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Chem.

-Biol Interacttons, 74 (1990)263-274

263

Elsevier Scientific Pubhshers Ireland Ltd.

COMPARATIVE EFFECTS OF THREE NATURALLY OCCURRING


F U R A N O C O U M A R I N S ON M I T O C H O N D R I A L B I O E N E R G E T I C S

OLUFUNSOO. OLORUNSOGO',ANTHONYO. UWAIFOb and SYLVIA O. MALOMOb*


Laboratory for Btomembrane Research and ~Oncology Research Sectton~ Department of B~ochemistry, College of Medicine, Universtty of lbadan, lbadan (Ntgerm)

(Received May 23rd, 1989)


(Revision received October 20th, 1989)
(Accepted November 20th, 1989)
SUMMARY
Oxygraphic measurements of the rates of mitochondrial respiration in the
presence of varying amounts of chalepin, imperatorin and marmesin, three
naturally occurring furanocoumarins, revealed that the oxidation of NAD linked substrates was inhibited by chalepin and imperatorin and less
significantly by marmesin. The order of potency being rotenone ) ) chalepin
imperatorin > marmesin, There was no effect whatsoever on succinate
oxidation by the furanocoumarins tested (up to 60 ~V[). State 3 respiration
was also inhibited by these furanocoumarins; by at least 800/0 by 10 ~
cha]epin and by 48 and 290/0 with 60 ~Vl imperatorin and 60/~M marmesin,
respectively. Consequently, ADP control of respiration was diminished by
those concentrations of furanocoumarins that inhibited respiration. At 60
~M, respiratory control ratio was reduced by about 88, 49 and 280/0 with
chalepin, imperatorin and marmesin, respectively.
A measurement of the rate of proton and Ca2-movements across the mitochondrial coupling membrane demonstrated that succinate-supported transport was not affected by these furanocoumarins. On the other hand,
pyruvate/malate-supported proton ejection was significantly inhibited by
chalepin, imperatorin and marmesin. The order of the degree of inhibition of
proton flux is rotenone > ) cha]epin ~ imperatorin ~ marmesin. The pattern of the inhibition of pyruvate/malate-supported Ca2-transport was identical to that seen during proton transport. A comparison of the effects of
chalepin to that of rotenone suggests that cha]epin might be about 10 times
less potent than rotenone.

K e y words: Furanocoumarins - Rotenone -- Mitochondrial bioenergetics

*Present address: Department of Biochemistry,University of florin, llorin, N]gerla.


0009-2797/90/$03.50
1990Elsevier Scientific Publishers Ireland Ltd.

264
INTRODUCTION

In tropical Africa, many plants of diverse species are used for curative
purposes. These medicinal plants include species such as clausena, crotalaria
and senecio [1]. For example, concoctions of the roots of Clausena anisata
(wild) Rutaceae are widely used in the treatment of haemorrhoids in most
West African countries including Nigeria [1,2]. Furthermore, in Central
American countries, different parts of the plant Arura (Ruta Chalepensis)
are also frequently ingested as herbal teas for the treatment of measles,
scarlet fever, headaches, and heart conditions [3]. In order to assess the biological activity and safety of these preparations, certain furanocoumarins
including chalepin (I), imperatorin (II), and oxypeucedanine (III) have been
isolated and purified to homogeneity from different parts of Clausena anisata
(wild) [4] (Fig. 1). Marmesin (IV) another furanocoumarin has been isolated
and purified to homogeniety from Afraegle paniculata (Rutaceae) [5]. It
seems likely that the frequent and uncontrolled use of these plants could
have adverse effects on mitochondrial energy metabolism, because of the
structural resemblance of these chemical compounds to (1) aflatoxin B1, an
uncoupler of mitochondrial respiration and a potent carcinogen produced by
certain strains of the fungus, Aspergillus flavus and (2) rotenone, a naturally
occurring fish poison and a potent inhibitor of mitochondrial respiration [6,7].

Ii

H'

AFLATOXI N B1

ROTENONE

'3=Cl"13

F~g. 1. Structures of four naturally occurring furanocoumarms, aflatoxm B, and rotenone: chalepm (I), ~mperatorm (II), oxypeucedamne (III) and rnarmemn (IV)

265
Toxicological studies have shown that although only chalepin has
significant necrotic and anticoagulant actions when intraperitoneally administered to rats [8], marmesin and imperatorin may be somewhat mutagenic [9].
We have investigated the possible effects of chalepin, imperatorin and marmesin on mitochondrial bioenergetics, in order to gain insight into the mechanism of interaction of these chemicals with mitochondrial metabolism. We
have reported elswhere the results of a preliminary investigation on the
inhibitory effect of chalepin on mitochondrial respiration [10].
In this paper, we present a detailed account of the interaction of these
chemicals with mitochondrial bioenergetics. Our results show that chalepin
and imperatorin inhibit the oxidation of NAD-linked substrates in the same
manner as rotenone. In addition, Ca 2 accumulation and proton translocation
are inhibited by these chemicals when mitochondria are respiring on NAD linked substrates. The order of potency is rotenone > > chalepin > imperatorin > marmesin.
MATERIALS AND METHODS

Materials
Pure samples of chalepin, imperatorin, marmesin were obtained from
Professors D.A. Okorie and K. Adesogan, Chemistry Department, University
of Ibadan, Nigeria. The purity of these compounds were determined by a
comparison of their spectra (IR, UV, NMR and mass spectroscopy) and
melting points with those of authentic samples [4,5]. All other chemicals
were of the highest purity available and were purchased from either Sigma
Chemical, London, or Fluka AG (CH-9470 Buchs), Switzerland. Mitochondria
were isolated in 250 mM sucrose essentially according to the method
described by Schneider and Hogeboom [11] from the livers of adult Wistar
strain albino rats obtained from pathogen-free colonies of the Pre-Clinical
Animal Breeding House, University of Ibadan. The respiratory control ratio
of each mitochondrial preparation was not less than 4.0. Mitochondrial protein was determined by use of the biuret reagent [12].
Methods
Polarogrpahic measurements of oxygen uptake. The rate of oxygen consumption by mitochondria was measured by use of the conventional Clarktype oxygen electrode (Yellow Springs Instruments, OH, U.S.A.) polarographic technique. When a steady-recorder tracing was achieved, an aliquot
of the mitochondrial fraction (final concentration 2 mg mitochondrial protein/
ml) was introduced into the reaction vessel which contained 1.1 ml reaction
medium (120 mM KC1, 20 mM Tris--HC1, pH 7.4 and 5 mM KH2P04}. After
the stabilization of the recorder pen, and aliquot of any of pyruvate/malate
{3.98 raM/1.9 raM} or succinate {3.98 raM) was carefully introduced by use of a
Hamilton syringe inserted through the opening in the glass stopper of the
reaction vessel. Aliquots of ADP (final concentration 50 nmol ADP/mg mitochondrial protein) or furanocoumarin were added at specific intervals after

266

obtaining satisfactory steady-recorder tracing. Aliquots of oligomyc~n or


FCCP were also added in some experiments. Rotenone was always added
together with succinate to block the transfer of electrons from 3-site
substrates. Respiratory rates and control ratios were computed as described
by Estabrook [13].
Measurement of proton transport. Proton ejection was measured according to the method described by Reynafarje et al. [14]. Pi-depleted mitochondria were prepared by pre-incubating washed mitochondria with 250 mM
sucrose, 10 mM KC1 and 3 mM HEPES (pH 7.1) for 10 rain in the cold, after
which the suspension was diluted with more of the incubation medium and
the mitochondria re-isolated and suspended finally in a small volume of the
250 mM LiC1, 10 mM KCI, 3 mM, HEPES, 4 taM rotenone and 100 ng valinomycin/mg protein (pH 7.2). Changes in the pH of the reaction medium were
followed by means of Philips pH-glass electrode connected through a bucking voltage box to a Perkin-Elmer Model 56 recorder. After 2 rain of incubation to ensure that endogenous substrates and ATP were depleted, 1 mM
potassium succinate or 1 mM pyruvate/malate was added to induce proton
translocation. In the test experiments with the furonacoumarins, an aliquot
of the compound to be tested was added after the addition of the substrate.
The rate of proton flux was computed as ng-ions H/min per mg protein. The
standard proton translocator used was FCCP.
Measurement of Ca~ movements across mitochondmal membrane.
Changes in the extra-mitochondrial concentration of free Ca 2 were followed
using a calibrated Ca 2 ion-selective electrode as modified by Reynafarje et
al. [15]. The electrode potentials were amplified by a Philips pH meter linked
through a bucking voltage box to a Perkin-Elmer recorder Model 56. The
reaction vessel contained, in final concentrations, 150 mM KCI, 3 mM
HEPES, 2 taM rotenone, 2 mM succinate and 100 taM CaC12. After obtaining a
steady-recorder tracing, 7.5 mg mitochondrial protein were added to initiate
the rapid uptake of Ca 2. This was followed by a retention of the accumulated Ca 2 by the respiring mitochondria; 1 taM FCCP or an aliquot of the
test compounds was immediately added to release the accumulated Ca ~. The
rate of flux of Ca ~ was computed as ng-ions CaZ/min per mg protein.
RESULTS
Effects of chalepin, marmes~n, and ~mperatomn on mitochondmal metabohc
state $ resp~ratzon
The effects of chalepin, lmperatorin, and marmesin on pyruvate/malatesupported state 4 respiration of rat liver mitochondria are shown in Tables I,
II and III, respectively. In this experiment, aliquots of the furanocoumarins
being tested were added to the reaction vessel on attainment of state 4 or
resting state respiration following ADP depletion in metabolic state 3 respiration. The effect seen consisted of a concentration-dependent inhibition of
state 4 respiration by the three furanocoumarins up to 60 taM which inhibited respiration by 69, 40 and 20% for chalepin, imperatorin and marmesin,

267
TABLE I
MODIFICATIONS OF MITOCHONDRIAL BIOENERGETICS BY CHALEPIN
Each value ~s a mean for five different mitochondrial preparations S.D. RCR or respn-atory
control ratio was computed as ratio of state 3 rate to state 4 rate ~n the absence of
furanocoumarin.
Chalepzn
(/aM)

State 4
State 3
(ng A 02 m-1 mg protein)

State 4
plus FCCP

RCR Rate of H*
Rate of Ca2.
ejection
accumulation
(ng runs H" or Ca2mm-1 mg
protein-1)

0
0r
5
10
15
20
30
40
60
60b
Rotenone

20.1
17.6
18.1
15.5
6.5
6.7
6.4
6.4
6.2
10.5
6.7

90.3
74.6
72.4
24.0
13.1
11.6
10.2
8.4
6.6
7.3
5.9

4.2
4.2
3.5
0.8
0.6
0.6
0.5
0.5
0.4
0.5
0.4

1.2
0.8
0.9
0.9
0.7
0.7
0.6
0.3
0.3
0.7
0.5

84.6
73.9
69.9
16.5
12.6
11.9
9.3
9.2
9.0
8.9
8.7

2.2
2.7
3.1
0.7
0.7
0.6
0.7
0.6
0.4
0.6
0.6

2.7
2.1
1.8
0.9
0.8
0.9
0.8
0.5
0.4
0.5
05

193.6
186.3
31.6
21.7
20.5
17.5
17.5
17.1
21.0

4.6
4.1
1.1
1.2
1.0
0.9
0.8
0.8
0.9

97.3
81.5
19.3
14.1
14.6
14.3
14.1
13.8
14.1

3.4

2.8
1.7
1.1
0.8
0.7
1.1
0.7

0.9

(1.5 ~v~)
Respn'atory substrate: pyruvate/malate
bRespiratory substrate: 5 mM/~-hydroxybutyrate

r e s p e c t i v e l y . T h e r e s u l t s also s h o w t h a t s t a t e 4 r e s p i r a t o r y r a t e w a s r e d u c e d
b y 68, 25, a n d 17/0 a t 30 /~M of c h a l e p i n , i m p e r a t o r i n a n d m a r m e s i n ,
r e s p e c t i v e l y . A t 10 /~lVl, o n l y c h a l e p i n s i g n i f i c a n t l y i n h i b i t e d s t a t e 4 r e s p i r a t i o n , b y 2 3 % . A l t h o u g h , a b o u t 10/0 i n h i b i t i o n w a s s e e n w i t h 5 wM c h a l e p i n ,
l o w e r c o n c e n t r a t i o n s of t h e t h r e e f u r a n o c o u m a r i n s (~< 5 ~M) did n o t s h o w a n y
a p p r e c i a b l e e f f e c t o n s t a t e 4 r e s p i r a t i o n . A s a s s e s s m e n t of t h e e f f e c t of t h e
f u r a n o c o u m a r i n s o n s u c c i n a t e - s u p p o r t e d s t a t e 4 r e s p i r a t i o n is p r e s e n t e d i n
T a b l e IV. H e r e , it is e v i d e n t t h a t n o n e of t h e t h r e e f u r a n o c o u m a r i n s t e s t e d
h a d a n y e f f e c t o n t h e r a t e of s t a t e 4 r e s p i r a t i o n w h e n s u c c i n a t e w a s u s e d as
an electron donor. Similarly, no inhibition was o b s e r v e d w h e n the furanocoum a r i n s w e r e p r e - i n c u b a t e d with fresh mitochondrial p r e p a r a t i o n s , in the
p r e s e n c e of s u c c i n a t e a l t h o u g h t h e r e w a s a m i l d s t i m u l a t i o n of s t a t e 4 r e s p i r a t i o n a t c o n c e n t r a t i o n s g r e a t e r t h a n 60 ~M c h a l e p i n ( r e s u l t s n o t s h o w n ) .

Effects of chalepin, marmesin and imperatorin on respiratory control by


ADP
T a b l e s I, I I a n d I I I s h o w t h e p r o f i l e s of t h e r a t e s of A D P - s t i m u l a t e d or
m e t a b o l i c s t a t e 3 r e s p i r a t i o n i n t h e p r e s e n c e of p y r u v a t e / m a l a t e a n d v a r y i n g
a m o u n t s of c h a l e p i n , i m p e r a t o r i n a n d m a r m e s i n , r e s p e c t i v e l y . T h e d a t a s h o w
t h a t A D P - s t i m u l a t e d r e s p i r a t i o n w a s i n h i b i t e d b y a t l e a s t 80/0 b y 10 ~M

268
TABLE II
INFLUENCE OF IMPERATORIN ON MITOCHONDRIAL BIOENERGETICS
Each value ~s a mean for f~ve different mitochondr~al preparations __ S.D. RCR or respiratory
control raho was computed as raho of state 3 rate to state 4 rate m the absence of furanocoumarin
Imperatorin
(~I)

State 4
State 3
(ng A O~ m-1 mg protein-~)

State 4
plus FCCP

RCR Rate of H*
Rate of Ca2
e~echon
accumulahon
(ng ~ons H or Ca2m~n-1 mg
protein-l)

0
0b
5
10
15
20
30
40
60
60b
Rotenone
(1.5 ~M)

23.5
19.2
22.1
21.6
20.0
188
17.6
16.5
14.2
10.9
5.6

91.3
87.5
83.4
77 6
70.3
675
60.1
52.1
45.7
80.6
7.1

41
4.2
3.8
3.6
3.4
3.2
2.7
2.4
2.1
2.4
0.4

1.1
0.9
0.7
0.7
06
0.7
0.6
0.7
0.6
04
03

96.5
82.6
89.7
84.1
79.3
744
63.9
57.8
50.1
46.3
9.7

2.1
1.9
2.1
2.2
1.8
1.5
1.9
2.1
1.1
1.3
0.7

1.8
21
1.6
21
1.4
13
1.5
0.9
0.7
2.3
0.6

189 7
-186.5
161.2
143.5
1336
119.6
117.6
114.5
-20 6

4.7
4.3
3.1
3.7
2.9
2.3
3.1
27
0.8

95.7
-94 6
83.7
76.6
69.3
60.7
53.6
51.2
-13.8

3.1

3.2
2.1
2.5
2.1
1.3
1.4
1.2

09

Respiratory substrate: pyruvate/malate.


bRespiratory substrate: 5 mM/~-hydroxybutyrate.

chalepin, indicating t h a t r e s p i r a t o r y control by A D P was almost totally lost


a t t h i s l e v e l of c h a l e p i n . W h e r e a s , a b o u t 13 a n d 8O/o i n h i b i t i o n o c c u r r e d w i t h
10/~M i m p e r a t o r i n a n d 10 ~M m a r m e s i n , r e s p e c t i v e l y . A l t h o u g h t h e s e v e r i t y
of i n h i b i t i o n i n c r e a s e d s t e a d i l y w i t h i n c r e a s i n g a m o u n t s of i m p e r a t o r i n a n d
m a r m e s i n u p t o 60 ~Vl, r e s p i r a t i o n w a s i n h i b i t e d m a x i m a l l y b y a p p r o x i m a t e l y 89o/o a t c o n c e n t r a t i o n s e q u a l t o or h i g h e r t h a n 60 pM c h a l e p i n . M a r m e s i n a n d i m p e r a t o r i n a t 60 /~M c o n c e n t r a t i o n i n h i b i t e d A D P - s t i m u l a t e d
r e s p i r a t i o n b y 29 a n d 4 8 % , r e s p e c t i v e l y .
R e s p i r a t o r y c o n t r o l b y A D P in a n i m p o r t a n t c r i t e r i o n for d e t e r m i n i n g t h e
c o u p l i n g of m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t t o A D P p h o s p h o r y l a t i o n . I t is
c o n v e n t i o n a l t o e x p r e s s t h i s c o n t r o l as a r a t i o of t h e r a t e of t h e A D P - s t i m u l a t e d or s t a t e 3 r e s p i r a t i o n t o t h e r a t e of t h e A D P - l e s s o r s t a t e 4 r e s p i r a t i o n .
T h i s r a t i o is t e r m e d t h e a c c e p t e r or r e s p i r a t o r y c o n t r o l r a t i o . V a l u e s for
mitochondrial p r e p a r a t i o n s r e s p i r i n g on p y r u v a t e / m a l a t e and in the
p r e s e n c e of v a r y i n g c o n c e n t r a t i o n s of t h r e e f u r a n o c o u m a r i n s a r e s h o w n i n
T a b l e s I - I I I . T h e r e s u l t s s h o w t h a t t h e r e s p i r a t o r y c o n t r o l r a t i o of r a t l i v e r
m i t o c h o n d r i a is r e d u c e d b y t h e s e f u r a n o c o u m a r i n s in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . T h e r e s p i r a t o r y c o n t r o l r a t i o of t h e m i t o c h o n d r i a l p r e p a r a t i o n
u s e d in t h i s s t u d y w a s n o t less t h a n 4.0. T h i s r a t i o w a s r e d u c e d b y 17, 7, a n d
5O/o b y a d d i n g to t h e r e a c t i o n v e s s e l , 5 pM c h a l e p i n , 5 pM i m p e r a t o r i n a n d

269
TABLE III
INFLUENCE

OF MARMESIN

ON MITOCHONDRIAL

BIOENERGETICS"

Each value is a mean for five different mitochondrlal p r e p a r a t i o n s S.D RCR or r e s p i r a t o r y


control ratio was computed as ratio of s t a t e 3 r a t e to s t a t e 4 rate in the absence of furanocoumarin.
Marmemn
(/~IVl)

State 4
State 3
(ng A 02 m -1 m g protein -~)

State 4
plus FCCP

RCR

Rate of H
Rate of Ca 2~
ejectlon
accumulation
(ng ions H or Ca 2 mln -~ m g
proteln -~)

0
0b
5
10
15
20
30
40
60
60 b
Rotenone

23.0
18.1
22.8
21.8
21 3
20.3
19.7
19.2
18.3
14.1
55.2

97.3
795
91.5
87.4
80.6
76.3
71.6
69.9
67.3
60.1
6.2

4.0
4.3
3.8
3.7
36
34
32
3.0
2.9
3.1
0.4

190.2
-183.7
180.6
179 6
171.2
163.4
160.2
157.3
-21.7

09
0.6
0.7
0.8
0.9
0.7
0.7
0.6
0.5
0.6
0.3

93.6
778
89.4
86.2
81.7
79.6
74.1
70.1
66.6
57.6
9.9

3.1
2.1
3.0
27
2.1
1.8
2.7
1.6
1.5
--+ 1.1
0.4

__

2.1
2.8
2.7
21
2.1
2.0
2.1
1.7
2.6
1.8
0.4

2.1

3.4
3.7
3.2
2.7
2.1
31
2.7

0.9

96.6
-94.6
92,6
92,3
89,4
83,6
79.1
73.6
-13.8

26

2.1
1.8
1.4
1.7
1.4
1.6
1.7

0.7

(1.5 ~V~)
' R e s p i r a t o r y s u b s t r a t e : pyruvate/malate.
bRespiratory s u b s t r a t e : 5 mM/~-hydroxybutyrate.

T A B L E IV
M I T O C H O N D R I A L B I O E N E R G E T I C S IN T H E P R E S E N C E OF C H A L E P I N , I M P E R A T O R I N
AND M A R M E S I N D U R I N G S U C C I N A T E O X I D A T I O N
Each value is a m e a n for four &fferent mitochondrial p r e p a r a t i o n s S.D
Additions

State 4
State 3
(ng A 02 m -1 m g protein-9

State 4
plus FCCP

RCR

Rate of H
Rate of Ca 2
ejection
accumulation
(ng ions H or Ca 2 m~n-1 m g
protein -1}

None
Chalepm

30.6 1.4
29.7 1.0

138.4 4.1
140.1 3.8

141.3 5.2
145.6 4.1

4.5
4.6

249.2 + 3.7
2449 41

1397 + 3 9
141.0 3 6

31.1 1.3

139.6 3.3

137.0 5 1

45

253.1 4.4

142 1 __ 4.1

31.7 1.7

141.7 3 7

142.6 4.7

4.6

250.3 4.6

144.1 3 7

32.1 0.9

140.1 4 0

140 7 3.7

4.6

2486 3.9

139.3 _+ 4 2

(60 ~M)
Imperatorin

(60 ~ )
Marmesm

(60 ~M)
Rotenone
(1.5 ~M)

270
5 ~M marmesin, respectively before adding ADP, the phosphate acceptor. At
10 ~M, this ratio was further reduced by 81, 13, and 8/0, with chalepin,
imperatorin and marmesin, respectively. The extent of inhibition of ADPsupported respiration with 40, 50 and especially 60 ~M chalepin was not less
than 88%, suggesting that the mitochondria were no longer able to synthesize ATP at these levels of chalepin. Respiratory control ratio was reduced by
49 and 28/o by 60 ~M imperatorin and 60 ~M marmesin, respectively.
Table IV summarises the results obtained when succinate was used as an
electron donor. As seen from the results, state 3 respiratory rate was not
affected by any of the three furanocoumarins at the highest concentrations
tested. Consequently, respiratory control by ADP was not altered by these
furanocoumarins during succinate oxidation.

Effect of furanocoumamns on respiration-dmven proton translocation by rat


liver mitochondria
The pattern of proton transport during the oxidation of succinate in the
presence of varying amounts of these furanocoumarins being investigated is
shown in Table IV. Succinate-supported proton translocation was unaffected
by the three furanocoumarins tested. The pattern of the effect of these furanocoumarins on proton translocation by mitochondria respiring on pyruvate/malate is summarized in Tables I - I I I . As seen from these results,
respiration-driven proton pumping was inhibited in a concentration-dependent manner by imperatorin and marmesin up to 60 ~M. Although chalepin
inhibited the process more than marmesin and imperatorin, no further inhibition was observed at chalepin concentrations higher than 15 wM. Thus the
degree of inhibition (89o/0) seen with 20, 30, 40 and 60 ~M chalepin was not
statistically different from that seen with 15 pM. The process was maximally
inhibited by 30 ~M marmesin, 30 pM imperatorin and 15 ~M chalepin by 14,
37 and 89o/0, respectively (Tables I--III).
Abolition of mitochondrial Ca~uptake by furanocoumarins
Ca e accumulation by mitochondria is supported by the membrane potential created either by respiration or by the hydrolysis of ATP. Because of
the relevance of the process in the mechanism of regulation of intracellular
free Ca ~ ion concentration as well as its sensitivity to respiratory inhibitors,
the influence of the three furanocoumarins of interest on the respirationdriven Ca~+-uptake by isolated rat liver mitochondria was studied. Changes
in extramitochondrial Ca e ion concentration during the oxidation of succinate or pyruvate/malate was followed by use of a sensitive Ca~-ion selective
electrode as described under Materials and Methods. The results obtained
with mitochondria respiring on pyruvate/malate are summarized in Tables I
--III. Again chalepin inhibited Ca ~ accumulation by over 85% at 15 ~M even
though the extent of abolition of Ca ~ uptake was not statistically different
at concentrations ~> 15 ~M chalepin. Maximal inhibiting of about 86/0 was
seen at 60 ~M chalepin. These results show that Ca ~ uptake was inhibited
almost totally at 15 ~M (Table I). Furthermore, the results are identical to

271
the effects of chalepin on respiratory control by ADP and respiration-driven
proton translocation.
The effects seen with imperatorin and marmesin differ (Table II and III)
however, from those seen with chalepin because the abolition of Ca~ uptake
was concentration-dependent up to about 30 ~M for these furanocoumarins
and was maximal at 60 ~ I imperatorin (Table II) which reduced the rate of
uptake by 460/0, and similarly at 60/~M marmesin which gave only 240/0 inhibition (Table III). The results show in addition, that at 15 pM, addition of
imperation and marmesin to the reaction medium gave 20 and 7/0 inhibition,
respectively. A comparison of the effects of chalepin with that of rotenone
revealed that the inhibition caused by the addition of 1.5 pM rotenone was
statistically equal to that produced by 15 ~M chalepin, thus indicating that
chalepin could be about 10 times less potent than rotenone.
Table IV shows the pattern of mitochondrial Ca2 transport supported by
succinate and in the presence of varying amounts of the three furanocoumarins. Clearly, succinate-supported Ca~-accumulation was not inhibited by any
of the furanocoumarins studied. Furthermore, an assessment of the interaction of these naturally-occurring chemicals with ATP-supported Ca2-accumulation revealed a no-effect response even at 60 ~M chalepin (results not
shown).
DISCUSSION

In this study, evidence is presented to show that chalepin and imperatorin


have significant inhibitory effects on the generation of proton electrochemical potential gradient during the NAD-linked oxidation of substrates by rat
liver mitochondria. An assessment of the interaction of chalepin, imp~eratorin, and marmesin with resting state respiration reveals that of the three
furanocoumarins, only chalepin and imperatorin significantly inhibited the
oxidation of pyruvate/malate; the degree of inhibition by chalepin (15 /~I)
being equal to that produced by 1.5 ~ rotenone (Table I). Interestingly,
succinate oxidation was insensitive to these chemicals even on pre-incubation
of mitochondria with high levels of the furanocoumarins. These results suggest therefore, that chalepin and imperatorin may be blocking electron transfer from NADH to coenzyme Q because electron transier from succinate via
FAD to coenzyme Q remains unaffected as in rotenone inhibition. The mechanism of this interaction is, however, not clear because of the bulky nature
of NADH co-enzyme Q reductase [16].
An important parameter for assessing mitochondrial bioenergetics is the
respiratory control ratio, which is a ratio of the rate of resting state respiration to the rate of ADP-stimualted respiration. A glance at the values
obtained for state 3 respiration at varying concentrations of furancoumarins
reveals that state 3 respiration is almost totally (at least by 80%) inhibited
by 10 ~M chalepin; indicating that ADP was not phosphorylated as such at
this amount of chalepin (Table I). Furthermore, imperatorin (60 ~M) inhibited
ADP phosphorylation by 480/0 while marmesin (60 ~M) gave a 29% inhibition.

272
Similar results were obtained when /~-hydrobutyrate was used as substrate
(Tables I--III), suggesting that the inhibition of phosphorylation of ADP by
chalepin and imperatorin is due to an interaction of the furanocoumarins
with the NADH-coenzyme reductase complex rather than with the NAD dependent dehydrogenase responsible for oxidizing pyruvate. Furthermore,
it seems likely that because state 4 respiration is almost completely annuled
by chalepin, enough protons are not translocated out of the matrix even on
addition of ADP, thus preventing the formation of sufficiently high
transmembrane pH difference necessary to form the proton electrochemical
potential difference. Thus the respiratory control ratios obtained in the presence of rotenone and/or chalepin are reduced by about 90O/o, whereas the
effect of imperatorin (60 ~M) is about 48/o that seen with chalepin. Marinesin has a slight inhibitory effect (28O/o) on state 3 respiration (Table III).
To confirm the effect of these compounds on electron transfer-linked
proton pumping, mitochondrial proton translocation was monitored by use of
a sensitive pH-glass electrode during exposure of mitochondria to varying
concentrations of the furanocoumarins. Our results demonstrate unequivocally that chalepin and imperatorin inhibit pyruvate/malate-supported proton
ejection (Tables I and II). Specifically, chalepin (15 ~M) almost completely
inhibited proton ejection while imperatorin (30 ~M) inhibited the process by
37O/o (Table II). Again, succinate-supported proton ejection was not affected
by any of the three furanocoumarins (Table IV). Clearly, electron transfer
proton pumping is obstructed by chalepin and imperatorin at the level of
NADH-coenzyme Q reductase. Although these compounds are likely to prevent pyruvate/malate-supported proton pumping indirectly by blocking electron transfer from NADH to coenzyme Q, the possibility that they could
interact directly with the hydrophobic proton translocating component of the
complex may not be completely ruled out.
However, because the proton electrochemical potential gradient is made
up of a pH difference and an inside negative membrane potential which is
normally used to drive the energy-dependent Ca 2 uptake by mitochondria,
the effects of chalepin, imperatorin and marmesin on the process of
mitochondrial Ca 2 uptake were investigated (Tables I--III). The results
obtained by following the rate of respiration driven Ca 2 accumulation by
mitochondria using an ion-selective Ca 2 electrode, reveal that chalepin
abolished Ca e uptake supported by pruvate/malate in the same manner as
rotenone while imperatorin only slightly reduced the ability of mitochondria
to accumulate Ca ~ (Tables I and II). Marmesin has a very mild inhibitory
effect on this process (Table III). The effect of vitamin K-3 on furanocoumarin-inhibited mitochondrial respiration is shown in Table V. The data
show that furanocoumarin-inhibition of pyruvate/malate oxidation was bypassed by adding catalytic amounts of vitamin K3 to mitochondria previously
exposed to chalepin, imperatorin and marmesin, respectively, in the same
manner as amytal [17]. This finding offers additional evidence that electron
transfer from NADH to Co Q was blocked by these furanocoumarins during
the oxidation of NAD-linked substrates.

273
TABLE V
EFFECT OF VITAMIN K-3 ON FURANOCOUMARIN-INHIBITED RESPIRATION"
Each value is a mean for three mitochondrial preparations standard deviation. Reaction
medium same as described under materials and methods. The additions made consisted of the
following in final concentrations: antimycin (0.22 ~g), rotenone (1.5 ~ I ) , vitamin K ~ (5 nmol),
dicumarol (10 tool), and amytal (1 mM),
Additions

State 4 rate
(ng A O~ mm -1 mg protein -1)

State 3 rate

Control
(no additions)
Vitamin K ~
Dicoumarol (a)
Antimycin (b)

17.3 0.9

79.6 2.1

17.4
17.1
3.2
4.7
4.9
10.4
13.3
17.2
9.5
5.1
17.6
17.3

80.3
80.1
4.1
50
5.5
59.2
66.4
80.1
14.1
6.3
81.3
81.6

Rotenone
Chalepin (60/~I)
Imperatorin (60 ~u~I)
Marmesin (60 ~lVl)
Chalep]n + Vit K-3
Chalepin + Vlt K-3 (a)
Chalep~n Vit K ~ (b)
Imperatorin + Vit K-3
Marmesin + Vit K ~

0.7
1.0
0.4
0.3
0.4
0.5
0.7
0.8
0.6
0.3
0.8
0.9

1.9
2.1
0.3
0.4
0.3
1.0
1.5
1.8
0.7
0.2
1.6
1.7

Pyruvate/malate was used as respiratory substrate.

Although the exact mechanism of inhibition of electron flow from NADH


to Co Q by these furanocoumarins is not known, it seems possible that they
could be acting like rotenone or piericidin [18] because (1) FCCP does not
relieve their inhibitory effects (Tables I--III), and (2) the chemicals possess
groups (Fig. 1) such as ),,),-dimethylallyl- in position 3 of chalepin, a 3-methylbut-2-enyloxy- in position 7 of imperatorin, and isopropenyl- in position 5 of
rotenone, in addition to either a furanocoumarin or coumarin ring which
could carry electrons/protons probably in the same manner as the 7,8-dimethyl-isoalloxazine moeity of FMN but unable to donate the electrons to the
next carrier due to the electron donating effect of the groups mentioned
above on the coumarin ring. Thus, aside from the hydrophobic characters of
these chemicals, the magnitude of the electron-donating effect of these
groups could determine the degree of potency of these compounds as inhibitors of electron transfer. Because millimolar amounts of amytal and about 1
and 0.3 nmol/mg protein of rotenone and piericidin are, respectively,
required to nearly totally inhibit the NADH oxidase of electron transport
particles [18], the order of potency of these inhibitors vis-a-vis chalepin may
be put as piericidine Y rotenone Y chalepin ~ amytal. It seems unlikely,
however, that chalepin and the other furanocoumarins studied will interact
with electron transfer in the same manner as rhein (4,5-dihydroxyanthra

274

quinone-2-carboxyhi acid), a compeititive inhibitor of NADH oxidation [19]


because vitamin K-3 restored respiration of furanocoumarin-inhibited mitochondria.
It may thus be concluded from the findings reported here that an
excessive and frequent ingestion of these substances could cause a serious
disturbance of energy metabolism in certain tissues. The relationship
between these effects and the toxicity of these substances and their metabolites should therefore be investigated.
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14
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