Xavier F. Figueroa and Brian R.

Duling
Am J Physiol Heart Circ Physiol 295:2001-2007, 2008. First published Sep 12, 2008;
doi:10.1152/ajpheart.00063.2008

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Dissection of two Cx37-independent conducted vasodilator mechanisms
by deletion of Cx40: electrotonic versus regenerative conduction
Xavier F. Figueroa1 and Brian R. Duling2
1
Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile,
Santiago, Chile; and 2Department of Molecular Physiology and Biological Physics, University of Virginia,
Charlottesville, Virginia
Submitted 18 January 2008; accepted in final form 9 September 2008
Figueroa XF, Duling BR. Dissection of two Cx37-independent
conducted vasodilator mechanisms by deletion of Cx40: electro-
tonic versus regenerative conduction. Am J Physiol Heart Circ
Physiol 295: H2001–H2007, 2008. First published September 12,
2008; doi:10.1152/ajpheart.00063.2008.—Conduction of changes in
diameter plays an important role in the coordination of peripheral
vascular resistance and, thereby, in the control of arterial blood
pressure. It is thought that conduction of vasomotor signals relies on
the electrotonic spread of changes in membrane potential from a site
of stimulation through gap junctions connecting the cells of the vessel
wall. To explore this idea, we stimulated a short segment of mouse
cremasteric arterioles with an application, via micropipette, of ACh,
an endothelium-dependent vasodilator, or pinacidil, an ATP-sensitive
K⫹ channel opener. Vasodilations were evaluated at the stimulation
site (local) and at 500, 1,000, and 2,000 ␮m upstream. The vasodilator
response evoked by direct arteriolar hyperpolarization induced by
pinacidil decayed rapidly with distance, as expected for the passive
spread of an electrical signal. Deletion of the gap junction proteins
connexin37 or connexin40 did not alter the conduction of pinacidil-
induced vasodilation. In contrast to pinacidil, the vasodilator response
activated by ACh spread along the entire vessel without decrement.
Although the ACh-induced conducted vasodilation was similar in
wild-type and connexin37 knockout mice, deletion of connexin40
converted the nondecremental conducted response activated by ACh
into one similar to that of pinacidil, with a decline in magnitude along
the vessel length. These results suggest that ACh activates a mecha-
nism of regenerative conduction of vasodilator responses. Connexin40
is essential for the ACh-activated regenerative vasodilator mecha-
nism. However, neither connexin40 nor connexin37 is indispensable
for the electrotonic spread of hyperpolarizing signals.
gap junction; connexin37; connexin40; conducted vasodilation; endo-
thelium; mouse cremaster arterioles
SYSTEMIC BLOOD PRESSURE is determined by cardiac output and
the resistance to blood flow in small arteries and arterioles in the
microcirculation. Precise regulation of blood flow distribution
depends on the well-integrated regulation of vasomotor tone
along the length of arterioles and on the coordination of
different microvessel segments (7, 15, 17, 47). Consequently,
longitudinal conduction of vasomotor signals has emerged as
an important physiological mechanism involved in the control
of blood flow distribution and coordination of vascular resis-
tance within the microvascular network (13, 14).
Conducted vasomotor responses have been associated with
the propagation of an electrical signal (7, 10, 15, 17, 47),
usually attributed to electrotonic spread of changes in mem-
Address for reprint requests and other correspondence: X. F. Figueroa,
Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas,
Pontificia Universidad Católica de Chile, Santiago, Chile (e-mail: xfigueroa
@bio.puc.cl).
http://www.ajpheart.org 0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society
Am J Physiol Heart Circ Physiol 295: H2001–H2007, 2008.
First published September 12, 2008; doi:10.1152/ajpheart.00063.2008.
brane potential via gap junctional connection between adjacent
cells (17, 34). Both dilator and constrictor signals propagate
electrotonically, but, in addition, endothelium-dependent vaso-
dilations have been observed to propagate along the entire
vessel without decay, suggesting the activation of a regenera-
tive mechanism (6, 7, 10, 15, 33). The molecular basis for the
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regenerative propagation of a vasomotor signal along the
arterioles remains to be determined.
Gap junctions are intercellular channels that connect the
cytoplasm of adjacent cells, allowing the passage of small
molecules (molecular mass ⬍1,000 Da) and current (13, 35).
Vascular gap junctions are assembled from one or more con-
nexin (Cx) proteins: Cx37, Cx40, Cx43, and Cx45 (27, 42).
Cx45 is expressed only in smooth muscle cells, whereas Cx37
is typically found only in endothelial cells. Although Cx40 and
Cx43 are expressed in both cell types (16, 29, 42), Cx40 is
located predominately in endothelial cells (16, 42), and, in the
mouse, this Cx has been observed solely in the endothelium (7,
15, 38).
Gap junctions are critical in cell-to-cell communication in
the cardiovascular system (13, 14). In particular, Cx40 seems
to play a key role in the control of microvascular network
function, because deletion of Cx40 has been associated with
irregular vasomotion (8) and a reduced conduction of vasodi-
lator responses induced by ACh, bradykinin (7, 46), or elec-
trical stimulation (15) in mouse cremaster arterioles, which
suggests that responses activated by endothelium-dependent
vasodilators are conducted through gap junctions connecting
endothelial cells. However, although Cxs have some overlap in
function, it has been demonstrated that these proteins may also
work in concert (13, 14, 36, 38). Therefore, we hypothesized
that Cx37 and Cx40 have different functions in the conduction
of vasodilator responses.
In this study, we used the ATP-sensitive K⫹ (KATP) channel
opener pinacidil and the endothelium-dependent vasodilator
ACh to examine characteristics of the conducted vasomotor
responses triggered by direct hyperpolarization of the vessel
wall or by a receptor-dependent mechanism in the mouse
cremaster microcirculation in vivo. It has been shown that the
vasodilation evoked by the activation of KATP channels is
conducted through endothelial cells (40). Thus, we also ana-
lyzed the participation of Cx37 and Cx40 in the conduction
mechanisms activated by pinacidil and ACh using Cx37
knockout (Cx37⫺/⫺) mice and Cx40 knockout (Cx40⫺/⫺)
mice. Our findings indicate that the conduction induced by the
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H2001
H2002 Cx40-DEPENDENT REGENERATIVE VASODILATION
Table 1. Characteristics of the mouse cremaster
arterioles studied
Maximum Resting Resting
n Diameter, ␮m Diameter, ␮m Tone, %
Wild-type mice 23 38.8⫾1.6 21.0⫾1.6 46.8⫾2.6
Cx40 knockout mice 19 30.4⫾1.4* 16.8⫾0.8 44.7⫾1.6
Cx37 knockout mice 12 35.4⫾2.5 16.2⫾1.7 55.3⫾1.9
Values are means ⫾ SE; n, no. of mice. Cx40, connexin40; Cx37, con-
nexin37. *P ⬍ 0.05 vs. wild-type mice by one-way ANOVA plus the
Newman-Keuls post hoc test.
two agonists is quite different. In contrast to pinacidil, the
vasodilation evoked by ACh does not decay along the arteriolar
length, and Cx40 seems to be critical only for the conduction
of the nondecremental component of the ACh-activated vaso-
dilator signal.
MATERIALS AND METHODS
Male C57 Bl/6 (wild type), Cx37⫺/⫺ (38), and Cx40⫺/⫺ (37) mice
between 22 and 30 g were used. Experiments were conducted
according to the Helsinki Declaration and the “Guiding Principles
in the Care and Use of Laboratory Animals” endorsed by the
American Physiological Society. All animal protocols were ap-
proved by the Animal Care and Use Committee of the University
of Virginia. Tail DNA was used to genotype the animals following
the experiment. The sequences of the primers used to detect Cx40
wild-type and knockout alleles were as follows: sense primer,
5⬘-TGGAGCCACAGTTGCAATGGT-3⬘; antisense primer, 5⬘-
TCTCTGACTCCGAAAGGCAAG-3⬘; and neo primer, 5⬘-
GCACGAGACTAGTGAGACGTG-3⬘. The sequences of the prim-
ers used to detect Cx37 wild-type and knockout alleles were as
follows: sense primer, 5⬘-TGCTAGACCAGCTCCAGGAAC-3⬘;
antisense primer, 5⬘-GTCCCTTCGTGCCTTTATCTC-3⬘; and neo
primer, 5⬘-AGAGGCTATTCGGCTATGACTG-3⬘.
Mouse cremaster preparation. Mice were anesthetized with pen-
tobarbital sodium (40 mg/kg ip diluted in isotonic saline to 5 mg/ml)
and placed on a Plexiglas board. Body temperature was maintained at
35–36°C with a heating pad throughout the experiment. The right
cremaster muscle was exposed, opened by a longitudinal incision on
its ventral surface, the testis and epididymis were excised after
ligation of the supply vessels, and the cremaster was pinned out on a
silicone rubber pedestal. The mouse was placed on the stage of an
Olympus microscope (BX 50 WI, Gibraltar Platform), and the cre-
master muscle was continuously superfused at 3 ml/min with bicar-
bonate-buffered saline solution [containing (in mM) 131.9 NaCl, 4.7
KCl, 2.0 CaCl2, 1.2 MgSO4, and 20.0 NaHCO3] kept at 35°C and
equilibrated with 95% N2-5% CO2. The preparation was allowed to
stabilize for 45– 60 min before the experiment was begun. Throughout
the experiment, supplemental doses of dilute pentobarbital sodium
anesthetic in isotonic saline (10 mg/kg ip) were administrated as
Fig. 1. Time course of local and conducted vasodilation induced by pinacidil in wild-type mice. A short segment of the cremasteric arteriole was stimulated with
a pressure-pulse ejection via micropipette of the ATP-sensitive K⫹ (KATP) channel opener pinacidil, and the resultant vasodilator responses were observed at
the stimulation pipette site (local) and at locations 500, 1,000, and 2,000 ␮m upstream. Note that the changes in diameter decayed rapidly along the vessel length.
Arrows indicate the time at which the stimulus was applied.
AJP-Heart Circ Physiol • VOL 295 • NOVEMBER 2008 •
appropriate. At the end of the experiment, animals were euthanized by
an anesthetic overdose.
Vessel diameters. The cremaster muscle was transilluminated, and
the microscope image was projected to a video camera (series 65,
Dage-MTI) whose output was displayed on a monitor (model
HR1000, Dage-MTI). Inner diameters of the arterioles were continu-
ously measured using Diamtrak software (15, 47).
Arterioles of the second and third order were stimulated focally
with a pressure-pulse ejection from a micropipette (inner diameter:
3– 4 ␮m) of either the KATP channel opener pinacidil (100 ␮M) or
ACh (10 ␮M). The duration of the stimulation period was set to
induce a local vasodilation of ⬃50% (pinacidil: 500 –700 ms and
ACh: 300 ms). In one group of experiments, the length of the pulse of
ACh was extended to 700 ms to elicit maximal vasodilation at the
stimulation site. Changes in diameter were measured first at the
stimulation site (local) and then at locations 500, 1,000, and 2,000 ␮m
upstream in four separate stimuli. To allow the recovery of the
vasodilator response and the stabilization of the control diameter, the
time interval between stimulations was set at 3 min for ACh and 5 min
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for pinacidil. Maximal diameter was estimated during superfusion of
1 mM adenosine. Variations in diameter were expressed as percent-
ages of the maximal dilation possible (%maximum), which was
calculated using the following equation: (Dst ⫺ Dcont)/(Dmax ⫺
Dcont) ⫻ 100, where Dst is the diameter after the stimulation, Dcont is
the diameter before stimulation (control diameter), and Dmax is the
maximal diameter.
Blood pressure measurements. Tail-cuff blood pressure measure-
ments were performed in a dark environment and determined in a
“blinded” fashion using a computerized tail-cuff system (Visitech
Systems, Cary, NC). Animals were conditioned to the measurement
protocol for at least 7 days, and typically three replicate blood
pressure measurement sets were averaged over the course of several
days.
Immunofluorescence. The vasculature of anesthetized mice was
perfused through the left ventricle with warmed MOPS-buffered
physiological salt solution (PSS) containing 1% FCS, 10 U/␮l hepa-
rin, 10 ␮mol/l ACh, and 10 ␮mol/l sodium nitroprusside and fixed by
perfusion of 2% paraformaldehyde in MOPS-buffered PSS. Cremaster
muscles were removed and postfixed overnight. Tissues were dehy-
drated, embedded in paraffin, sectioned (5 ␮m), placed on charge-
coated slides, and deparaffinized using standard procedures. Antigen
retrieval was carried out by microwaving the slides in a citrate buffer.
Sections were blocked with 0.5% BSA in PBS and incubated over-
night at 4°C with rabbit primary antibody directed against Cx37 (ADI,
TX) or Cx40 (ADI, TX) and then with Alexa 568-labeled goat
anti-rabbit secondary antibody (Molecular Probes, OR) for 1 h at
room temperature. The fluorescence signal was examined using an
Olympus Fluoview confocal microscope.
Chemicals. Adenosine, ACh, pinacidil, and chemicals of analytical
grade were purchased from Sigma Chemical (St. Louis, MO). Pinaci-
dil was prepared fresh daily in DMSO and further diluted in the
superfusion solution to the final working concentration (DMSO
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 Cx40-DEPENDENT REGENERATIVE VASODILATION
Fig. 2. Analysis of the maximal response induced by pinacidil in wild-type
(WT), connexin40 (Cx40) knockout (Cx40⫺/⫺), and connexin37 (Cx37)
knockout (Cx37⫺/⫺) mice. A short segment of the arteriole was stimulated
with a pulse of the KATP opener pinacidil ejected by pressure from a micropi-
pette. The maximal vasodilator response induced by pinacidil was evaluated at
the stimulation pipette site (local) and at locations 500, 1,000, and 2,000 ␮m
upstream.
⬍0.1%). The vehicle of pinacidil did not affect the vasomotor tone of
the arterioles (data not shown).
Statistical analysis. Results are presented as means ⫾ SE. Com-
parisons between groups were made using one-way ANOVA plus a
Newman-Keuls post hoc test. P ⬍ 0.05 was considered significant.
RESULTS
The maximum diameter of the mouse cremaster arterioles
studied ranged from 19 to 58 ␮m. Although the vessels
selected were of the same branching order in the three groups
of animals, the maximum diameter of the arterioles from
Cx40⫺/⫺ mice was smaller than that of wild-type animals but
not significantly different from that of Cx37⫺/⫺ mice (Table 1).
However, the mean resting diameter and degree of resting tone
were similar in wild-type, Cx37⫺/⫺, and Cx40⫺/⫺ mice (Table 1).
Vasodilation induced by direct K⫹ channel activation. In
wild-type animals, activation of KATP channels by a pulse of
pinacidil evoked a slowly developing vasodilation that attained
AJP-Heart Circ Physiol • VOL 295 • NOVEMBER 2008 •
H2003
a maximum at ⬃10 s after stimulation (Fig. 1) and returned to
baseline gradually within 30 – 40 s (data not shown). The
pinacidil-induced vasodilation was conducted along the vessel
but decayed rapidly with a mechanical length constant (47) of
1.5 ⫾ 0.4 mm (Fig. 1). Deletion of Cx40 or Cx37 did not affect
the time course of the response to pinacidil (data not shown),
and the mechanical length constants observed in arterioles
from either Cx40⫺/⫺ (2.0 ⫾ 0.4 mm) or Cx37⫺/⫺ (1.3 ⫾ 0.6
mm) mice were similar to those of control animals (Fig. 2).
Vasodilation induced by ACh. Stimulation with a pulse of
ACh (300 ms) induced a very fast and transient vasodilator
response that peaked at ⬃6 s and rapidly returned to control
diameter within 5–15 s (Fig. 3). The response to ACh was
reduced from the local site to the 500-␮m conducted site, but
from this conducted site, the vasodilation was propagated
along the entire arteriole without decay (Fig. 3), suggesting the
activation of an endothelium-dependent regenerative vasodila-
tor mechanism.
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As observed with pinacidil stimulation, deletion of Cx37 did
not affect the ACh-induced local or conducted vasodilator
response (Fig. 3A). In contrast, endothelial cell communication
via Cx40 seems to be critical for the regenerative propagation
of the vasodilation. The conducted vasodilator response acti-
vated by ACh decreased rapidly with distance in Cx40⫺/⫺
mice (Fig. 3B), and the mechanical length constant of the
response to ACh in these animals (2.1 ⫾ 0.3 mm) was similar
to that observed with pinacidil stimulation in wild-type mice
(Fig. 4). In addition, although the magnitude and time of the
peak of the ACh-induced vasodilation were similar in Cx40⫺/⫺
and control mice, the arteriolar diameter returned to baseline
faster in Cx40⫺/⫺ animals (Fig. 3B).
The pulse stimulation with ACh was set to induce an ⬃50%
vasodilation, and the deletion of Cx40 may have affected the
threshold for triggering the regenerative propagation of the
vasodilator response. To evaluate this possibility, we extended
the pressure-pulse ejection of ACh from 300 to 700 ms to elicit
a larger dilation. The increase in the intensity of the stimulation
resulted in near-maximal vasodilation at the ACh application
site (Fig. 5). Although the increase in diameter at the local site
Fig. 3. Time course of the local and conducted
vasodilation induced by ACh in WT, Cx37⫺/⫺,
and Cx40⫺/⫺ mice. ACh was ejected by a
pressure pulse (300 ms) via a micropipette to
stimulate a short segment of the cremasteric
arteriole, and the vasodilator response was an-
alyzed at the stimulation site (local) and at
locations at 500, 1,000, and 2,000 ␮m up-
stream. The vasodilator responses initiated by
ACh in two different groups of experiments
performed in WT animals were compared with
the vasodilation observed in arterioles from
Cx37⫺/⫺ (A) and Cx40⫺/⫺ (B) animals. Arrows
indicate the time at which the stimulus was
applied. *P ⬍ 0.05 vs. WT mice by one-way
ANOVA plus the Newman-Keuls post hoc test.
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H2004 Cx40-DEPENDENT REGENERATIVE VASODILATION

Fig. 6. Systolic blood pressure (SBP) of WT, Cx37⫺/⫺, and Cx40⫺/⫺ mice.
Fig. 4. Comparison of the peak response induced by pinacidil with that Deletion of Cx37 did not alter the arterial blood pressure, but elimination of
initiated by ACh in WT and Cx40⫺/⫺ mice. Maximal vasodilator responses of Cx40 resulted in marked hypertension. Numbers inside the bars indicate n
values. *P ⬍ 0.05 vs. WT or Cx37⫺/⫺ mice by one-way ANOVA plus the

Downloaded from ajpheart.physiology.org on November 12, 2008

the data shown in Figs. 1 and 3 were compared. For this analysis, all the
ACh-induced vasodilator responses shown in Fig. 3, A and B, were pooled
together. *P ⬍ 0.05 vs. ACh-evoked vasodilation in WT mice by one-way
ANOVA plus the Newman-Keuls post hoc test.
was similar in Cx40⫺/⫺ and control mice, after deletion of
Cx40, the response gradually became shorter and decayed
along the vessel length as observed with the submaximal ACh
stimulation (⬃50% of maximum; Fig. 5).
Systolic blood pressure. Wild-type and Cx37⫺/⫺ mice were
normotensive (Fig. 6), showing a blood pressure comparable
with that described for C57 Bl/6 mice (28). In contrast,
Cx40⫺/⫺ animals exhibited marked hypertension (Fig. 6),
which is consistent with pervious reports (7, 8, 25, 44) and
supports the idea that Cx40-mediated coordination of vascular
function contributes to the control of peripheral vascular resis-
tance.
Immunocytochemical analysis. The cellular distribution of
Cx37 and Cx40 in cremaster muscle arterioles was assessed by
immunofluorescence. The signal for both gap junctional pro-
teins Cx37 and Cx40 was apparently restricted to the endothe-
lium, with no evident, positive label of arteriolar smooth
muscle cells. As control, we also analyzed arterioles from
Cx37⫺/⫺ and Cx40⫺/⫺ mice, which confirmed the deletion of
Cx37 or Cx40 and the specificity of the immunocytochemical
analysis (Fig. 7).
DISCUSSION
The endothelium provides a preferential intercellular path-
way for the conduction of vasodilator signals (3, 7, 11, 12, 15),
Newman-Keuls post hoc test.
and gap junctions are thought to be essential for the functional
integration of endothelial cells. Cx37 and Cx40 are the main
gap junction proteins present in the endothelium (7, 13–16, 38,
42), and we used two strains of knockout mice to study the
participation of these proteins in the conduction of vasodilator
responses induced by the KATP channel opener pinacidil and
the endothelium-dependent vasodilator ACh. Our results indi-
cate that neither Cx37 nor Cx40 plays a determining role in the
conduction of the vasodilation evoked by opening of KATP
channels. However, Cx40, but not Cx37, is essential for the
efficient, nondecremental propagation of the vasodilator re-
sponse activated by ACh.
Activation of KATP channels. The vasodilation induced by
openers of KATP channels, such as pinacidil, is associated with
direct hyperpolarization of the vessel wall (2, 32, 40, 43), and
the conduction of the resulting vasomotor responses is thought
to rely on the passive, electrotonic spread of changes in
membrane potential through gap junctions connecting cells
along the length of the vessel wall (17, 34). Consistent with this
idea, the local vasodilation evoked by pinacidil was conducted
decrementally along the longitudinal axis of the stimulated
arteriole (Fig. 1) with a mechanical length constant quite
consistent with the electrical length constant determined by
current injection in arterioles in vitro or in vivo (0.9 –1.6 mm)
(10, 18, 19).
Although KATP channels are known to be in both smooth
muscle and endothelial cells (2, 4, 23), the hyperpolarization-

Fig. 5. Time course of the conduction of maximal vasodilation induced by ACh in WT and Cx40⫺/⫺ mice. ACh was applied as described in Fig. 3, but
the duration of the pressure pulse was increased to 700 ms to stimulate maximal vasodilation in the segment of the arteriole just underneath the micropipette. The
vasodilator response was observed at the stimulation site (local) and at locations at 500, 1,000, and 2,000 ␮m upstream. Arrows indicate the time at which the
stimulus was applied. *P ⬍ 0.05 vs. WT mice by one-way ANOVA plus the Newman-Keuls post hoc test.

AJP-Heart Circ Physiol • VOL 295 • NOVEMBER 2008 • www.ajpheart.org

 Cx40-DEPENDENT REGENERATIVE VASODILATION
mediated vasodilation elicited by the opening of KATP channels
has been shown to spread through the endothelium of rat
mesenteric arteries (40), and, in the mouse cremaster micro-
circulation, Cx37 and Cx40 appear to be localized exclusively
in endothelial cells (Fig. 7). However, deletion of either Cx37
or Cx40 did not affect the local or conducted vasodilator
response induced by KATP channel activation with pinacidil
(Fig. 2). This finding may indicate that these Cxs are redun-
dantly expressed and can compensate for each other, but the
involvement of Cx43 in this process cannot be ruled out.
Further experiments using knockout mice in combination with
Cx mimetic peptides to study electrotonic potentials produced
by current injection may help us to elucidate this problem.
Conducted vasodilation activated by ACh. In contrast to the
response induced by pinacidil, ACh stimulation activated a
very fast vasodilation that dropped off only from the local site
to the conducted site located 500 ␮m upstream, but it is
noteworthy that from the 500-␮m site, the response was
propagated along the entire arteriole without apparent decay
(Fig. 3), indicating that the response observed at the local site
is a combination of two components: one that was restricted to
the stimulation site and another that was conducted. The
conducted vasodilation must then be assessed excluding the
stimulation site, since the difference between the responses
observed at the local and conducted sites may be misinter-
preted as decay.
The conducted vasodilator response triggered by ACh has
been associated with the electrotonic spread of the hyperpo-
larization initiated by the release of an endothelium-derived
hyperpolarizing factor at the stimulation site (17, 20, 45).
However, the response initiated by ACh was propagated over
distances much longer than those predicted by the electrotonic
model (Figs. 3 and 4), which suggests that ACh activated a
regenerative vasodilator mechanism. Consistent with this idea,
computer simulations have led to the proposal that activation of
AJP-Heart Circ Physiol • VOL
H2005
Fig. 7. Cellular distribution of Cx37 and Cx40 in the
arteriolar wall. Cx40 (top) and Cx37 (bottom) were
detected in endothelial cells of the arterioles of WT
mice (left). There was no immunoreactivity for Cx40
or Cx37 in arterioles of Cx40⫺/⫺ and Cx37⫺/⫺
animals, respectively (right). The fluorescent signal
was not evident in smooth muscle cells of WT or
knockout mice. Arrows indicate the internal elastic
lamina (IEL).
Downloaded from ajpheart.physiology.org on November 12, 2008
voltage-dependent conductances may be involved in the con-
duction of the responses initiated by ACh (5), and, interest-
ingly, focal electrical stimulation of mouse cremaster arterioles
activates a very rapid conducted vasodilation of similar char-
acteristics of those induced by ACh (12, 15).
Direct measurements of membrane potential also support the
idea that ACh activates a regenerative mechanism of propaga-
tion of vasodilator signals. In feed arteries of the hamster
retractor muscle, analysis of the conduction of changes in
membrane potential along the vessel has shown that the elec-
trical length constant of ACh-induced hyperpolarization is
longer than that for current injection (10). In addition, recently,
Crane et al. reported that the hyperpolarizing current initiated
by ACh in hamster cheek pouch arterioles in vivo can even
increase during the first 1,000 ␮m of longitudinal conduction,
which has led to the proposal that the regenerative mechanism
may involve the opening of inward rectifier K⫹ (Kir) channels
by hyperpolarization and by an elevation in extracellular K⫹
concentration (6, 24). However, conduction of the changes in
diameter evoked by pinacidil argues against this hypothesis,
since the hyperpolarization and the increase in extracellular K⫹
concentration produced by the activation of KATP channels
would have been expected to have a similar effect on Kir
channels and result in extended dilation. However, pinacidil-
evoked conducted vasodilation decayed just as expected for the
electrotonic spread of a hyperpolarizing current (Figs. 1 and 2).
In addition, activation of Kir channels should also be expected
during the hyperpolarization induced by current injection, as
predicted by computational modeling of cell-to-cell communi-
cation (24). Taken together, these results suggest that the
hyperpolarization of the vessel wall contributes to the electro-
tonic conduction of vasodilation but not to the propagation of
the nondecremental component of the vasomotor signal acti-
vated by ACh. Probably, the ACh-induced hyperpolarization is
enhanced along the vessel length by the activation of a regen-
295 • NOVEMBER 2008 • www.ajpheart.org
H2006 Cx40-DEPENDENT REGENERATIVE VASODILATION
erative vasodilator mechanism as has been recently described
for the electrically induced conducted vasodilation (12).
The response to ACh might be conducted by either the
endothelium or smooth muscle in arterioles (1, 3). However,
the regenerative propagation of the vasodilation induced by
ACh depended on the expression of Cx40 (Figs. 3 and 4), and,
in cremasteric arterioles, Cx40 is only expressed in the endo-
thelium (7, 15), which we confirmed in the present work (Fig.
7). Interestingly, deletion of Cx40 converted the nondecremen-
tal response activated by ACh into a rapidly decaying vasodi-
lator signal (Figs. 3 and 4). In addition, the longitudinal
reduction of the vasodilation elicited by ACh in Cx40⫺/⫺ mice
was similar to that observed with pinacidil stimulation (Fig. 4).
Therefore, these results suggest that ablation of Cx40 selec-
tively disrupted the regenerative propagation of a vasodilator
signal via endothelial cells but without affecting the electro-
tonic component of the conducted response triggered by ACh,
which may be mediated by others Cxs expressed either in the
endothelium or smooth muscle. It is important to note, how-
ever, that Cx40 has been detected at myoendothelial junctions
(22, 31), which raises the possibility that the gap junction
communication between smooth muscle and endothelial cells
may be involved in the regenerative mechanism. Although
deletion of Cx40 may also affect the expression of Cx43 or
Cx37 (21, 39), these Cxs probably are not involved in the
regenerative vasodilator mechanism because the absence of
Cx37 did not alter the conducted response induced by ACh
(Fig. 3) and, in mouse cremaster muscle, Cx43 has been
detected only in endothelial cells of large arterioles (30).
Deletion of Cx40 might have changed the axial resistance of
the endothelial layer and, thus, the minimum threshold poten-
tial for triggering the activation of the regenerative vasodilator
mechanism along the vessel length. To test this possibility, we
stimulated arterioles with a longer pulse of ACh (700 ms) to
evoke a larger response. Although the stronger ACh stimula-
tion produced near-maximal vasodilation at the local site (Fig.
5), this response decayed along the vessel length in Cx40⫺/⫺
animals exhibiting the same characteristics as those observed
with ⬃50% of maximal dilation induced by shorter pulses (300
ms) of ACh (compare Figs. 3 and 5), which supports the idea
that Cx40 is an integral part of the regenerative vasodilator
mechanism.
Cx37 and Cx40 play an important role in the development
and function of the vascular system (26, 38, 39). However,
only Cx40 seems to be involved in the coordination of vaso-
motor tone in the microcirculation and control of arterial blood
pressure (Fig. 6). Although deletion of Cx40 has been associ-
ated with an increased plasma renin concentration, the hyper-
tension observed in Cx40⫺/⫺ mice appears to be mostly inde-
pendent of angiotensin II (8, 44). These results support the
notion that Cx40 is essential for the well-integrated regulation
of peripheral vascular resistance.
In arterioles of the hamster cheek pouch, Budel et al. showed
that the conducted vasodilation initiated by ACh is coupled to
nitric oxide (NO) production (3), and, most recently, in mouse
cremaster arterioles, this response was also observed to be
followed by a slow, rapidly decaying vasodilator component
that was associated with the spread of a Ca2⫹ wave (41).
Although the Ca2⫹ wave-dependent vasodilation seemed to
prolong the response to ACh observed at short distance from
the stimulation site (300 – 400 ␮m), it was not involved in the
AJP-Heart Circ Physiol • VOL
vasodilator response propagated beyond 500 ␮m from the local
site (41). It is important to note that the vasodilation induced by
NO is not conducted along the length of arterioles (9, 20), and
the hyperpolarization of endothelial cells is not coupled to an
increase in intracellular Ca2⫹ in intact vessels (32, 40), as was
proposed by Budel et al. (3) to explain the activation of
endothelial NO synthase (eNOS) at the conducted sites. In
addition, as mentioned above, focal stimulation with depolar-
izing pulses of current activates a nondecremental conducted
vasodilator response of similar characteristics to those initiated
by ACh (15). Interestingly, the conducted response induced by
electrical stimulation of mouse cremaster arterioles is also
coupled to NO production and is associated to the activation of
voltage-dependent Na⫹ and Ca2⫹ channels in endothelial cells
(12). Therefore, these data are consistent with the activation of
a voltage-dependent regenerative vasodilator signal by ACh
that is, in turn, coupled to eNOS activation and endothelial cell
hyperpolarization.
Downloaded from ajpheart.physiology.org on November 12, 2008
In summary, direct hyperpolarization of the arteriolar wall
by the opening of KATP channels induces a vasodilation that
decays along the vessel length in a manner consistent with the
electrotonic spread of an electrical signal. Conduction of this
vasodilator response does not depend on the presence of Cx37
or Cx40. In contrast, the vasodilation induced by ACh is
propagated along the entire vessel without an apparent reduc-
tion in magnitude or duration of the response, strongly sug-
gesting that ACh receptors are coupled to the activation of a
regenerative vasodilator mechanism. The regenerative compo-
nent of the response initiated by ACh was selectively blocked
by deletion of Cx40 but not Cx37, which indicates that Cx40
gap junctions play an essential role in the function of the
regenerative vasodilator mechanism activated by ACh.
ACKNOWLEDGMENTS
Cx40 and Cx37 knockout mice were generously provided by Alexander M.
Simon and David L. Paul. We thank Kathleen H. Day for excellent technical
assistance.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-53318 (to B. R. Duling), a grant from Vicerrectorı́a Adjunta de
Investigación y Doctorado de la Pontificia Universidad Católica de Chile (to
X. F. Figueroa), Fondo Nacional de Desarrollo Cientı́fico y Tecnológico Grant
11060289 (to X. F. Figueroa).
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