Riede l , 20 1 1 ). Although both m uscari nic and n i coti n i c chol i nergic

receptors are known to modulate brain electrical activity, i n recent years, there has

been increasing i nterest i n de elop i ng drugs targeted to brain nAChRs (Ashoor,

2 0 1 1 ); Among the nAChRs, the arnAChR has maj or c l i n ical and pharmacological

i m p l i cations for AD. This receptor also appears to trigger altemative signal pathways

i n neuronal and non-neuronal cells. I n neuronal cel l s activation of the arnA C h R

causes an increase i n i ntracell ul ar Ca2+ directly t hrough voltage activated channels

(activation of ERK l /2 in a Ca2+ and PKA dependent maruler) ( F A Daj as-Ba i l ador,

Sol i akov, & Wonnacott, 2002) and indirectly from i ntrace l lular sources fol lowi ng

nicot i n i c ryanodine receptor channels activation (Federico A Daj as-Bai l ador, Mogg,

& Wonnacott, 2002) . In astrocytes, the arnAChRs appears to modulate Ca2+ rel ease

from intracel l ul ar stores ( Sharma & V ij ayaraghavan, 200 1 ) . In neurons, nicot i n i c­

i nduced i ncrease i n i ntracel l u l ar Ca2+ modulates glutamate and GABA activity

(Maggi, Sher, & Che rubini, 2 00 1 ; Radcl i ffe & Dani , 1 998) and regul ates CREB, the

transcription factor invol ved i n the fonnation of the LTP ( Lynch 2004 ; S i l va, Kogan,
 20

Frankl and, & Kida, 1 998 via gl utamatergic transm ission ( H u, 2002). Furthermore,

the <Xrn ChR are widel distributed in brain. Radioligand binding of [ I 1 25 J <X­

bungarol x i n how a hi gh to moderate even di stri bution of the <X7-nAChR in the

A1 region of the hi ppocampu and entorh inal cortex ( 1 . . Court, Martin-Ruiz,

Graham, & P rry, 2000 ) . I n the prefrontal and the motor cortex, the <XrnAChR

local i zes in the pyram idal nemon of tile layers I lII n , V and VI ( Wevers et a! . , 1 994).

AI o. <xrmRNA has high and intermedi ate expression in tbe nuc leus reticu1aris and

lat rallmedial geniculate bodie as opposed to the low expression in the thalamu

( B ree e et a1 . , 1 997; 1 . . Court e t al . , 2000; Rubboli et al . , 1 994 ; Spmden et al . ,

1 99 7 ) . I ntere tingl , basal gangl ia have h i gh expression of <XrnAChR in substantia

nigra, i nternlediate i n caudate and putamen and a lower expression in tri ahlm

( Court et al . , 2000). Cerebe l l um exhibits strong expression of <XrnAChR in

selectively Purkinge cel l s and shows higher density i n the molecular than in the

granular l ayer (Court et aI . , 2000).Decl i ne in nicotin i c receptors, particularly the <Xr

nAChR in the frontal cortex, is associ ated with aging ( Utsugisawa et a l . ,

1 999). Herein, CHRNA 7 knock-out m Ice have been found t o show cognitive

dysfunction, i nc l uding attention and worki ng memory deficiencies ( Fernandes,

Hoyle, Dempster, Schal kwyk, & Coll ier, 2006; Sousa, Fernandes, & Ramos 2006;

Young et a l . , 2007); L ikewise there is general consensus that the <XrnAChR plays an

i mportant role in inform ation processing in humans. Epidemiological studies show

that nicotine decreases the risk for Parkinson's disease ( P D ) and AD ( Fratiglioni &

Wang, 2000). Thi s negative association is i n agreement with postmortem studies

showing d i m i nutions of amyloid p l aque deposits in former smokers with AD

( H e l l strom-Li ndahl et a l . , 2004). Ni cotine treatment appears to i nterfere with the

formation of the amyloid plaques i n vitro and i n vivo ( H e l l strom-Lindah l , Motlsavi ,

 21

Ravid. & rdberg, 2004; Ono, H a egawa, Yamada, & aiki, 2002; Ut uki et a1 . ,

2.002 ) and t o reduce t h accumulation of insoluble b42 peptides(Nor dberg et a I . ,

2002)through a mechani m medi ated b y arnAChRs a s sho\'" n with mecamy l am i ne

that blo k the increa e f PP produced by treating S H - Y5Y neuroblastoma cel l s

with nicot ine ( H e l l tram - L i ndahl e t a1 . , 2004). Several c l i nical tri als i ndicate that

chronic adm i n i, tration of nicot i ne in i ndividuals with age-associated memory

im pairments and A D improves cogn ition i n attention but not in memory (Snaedal et

aI . , 1 996: White & Levin, 1 999,2004 ; W i l son et a1. 1 99 5 ) . Simi lar studies indicate

that ABT 4 1 8 . a nicotinic a4p2-agon ist, improves memory and learning ski l l s in AD

patient ( Potter et a I . , 1 999). I n conc l usion, for AD, nicotine improve performance

in attention through thea7-nAChR , wh i l e memory improvements seem to be dri ven

through th a4P2-nAChRs.Nicotine seems to protect agai nst the dev lopment of AD

and P D through anti-i nflammatory mechanisms. Both AD and PD are characterized

by l ocal i n fl amm a tory responses sustai ned by activated microgl ial cel l s. Nicotine

i nduces anti i nfl ammatory mechanisms that dimi nish local i n flam matory responses

( H e l l strom-L indahl et a I . , 2004; Streit 2002 ; Wang et a I . , 2000). Among other,

n icot i ne abrogates the production of TNF i n culture of microgl ia t hrough a

mechanism dependent on ERK and p 3 8 MAPK ( S hy t I e et aI . , 2004; S uzuki et a I . ,

2006). Experi mental mode l s of AD i ndi cate that arnAChR i s the central core of the

nicotine-medi ated neuroprotection. N icotine decreases accumulation of beta-amyloid

i n the cortex and hippocampus of APP (V7 1 7 I ) transgenic mice. These studies also

indicate that nicotine prevents the acti vation of the N F-kB and c-Myc pathways by

inhibiting the ERK and p3 8 MAPK ki nases via arnAChRs ( L i u et a I . , 2007). This

mechanism of the arnAChR function was con fi rmed by using RNA i nterference in
 22

the e. peri ment f nicoti ne-mediated neuroprotect ion ( For a reVl \ ee Conejero-

Goldberga et a I . , 2008).

1 .6 cety lcholi n e Receptor

There are two type of acetyl chol i ne receptors, muscan mc ( mAChRs) and

nicotinic receptor ( nAChRs). Both mu cari nic and nicoti n i c acetylcho l i ne receptors

are not pec i fi c of the c ntral sy tern a they al 0 appear in the peri pheral nervous

tem.

euronal mAChRs are metabotropic G-protein coupled receptors stimulated

b acet lcho l i ne and muscari ne and bl ocked by atropine. They also activate other

ionic channels via a second messenger cascade.

The nicot i n i c receptors ( nAChRs) are cation-selective, l igand-gated ion

pentameric channels composed out of selected al pha and/or beta subunits. The

combination of these subunits defines the function and affi n i ty of the receptor for

speci fic l igands. N i cotinic receptors are stimulated by acetyl c ho l i ne and nicotine, but

blocked by c urare, a-conotox in, a-bungarotoxi n or mecam y l am i ne (See I tier &

Bertrand. 200 1 ; Jones, Sudweeks, & Yakel 1 999)

1 .6. 1 Nicotin i c acety l c h o l i n e rec e p t o rs

"The n i cot i n i c acetyl c ho l i ne receptor is a transmembrane al losteri c protei n

that mediates transduction of chemoelectric si gnal s throughout the nervous system by

openi ng an i ntrinsic ionic channel . This rapi d pore open ing enables flow of Na+ , K + ,

and. i n several i nstances, Ca2+ ions across the c e l l membrane. As a consequence,

nicot i n i c acety l c ho l i ne receptors e l i c i t fast changes in the membrane electric

potential, but they also regul ate transmission of electric signals by c losing the pore

through slower desensiti zation transitions. As such nicotin i c acety lchol i ne receptors
 23

play c rucial physi ological ro le and, when altered, they cause pathologies 1 0

human . J .-P.
" hangeux, Encyclopedia of euro cience.

icotinic acetylcho l i ne receptors (n ChRs), ar members of the Cys-Ioop

J igand- gated ion channel superfam i l y whichshares structural and functi onal

properties with other ionotropic receptor , such as 5-hydroxytryptam ine type 3, }'­

ami nobutyric acid A receptor, glyc i ne receptorsand gl utamate-gated chloride

hannels (GluCI); The fi rst two are cation-selecti e, whereas the last three are anion-

elective. In this fam i l , channels are constructed of at l east an extrace l l ular

domain that i the l i gand-bi nding domain ( L B D ) and a transmembrane domain

(TMD) that contain the pore for ion i n fi l tration. Addi tional domains m ight be

pre ent that play otherfunctional rol es such as regulation of channel activity and

trafficking; Cys-Ioop receptors l i gand binding sites are located in the i nter-protomer

interfaces(Du, Dong, & Zhou, 20 1 2 ) . nAChRs are penterm ics composed of fi ve

subunits (2a,�,y and 0 or c) ; M amm als have 1 6 nACh receptor subun it-encoding

genes. fi ve of which function at the neuromuscular j unction whi l e the remai ning

subunits are neurona l · Depending on the location of the receptor, i t has a different

combination of subunits; A l l these subtm i ts share 3 5-50% homology with one

another. At the neuromuscular j unction (N I or N m ), the AChR is composed of two 0.

(0. 1 ) subunits; one /3 (/31 ); one b; and i n embryonic or denervated musc le, one y

subuni t . I n the adult i nnervated m uscle the y is repl aced with one ( subunit.

Functional neuronal nAChRs (eNS No) also ex ist as pentamers composed of two 0.

and three /3 subunits. Autonomi c ganglia (N2) and the adrenal medul l a form

homomeric0.7 and heteromeriw31/34, ( Westfal l , 2009). The characteristics of subtypes

of nAChR are descri bed in Table 1 -3 . Each subunit is composed of four

transmembrane spanning regions, M 1 , M2, M3, M4, M 2 form ing the i nner portion of
 24

the i n channel . The ag ni t binding site is located at th i nterfacia l region of one

al pha ubun it (prima com ponent and one non alpha subunit (complementary

component). icotinic rec ptor act as cation channels at the pre orpost-synapse of

the neuron to sti mulate act ion potentials and at the neuromuscular j unction to

timul ate mu cle contractions: The phatmacological behavior of the nAChR in three

basic teps, tat1s with the neurotransmitter acetylcho l i ne (ACh) binding to the

receptor. fol lowed b the opening of the i ntri nsical ly coupled i on channel with

ub equent ion n ux activity. Finally, the AChR, becomes desensitized in a process,

where the ion channel becomes closed in the prolonged presence of ACh ( K i m et a!.,

2006).

Table 1 -3 : Adrenergic agonists and antagonists

R e c e p t o r ( p r i m a ry M a i n s y n a pt i c M olecular
I\ l e m b r a n e re p o n s e A g o n ists A n t a go n ists
receptor u b ty p e ) location mechanism

Atracurium V

I Skeletal m uscle Skeletal Increased ecuronium d-

I(NOl) (a l )2� 1 ( 0 neurom usc u l Excitatory; e n dplate

ar j unction
cation
depolarization; skeletal penneabi l i
ACh N icoti n e
Tubocurarine
Pancuronum
Adult ( U I )2 � 1 y o Succinylcho l i ne
( postj unction m uscle contraction ty ( N a+ ; a-conotoxi n
Fetal
al) K) u-
bungarotoxin

Excitatory;
I ncreased
Auton o m i c depolarization fi ri n g of ACh N icotine
Peripheral cation Trimethaphan
ganglia; postganglion n e uron ; Epibat i d i n e
neuronal (N,,) permeab i l i Mecamy lam i

(u3h (4)3
adrenal depolarization and D imethy lpheny l p i
ty ( Na + ; ne
medu l l a secretion o f per-aZlllum
K)
catecholamines

Pre-and postsynapt ic
I ncreased Mecamy l a m i
Central neuronal CNS; pre- excitation
cation Cyt isine, neDi hydro-�-
(CN S) (u4h and Prej unctional control
penneab i l i epibatidine erythrodineEr
(�4h ( a-btox- postj unctiona of transm itter release
ty (Na + ; A natox i n A ysod ineLoph
insensitive) I Prej unctional control
K) otoxi n
of transm itter release

Adapted from Westfal l TC and Westfa l l DP (2006 ) ; l n : Brunton LL, Lazo JS , and
Parker KL (eds . ) Goodman and G i l man' s the Pharmacological Basis of Therapeuti cs,
1 1 th edn . , pp. 2 3 7-296. New York : McGraw- H i l l .
 25

1 . 6. 1 . 1 c u ro n a l n i cot i n i c rece p t o r

eur nal n A h R s are cation- el ective, l i gand- gated i o n channels expressed

in b th the and the P icotinic AChR are the main mediators of gangl ionic

fa t ynaptic tran m i ssion. and, i n thi re pect, the function a an i nterface between

nervous system and peri pheral organ Langley in the 1 800s, e tabli shed the key role

[ nA ChR in the that autonom ic ganglia are fi rst activated and then bloc ked by

nicotine. The neces ar ro le of nAChRs for synaptic transmission in the peripheral

nervou y tem ( P ) contrasts with the role of the receptors in the central nervous

ystem (C ). v,'here the most conm10nly observed nAChR rol e is to act

presynaptica l l y to enhance neurotransmitter rel ease. The manm1al ian nervous system

expre es transcri pts for al l ubunits except a8, which is found only in avians. I n the

rat there are two functional splice variants for the a7 subunit. The a7-2 isoform

contains an 8 7-base-pair i nsert that represents the incorporation of a novel exon

between exons 4 and 5 of the conventional a7 gene product (a7- 1 ). The two isoforms

are found in both the CN and PNS. a7 can form e i ther homopentamers or alb

heteropentamers. The a}-nA ChRs subun it expressed i n autonomic gang l i a has been

sho\\TI to form homomeric channels i n heterologous expression systems, but the

l iterature suggests that it m ight also form heteromeric channels in native ganglionic

neurons( De B iasi. 2 009). The presence of the heterologous a7b2nAChR in the

cholinergic basal forebrain and its sensitivity to beta-amyloid (Ab) suggest that this

nAChR subtype may have rel evance to AD i n particular ( Wal l ace & Porter,

20 1 1 ) . The homomericarnAChRs receptor is one of the most abundant forms of

nACh receptors in the m amm alian brain and appears to play rol es in the

development, d i fferentiation, and pathophysio logy of the nervous system ( Dani, 1 .

. . & Bertrand, 2007; Lindstrom, 2003 ) .

 26

The cat ion permeabi l i t of t h e ubt p e (nACh receptors are pem1eable t o the

m n valent a " and K + ion , a w II a to 2

a ) is i n fl uenced by the i r subun it

compo ition ( Couturier, .. Bertrand, D . , Matter. J. M., Hernandez, M. c . . Bertrand,

., M i l l ar. " 1 990; eguela. P., Wadiche, L Dineley-M i l ler, K., Dan i , J . A., &

Patrick, 1 993 ) ; hetero pentameric nACh receptors, comprising u and � subuni ts, have

2!-
lower measured a permeabil ity. Fol lowing nACh receptor stimulation, Ca2+ can

enter cel l s ei ther directly, thl'ough the intrinsic ion channe l , or indirectly fol lowing

ol tage-gated channel acti vation by nACh receptor-induced

depol arization( shoor, _ 0 1 1 ) . The unique characteristic of the ionotropic neuronal

2
urnAChRs in its preference for Ca + over other cations is one of the most interesti ng

2
with regard to it potential for cognitive enhancement in that Ca + has wel l

recogni zed i nvolvement i n mediating intracel lular signal ing cascades and synaptic

2
pIa ticity . The urnACh Rs has a cal c i um to sodi um permeabil ity ( PCa + : PNa+ ) ratio

of approxi matel y 1 0 as demonstrated i n Xenopus oocytes expressing the U7-nAChRS,

which is greater than any of the other nAChRs and the M DA receptor which shows

2+
a PCa :p a+ ratio of approximately 5 . The a7nAChR-mediated Ca2+ current is

2 2
regul ated by activation of Ca + -induced Ca + rel ease from internal stores, and is
7+
d i fferent than other nACh Rs which are coup l ed to oltage-operated Ca- channels

( Wal l ace & Porter, 20 1 1 ) .

1.6. 1. 1 . 1 a7- n AC h Rs

U7-nAChRS are mostly homopentamers of the a7 subunit with both ACh and

chol ine acting as its endogenous l i gands; These receptors have some unique

properties: an extremely low open probab i l ity ( Pest i , Szabo, M ike, & Vizi, 20 1 4 ) ; I n

contrast t o heteromeric nAChRs, when a population of homomeric a 7 receptors are

stimulated by a rapid increase i n ACh concentration, as occurs at a synapse each

 27

acti vatable rccept r ha o n l y a 0 . 3 % probabil ity of opening, and under steady-state

conditions the probab i l i ty of any ingJe a7 receptor being open is less than one in a

m i l l i on. The a erage t i mes � r a7 receptors openings are typical l y less than 1 00 �s

and usua l l y occur in i olation. When a rapid appl ication of a concentration of ACh

suffi cientl high to aturate the agonist bind ing sites is made to a popUlation of a7

receptors, the maxi mal nchronous transient acti vation occurs when on ly a fraction

of the agoni t binding si tes are occupied that is suggestive of the allosteric effects of

binding to j u t one or two of the fi ve possible binding sites promoting channel

open i ng which is the same case ob erved with other heteromeric receptors, but

unique to a7 i that, at higher levels of binding, the receptor is most l i kely to adopt

nonconducting confo rmations. Though the desensitized states of a7 receptors do not

how sign i ficantly increased affin ity for agonists compared to the resting state of the

receptor highly selecti ve po iti ve al losteric modulators ( PAMs) can destabi l i ze this

concentration-dependent form of desensitized state and thereby greatly increase the

probab i l i ty of channel openi ng ( Papke 20 1 4). Another unique property of alpha7

receptors IS a high Ca2+ penneab i l ity counterbalanced by extremely fast

deseensitization, causing a fast onset and decay k inetics(Pesti et aI., 20 1 4 ) However,

whi l e the prolonged presence of a low concentration of ACh (or other agonists), w i l l

i nduce h i gh l evels of heteromeric receptor desensitization, a7 receptors wi l l remain

responsive to fluctuating stim u l i ( Papke, 20 1 4).The short open t i me and rapid

desensitization act as mechanisms that protect urnAChRs expressing cells from

excessive and thus, damaging Ca2 + i n fl ux (Uteshev, 20 1 2 )

The i m portance of urnACh Rs can b e impl ied b y highlighting their

expression pattern :
 28

• a7-containing n ChR are l i kely the mo t abundant neuronal-

type r c ptor in the bronchial epithelial cel l s and ha e been reported to ha e

phannacological and bi ophysical properties simi lar to those of ganglionic

receptor and are invol ved i n mai ntai ning the flat shape necessary for those

I l s to l i ne the bronchial surface. Those receptors cem to modul ate the

synthe i f the extracel l u lar matrix glycoprotein fi bronectin that is

impl icated i n tis ue i nj ury and repair and is highly expressed i n tobacco­

rel ated I lmg diseases.

• The a7 subunit was found in al l vessels exam i ned (except

tho e in the kidney) in mechanisms w1derly i ng the m i gration of endothe l i a l

cel l s i nduced b y angiogenic grow1h factors such a s vaccinia i rus growth

factor (VGF). Thi sho s part i c i pation of a7-contai ning nAChRs in these

mechanisms.

• Vagus nerve sti m u l ation attenuates tumor necrosIs factor

(TNF) rel ease from macrophages, and this effect is abol i shed in a7 null mice.

ACh released from the vagus nerve IS thought to modulate

monocyte/macrophage activity by i n hibiting proinflammatory cytok i ne

production and systemic i n fl ammation. I n the presence of an i nflammation

site, monocytes quickly m i grate from the blood vessel and start an i n tense

phagocyt i c activity accompanied by the release of a number of cytokines. The

balance between the levels of pro- and ant i - i n flammatory cytokines and their

sequential rel easemay i n fl uence the degree of i n fl an1lTI atory responses. A

central rol e i n these mechan i sms i s played by a7-containing nAChRs found at

the plasma membrane of monocytes/macrophages.

 29

• The primal cel lular component of the derm is, also expres

nAChRs subtype a3,a7, [J2, a5 and {34 : I n addition to i nh i bi t i ng the release

of TN F, a7-contai rung nA h Rs may uppress the reJea e of high-mobi l ity

group box 1 (I IMGB 1 ) a cytok ine i nvol ved i n severe i n flammatory responses

such a those ob erved during sepsis Dermal fibroblasts.

• m RNA for the a7as wel l as a3, a5, [J2, and {34 nAChR

subun i ts has been ident i fi ed i n rat urothel ial cells using reverse transcription

polymera e chain reacti o n ( RT-PCR). I nterest ingly, in hwnan bladders the

I vels of expression of nAChR subunits seems to be l ayer spec i fic, with a

gradient decreasing i n i ntensity from luminal to basal . The signi ficance of this

anatom i cal arrangement for bladder physiology remains unknown; Functional

studies conducted in rodents suggest that a7 and a3- conta i n i ng nAChRs

might have opposite roles in contro l l i ng bladder fi l l i ng and voiding by

promoting the release of medi ators with anti thetic functions. I nvestigations on

the hwnan urothel i wn have so far indicated for it a rank order of expression

of a7 >a 1 O>a9 subunits; ganglionic-type nAChRs are l i kely to be expressed

i n the urotheli um as wel l .

• F i nal ly, the a3, a5, a7, a9, a I D, [J2, and (34 nAChR subuni ts

are also expressed i n hair fol l icles as wel l as sebaceous and sweat gl ands (De

B iasi , 2009).

Several i mportant findings on the nicot i n ic chol i nergic transm i ssion in the

h ippocampal structure have been reported i n recent years. F i rst, there are high levels

of a7 conta i n i ng nACh receptors i n the h ippocampus (J. Court & Clementi , 1 995;

Seguel a, P . , Wadiche, J . , D i neley- M i l ler, K., Dan i , J . A . , & Patrick, 1 993 ) . Second,

rapidly desensitizing currents and nicot i ne-i nduced gl utamatergi c rel ease i n
 30

hi ppocampal culture are mediated by pre ynaptic 0.7 receptors. Final ly, there are

[a L rapidl} d en iti zing inward currents present in CA l intemeurons that respond

to h through o.rn ChRs receptor-medi ated mechanisms ( Kenney, J. & Gould,

2008). suggesting that 0.7 subun i ts are critical for various types of chol i nergic

ynaptic transmi ssion in the hi ppocampus, and that 0.7 mechani ms may be important

[or cer1ui n t) pes of learn ing and memory .

 31

2 M A TE R I A LS AND M E T H O DS

2.1 M a te r i a l

2. 1 . 1 F e m a l e X e n o p u laevi

Matur fem a l e frican c l awed frog (Xenopu lael'i ) were purchased from

Xenopu Expre . Haut - L o i re . France . They v,'e re housed in a A q uarium (66 cm

width. 1 30 c m breadth and 320 c m height) fi l led to a hei ght appro x i mate l y 50 cm

\';i t h dechlori nated tap water. The storage env i romnent wa mai ntai ned a t 1 9-2 1 °C

with a 12 hour alternate l i ght and dark c c l e o Frogs were fed twice a week \ ith frog

food pellets. uppl i e d by Xenopus E xpress. Fran c e . Tank-water was chan ged twi c e a

week. A n i m al care and hand l i n g were i n accordance with i nstitutional guide l i nes.

F i gure 2- 1 : Xenopus L eavi A frican Frog

 32

2. 1 .2 C h e m i c al

Table 2- 1 : hemical req uired for e peri ments.

Compounds Formula Manufacturer / Catalogue
weight number

Acet} !cho l i ne c h loride 1 8 1 .7 Sigma, USA / A6625

Calcium ch lorid (CaCh) I I 0.9 Sigma-Aldrich, USA / C-490 1

Bari um chl oride ( BaCl 2 ) 244.28 B D H , EnglandlR-20-25 S :45

H EPES 238.3 igma, U S A / H 3 3 75

Pota' ium chloride (KCI ) 74.55 Mal l inckrodt USA / 6858

Magnesium chloride 95.22 Sigma, USA / M 8266

Magnesium ul phate 246 . 5 Sigma, USA / M93 97

Sodium chl oride aC I ) 5 8 .4 Sigma, USA / S-30 1 4

odium bi carbonate 84. 0 1 S i gma, U SA / S60 1 4

aHC03)

Sodium hydroxide (NaO H ) 40 Amresco, U S A /

Lot. # 2 1 46 1 3 0 1 0

Hydrogen chloride ( H C l ) 3 7% Sigma-Al drich, U S A /25 . 8 1 4. B

Pen ici l l in G 3 56.4 Sigma, USA / P3032

Gentami c i n reagent solution Conc. 50mg/m l I nvitrogen Corporati on/China

Cat. number 1 5 750-060

Streptomycin 1 45 7 .4 S i gma, USA / S9 1 3 7

Sodium pyruvate 1 1 0.04 Sigma-Aldrich, U S A 1P2256

Benzocaine 1 65 . 2 Sigma, USA! E- 1 5 0 1

u- B ungarotoxi ns -8500 Sigma / T-0 1 95

Col iagenase-A (From ------ - -

- - Roche Diagnostic Corporations
Clostrid i um h i stolyticum U S A! Lot number 93 2 1 1 52 1
E C . 3 .4.24 . 3 )
 33

2 . 1.3 T h e E x p e ri m e n t a l rig

The experimental rig � r el ectrophysiological recordings is shown In the

bel V\ figure. Two-electrode vol tage c lamp technique was empl oyed USing

GeneC lamp-500 amp l i fier ( xon I n truments, Molecular Devices, Inc., Bur l i ngame,

, U A) Recording setup included magneti c holding devices ( Kanetec USA

C rporat ion, Bensen i l k I L, USA). manual m i c romanipulator (M3 3 ; Marzhuuser,

Wetzl ar, Gelmany) . H ead- tage [or ol tage (H -2A Headstage, Gain I MG, Axon

In truments. Molecular De ices, Inc., Sunnyvale, CA, USA) and current ( H S -2A

Head tage. Gain l OM G ), wer attached to these manipulators. Electrodes were

inserted in electrode holders and connected to the headstages.

light

1 v
solution
::=��- Jn

objective

Fi gure 2-2: Experi mental rig for electrophysi ological recordings.

(Adopted from ionchannel .ku.dk)

The perfusion apparatus consi sted o f perfusion tubes and bottles containi ng

extrac e l l ular sol utions (bottles with si l i con tubi ng, Cole Palmer I nstmment

Company, I . D. 1 1 1 6 i nch, 0 . 0 . 1 18 inch and WALL l /3 2 , Vernon H i l l s, I l l inois,

 34

). Multichannel econd perfu i on et up for drug app l i cations included tubing

(indigenousl) made with C-Flex tubing, Cole-Parmer I n trument Company, I . D. 1 13 2

i nch, O . D . 3/3 2 inch and W A L L l /32 inch, Vernon H i l l , I l l inois, U A ) , 50mL glass

syri nge , pia tic al ves and coupl i ng devic . The system wa based on gravity fl ow

via a micropipette, po iti oned about 2-5 mm from the oocyte perfusion

cham ber/recording chamber ( Warner I nstruments LLC, Hamden, CT, UK) for

placing oocyte and impal i ng v ith electrodes. Opt i c fiber l i ght source was employed

for i l l um i nation of the recording chamber ( Fi berLite, H i gh I ntensity I l l umi nator

erie ] 80, Dolan-Jenner I ndustries Inc. Boxborough, MA, USA). A Low-power

tereo-dissection micro cope (Olympus, Tokyo, Japan, SZ-STB 1 , 1 00 ALO.S X,

WD 1 86) was used for vi sual observations. Computer set up for record i ng consisted

of Compaq computer, (Compaq Corporation, Product of UK, C i ty of Wynyard) and

analog-digital con e rter, BNC 208 1 (National I nstruments, A ustin Texas, USA).

2. 1"'. O t h e r m ateri a ls

Petri dishes ( Steri l i n , Newport, U K . Catalog nWl1ber: 1 2 7, 60 rom )

tirrer ( Rotomix, Type S0800, Barnstead/Therrnolyne, Dubuque, I A , U SA Model

number: M S0825).

pH meter (Coming p H meter model 450, Albany, NY, U S A ).

Picofuge ( Stratagene, Santa C lara, CA, USA, Catalog number: 400 S S 0 )

Borosi l icate G l ass tubing for microelectrodes (Glass Thin-wal led w/fi lament 1 . S mm,

Catalogue number: TW l S 0F-4, World Precision I nstrument, Sarasota, FL, USA).

Automatic nano l i ter i nj ector (Nanoj ect, D nunm ond Scient i fi c Company, Broomal l ,

PA, USA).
 35

icro-4 Mi cros ri nge pump control ler (Model U MC4-C, World Precision

In trument , ara ota. FL, USA).

R a fr�e water in 1 . 8 mL Eppendorf tubes ( Lot number: M2S-80S 02, Epicentre

Biotechnol ogies, Madi on, Wiscon in U ).

Micro fi l fi l l ing s ringe ( World Precision I nstrwnents, Sarasota, FL, U S A ) .

El ectrode holder ( World Precision I n trument, Sarasota, FL,USA)

fagnetic stand and m anipulators (Catalogue number: 7739 Narishige, Tokyo,

Japan).

i l ver wires ( World Prec ision I nstruments, Sarasota, FL, USA).

urgical utures (Catgut c hromo rever e cutti ng 3/8 c i rcle, USP 4/0, S M I , DemeTech

corporation , M i am i , Florida. USA).

urgical acce sories (scissors, forceps, scalpels; WorldPrecision I nstrument,

Sarasota. FL, U A ) .

Vertical p u l l e r (Model 700D, Dav i d Kopf I nstruments, Tuj unga, C A , USA). Heater

and solenoi d settings were adj usted to 48 and 70, respectively, to get optimal

resistance ( 1 -3 MQ) glass m icroelectrodes.

 36

2.2 M et h o d

2.2. 1 Prepa ra t i o n of o l u t i o n

2 . 2. 1 . 1 P r e p a ra t i o n of m o d i fied b a rt h o l u t io n ( M B S )

The compo ition of Modi fied Barth olution is gi en in tabular form in TabJe 2-2.

a. Calc i um-free sol ution

Tabl e 2-2 : Cal cium-free sol ution .

Compounds Concentration(mM) lx l Ox
(weight in (weight in
gram ) gram)

NaCI 88 5. 1 4 5 1 .4

HEPES 10 2.38 23.8

NaHC03 2 .4 0.20 2.0

KCl 1 0.075 0.75

MgS04 0.8 0.20 2.0

The above substances were d i ssolved i n 1 L of disti l led water and the pH of the
o l ution was adj usted to 7.5 using NaOH .

b. Modified Barths Solution ( M B S ) with Calc i um

I n addition to the above mentioned substances, CaCb 2mM; 0.22 g for I x and 2 . 2 g

for l Ox stock sol utions were prepared, and the chemicals were added to make the

fi nal sol ution with vol ume 1 L of disti l l ed water and kept at pH 7 .4-7.6.
 37

2 . 2 . 1 . 2 P re p a ra t i o n o f frog r i n ge r o l u t io n ( N D96)

The c m po ition of the D96 bathi ng s l ution i gi en in tabular fom1 i n Table 2-3 .

Table 2-3 : N D96 sol ution.

Compounds Concentration (m 1) 1 x (weight in l O x (weight in
gram) gram)

NaCI 96 5.61 56. 1

H EPES 5 1.19 1 1 .9

KCI 2 0. 1 5 1 .5

MgCh 1 0. 1 0 1 .0

CaCh 1 .8 0.20 2.0

OR BaC h 1 .8 0.439 4.39

The a b v e ubstances were dissol ved I n 1 L of dist i l l ed water and t h e pH was

adj usted to 7 . 5 .
 38

2 . 2 . 1 .3 P re paration of O O C. te torage o J u tion

Table 2-4 : Oocytes storage sol ution.

Com pounds Concentrat ion Weight i n gram
(mM)

NnC l 88 5. 1 4

HEPES 10 2.38

NaHC03 2.4 0.20

KCl 1 0.075

MgS04 0.8 0.20

CaCh 2 0.22

Penic i l l i n G 1 0000U/L 0.0 1 9

Streptomyci n 1 0 mg/L 0.0 1

Gentamyc i n 50 m g/L 2mL

Sodium pyruvate 2 0.22

Theophy l l i ne 0.5 0.09

2.2.2 I so l a tion of oocy tes from Xenopus laevis

Individual frogs were anestheti zed by i m mersion i n 0.03% wN benzocai ne.

3 00 mg of ethyl p-an1i nobenzoate was dissol ved in 1 5 mL of 70% ethanol and then

added to 1 L of cold tap water. The frog was then i mmersed in this 1 L of anesthetic

sol ution. Fai l ure to respond to noxious stimuli was used as the end point of

anesthesia. Thi s was determ i ned by p i nching of the l ower l i mbs and it took on

average of 5 minutes to anesthetize the frog under bathing conditions. A l l the

 39

procedures canied out JTI this study ha e been appro ed by the Ani mal Ethics

mmittee o f the FMH , UAE n i versity.

The anesthetized frog was placed on a bed of crushed ice to maintain a l ow

core bod temperature during surgery. teri le surgical procedures were used to

remov oocytes. teri l i ty was mai ntained by regul ar use 70% ethan o l . A sma l l

incision (about 1 .5 cm) as made with a surgi cal scal pel through the epidermal l ayer

of the lower abdomen to the ri ght or l e ft of the m i d l i ne. An inc ision of simi lar size

was then made through the m uscle l ayer using a surgical scal pel and a pair of fine

surgical sc i ssor . One to two lobes o f ovary were removed and placed i n a petri dish
2+
contain i ng Ca _ free M B S (Figure 3 .4 ) . Upon removal of oocytes, the musc le l ayer

of the abdomen was sutured with absorbable Catgut sutures. Immediately after

surgery the frog \'I'as placed in a water container and mon i tored for recovery based on

free swim m i ng behavior. After 3 -4 hrs moni toring, the frog was transfe rred to the

mai n frog aquarium. Two to three months after the fi rst surgery the frog underwent a

second surgery for oocyte col lection.

2 .2. 3 Oocyte p re p a ra t i o n

The preparation o f oocytes was done according t o procedures descri bed

earl ier (Oz, Ravindran, D i az-Ruiz, Zhang & Morales, 2003 ) . After thecal, epithelial

and fol l icular l ayers were manual l y dissected from oocytes using fi ne forceps then

treated with collagenase at room temperature. F i fty mg of Col l agenase-A was

d issol ved i n 2 5 mL Ca+2 free M B S . Then oocytes were transferred i nto 1 2.5 m L of

col l agenase sol ution i n a smal l conical flask and kept stirri ng slowly (60-80

rotations/mi n ute) for one hour. A fter one hour, the oocytes collagenase solution was

repl aced with the rema i n i ng fresh col l agenase solution ( 1 2 . 5 mL) and kept for an
 40

additional hour i n collagena e solution. ub equently , the oocytes were wa hed with

MB oluti n for 6 times, fol l ow d by 6 ti mes 'V ashing "" ith MBS

containig Ca+2 solution . ince collagena e damages the vite l l ine and plasma

membrane, the oocyte w re wa h d gently, and then tran felTed in a petri dish

contai ning M B for oocyte s e l e tion. Only V and V I stages oocytes were selected

under dis ecting micro cop ( Bunton I nstruments Co Inc., Model GSZ, Rockvi l le,

MD, U . S . A . ) . These oocytes are l arge in size, roLmd i n shape, about 1 . 1 to ] . 2 mm i n

d ian1eter and h a characteristic brown and white colored poles, a dark bro'vv n animal

pol e and a y l l owish vegetal pole.

2 . 2 .... Sy n t h esis of c RN A

The cD A c lone of humana7-nACh was k i ndly provi ded by Dr. J. LindstolTn

(University of Pennsy l vania, PA, U.S.A.). Capped c RNA transcri pts were

synthesized in 1'ilro using a m M E S SAGE m M E S SAGE kit (Ambion, Austin, TX,

U . S . A . ) and analyzed on ] .2% formal dehyde agarose gel to check the size and

qual i ty of the transcripts.

2.2.5 I n j ec t i o n of c R N A i n t o oocy tes

The concentration of human a7 nAC h receptor mRNA synthesized was 3 . 7-

�g/�L . I t was stored i n 1 � L a l i q uots i n -80 DC freezers. On the day of inj ection onl y

o n e a l i q uot i s transferred t o t h e l aboratory on i c e . Before handl ing of t h e c RNA,

gloves and face m ask were put on and the bench surface was cleaned with 70 %

ethanol to m i n i m i ze RNase contami nation. The tube was centri fuged i n a

microcentri fuge for few seconds to separate and settle the cRNA i n tube, then 8 �L
 41

o f R a e and 0 ase- free water was added to the cRNA pol l et u ing a sterile pi pette

and R a e free and 0 a e fr e tips (D nvi l l e c i enti fic Inc . , Metuchen , N J , U . . A.).

horizontal puller PUL- l ( World Precision I nstruments, Sara ota, FL, SA)

was u ed to pul l a 3 . 5 nL autoc l a ed gl ass capi l l ary ( World Precision I nstruments,

ara ota, FL, U ) to make a needle ith a long shank. The tip of needle was

broken by applying pressure using fi ne forceps ( Fine Sc ience Tool s I nc, Vancouver,

BC. Canada) under di ecting microscope ( Bunton Instrument Co, Rockvi l le, MD,

) . The needl e was back fi l led with mi neral oil (S igma, St. Louis, MO, USA)

u ing a glass 1 m L syri nge. The need le was then secured i n the micro-di spenser,

\-\- hich wa manipulated b a microman i pulator under the microscope. The inj ection

procedure began b swabbing the micro dispenser, surface and m icroscope by 70 %

ethanol. 3 ilL of d i l uted c RNA or disti l led water was transfened to the middle of a

mineral o i l drop on the parafi l m ( American National Can, Greenwich, CT, USA).

Using the withdrawal option on the micro-di spenser contro l l er, the aqueous phase

alone was drawn i nto the needle. This procedure was perfom1ed careful l y under a

microscope to ensure the tip of needle was always in the centre of the aqueous

dropl et during the procedure.

Once the micro dispenser was fi l led, the sel ected oocytes were placed in a U

shaped pattem i n a smal l petri dish with a mesh bottom to hold the oocytes i n place.

Each oocyte was i mpaled by the needle and 1 0nL of mRNA or dist i l l ed water was

inj ected i nto each oocyte cel l by the nanol iter i nj ector, driven by a M icro-4, m icro­

syringe pump contro l l er. F i nal ly, i nj ected oocytes were incubated at 1 8 °C i n 25 m L

petri dish containing oocyte storage sol ution for 4 8 hours for m aximal expression of

a7 nACh receptors. The i ncubation time al lowed for optimal expression of protei n

from c RNA. The i nj ected oocytes were usua l l y used for 4-5 days after expression.
 42

fhe torage o l ution wa changed dai ly and OOCy1es with poor qual ity were remo ed

every da under microscope. Oocyte were sorted i nto tv.·o groups, one wa inj ected

\\ ith human 0.7 nA h receptor c RNA. whi le the other group was either injected with

di ti l l d v. ater or kept uni nj ected as controls.

The am procedure v, as appl ied for i nj ection of human GlyR and human 5 H T3 R .

2 . 2.6 Drug p re p a ra t i o n

tock oluti n (mM concentration) of test compounds were prepared i n

D96 solution u ing the fol l owing fOlmula: Wei ght i n mg = ( M W ) x (vol ume i n L ) x

(mM concentration). FUl1her d i l utions were prepared by using Charles equation :

Cl x VI = C2 x V2.

Where C l is the concentration of stock sol ution, V I is the vol ume of stock solution

C2 is the concentration to be prepared, and V2 is the volume to be used for d i l ution.

2.2.7 E lectro p hys i o\ogica\ reco rd i n g

The oltage c lamp technique is a method that al lows ion flow across the cell

membrane to be measured as an electric current whi le the transmembrane potential is

held constant (clam ped) with a feedback ampl i fi er. The functional properties o f i on

channels expressed in Xenopus oocytes can be studied effective l y usi ng the two­

microelectrode voltage c lamp. The membrane of the oocyte is penetrated by two

m icroel ectrodes. one for voltage sensing and one for current i nj ection. The

membrane potential as measured by the voltage-sensing el ectrode and a high i nput

i mpedance amp l i fier was compared with a command voltage, and the d i fference was
 43

brought to zer b a feedback ampl i fier. The inj ect d cUlTent by the amp l i fier

provides a mea ure of the total membrane CUlTenl. The e periments were performed

u ing the tv\. o-eJ ctrode oltage-c lamp technique. I ndividual oocytes were placed in

th p rfu ion or record i ng chamber and constantly bathed with ND96 olution at a

perfu ion rat of 5 - 7 or 2-3 m llm inute. The ooc te wa impaled (at the animal pole)

with two gl ass microelectrodes ( � 0.5-3 MD el ectrode resi stance) prepared using

vertical microel ectrode pul ler ( heater and solenoid val ues were adj usted to 50 and 70,

re pectively; David Kopf I nstrum ent Tuj unga, CA, USA) and fi l led with 3M KCJ

o l ution. Drug were appl ied by a gra ity-ba ed multi channel appl i cation system via

a micropip tte. The perfusion by D96 was stopped at the time of drug appl ication

and immediately opened after drug application. The tip of the drug deli very system

was positioned about 2-4 mm from the oocyte. A Two-electrode voltage-c lamp was

achieved using a Geneclamp 500 ampl i fi er (Axon I nstruments, Molecular Devices

I n c . . Sunnyvale, CA, USA), i nterfaced to a PC computer equipped with an

electroph siology software, Win WCP (University of Strathclyde, Glasgow, U K ), for

data acquisition. The fol lowing parameters were tested by electrophysiological

recordings:

2 . 2 . 7 . 1 C o n c e n t ra t i o n s response c u rv e ( I Cso d eterm i n a t i o n )

The oocytes were voltage-clamped a t a holding potential of - 7 0 m V using a

GeneCl amp-500 ampl i fi er (Axon I nstruments, Molecular Devices, I nc . , S un nyvale,

CA, USA), and current response induced by 1 00 ilM of acetylcholine chloride (ACh)

were recorded digital l y on an I BM Pc. It is i mportant to note that ACh is the

endogenous agonist for n i cotinic receptors. It was pre felTed over other agonists

mostly because, compared to nicotine and other agents, it has less desensitizing effect
 44

(Ku an , Mi ledi , & tinnakre, 1 98 2 ) . Furth rmore, the low hydrophobi city of ch

make it easy to be a hed out.

typical experi ment began with three to five recordings of ion currents

i nduced by 1 00IJ-M of ACh with fi ve mi nutes i ntervals of washing with ND96

sol ution. ontrol r cord i ng were obtai ned during this period. The average of three to

four table readi ngs before the commencement of the experi ments was calculated as

the control value. Fol lowing the control recordi ngs the oocyte was perfused for a

total of 1 5 min ute vvith ary i ng concentrati ons of drugs. The averages of two to

thr e responses at the end of \ 5-20 min drug appl ication were calculated to determi ne

the effect o f the dmg used. Subsequently, the appl ication of the dmg was stopped and

ooc 'te \\'ere \.\'a hed with recovery sol ution (ND96 alone) .

The percent i nh i bition was calculated by dividing the average of currents i n

the presence of the dmg b y t h e control values obtained before dmg appli cation.

These val ues were nonn a l i zed and displayed as percent changes (compared to

controls).The concentration of a dmg which produced 50% i nhibi tion of ACh­

i nduced currents ( I Cso) was detemli ned by nonl i near curve-fi tting and regression fits

( logistic equation) using computer software Origi n (Origin Lab Corp . , Northampton.

MA, U . S . A . ) . D i fferent concentrations of drugs were p lotted on the X-axi s and %

i nhibition of ion current on respective concentrations was pl otted on the Y-axis.

Concentrations of drugs c lose to their ICso val ues were employed for further studies.

With the excepti on of JNJ , a l l compounds were sol uble in water. JNJ was dissolved

i n D M S O . At the concentration of 0. 1 % (v/v), D M S O caused a signifi cant inhibition

of a7 nACh receptors. However, DM SO, at the concentration of 0.00 1 % ( v/v) , did

not i n duce a statistically signi ficant effect on the maximal ampl itudes ACh-induced
 45

current . Therefore. fi nal concentration of 0.00 1 % DM 0 was used to di ssol ve IN]

in th e. peri ment.

2 . 2 . 7 . 2 Ex peri m e n t on the c o m p e t i t i v e a n d n o n-com p e t i t i v e n a t u re o f d ru g

inh ibition

Concentrati on-response curves for ACh were determ i ned by i ncreasing ACh

concentration i n the range of 1 0 �lM to 3 mM. During these e periments oocytes

were voltage-clam ped at a holding potential of - 70 m V . For each data point

calculated. the experi ment was conducted in �5 to 7 d i fferent oocytes and percent

i nh i bition wa calculated a de cribed earl ier.

2 . 2 . 7.3 E x p e ri m e n ts o n t h e voltage-d e p e n d e n cy o f d ru g i n h i b i t i o n

Voltage-dependence of t h e drug action was determ ined b y holding the

membrane potential at d i fferent val ues for 30 s. At each poi nt, membrane potential

\vas returned to -70 m V, and subsequent readings were taken every five mi nutes.

During these experi ments, ACh was used at a concentration of 1 00 �LM. Current­

voltage ( I - V ) relationshi ps for ACh-induced currents were determ i ned in the absence

and presence of drugs.

2 . 2 . 7 --' E x peri m e n t on tbe d eterm i n a t i o n o f Ca + 2 c o n t ri b u t i o n to observed d ru g

action

I n this series o f experi ments, oocytes were placed in a ND96 sol ution

containing 2 m M Ba2 + (ND96 sol ution contained 2 m M B aH i nstead of 2 mM Ca+2 ).

I n order to determine the contribution of intracel l ular Ca2+ l evels. the effect of the

2
drug on the ACh-induced currents was tested in the presence of either Ca2 or B a +

containing D96 solutions, and the extent o f drug inhibi tion was compared.
 46

2 . 2 .8 Docking tudie

The MR protein structure was obtained from the protein data bank and then

wa prepared using the MOE software package. The 3 D protonate module ( P . Labute

& Protonate. 2009) was empl oyed to add hydrogens, predict ionization states and

then to a sign partial charges on each atom based on the MM FF94 forcefield

( Hal gren & Nachbar, 1 996; Halgren, 1 996a, 1 996b 1 996c, 1 996d). The fol lowing

re idues were employed to define the bind i ng site: Phe2 3 0, Ser2 85 and Phe45 3 .

Ligands were prepared by identi fYing their ionizat ion state at p H 7 via the wash

module in MOE. They were given partial charges and were energy m i n i mized usi ng

the M M FF94. forcefield ( Halgren & Nachbar, 1 996; Halgren, 1 996a, 1 996b, 1 996c ,

1 996d). ext. the l i gand molecules were docked i nto the previously defi ned binding

site using the MOE-Dock program . Triangle Match was used to place the l i gand

i nside the bind i ng pocket by creating two sets of triplet atoms for both the l i gand and

the active site; each l i gand triplet is then matched to each active site triplet to

generate multiple docking solutions. The London llG, which i n c l udes terms for

hydrogen bonding, l i gand entropy, desol vation and geometric fit, was used to score

generated poses. The best 30 poses were refined using the M MF F94x forcefield

( Halgren & Nachbar, 1 996; Halgren 1 996a, 1 996b, 1 996c, 1 996d) and then rescored

via the GBV I IW S A llG scoring function ( Paul Labute, 2008) ; it incl udes tenns for

l i gand entropy, desolvation, electrostati c and V ander- Waal i nteractions. The best 1 0

poses were retained to be vi suali zed v i a the PyMol program .

2.2.9 Data a n a lysis

For the non l i near curve-fitting the computer software Origin (Origin Lab

Corp. Northampton, MA, U . S . A . ) was used. Average val ues were calculated as the
 47

mean ± standard error mean ( E M ) . tati tical signi ficance was analyzed USing

tudent' t-test or OV a indicat d. Concentration- re ponse curves were

obtai ned by fi tting the data to the logistic equati n,

Where x and are drug concentrat ion and response, respectively, Em ax is the maximal

re ponse. EC-o is the hal f-maxi mal concentration, and n is the slope factor (apparent

H i l l coefficient).
 48

3 R E S U LTS

3. 1 Functional Exp" c i o n of t h c H u m a n (lr n A C h Rece p t o r i n Xenopus

Oocyte

cel lcho l i ne ( Ch), at the highe t concentration of 1 0 mM used in this

tudy, did not cau e detectabl e currents in uninj ected oocytes (n=5 ) or in oocytes

inj ect d 'with dist i l led water (n=7). Appl ication of 1 00 11M ACh for 3 to 4 second

activated fa t inward currents that desensitized rapidly (within 2 to 3 s) in oocytes

i nj ected with cRNA transcribed from cDNA encod ing the a7subuni t of human nACh

receptor : Furthermore, ACh-induced imvard currents were abol i hed completely

\",ith 1 00 nM a-bungarotoxin a potent antagonist from a poisonous component of

snake venom ( n=9). indicati ng that these re ponses are mediated by a-bungarotoxi n­

sensitive neuronal a7 nACh receptors (Figure 3 - 1 ) .

 49

\, h
I I It ,.. ( I

B :""" ACb
( 1 00 � 1 ) ( 1 00 Ji M )
�n .H5 1
")11
T
'1
� II J oj )
�
-< T
$:: I 'UP
----
......
c
V I I �l)fJ
>-
>-
:::I
::....; '''''
In IhI
-r-

"
In -ll'.l
I I
�U'II11J 1 .;nnlTnl ( , · HTX
< , , - ·nA ' h) I , � J n�l .l

Figure 3 - 1 : Functional expression o f the human a7-nACh receptor in Xenopus

oocytes.

3.2 Th e E ffect of S el ec t ed H R B l oc kers o n t h e F u n c t i o n of H u m a n 0. 7- n A C h

Rece p t o r

In the initial experiments, effects of various nominated histam ine receptor

antagonists (chemical structures in F i gure 3-2), namely the H I R antagonists ( PMZ,

DPH, P Y R, ORP, CLP. TRP, CTZ) (Figure 3 - 3 ), the H2R antagonists ( CMT, RNT,

ZT), the H 3 R antagonists (CLB, C P R, P I T), and the H4R antagonist (JNJ ) were

te ted on the function of human arnACh receptor ( Figure 3-4). The effects were

observed by 1 0 m i n incubation with 1 0 �lM of these various antagonists on 0.7-

nAChR mediated currents. Although H2R- H4Rantagonists did not alter the maximal

amp l i tudes of ACh-induced currents, many H I R antagonists (anti histami nes),

espec i a l l y P Y R, caused an i nh ibition o f ACh-induced responses with the order of

their potencies being PYR > CLP > TRP > PMZ > ORP � DPH � CTZ ( Figure 3 - 3 ) .

Moreover, recordings of the currents mediated b y 1 00 � M ACh, i n the absence and

presence of 1 0 �M P Y R and after 1 0 m i n recovery and time-courses of effects of

 so

PYR applicati n n the amplitude of ACh-i nduced currents are presented in Fi gure

3-5.

ill antagonists

Promethazine Diphenhydramine Pyrilamine Orphenadrine

( PMZ) (DPH) (PYR) (ORP)

CI
?; CH 3
I N CH
3
�

"N
1 .0

Chlorpheniramine Triprolidine Cetirizine

(ClP) (TRP) (CTZ)

H2 antagonists

Cimetidine Ranitidine Nizatidine

(CMT) ( RNT) (NZT)

H3 antagonists

N
f j}� o
�
)l) V O
HN O� ��CI

Clobenpropit Ciproxifan Pitolisant

(ClB) (CPR) (PIT)

H4 antagonist
CHJ
,

H ()
�N N
CI
I A
� 0
JNJ ·7777120
(JNJ)

F igure 3 - 2 : Chemical structures of H l -H4R antagonists used i n the current study.

 Sl

1 00

80
c
0
( n=6 )
. �
�
.- 60
�. (0=6 )
-
( n=6)
.

,..c
s:: 40
. - (n=6)
0
0
20 ( n=6) ( n=6) (n=6 )

0
PMZ DPH PYR ORP eLF TRP

Figure 3 - 3 : Relative potencies of H l R antagonist tested on human a7-nAChR.

1 00

I()
-
r

0
.-
�
.- 60
.!)
. ....
..c
r 40
-
�

�
0
_0
( 11 4- )
( I I =� )

Figure 3-4: Relative potencies o f H 2 R-H4R antagonists tested o n human a7-nAChR.

 52

A u h
.�.

\Ch
J fJ:'I1 I PYR J .\1 I
1 1
1. 1 1 ' 1 P ,' 1

51)1 lL.\.

B 1 00
-
-
/. - 0 - 0 o,.. O --� :;i ;:i
e/
r.f)

0
l-
......
:-to

(i0
\ .I
t::
0
0 -+f.)
."e - ./
,
"-

Co 20

� 0
0 10 _0 30 40
] i ll1e I11i tl )
Figure 3-5 : E ffects of pyrilam i ne on a7 nAChR-mediated ion currents.
(A) Records o f currents activated by acetylchol i ne (ACh 1 00 /-lM ) i n contro l
conditions ( l eft), duri ng co-appl ication of 1 0/-lM pyril amine a n d acetylchol i ne after
1 0 m i n pretreatment with 1 0 �lM pyri l amine ( middle), and 1 5 min fol l owing
pyril am i ne washout ( right). ( B ) Time-course of the e ffect o f pyri l am i ne ( 1 0 /-lM) on
the peaks of the acetyl c ho l i ne-i nduced CUlTents. Each data point represents the
normal ized mean ± S . E . M . of 6 to 7 experiments. D uration of pyril amine app l ication
i s i ndicated by the horizontal bar i n the fi gure.

Noteworthy, the inhibitory effect observed for PYR was dependent on the

app l ication mode. For example, without PYR prei ncubation, the coappl ication of

PYR ( 1 0 /-lM ) and acetyl c ho l i ne ( 1 00 /-lM ) did not alter the amplitudes of maxi mal

currents (0 time point in Figure 3-6. However prei ncubation of oocytes with PYR

inhibi ted maximal responses in a time-dependent manner. Also the prolongation of

incubation time significantly enhanced PYR i nh ibition which reached a maximal

l evel within 1 0- 1 5 min (with a hal f-time (-r l l2 ) of 1 .9 ± 0.2 min). S ince the magnitude
 53

of the PYR e ffect wa ti me-dependent, app l icat ion time of 1 0- 1 5 min wa used to

en ure equi l i brium c ndi ti ons.

1 00

1 . 9 ll1in
-

0 't l 2
$-.
=

GO
�

�
0
u
4-0
0 40

�
20

o �----�----��-
o 5 10 15
Pre i ncubat i o n t i me
Figure 3-6: Time and concentration-dependence of P Y R i nh i bition of a7-nACh R-
mediated ion currents.
I nhibition of the a7-nAChR increases with the prolongation of P Y R
preappl icationtime. Each data poi nt represents t h e mean ± S . E . M . of 6 - 7 oocytes. The
curve is the best fit of the data to the logistic equation desclibed in the methods
section.

I nterest i ngly, PYR inhibi ted the function of arnAChR in a concentration-

dependent manner with respect i ve I C s oand slope values of 6.2 ± 0.4 11M and 1 . 2,

respecti vely ( F i gure 3-7). Based on the I C s o val ues, PYR was the most potent

inhibitor of ACh-i nduced currents. Therefore, 1 a �lM (to round up the value for

easier calculations) of PYR was employed routinely i n the rest of the experiments.
 54

Ie (, . 2 �l rvl
. IJ
-

c
o

. - 40

0. 1 10 10

Conc�n trati o n ( �l f\/f )

Figure 3 - 7 : PYR i n h i bits a7-nAChR function in a concentrati on-dependent manner.
Each data poi nt represents the mean ± S . E . M . of 6-7 oocytes. The curve is the best fit
of the data to the logistic equation descri bed in the meth ods section.

3.3 T h e Role of I n tracel l u la r C a l c i u m Level in Py ri l a m i n e- I n h i b i t i o n of 0.7

n A C h Receptor

Activation of a7 nACh receptors are k nown to allow i nfl ux of sufficient Ca2 +

to activate endogenous Ca2+-dependent Crchannels i n Xenopusoocytes (Sands,

Costa, & Patrick, 1 993 ; Seguel a, P., Wadiche, 1 . , D i neley- M i ller, K . , Dan i , 1. A . , &

Patrick, 1 99 3 ) . Therefore, it is important to dete1111 ine whether the effe ct of

pyri l am i ne was mediated by its direct actions on arnACh receptor or on the cr

currents i nduced by Ca2+ entry though nACh receptors. I n this set o f experiments,

extracel lular Ca2- was replaced with BaH since BaH can pass through a7-nACh

receptors but causes l ittle if any, activation of Ca2+ -dependent Crchannels (Sands et

aI . , 1 99 3 ) . PYR (10 �M ) produced the same level of i nhibition of ACh-i nduced

currents ( 5 9 ± 6 in controls versus 63 ± 7 in B a2+ containing sol utions, ANOV A,

 ss

n 6-7. P>0.05 : Fi gure 3 - 8 ) . I n the ooc te expre ion ystem. an increased level an

increa cd level f intracel l ular a2+ can be detected by Ca2+-acti vated Cl- channels

and concom itant alteration in the holding currents and membrane input resistance.

However. in control experi ment . PYR used in this study ( 1 0 flM for 1 5 m i n ) did not

alter the magni tude of holdi ng-currents (47 ± 5 m V in control versus 42 ± 4 m V i n

PYR) o r input resistanc ( 1 . 8 ± 0.4 MD. in control versus 2 . 1 ± 0 . 3 MD. in PYR) of

o C) tes vol tage-clamped at -70 mV (n= l l ). indicat ing that intracel l ul ar Ca.2+ levels

were not altered by PYR.

1 00 .

contro l 1 11 B a' r
80

�
� T
0 60
"--'

..0

� 40
c
. ....

0'
20

Figure 3 - 8 : E ffect o f P Y R on ACh-induced currents is independent of Ca2 + -activated

C I - channels.

304 T h e V ol t a ge- De p e n d e n ce of Py ri la m i n e A c t i o n o n the a7nACh Recep t o r

Pyri l am i ne i s h ighly l i poph i l i c and a weak base with a pKa val ue of 8 . 8 5

( rangi ng from 7 . 5-9 . 5 ). It can easily pass cel l membranes and accumulate i n

intracel l ular organelles i n protonated forms (Haley, 1 98 3 ; Levin e t aI ., 20 1 1 ).

Therefore, i t is l i kely that i nhibition by the protonated (charged) form of Pyri lamine

can be affected by changes i n membrane potential and that blockade o f the nicotinic
 56

acetylch l i ne receptor-ion channel c mplex i voltage-dependent. For this rea on.

vol tage-dependence of pyri lamine-inhibition \Va exam l ned on a7nACh receptors.

Each te ted membrane potential was he ld for 30 s and then returned to -70 mY . As

indi cated in Fi gure 3 -9. the inh ibition of h ( l 00 IlM )-i nduced currents by

pyri lamine ( 1 0 11 M ) doe not appear to be vol tage-dependent. The extent of

p ri l am i ne inhibition wa imi lar at a l l te ted membrane potentials from - 1 00 to -20

mY. Eval uation of data from current-vol tage relationship (Figure 3-9) i ndicated that

the extent of the i n h i bi tory effect of pyrilamine did not change signi ficantly at

d i ffer nt holding potential (P>0 .05, n=7. ANOVA).

 57

A V o l ta ge ( m\' )

-)0 -60 --to

. /E9'; -0.2
......

/Q O
__
�� ·
;:> �

1;
....
....
:::
-0.4 u

Q -0.6
-0
�
,/ cj
c
-0. ....
c
• C ont r Z
• 0 PY R - 1 .0

B 1 00

a
=
::
.....

..0
60 --!
...:;:;:
= -+0
�
e

20

O �---r-""""--r--"-'---"-r-""'T"
- 1 20 - 1 00 - 0 -60 -40 -20
Volt age (m\r)

F igure 3-9: Pyri l amine inhi bi tion of acetylcho l i ne-induced currents is i ndependent of
membrane potenti a l .
(A) Current- oltage rel ationships of ACh-activated currents in t h e absence and
presence of PYR ( 1 0 11M ) . Normalized currents activated by 1 00 11M ACh before
(control , e ) and after 1 5 m i n treatment with PYR ( 0 ). Each data point presents the
normalized means and S . E . M . of 4-6 experi ments. ( B ) Quantitative eval uation of the
effect of PYR as percentage i nhibition at di fferent voltages.
 58

3.5 t u d ie o n t h e B i n d i n g i t e o f Py d la m i n e o n a 7 n A C h Recep t o r

Pyri lam i ne ma decrease the binding of the agonist to the receptor by acting

a either a om petitive or non-competitive antagonist. For this reason, the effect of

pyri lamine was examined at d i fferent concentrations of Ch. Concentration-response

curves for h i n the ab ence and pre ence of I OIlM pyri lamine are presented i n

Figure 3 - 1 0. I n the pre ence of PYR, the concentration-response relationship

remai ned unchanged, but the rn a ' i mal response i nduced by acetylcho l i ne decreased

igni ficantly ( n=6-8 ) . I n the ab ence and presence of P Y R, the EC50 val ues were 93

± 1 1 �lM and 86 ± 9 11M (P>0.05, A OVA n=6-8 ) and slope values were 1 . 7 ± 0.4

and 1 .9 ± 0.3 , re pectively. uggesting that pyri l am i ne i nhibits the ACh responses in

a non-competitive manner.
 59

1 00 • Control
0 0 PY R

80
1-.
---
h
0
u

ro 60
E
x
co 40
�
.....

�
0 20
�
0

0
10 1 00 1 000
Concentration of A C h ( �l M )

Figure 3 - 1 0 : Concentration-re ponse curves forACh-i nduced currents.

Effect of PYR on the ACh concentration-response rel ationship. Oocytes were
vo ltage-clamped at - 70 m V and CUlTents were activated by appl ying ACh ( 1 11M to 3
m 1 ). Oocyte were expos d to 1 0 �lM P Y R for 1 0 min and ACh was reappl ied.
Paired concentration-respon e curves were constructed and responses normal i zed to
maximal response under control conditi ons. EC50and slope val ues were determ i ned
by fi tt i ng the curves from 5-7 oocytes to the standard logistic equation as described
in the methods section. Data points obtained before (contro l ) and after 1 0m i n
treatment with P Y R ( 1 0 11M) were i ndicated b y fi l led and open c i rc les, respectively.
Each data point presents the normal i zed means and S . E . M . of 5-7 experi ments.

3.6 D ocki n g S t u d ies for Py r i l a m i n e a t (l7- n A C h R

I n l i ne with functional experiments, docking studies indicated that Pyri l am i ne

has the features needed to bind to the allosteric site of the u7 transmembrane domain

that was previously di scovered to be the site of bind i ng of the known non-

competitive antagonist ketamine ( Bondarenko et a l . 20 1 4). Pyri lam i ne and ketam ine

demonstrated favorable binding energies, as their scores have negative val ues (Table

3- 1 ). PYR showed to bind in a simi lar fashion to ketami ne, mai n l y through extensive

hydrophobic contacts with the side chains of I le222, Ala226, Phe2 3 0 Leu242,

Va1246, Val282 Ser2 8 5 and Val 286 along with a cation-n i nteraction, through their
 60

protonated amine , \.\ i th Phe4 5 3 (Fi gure 3 - 1 1 ). I n order to test whether Pyri lam ine

and ketam ine share a functionally i nteract ive common binding site on the nicoti nic

receptor, we ha e conducted further electrophysiological experiments. Within the

same oocytes we have compared the e ffe cts of Pyrilam i ne alone ( 1 0 flM ) ketamine

alone (20 flM ) and Pyri l am ine + ketam ine on the maximal amplitudes of ion currents

mediat d by a7 nAChR (Figure 3 - 1 2 ) .

The results i ndi cated that extent of i nh ibition induced b y coappl ication o f

Pyri l am i ne + ketam ine was not sign i ficantly d i fferent than those of Pyri lam i ne alone

or ketamine alone (P>0 .05, ANOVA, n=7) suggesting that and ketam i ne occlude

each other's inhibitory effect by compet ing for the common binding site on the

nicotini receptor.

Table 3 - 1 : The WSAt1G scores of the pyri lam ine and ketami ne
Best three poses generated from docking i nto the human a7 nAChR transmembrane
domain.

Score

Molecule (kcal/mol)
2 nd 3 rd
1 51 pose pose pose

Pyri lam ine -6.4 -5.9 -5.8

Ketam in e -4 . 5 -4 . 3 -4 .2
 61

Figure 3 - 1 1 : The binding mode of the pyrilam i ne ( A) and ketamine ( B ).

3 rd
best pose obtained from docking onto the human a7 nACh R transmembrane
domain . Yel low dotted l i nes i ndicate for cation-1t i nteractions along with distance
(A). Some protein residues are not shov;n for c larity.

3.7 I n h i b i t i o n of PY R a n d i ts C o m p e t i t i o n w i t h Keta m i n e o n (l7-nACh R

I n order to test whether PYR and ketamine share a functionally i nteract ive

common binding site on the arnAChR, we ha e conducted further

electrophysiological experiments. Within the same oocytes we ha e compared the

effects of P Y R alone ( l 0 11M), ketamine alone (20 11M) and P Y R+ ketamine on the

maximal amplitudes of ion currents mediated by arnA ChRs ( Figure 3 - 1 2 ) . The

results i ndicated that extent of inhibition i nduced by coappl ication of PYR +

ketami ne was not signi fi cantly different than those of PYR alone or ketamine alone

(P>0.05, A OVA, n=7 ) suggesting that PYR and ketamine occl ude each other' s

inhibitory effect b y competing for the common binding site on t h e nicotinic receptor.
 62

1 00

C
\.....
.......
C
T
T T
60 .
0
u
4-
0
C
0
....... 4
.D
....c
c

0 10
0

0
P YR kewmine P 'YR
+
ketamine

F igure 3 - 1 2 : Compari on of tbe inhibitory effects of PYR, ketamine, and

PYR + ketam ine on u7-nAChR mediated ion CUlTents.
The effects of PYR ( l 0 !AM ) alone, ketamine (20 !AM ) alone, and coappl ication of
PYR + ketami ne \vere tested i n the same oocytes. Bars represent the means ± S. E. M.
of 7 experi ments.

3.8 P Y R Effect o n G ly c i n e a n d 5 - H T3 Recep t o rs

As mentioned earli er urnAChR is a subtype of n i coti nic receptors belonging

to c s loop receptor fam i ly which includes glyc i ne receptor (GlyR) and serotonin

( 5 H T3 ) receptor that are l i gand gated ion channels .To test whether the i nh ibitory

effect of PYR on urnAChR is also effective on Gly receptor and/or 5-HT3receptor,

the same voltage c l amp technique was used in oocytes i nj ected with c RNA of those

receptor ion channels. As shown in Figure 3 - 1 3 , PYR did not inh ibit functional

properties of either Gly receptor or 5-HT3 receptor.

 63

1 00 -

80 -
�
-

c
....
60 -
...c
�
-

40 -
,..-
-

0
c....

20-

o �------��--�-

Ct.7 5-I 1T3 G ly

Figure 3 - 1 3 : Comparison of the effect of 1 0 �M PYR on arnicotinic, 5-HT3, and

glycine receptor-mediated currents. Bars represent the means ± S . E . M . of 6-
8experiments.
 64

4 DI C U SS I ON

Pyri l am i ne (PYR), al 0 knovm a Mepyram ine, a fi rst generation

anti hi tam ine targeting H J R with add itional antichol inergic properties (Chen, hen,

& Kamei, :200 1 ).

i cotine T) ha H J R antagonistic property with a low r activity than PYR

for H J R ( E rcan & Turker, 1 98 5 ) . B a ed on these i nteresti ng studi es, the necessity of

biochem ical study on r lation hip between the ni cotine and PYR on spec i fi c target

protein ha been rai ed as an issue. The present study identified the no el action

mechan i m of PYR targeting on the nAChR and i nvestigated the l igand binding site

of pyri lam i ne on nAChR's.

In a very recent report, K i m et aI, represents PYR as a prOlTIlSmg new

candidate compound that may be useful in a search for sma l l -molecule

pharmacotherapies for nAChR-related brain diseases which i n addition can be a

u eful regul ating tool i n the study of nAChR-mediated si gnal transduction i n

neuroendocrinal systems. PYR pretreatment signi ficantly inhibited NCT-i nduced

v,;hol e cell chromaffi n (CA) secretion; when CA cel l s were treated with 30 11M PYR

for 5 m i n , the fl uorescence intensity i n response to 1 0 uM NCT was markedl y

reduced. I n contrast, PYR had no effect o n KC1 -evoked calci um entry. PYR

sel ecti ve l y i n h i bited nAChRs and thereby caus i ng reduction of CA. I t inhibited al l

the NCT-induced responses tested, i n c l uding CA secretion and i ntracel l ular Ca2 + .

i ncreases, thus i ndicati ng that PYR sel ectively suppresses the activity of nAChRs

(Kim et a I . , 2 0 1 4 ) .

Another study b y Tega e t al . , suggested PYR a s an effective drug for smoki ng

cessation d ue to its i nhibitory effect of NC i n fl ux transport across the B B B . PYR

inhi bited C uptake in a competitive manner with a K, value of 1 5 11M , which is i n

 65

an agreement \\ ith it Km value for uptake by TR- B B B 1 3 cel ls (28 11M ) (Okura et ai . ,

2008). The e re ult tr ngly sugge t that C is transpOJied across the BBB via

PYR-sensitivt; organ ic cation tran port process(es); a catTier-mediated blood-to-brai n

tnm port y tern, \\ hich i i nh i bited by PYR, and plays a rol e in i n fl ux-predomi nant

transport at the B B B . The uptake of eH ] nicotine by brain sl ices was inhibited by

PYR l i ke the i nhibition of C by TR-BBB 1 3 cel l s. The l atter result i mpl ies that NC

in the erebral i nter titial fl uid i concentration-dependently taken up i nto brain

parenchymal cel ls ia PYR-sensiti e organic cation transport process(es) as is tbe

ca e at the BBB (Tega, Akanuma, Kubo, Terasaki & Hosoya. 20 1 3 ) . Moreover.

Levin et a I . , reported that high-dose administration of PYR reduced sel f­

administration of C in rat : this mechanism is thought to explain the effect on

motivated beha ior ( Lev i n et a l . , 20 1 1 ). Moreover. P Y R was even more effective i n

reducing operant l icking for I V C sel f adm inistration than has previously seen for

reducing lever pressing for I V NC sel f-administration(Cousins, Rose, & Lev i n,

20 1 4) .Accordingly, further c l i n i cal studies targeti ng smokers wi l l be helpful i n

determ i n i ng t h e efficacy and safety of PYR treatment i n promoti ng smok ing

cessation ( Ki m et a ! . , 2 0 1 4).

I n the present study, we provide electropbysiol ogical evidence i ndicat i ng

tbatol d-generation antagonists of H J R subtype (antihistam i nes) such as P Y R, CLP,

TRP and PMZ i nhibit human a7-nAChRs. The inhibitory effect observed for PYR

was reversible and concentration-dependent since i ncreases i n the concentration of

acetylcholine did not overcome PYR i nhibition of acetylcho l i ne-induced ion currents

suggesting that PYR i nhibits a7- nAChRs in a noncompetitive manner.

 66

During voltage-c lamp experi ment , al terations i n i ntrace l l ular Ca2+ levels can

be monitored by ob erving change i n the ho lding currents. si nce Ca 2+-activated r

channel are highly en itive to change i n i ntracel l ular Ca2+ level i n Xenopu

oocyte ( for a rev iew: ( Fra er & Dj amgoz. 1 992)). I Iowever, during our experiments.

appl ication [ PYK v n at the highest concentration ( 1 00 �M) used i n this tudy,

did not cau e any alteration on the hol ding currents. suggesting that PYR did not

affect intracel l ular Ca2 concentrat ion .

I mportant! . the activity of an I on channel can be inhibited by several

d i fTerent mechani m . For i nstance, channel act ivity can be inhibited by preventing a

c l o ed channel from openi ng or promoting the c losure of an open chann e l . Another

\\ a)' of inhi biting current i n fl ux or efflux through an open ion channel is to sterically

occlude the open pore by a mechani sm cal led op n-channel blockade. I n this

mechani m, an open channel blocker enter the trans-mem brane l umen of a channel

and i nteract non-covalently with the pore- l i ning resi dues. Unl ike other channel­

i nh i bition mechani sms, open-channel blocker al lows a channel to open and close

nom1al l y i n response to sti mu l i but transiently and reversibly impedes current

fl owing through the open pore. Depending on the k i netics of blockade, open-chann e l

blockers c a n cause d i fferent effects o n the current mediated b y t h e opening of the

channel ( for further discussion see H i l le, 200 1 ).

I nterestingly, it has previously been reported that up to 90% of PYR i s

protonated i n aqueous solutions a t physiological pH ( Haley. 1 98 3 ) . However, this

situation is com p l icated due to h ighly hydrophobic structure of PYR with a

predictively calculated LogP of 3 .5 3 (Molinspiration software, Nova U l i c a, Slovak

Republ i c ) . The combination of a h i gh l i pophi l ici ty and charged molecular structure

makes PYR a suitable candidate for open channel blockade, which is a widely u ed
 67

m del to de cri be the bl ockade [ i on channels ( H i l le, 200 1 ) . H owe er. open channel

bl ockade does not seem to account [or the results obtai ned in the pre ent study.

F i rstl , for pen channel bl ocker , the pre ence of the agonist is requi red to let the

blocker enter the channel after the receptor has undergone an agon ist-i nduced

con [omlat ional change to open the channel. I n contrast to open channel bl ockers,

prei ncubation of PYR cau ed a further i nh ibition indicati ng that PYR can interact

with the clo ed state of the 0.7 nAChR. Secondly, i nh i bition by PYR is not voltage

sen itive, uggest ing that the PYR-binding site i s not within the transmembrane

electric field. F i nal ly, PYR did not signi ficantly affect the reversal potential of ACh­

induced currents, i ndicati ng that inhi bition is not due to an alteration in the ion

selectivi!) of the chanl1els.

Moreover. PYR did not ign i fi cantly change the potency of acetylchol i ne, the

natural l i gand (agonist) for this receptor, in electrophysiological studies conducted in

the current study. H owever, PYR signi ficantly reduced the efficacy of ACh,

i ndicat i ng that PYR acts in a noncompetitive manner. The structure of the h uman 0.7-

nAChR transmembrane domain has recently been solved and, also, the binding site

of ketami ne, an al l osteric modulator of a7-nAChR has been i denti fi ed; Using NMR,

ketamine was sho\\11 to bind to the i nternal cavity of thea? domain ( Bondarenko et

a1 ., 20 1 4). As PYR possesses s i m i l ar structural characteri stics and features to

ketam ine, such as the presence of a protonated am i ne group in their structure, we

hypothesized that both drugs may share the same binding pocket. Accordingly, PYR

and ketami ne were docked i nto the 0.7 domain i nternal cavity and the best three poses

were retained for v isual i n spection. As shown in Table 3 - 1 both PYR and ketam ine

were able to score favorable binding energies. The role of the protonated am i nes was

wel l i l l ustrated by the third ranked pose of Pyri l am i ne (-5 . 8 kcal/mol) as wel l as
 68

ketam ine (-4.2 kcal/m I ) . I ndeed, the positi ely charg d ami nes in the e pose 'were

able t form a cation-n interacti n vvith the aromatic ide chain of Phe453 ( Figure

3 - 1 j ). Interestingly, Phe453 wa con fi m1ed to play an important role in ketam ine

binding a it showed a change i n it c hemical h i ft when ketam ine was added to the

a7 domai n sol ution ( Bondarenko et aI . , 20 1 4). The binding of PYR and ketam ine

appeared to be further stabi l i zed by fom1i ng extensive van der Waals interactions

with the sunounding hydrophobic residues. Moreover, it seems that the larger size of

PYR ha re ulted in a uperior shape complementari ty with the a7 al l osteric site and,

hence, in a better docking score compared to ketam ine. Therefore PYR appeared to

ful fi l l al l tructural req uirements to bind allosterically with the transmembrane

domain of the a7nAChR c hannel . Importantly, it appeared that PYR and ketam ine

occl ude each other inhibitor action suggest ing that these compounds can compete

for a common binding site on the nicotinic receptor.

I t is l i kely that the anti-nicoti nic receptor effects of antih i stam ine tested in

the present tudy are rel evant to side and/or toxic effects of these drugs. For example,

plasma concentrations of PMZ, has been shown to be i n the range I -2 11M (AmaraL

Kristiansen. V iveiros, & Atouguia, 200 1 · Ter Laak, Donne-Op den Kel der, Bast, &

Timmerman, 1 99 3 ) .

otably, fi rst-generation anti histam ines are l i pid-so l uble drugs which can

freel y cross the blood-brain barrier at therapeutic doses and can affect the eNS with

resulting drowsi ness and the worsening of epi leptic symptoms ( Sadek et a I . , 20 1 3 ;

Yokoyama, Onodera, I inuma, & Watanabe, 1 993 ). I t is reported that in tissues basic

ambiph i l i c substances such as H I R antagonists used i n this study can reach up to 25-

fol d h igher plasma concentration ( Dan i e l , 2003 ; Jonkm.an, Westenberg, Rij ntj es, van

der, & Li ndeboom, 1 98 3 ; Mul ler, 1 996). To this end, the pl asma concentrations of
 69

the anti histam ine LP which has the second highest potency in our study were found

to reach a pia ma concentrations of 3 0- 1 00 �lM in patients who have received tox ic

overdo e ( Wogoman, tei nb rg, & Jenkin , 1 999); suggesting that inhi bition of

nic tinic receptor can play a role dur i ng chronic use or in tox ic effects of these

drugs.

In conclu ion our results indicate that fi rst-generation histam ine H I receptor

antagonists such as PYR, LP, PMZ and TRP inhibit directly the function of human

a7 nAChR, possi bly by an al losteric modulati on. This i nhibition may be associated

with ad\' r e neurological effects, especial l y seizures, in conditions of antih i stam ine

therapeutic and/or overdo e.

 70

B I B LI OG R A P H Y

\maraL L. . Kri tiansen. 1 . E . . l ve l ro . 1 . , & touguia. 1 . (200 1 ). Activity of

phenothiazi ne agai nst anti biotic-re i tant Mycobacterium tuberculosi : a
reyic\.\ 'upporting further tudie that may e l ucidate the potential use of
thioridaLi ne a anti-tuberculosis therap . Journal of A ntimicrobial
Chemotherapy , · r (5 ). 505-5 1 1 . doi : 1 0. 1 093/jac/4 7.5 .505

;\ hoor. . (20 1 1 ). Phenothiazine class antipsychotic inhibit the function oj the

humal7a � nicotinic.: acetylcholine receptor. UAE U ni versity .

Blandina. P . . Efoudebe. L enni G., Mannaioni, P . . & Passan i, M . B . (2004 ) .

Acet} Ichol i ne. I I i tami ne, and Cogn ition : T\vo ides of the Same Coi n.
I (!amil1R & .\ feIllOf)' . 11 ( 1 ), 1 -8 . do i : 1 0. 1 1 0 I /lm . 68004

Bondarenko. V . . f owrey. D. D .. Ti l l man. T. S . . eyoum . E .. Xu. Y . , & Tang. P .

(20 1 -1-). MR structmes of the human 0.7 nAChR transmem brane domain and
a soc iated anesthetic binding sites. Biochimica el Biophysica A cta. 1 838( 5 ),
1 3 8 9-95 . doi : 1 0. 1 0 1 6/j . bbamem .20 1 3 . 1 2 . 0 1 8

Bover. D . . Horcloi . R., & Wal thert. F. ( 1 944). Ami nopyridine, Proprietes
antihistanliniques de l a -p-methoxybenzyl - -di methylami noethyl al pha. CR
Soc BioI. ( 1 3 8 ). 99- 1 08.

Bree e. C R . , Adams. C , LogeL J.. Drebing, C , Rol l i ns Y. Barn hart, M., . . .

Leonard. . ( 1 997). Comparison of the regional expression of nicotinic
acetylcholine receptor 0.7 m RN A and [ 1 2 5 I J -o.-bungarotoxi n binding i n human
postmortem brain. The JOZlrnal of Comparative Neurology, 38 7(3 ), 3 8 5-39 8 .
doi : 1 0. 1 002/( S r C r ) 1 096-986 1 ( 1 997 1 027)3 8 7 : 3 <3 8 5 : : A I D-CNE5> 3 . 0.CO; 2-X

Chan-Tack, K. ( 1 999). euroleptic mal ignant syndrome due to Promethazi ne. South
:vfed J , 92( 1 0), 1 0 1 7- 1 0 1 8 .

Chen. Z . . Chen, J . Q., & Kamei , C (200 1 ). E ffect of H I -antagonists on spatial

memory deficit eval uated by 8-arm radial m aze in rats. A cta Pharmacol. Sin.
(22). 609-6 1 3 .

Conej ero-Goldberga, C . Daviesa. P., & U l loa, L (200 8 ) . A l pha7 n icotinic

acetyl chol i ne receptor: A l ink between inflammation and neurodegeneration.
Neuroscience and Biobehavioral Revie"vps, 32, 693-706.

Court. 1. A .. Mart i n-Ruiz, C. Graham, A, & PelTY, E. (2000) . N icotinic receptors in

human brain: topography and pathology . Journal of Chemical J euroanalomy,
20( 3 -4), 28 1 -298. doi : 1 0 . 1 0 1 6/S089 1 -06 1 8(00)00 1 1 0- 1

Court 1 . . & C lementi, F . ( 1 99 5 ) . Distri bution of n icoti nic subtypes in human brai n .
A l::.heimer Dis A ssoc Disord. . 9 ( Supp/, 6- 1 4 .
 71

ollsi ns, V . , Ro e . 1 . E . . & Le\ ' in, E . D . (20 1 4). I V ni cotine e l f-admini tration i n
rate; u ing a con ummatol) operant licking re pon e: ensi tivity to serotonergic,
gl utam inergic and histam i nergic d rugs. Progre 's in Nellro-P. )'chop!wrmaco!ogy
& Biological Psychiatry. 5-1. 200-5 . doi : l 0. J O I 6/j .pnpbp.20 1 4 .06 .004

Couturier, . . Bertnmd, D . , Matter, J. L I Iernandez, M. c . . Bertrand. . . M i l l ar, . ,

c t al . ( 1 990). n uronal nicoti nic acety lcho l i ne receptor subunit (al pha 7 ) I S
d e \ elopmentaJl) regulated and form a homo-ol igomeric charmel bl ocked by
al pha- BTX . Xel/ron, 5( 6 ) . 8-+ 7-8 56.

Daj a -Bai lad r, F. f ogg. A. 1 . . & Wormacott, . (2002 ). I ntracel l ular Ca2+
ignal ev ked b) sti mulation of ni cotinic acetylcholine receptors in SH-SY5Y
cel l : contri bution o r vol tage-operated Ca2 channel and Ca2+ stores. Journal
(�fXelirochell7i /IY, 8 1 ( 3 ) . 606-6 1 4 . doi : l 0. 1 046/j . l 4 7 1 -4 1 59. 2002. 00846.x

Daj as-Bai bdor. F. A. . ... oliako . L . , & Wonnacott. S . ( 2002 ). icotine activate the
extracell ular signal-regulated ki nase 1 12 v i a the a7 nicotinic acetylchol i ne
receptor and protein kina e A. i n SH-SY5Y cel ls and h i ppocampal neurones.
JOllrnal oj Xeurochellli try, 80( 3 ), 520-530. doi : l 0. 1 046/j .0022-
30-+2. 200 1 .00725.x

Dan i , J . . . & Bertrand, D. (2007). N icotinic acetylcho l i ne reseptors and n icotinic

chol inergi c mechan isms of the central nervous system. A nnllal Review
Pharmacology and Toxicology. -1 7, 699-729.

Danie l . W . . (200 3 ) . Mechanisms of c e l l ular d i stribution of psychotropic drugs.

ign i ti cance for drug action and i nteract ion . Prog. Neuropsychopharrnacol.
BioI Psychiatl}', ( 2 7 ), 65-7 3 .

D e Biasi , M . (2009). Encyclopedia oj Neuroscience. Encyclopedia oj Neuroscience

(pp. 1 1 3 5- 1 1 3 9). E l sevi er. doi : l 0 . 1 0 1 6/B978-008045046-9.0069 1 -4

Druce, H . ( 1 990 ) . Impaim1ent of function by Ant i h i stami nes. A nn A llergy, 6-1( 5 ),

403�0 5 .

D u , L Dong. H . , & Zhou, H.-X. ( 2 0 1 2 ) . Size matters in activation/inhibition o f

l i gand-gated i o n channels. Trends i n Pharmacological Sciences. 33(9), 4 8 2-93 .
doi : 1 0. 1 0 1 6/j . tips.20 1 2.06.005

Endo, Y. ( 1 982). imultaneous induction of h i stidine and omithi nedecarboxylases

and changes in their product amines fol lowing the injection of Escherichia col i
l ipopolysaccharide i ntom ice. Biochem Pharmacal, 3 1 , 1 643- 1 647 .

Ercan, Z . S . , & TurkeL R. K . ( 1 9 8 5 ) . N icotine is a competitive antagonist for H l ­

receptors i n smooth m usc les. IArcg. Int. Pharmacodyn. Ther. , ( 2 74), 24-3 0.

Fernandes, c.. Hoyle, E., Dempster, E . , Schalkwyk, L. C . , & Coll ier, D. A. (2006).
Performance deficit of u.7 nicotinic receptor knockout m ice in a delayed
matchi ng-to-place task suggests a m i ld i mpai rment of worki ng/episod ic- l i ke
 72

memory . Gene ,'. Brain and Behavior, 5(6), 43 3-+40. doi : 1 0 . l 1 1 1 lj . 1 60 1 -

J 8 3 . . 2005 . 00 1 76.x
Fraser. L . &
Dj amgoz,
• • L B . . ( J 992 ). Xenopus oocytes: endogenous
electroph) iological characteri st ics. N J\ . Osborne (Ed. ). Current Aspects of the
SClIroscfence . The .'vfacmillan Pres. . Ba. ings/oke, 267-3 1 5 .

Fratiglioni, L. , & Wang, H .- X . (2000). Smoking and Parki n on' and Alzheimer's
di ea c : revie\\ f the epi dem iological tudies. Behavioural Brain Research.
1 13( 1 -2). 1 1 7- 1 20. doi : 1 0. 1 0 1 6/S0 1 66-4328(00)00206-0

l I ak) , T. J. ( J 9 8 3 ) . Ph) sical and bio logical propeliies of pyri lamine. JOZlrnal of
Pharmaceutical Science . 72( 1 ), 3- 1 2 . doi : l 0. 1 002/j ps .2600720 1 03

l Ial gren, T. . ( 1 996a). Merck molecular force field. 1. Basis, [om1, scope.
parameteri zation, and performance of I M FF94. Journal of Computational
Chemisl/y, r '( 5 - 6 ). 490-5 1 9 . do i : 1 0 . 1 002/( I C I ) 1 096-
987X( 1 99604 ) 1 7 : 5/6<.A 90: : A I D-JCC 1 >3 .0.CO:2-P

Halgren. T. . ( l 996b) . Merck molecular force field. I I . MMFF94 van der Waals and
electrostatic parameters for i ntermo lecular i nteractions. Journal of
Complltational Chemistry, } 7(5-6). 520-552. doi : 1 0. 1 002/( S l C I ) 1 096-
987X( 1 99604 ) 1 7 : 5/6<520 : :A l D-JCC2>3 . 0.CO;2-W

Halgren, T. . (l 996c ) . Merck molecular force field. I I I . Molecular geometries and

vi brational frequencie for M MFF94. Journal of Computational Chemistry,
1 7(5 -6). 5 5 3-586. doi : 1 0. 1 002/( S I C I ) 1 096-987X( 1 99604) 1 7 : 5/6<5 5 3 : :A I D­
JCC3>3 .0.CO;2-T

H a l gren. T. A ( l 996d ) . Merck molecular force field. V. Extension of MM FF94

using experi mental data, additional computati onal data, and empi rical rules.
Journal of Computational Chemish-y, 1 7(5-6), 6 1 6-64 1 .
doi : 1 0 . 1 002/(S I C J ) 1 096-987X( 1 99604) 1 7 : 5/6<6 1 6 : : A l D-JCC5> 3 . 0.C0;2-X

Halgren. T. A , & Nachbar, R. B . ( 1 996). Merck molecular force field. I V .

confom1ational energle and geometries for M MFF94 . Journal of
Computational Chemistry, 1 7(5-6). 5 8 7-6 1 5 . doi : 1 0 . 1 002/( S I C I ) 1 096-
987X( 1 9960-i ) 1 7 : 5/6<5 8 7 : : Al D-JCC4> 3 .0.CO:2-Q

Han uskova, E . . & P levkova, 1 . (20 1 3 ) . H I STAM IN E AND I TS EFFECTS

M E D I ATED VIA H3 RECE PTOR POTENTIAL CLI lCAL
A P P L ICATIONS OF H 3 A TAGON I STS . A C TA AfEDICA A1A R TIN1ANA ,
Suppl. 1 . doi : 1 0.2478/acm-20 1 3-0004 A d

H e l l strom-Lindah l . E . , Court, Keveme, Svedberg, Lee, Marutle, . . . Nordberg.

(2004) . i cotine reduces A beta in the brain and cerebral vessels of APPsw
M i ce. European Journal ojNeuroscience, 1 9. 2703-2 7 1 0.

H e l l strom- Li ndahl . E., Mousavi , M., Ravid, R . , & Nordberg, A (2004 ) . Reduced
l evels of Abeta 40 and Abeta 42 in brains of smok ing controls and Alzheimer's
 73

patients. Xellrohi% KY Disca e. 1 5( 2 ). 3 5 1 -60.

do i : 1 0. 1 0 1 6 j .nbd .200 ... . J 1 .024
I fill. . J.. & Baker J. G. ( 2004 ). Encyclopedia of Biological Chemistry.
EI7(l'c/opedia o{ Bi% gical Chemistl), ( pp. 3 7 8-3 83 ). El evier. doi : 1 0 . 1 0 1 61B0-
1 2-443 7 1 0-9/00309-4

I I indmarch, L J hn ·on. ., Meadov. . R., Kirkpatrick, T., & hamsi, Z. ( 200 I ). The
acute and sub-chron ic effects of Ie ocet iri zine, cetirizine, l oratadine,
promethazine and placebo on cognitive function, psychomotor performance, and
\\ eaJ and f1 are. Curr Afed Res Opin 1 7(4) 24 1 -2 5 5 .
. ,

I Io\,,·arth. P. ( 1 999). Asse sment of antihi stam ine efficacy a n d potency. Clin Exp
rt f/erg)" 29. 8 7-97.

I lu. M. (2002 ). icotinic Regulation of C REB ctivation in Hi ppocampal Neurons

b Gl utamatergic and Nongl utamatergic Pathways. j40leclilar and Cellular
Xeuro eimce, _ 1 (4), 6 1 6-625. doi : l 0. 1 006/mcne. 2002 . 1 202

T r i er. Y . , Bertrand, D. (200 1 ). eur'onal nicotinic receptors : from protein structure

.

to function. FEBS Leffen , 50-/(3 ), 1 1 8- 1 2 5 . doi : 1 0. 1 0 1 6/S00 1 4-

5 793(0 I )02702-8

Jone , ., Sudweeks. .. & Yakel. 1. L. ( 1 999). N icotinic receptors in the brain :

correlating ph) siology with function. Trends i n j eum. ciences, 22( 1 2) . 5 5 5-
5 6 1 . doi : 1 0. 1 0 1 6/S0 1 66-2236(99)0 1 47 1 -X

Jonkman, 1 . Westenberg. H . . , Rij ntj es, N . . , van der, K . , & Li ndeboom, S . .

" .

( 1 983 ) . Whole body distribution of the quaternary ammonium compound

t h i azinam i um (N-methyl promethazine) and promethazi ne in monkey and mice.
A r:;neimittelforschllng. ( 3 3 ). 223-228.

J utel , lvL Akdis. M., & Akdis. C . ( 2009) . H istam ine. histamine receptors and their
rol e i n immune pathol ogy. Clin Exp A llergy, 39, 1 78 6- 1 800.

Kavanagh. 1. J . , Grant. G. D .. & Anoopkumar-Dukie. S. (20 1 2 ). Low dosage

promethazine and loratadine negat ively affect neuromotor function. Clinical
Xeurophy iology : Official Journal of the international Federation of Clinical
Xeurophysio!op:y, J 23(4). 780-6. doi : 1 0. 1 0 1 6/j .cl inph.20 1 1 .07.046

Kenney , J . , & Gould. T. (2008). Modulation of hippocan1pus-dependent l earn i ng and

synaptic plasticity by n icotine. l'vfol Neurobiol. , 38( 1 ), 1 0 1 - 1 2 1 .

Kholdebarin , E . , Caldwe l l , D. P . , B l ackwelder, W. P . , Kao, M . , Christopher. N . c . , &

Levin. E. D. ( �007). I nteraction of nicoti nic and histamine H ( 3 ) systems in the
radial -arm maze repeated acq uisition task . European Journal of Pharmacology,
569( 1 -2), 64-9. doi : 1 0. 1 0 1 6/j .ej phar.2007.04.05 1
K i m , D . - C . . Park, Y - S . , Jun, D.-1., Hur, E.-M., Kim, S .- H . , Choi , B . - H . , & K i m , K.­
T. ( 2006). -(4-Tri fl uoromethyl phenyl )am i de group of the synthetic hi stam ine
receptor agonist inhibits nicot i n i c acetylcho l i ne receptor-mediated
 74

catechol am i ne ecretion. Biochemical Phannac% gy, 7J ( 5 ) , 670-82 .

doi : 1 0. 1 0 1 6/j . bcp. 2005 . 1 1 .02 1
K i m , D.-C., Y U I1 , . .J . . Park, Y . - . , Jun, D .-J ., Kim, D . , J iten i ngh, N . , . . . Kim, K . ­
T. (20 1 4 ). P ri l am i ne inh ibi ts nicotine-i nduced catecholamine sec retion.
XClIroch 'mi, II)' international, 7-1, 42-5 . doi : l 0. 1 0 1 6/j . neui nt.20 1 4 .04.0 1 5

K u ano, K . , 1 i led i . R., & ti nnakre, 1 ( 1 982). Chol i nergic and catecholami nergic
receptors i n the Xenopus oocyte membrane. 1 -/3- 1 70, ( 3 28), 1 43- 1 70.

Labute, P . (�008). The general ized Bom/vol um e integral impl i cit sol vent model :
Esti mation of the free energy o f hydrat ion using London dispersion i nstead of
atomic urface area. Journal of Computational Chemistry, 29( 1 0). 1 693- 1 698.
doi : I 0. 1 002/j cc.2093 3

Labute, P . . & Protonate, D. (2009) . Assignment of ionization states and hydrogen

coordi nates to macromolecular structures Protei ns: truct. Funct. Bioinfonn . ,
( 7 5 ), 1 87-205 .

LEE. F . , MATTH E W , L . . M ILLS, L DUBNO, J . , & A DK IN S , W . ( 1 998 ). Gender­

spec i fi c effects of medici nal dmgs on hearing l evels of o l der persons.
O{olw�1'l1g01ogy Head and Neck Surger)'. J 1 8( 2 ), 22 1 -227.
doi : 1 0. 1 0 1 6/S0 1 94-5998(98 )80020-X

Levin. E. D . . S l ade, S . , Wel l s, c . , Pruitt, M . , Cousi ns, V . Cauley. M . , . . . Rose, 1

.

( 20 1 1 ). H i stamine H ( 1 ) antagonist treatment with pyri lamine reduces n icot i ne

sel f-ad m i nistration i n rats. European Journal of Pharmacology, 650( 1 ), 256-60.
doi : I 0 . 1 0 1 6/j .ej phar.20 1 0 . 1 0.0 1 3

L i ndstrom, J . (2003 ). N i cotinic acetylchol i ne receptors of m uscles and nerves:

comparison of their structures, functional ro les, and vul nerab i l i ty to pathology.
A nn N Y A cad Sci. , 998, 4 1 -5 2 .

L i u, Q . , Zhang, J . , Zhu, H . , Q i n, C., Chen, Q . , & Zhao, B . (2007). D issecting the

signal i ng pathway of n icotine-mediated neuroprotection in a mouse Alzheimer
disease Model . FASEB Journal, 2 1 , 6 1 -7 3 .

Lowe E . , Mac M i l l an , R . , & Katser, M . ( 1 946). The anti histam i ne propelties o f

Benadryl. beta-dimethyl -aminoethyl benzhydry l ether hydrochl oride. J
Pharmacal Exp Ther, (86). 229.

Lynch, M. A. (2004) . Long-term potentiation and memory. PhYSiological Reviews,

8-/( 1 ). 8 7- 1 3 6 . doi : l O . 1 1 5 2/physrev . 000 1 4 .2003

M aggi , L, Sher. E . , & C herubini, E. (200 1 ). Regulation of GABA release by

n icotin i c acetylcho l i ne receptors in the neonatal rat hi ppocampus. The Journal
a/Physiology, 5 3 6( 1 ) 89- 1 00. doi : l 0. 1 1 1 1 /j . 1 469- 7793 . 200 1 .00089.x
,

Mahdy, A . M., & Webster, N. R. (20 1 1 ). H i stam ine and antihistam ines. A naesthesia
& intensive Care MediCine, 1 2(7), 324-3 29. doi : 1 0. ] 0 J 6/j . mpaic.20 1 1 .04 .0 1 2
 7S

1a kos, . (2008). The cho l i nergic me opontine tegmentum i s a rel atively neglected
ni coti ni master m dulator or th dopam inergic ) stem : relevance to drugs of
abu' C and pathol og:; . British Journal of Plwrll1C1colot-,y) ', i 53 (Suppl, S43 8-445 .

lena- egovia. 1., Winn. P., & B lam. J . P. ( 200 8 ) . (2008). Cho l i nergic modulation
or midbrai n dopami nergi c system . Brain Rescarch Reviews. 58(2). 265-27 1 .

l erk. 1 1 . (200 I ). tandard treatm nt : the role of antih istam ine . J ime, fig Derma/ol
�yl/1p Proc. 6 .

.l leyler '.� Side Effe cts 0./ Drugs: The international Encyclopedia of Adverse Drug
Reactiol1s and Interaction . (2006a). ,'vieyler 's Side Effects of DrZ/gs: The
Intcrnational Encyclopedia of A dverse Drug Reactions and interactions (pp.
:2 3 8-2939). El sevier. do i : 1 0. 1 0 1 6/BO-44-45 1 005 -2/00672-0

.\Jeylcr · ide Effect · of Drug ': The international Encyclopedia of A dverse Drug
Reactions and interactions. (2006b) . .'vJe)'/el" 's Side Effect of Drug : The
il1ternatiollal Encyclopedia (�f Adrerse Drug Reactions and interactions (pp.
3 05-3 1 6) . E l sevier. doi : 1 0. 1 0 1 6/BO-44-45 1 005-2100634-3

Mul ler, T. ( 1 996). E l ectron microscopic demonstration of intrac e l l uar promethazi ne

accumulation sites by a prec ipitation technique: appl i cation to the cerebel l ar
cortex of the mouse. J His/ochel71. CytocheJl1. (44), 53 1 -5 3 5 .

akam ura. T . . I tadani . H. . H idaka, y . , Ohta, M . , & Tanaka. K . (2000). Molecular

c l o n i ng and characterization of a ne" hlU11an histamine receptor, H H4R.
Biochem BiophJ's Res Cornmllil. ] 79. 6 1 5-620.

ordberg, A., H e l l strom - Lindah l . E . . Lee. r . . Johnson, M., Mousavi , M., Hal l, R.,
. . . Court, J. (2002 ) . Chronic nicotine treatment reduces �-amyloidosis i n the
brain of a mou e model of Alzhei mer' disease (APPsw). Journal q(
Neurochemistl:v. 8 1 ( 3 ). 655-65 8 . do i : 1 0 . 1 046/j . 1 4 7 1 -4 1 59.2002.00874.x

Oda. T .. Morikawa. .. aito. y . . M asuho, Y . . & Matsumoto. S. (2000). Molecular

c l o n i ng and characteri zation of a novel type of histam i ne receptor preferentially
expressed in l eukocytes. J BioI Chem. 2 75. 3678 1 -3676.

Okura. T . . Hattori. A . , Takano, Y .. Sato, T., Hammarl und-Udenaes, M., Terasaki , T . .

& Deguchi. Y . (2008). I nvolvement of the pyri lam i ne transporter, a putative
organic cation transpOlter, i n b lood-brain barrier transport of oxycodone. Drug
,\tIelab. Di po (3 6). 2005-20 1 3 .
. .

Ono. K . . H asegawa, K . , Yamada, M . , & Naiki. H . ( 2002 ). N i cotine breaks down

preformed A lzheimer's �-amy loid fibri l s in vitro. Biological Psychiatry, 52(9),
8 80--8 86. doi : 1 0. 1 0 1 6/S0006-3223 (02)0 1 4 1 7-8

Oz. M . , Ravindran, A . . D i az-Ruiz, 0., Zhang, L. , & Moral es, M . (2003 ). The
endogenous cannabinoid anandamide inhi bits alpha7 nicotinic acety lcholine
receptor-mediated responses in Xenopus oocytes. J Pharmacol Exp Ther,
306( 3 ) . 1 003- ] 0 1 0.
 76

Papke. R. L. C�O I ··n. Mergi ng old and ne"" perspecti ves on nicotinic acety l choline
receptors. Biochemical Pharmacology. doi : 1 0. 1 0 1 6/j .bcp.20 1 4.0 1 .029

Parson ', L E .. & Ganel l i n , 2 . Robin. (2006). I Ii tam ine and its receptors. British
Joumal oj Pharmacolo[!J:, 1 -1 , , 1 2 7- 1 3 5 .

P e t i , K . , , labo, A K . . f i ke. A , & Vizi, E. . (20 1 4). Kinetic properties and open
probabi l i t) of u7 nicotinic acet) !cho l i ne receptors. j cliropharl71ocology X l ,
1 0 1 - 1 1 5 . doi : l 0. 1 0 1 6/j . neurophaml . 20 1 4 . 0 1 .034

Potter. A . . Com in, J., Lang. .T Pia ecki, M., Lenox, R., & Newhouse. P. A. ( 1 999 ).
. •

Acute e rfect or the selective chol inergic channel activator (nicotinic agonist)
AB [-4 1 8 in Izhei mer' disease. Psychopharmacology, 1 42(4), 3 3 4-342.
doi : 1 0. 1 007Is002 1 ., 0050897

Radc l i ffe. K. . , & Dan i, J. A . ( 1 998). icotinic stimulation produce mUltiple fonns
of increased gl utamatergic synapt ic transmis ion. J nellroscience, ( 1 8), 7075-
7083 .

R b i n on. . (20 1 3 ) . 2 2 Twenty TlI'o Nol a new l!(esty/e For those 11'ho thirst for
hllJJlUmll'. Lulu.com . Retrieved from http ://books.google. comlbooks?i d=qzC­
BAAAQBAJ&pgis= l

Robin on. L . , P l att. B . , & Riede l , G. (20 1 1 ). I nvolvement of the cho l i nergic system
in conditioning and perceptual memory . Beha\' Brain Res.

Rubbo l i . F . , Court, 1 . A . . ala, c. , Morris, c . . C h i n i , B . , Perry, E . , & Clementi, F.

( 1 994) . Distribution of Nicotinic Receptor i n the Human H ippocampus and
Thalamus. European Journal of Neuroscience. 6( 1 0), 1 596- 1 604.
doi : 1 0. 1 1 1 1 Ij . 1 460-9568. l 994 .tb005 50.x

adek, B . . Shehab. . . Wiycek, M., Subramanian, D., Shafi u l lah, M., Kiec-
Kononowicz, K., & Adem, A. (20 1 3 ) . Anti convul sant properties o f hi stam ine
H 3 receptor l i gands belongi ng to N-substituted carbamates of imidazopropanol .
Bioorganic & ,\dedicinal Chemistry Letters, 23( 1 7), 4 8 8 6-9 1 .
doi : 1 0 . 1 0 1 6/j . bmc I . 20 1 3 .06.075

Sands. . B . . Costa. A. c . . & Patrick, J. W . ( 1 993 ). Barium permeab i l i ty of neuronal

n icot i n i c receptor alpha 7 expressed in Xenopus oocytes. Biophysi(;({l Journal,
65(6). 26 1 4-2 l . doi : 1 O. l 0 1 6/S0006-3495(93 ) 8 1 296- 7

Seguela. P . . Wadiche. 1 . , Dineley-Mi l Ier, K . , Dani J . A., & Patrick. 1 . W. ( 1 993 ).

( 1 993 ). Molecular clon i ng, functional properties, and distri bution of rat brain
alpha 7: A nicoti nic cation channel highly permeable to calcium. The Journal of
Neuroscience, 1 3(2), 596-604 .

esack, . R . , & Grace. A . A . (20 1 0). COIiico-Basal Ganglia Reward etwork :

M icroc i rcuitry. Neuropsychopharmacology, 35( 1 ), 2 7-4 7 .

harm a , G . , & Vij ayaraghavan, S . (200 1 ). Nicot i n i c cho l i nergic signal ing in
hi ppocampal astrocytes invol ves calc i um-i nduced cal cium reJease from
 77

i ntracel l ular tores. Proceedings of the Xafional Academy of Sciences . 9R (7 ),

4 1 48-4 1 5 3 . d i : l 0. 1 073 pna .07 1 540 1 98
S hyt!c, R. D . , Mori , T. , TO\,\ n end, K . . Vendrame, L un, " Zeng, J . , . . . Tan. J .
(2004 ). Cho l i nergic modulation of microgl ial acti vat ion by a7 nicotinic
receptors. Journal 01 1 eurochemi, flY, 89( 2 ), 3 3 7- 43. doi : 1 0. 1 046/j . 1 47 1 -
4 1 59.2004.02347.x

i l va, A . . , Kogan. 1. . . Frankland, P . . , & Kida, . ( 1 998). CREB and Memory .

A nnual Review q( Neuroscience, 2 1 , 1 27- 1 48 .

, I 10 . F. ( 1 989). I I- receptor antagonists: Cl inical pharmacol ogy and therapeutics.

Journal of .rJ //erg)' and Clinical Immunology, 8./(6), 845-86 1 .
doi : 1 0. 1 0 1 6/009 1 -6749(89)90 3 7 7- 1

ou a. . F . , Fernandes. P . a. & Ramos, M . J . (2006). Protei n l i gand dock ing: current

tam and future chal lenges. Bioit?(orll7atics. 26( February ), 1 5-26.
doi : 1 0 . 1 002 prot

pUI'den, D. " Court, J . . , Lloyd. S .. Oakl ey, A . , Perry, R . , Pearson, C . . . . . Perry. E . .

( 1 99 7 ) . N icoti nic receptor distribution In the human thal am us:
autoradiograph i cal localization of [ 3 H ] n icotine and [ 1 25 I ]a-bungarotoxin
b i nd i ng. Journal of Chemical J\ euroanafomy. /3(2). 1 05- 1 1 3 .
doi : 1 0. 1 0 1 6/S089 1 -06 1 8(97)0003 8-0

treit. W. J. (2002). M ic rog l i a as neuroprotective, i m munocompetent cel l s of the

C . Glia. ./0( 2 ). 1 3 3- 1 3 9. doi : 1 O. 1 002/g1 ia. l 0 1 54

uzuki . T.. H i de. 1 . , [at ubara, A., Hama, Harada, K . . M i yano. K . , . . . Nakata.
(2006). Gcrogl ial al pha7 n icotinic acetylcho l i ne receptors drive a
phosphol i pase CIIP 3 pathway and modulate the cel l activation tov,' a rd a
neuroprotective role. Journal ofNeuroscience Research. 83, 1 46 1 - 1 470,

Tega, Y . Akanuma, S . - I . . K ubo, Y, Terasaki . T., & Hosoya, K . - I . (20 1 3 ). B lood-to­

brai n i n fl ux transp0l1 of nicotine at the rat b lood-brain barrier: involvement of a
pyri lamine-sensitive organi c cation transport process. Neurochemistry
international. 62(2). 1 73-8 1 . doi : 1 0. 1 0 1 6/j .neuint.20 1 2 . 1 1 . 0 1 4

Ter Laak. A . M . . Donne-Op den Kelder, G. M . , Bast, A . . & T i mmerman, H . ( 1 993).

I s there a di fference in the affinity of histam i ne H I receptor antagonists for eNS
and perip heral receptors? A n in vi tro study. European Journal vf
Pharmacvlvgy. 232(2-3 ). 1 99-205 . doi : 1 0. 1 0 1 6/00 1 4-2999(93 )90774-C

Thurmond. R., Pl . D .. Pl, D., WP, F . - L . , CL, H . , W, J., . . . A, K. L. (2004 ). A potent

and sel ective h i stamine H4 receptor antagonist \.... ith ant i i nflammatory
properties. Pharmacal Exp Ther, 309, 404-4 1 3 .

Timmerman, H . , & van der Goot. H . (2009 ) . Encyclopedia of Neuroscience.

EncJ'clopedia of Neuroscience (pp. 1 1 49- 1 1 66). El sevier. doi : l 0 . 1 0 1 61B978-
008045046-9.0 1 1 49-9
 78

rueciako\\"'k i . L & Lc\ i . R. ( 1 983 ). A lIerr) '. Princ iples and Prac!ice . (E.
:

1 iddlcton. . Reed, ' E . I I i . d . ) ( 2 ml eel.). Mosby, 1. Louis.

te'hc\ . V . V. ( 20 1 2 ). ??7 nicoti n ic ACh receptor as a l i gand-gated source of a 2+

ions: The 'earch for a Ca 2+ optimum. A dl'Onces in Experimental lv/edicine and
Bi% ,f!J', 7..fO. 603-6 3 8 .

Utsugi sa\\ a. K.. agane, Y . . Tohgi . H. , Yoshim ura, M . , Ohba, H . & Genda, Y.
.

( 1 999). Changes "" ith agi ng and i chemia in n i cotinic acetylcholine receptor
'ubun it a7 mR expre ion in po tmortem human frontal cortex and putamen .
.Yelfroscience Lefler . 2 �OC' ), 1 45- 1 48 . doi : 1 0. 1 0 1 6/S0304-3 940(99)00473-5

tsuk i . T . hoaib. f . . H o l l oway, H . I n gram, D., Wal bce. W .. Haroutunian, v . , . . .

.

re l g, . ( 2002 ) . icoline lowers the ecretion of the A lzheimer's amy loid

beta-protein precursor that contains amyloid beta-pept ide i n Mouse. Journal of
A I:::h eimcr " Di eo,\ c . ..f, 405-4 1 5 .

\,: allace. T L . . & Por1er. R. H . P. ( 20 1 1 ). Target ing the n i cotinic alpha7

ncety ! c ho l i ne receptor to enhance cognition in disease. Biochemical
Plwrmw:ology, 82, 89 1 -903 .

Walsh. G . , Arumnziato. L. , Frossard. . , KnoL K . , Levander. S . , Nicolas, l-M . . . . .

Timmem1Cln H . (200 1 ). New I nsights i nto the Second Generation
ntihi tami nes. Drugs. 6 J ( 2 ). 207-236. doi : 1 0.2 1 65/00003495-200 1 6 1 020-
00006

Wang. H . - Y . . Lee, D. H. S .. D ' Andrea, M. R., Peterson, P. A., Shank, R. P., & Reitz,
A . B . (2000). �-Amyloid l -42 B i nds to a7 icot i n i c Acetyl c ho l i ne Receptor
with H igh Affi n ity : I M P L I CATIONS FOR A LZH E I M ER ' S D I S EA S E
PATHOLOGY . JOlfrnal of Biological Chemisfly 2 75 ( 8 ), 5626-5632.
.

doi : 1 0 . 1 074/jbc .275 . 8 .5626

WestfalL T C . ( 2009). Encyclopedia of Neuroscience. Encyclopedia ofNeuro cience

(pp. 8 1 7-834). E l sevier. doi : 1 0. 1 0 1 6/B978-0080-+5046-9. 0 1 1 3 3 - 5

Weyers. A . . Jeske. A . , Lobron, c . , B i 11sch. c . . Hei nemann, S . , Mae l icke A., . . .

Schroder. H . ( 1 994) . C e l l ular distribution of ni coti nic acetylcho l i ne receptor
ubuni t m RNAs in the human cerebral cortex as revealed by non-isotopic in situ
hybridization. j\;foie C1l iar Brain Research, 25( 1 -2), 1 22- 1 2 8 . doi : 1 0. 1 0 1 6/0 1 69-
3 2 8X(94 )90286-0

White. H. K .. & Levin, E. D. ( 1 999). Four-week nicotine ski n patch treatment effects
on cogn it i ve perfOlmance i n Al zhei mer" s disease. Psychopharmacology. 1 ..f3(1).
1 5 8- J 65 . doi : l O . 1 007/s002 1 3 00S093 1

W i l son. A . L . , Langley, L. K . , Monley. 1 . , Bauer. T . Rottunda, S . , McFal ls E . . . .

McCarten. J . R. ( 1 99 5 ) . Nicotine patches in A lzheimer' s disease : Pi lot study on
learni ng, memory, and safety . Pharmacology Biochemistry and Behavior, 5 1 (2-
3). 509-5 1 4. doi : 1 0. 1 0 1 6/009 1 -305 7(95 )00043-V
 79

Wog man. I I .. tei nberg. 1.. & Jenkins. A. . ( 1 999 ) . Acute i ntoxication with
guaifenesin. di phenhydram ine. and chlorpheni ramine Pathol. d in. J Forensic
.Hec/ . (20). 1 99-202.

Yokoyama. H . . Onodera. K .. I i numa. K . . & Watanabe, T. ( 1 99 3 ) . Proconvulsive

. .
e ffects of hi tamine H I -antagonist on electrica l l y-induced selzure In
de\el ping m i ce. PsychopharmacologJ. 1 1 2(2-3 ) . 1 99-2 03 .
doi : 1 0. 1 007tBF022449 1 1

Yonkman. r . . hess. D . . Mathi eson. D . . & H ansen. . ( 1 946). Pharmacodynam ic

tudics of an ne\ antihistam ine agent, N '-pyridyl- -benzyl -N-dimethylethyl ene
diamine H C ! . pyribenzamine H C l . J Pharmacol E\p The/". ( 8 7 ). 256.

Young. J . W .. Cra\\ D rd, .. Kell , J . S . , Ken, L. E .. Marston, H. M . , Spratt, c. . . . .

harkey. J . (2007). I mpaired attention is central to the cognitive defi c i ts
ob erved i n al pha 7 deficient m ice. European J ellrop:,�n.'hopharmClcology : The
JOllrnal o/ fhe European College of europ .vc!Jopharmacology. r '(2). 1 45-5 5 .
doi : 1 0 . 1 0 1 6/j .euroneuro. 2006.03 .008

Zhou. F. tv1. W i l son, C. 1 . . & Dan i . J. A. (2002 ). Cholinergic I ntemeuron

,

Characteri stics and nicot i n i c properties in the stri atum. Journal (�INeurobi()l()gy,
53(4). 590-605.
 79

Wogoman. H . . teinberg, L & Jenkin , A. . ( 1 999). Acute i ntox ication with

guai fene in. di phenhydram ine. and chl orphen i ramine Pathol. Am .J Foremic.

,'vied , ( 20), 1 99-202.

Yokoyama, H. . Onodera. K . I i numa, K . , & Watanabe. T. ( 1 993). Proconvul ive

.

. .
e lTects of histam ine I I l -antagoni t on electrical l y-i nduced se I zure 1 11
de eloping m I ce. Psychopharmacology. 1 1 2(2- 3 ). 1 99-203 .
ooi : 1 0. 1 007 B F022449 I 1

Yonkman. F . . Ches , D., athie on. D . . & Hansen, . ( 1 946). Pharmacodynam ic

stlld ie of an new antihistam ine agent, '-pyridyl -N-benzyl-N-di methylethylene
diam ine HeL p; ribenza m i ne H C ! . J Pharmacol Exp Ther. ( 8 7 ), 256.

YOllng. J. W . . C rawford, N . , Kelly, J . S., Ken L. E . . Marston, H . M., pratt, C . . . . .

harke)', J . (2007 ). Impaired attention is central to the cogn itive deficits
ob erved in al pha 7 defi c ient mice. European J europsychopharnwcology . The
Journal a/ the European College of Neurops)'chopharmacalogy. 1 7( 2 ), 1 4 5-5 5 .
doi : 1 0. 1 0 1 6/j .euroneuro . 2006.03 .008

Zholl, F. 1. , W i ! on. C. L & Dan i , J. A. ( 2002 ). Cholinergic I nterneuron

Characteristics and nicotinic propeliies in the st riatum . Journal of Neurobiology,
53(4). 5 90--6 05 .
 79

\: ogoman, I I . . teinberg, L & Jenkin , A. . ( 1 999). cute intoxication with

guai fene in, di phenhydram ine, and chl orphen i ramine Pathol . Am . .J Forensic
.\fed. , (20). 1 99-202.

Yokoyama. H . , nod era. K . , I i numa, K . , & Watanabe, T. ( 1 993 ) . Proconvulsive

effect of histrun ine I l l -antagonist on electrical ly-induced sejzure i n
developing mice. Psychopharmacology. J J 2(2- 3 ). 1 99-203.
d i : 1 O. I 007 BF022449 1 I

Yonkman, F . . Chess, D . Mathie on. D . , & Hansen. . ( 1 946). Pharmacodynami c

.

tudies of a n ne\-v antihi stam i ne agent, '-p_ ridy l -N-benzyl -N-dimethylethylene

diamine HCL pyri benza m i ne f IC ! . J Pharmacal Exp Thcr, ( 8 7 ), 256.

Young, J. W . . C ra\\ ford, N . . Kel ly, 1 . S . KelT, L. Eo, Marston, H . M., Spratt. c . , . . .
harke , J . (2007). I mpaired attention is central to tbe cogn i ti ve defic its
observed in al pha 7 deficient mice. European Neurop.'(vchopharmacology : The
Journal a/ fhe European College of Neurop ) 'chopharmaca/ogy. J �( 2 ) , 1 45-5 5 .
doi : 1 0. 1 0 1 6/j .euroneuro . 2006.03 .008

Zhou, F . ! . , Wil on, C. 1 . . & Dani , J. A. ( 2002 ). Cho l i nergic l ntememon

haractcristics and nicotinic propeli ies i n the striatum. Journal o/Neurobiology,
53(4). 590-605 .

Digitally signed by
Shrieen
DN: cn=Shrieen, o=UAE
University, ou=UAEU
Libraries Deanship,
email=shrieen@uaeu.a
c.ae, c=US
Date: 2016.02.24
'13:38:23 +01'00