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Circulation Research. 2003;92:793-800
Published online before print March 13, 2003, doi: 10.1161/01.RES.0000065918.90271.9A
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(Circulation Research. 2003;92:793.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Central Role of Connexin40 in the Propagation of Electrically Activated Vasodilation in Mouse Cremasteric Arterioles In Vivo

Xavier F. Figueroa, David L. Paul, Alexander M. Simon, Daniel A. Goodenough, Kathy H. Day, David N. Damon, Brian R. Duling

From the Department of Molecular Physiology and Biological Physics (X.F.F., K.H.D., D.N.D., B.R.D.), University of Virginia, Charlottesville, Va; the Department of Neurobiology (D.L.P.) and Department of Cell Biology (D.A.G.), Harvard Medical School, Boston, Mass; and the Department of Physiology (A.M.S.), University of Arizona, Tucson, Ariz.

Correspondence to Dr Brian R. Duling, Dept of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, PO Box 800736, Charlottesville, VA 22908-0736. E-mail brd{at}virginia.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40-/-) mice to study vasomotor responses induced by 10-second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 µm upstream). In wild-type (Cx40+/+) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40-/- mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 µmol/L) had no effect on the local vasoconstriction in either Cx40+/+ or Cx40-/- mice, but enhanced the conducted vasodilation in Cx40+/+ animals. In Cx40-/- mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40-/- mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40+/+ mice and complete elimination of this connexin in the Cx40-/- animals. These results indicate that focal current stimulation causes vasoconstriction by a combination of perivascular nerve stimulation and smooth muscle activation. Moreover, electrical stimulation activates a nonneuronal, Cx40-dependent vasodilator response that spreads along the vessel length without decay.


Key Words: connexin40 • conducted response • vasodilation • electrical stimulation • cremaster microcirculation


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Coordination of microvascular function plays a pivotal role in the regulation of blood flow distribution, and conduction of vasomotor signals along the vessels may play a key role in integrating function of arterioles and feed arteries.R1-126738 R2-126738 1–3 Restricted application of vasoactive substances to short arteriolar segments induces both direct effects and secondary conducted vasomotor responses.R4-126738 R5-126738 4–6 The latter are thought to rely on electrotonic spread of membrane potential from the site of agonist application through gap junctions connecting cells of the vessel wall.R5-126738 R7-126738 5,7,8

Electrical stimulation has also been used to study conducted vasomotor responses in arterioles,R9-126738 R10-126738 R11-126738 9–12 but the pathways and modes of activation of the responses induced by electrical stimulation remain to be determined. Endothelial cells are thought to be electrically unexcitable,13 although field electrical stimulation has been reported to evoke an endothelium-dependent relaxation in isolated vessels.R9-126738 R14-126738 R15-126738 R16-126738 9,14–17 Consequently, changes in diameter observed in response to electrical stimulation could be the result of activation of different cell types and the conduction along the vessel axis of these vasomotor responses can depend on the summation of multiple vasomotor signals both by chemical interactions and via gap junctions.

Conducted signals spread over vessel segments spanning many cells, and thus gap junctions are likely to form the pathway for cell-cell conduction. Gap junctions are intercellular channels formed by proteins known as connexins (Cxs). Four Cx proteins have been identified in vessels: Cx37, Cx40, Cx43, and Cx45.R18-126738 R19-126738 18–20 Cx45 is reported to be only in vascular smooth muscle cells, whereas Cx37 is thought to be only in endothelial cells. Cx40 and Cx43 are expressed in both cell types,R20-126738 R21-126738 R22-126738 20–23 but Cx40 expression is more abundant and uniformly distributed in endothelial cells, whereas Cx43 is found predominately in smooth muscle cells.R20-126738 20,21

The development of Cx knockout animals represents an important tool to disclose the role of gap junctions in vascular physiology. Early data suggest that the roles of Cxs in the vasculature may be selective and diverse. Cx43 endothelial cell-specific knockout mice are reported to be hypotensive,24 whereas Cx40 knockout (Cx40-/-) mice are hypertensive and manifest an impaired conduction of responses induced by acetylcholine or bradykinin.25

In the present study, we assessed the functional role of Cx40 gap junctions in the conduction of vasomotor signals induced by electrical stimulation, using Cx40-/- mice. We analyzed the vasomotor responses induced by focal electrical stimulation of second and third order arterioles in the mouse cremaster microcirculation. Our results show that depolarizing current injection activates local and conducted vasoconstrictor signals mediated by a combination of perivascular nerve stimulation, smooth muscle stimulation, and a nonneuronal, endothelial cell Cx40-dependent vasodilator response that propagates along the vessel length without decay.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Adult, 22 to 30 g male C57 bl/6 (n=28, Hilltop Laboratory Animals Inc, Scottdale, Pa) or Cx40-/- (n=13) mice26 were bred and maintained in the Research Animal Facilities of the University of Virginia. Cx40-/- mice were backcrossed with C57 bl/6 mice a minimum of 6 generations. 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.

Mouse Cremaster Preparation
Mice were anesthetized with pentobarbital sodium (80 mg/Kg, IP, diluted in isotonic saline to 10 mg/mL), and the cremaster muscle was prepared as described previously.27 The cremaster muscle was spread and pinned on a Sylgard pedestal. The mouse was placed on the stage of an Olympus microscope (BX 50 WI, Gibralter Platform), and the cremaster muscle was continuously superfused at 3 mL/min with a bicarbonate-buffered saline solution (in mmol/L: 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 20.0 NaHCO3) kept at 35°C. The buffer solution was equilibrated with 95% N2/5% CO2 to yield a pH 7.35 to 7.45. The preparation was allowed to stabilize for 45 to 60 minutes before starting the experiment. Throughout the experiment, supplemental doses of anesthetic in isotonic saline (20 mg/Kg, IP) were administrated as appropriate. At the end of the experiment, the animals were euthanized by an anesthetic overdose.

Vessel Diameters
Arterioles were visualized by transillumination using a 10x eyepiece and a 50x Leitz UMK (NA=0.60) objective. The microscope image was projected to a video camera (Dage-MTI Series 65) whose output was displayed on a monitor (Dage-MTI Model HR1000) and recorded on videotape (Panasonic Omnivision). The inner diameter of the arteriole was continuously measured using Diamtrak software.28

Electrical Stimulation
Arterioles were stimulated electrically for 10 seconds with a micropipette (inner diameter 3 to 4 µm) filled with 1 mol/L NaCl connected to the cathode of a Grass SD9 stimulator (Grass Instrument). To minimize heterogeneity in the electrical field around the pipette tip, an Ag/AgCl reference electrode was positioned symmetrically around the cremaster in the superfusate covering the tissue. A variety of electrical stimuli with an amplitude of 30 to 50 V were used, including a pulse train of 1 ms duration at 30 Hz, 100 ms duration at 5 Hz, and direct current (DC).

Immunocytochemistry
Anesthetized mice were perfused through the left ventricle with a warmed MOPS-buffered PSS containing 1% fetal calf serum, 10 U/µL heparin, 10 µmol/L ACh, and 10 µmol/L sodium nitroprusside followed by a 2% paraformaldehyde perfusion in MOPS-buffered PSS to fix the vasculature. The cremaster muscles were removed and postfixed overnight. The tissues were dehydrated, embedded in paraffin, sectioned (5 µm), placed on charge-coated slides, and deparaffinized using standard procedures. Antigen retrieval was performed by microwaving the slides in a citrate buffer. The sections were blocked with 0.5% BSA in PBS, incubated with rabbit primary anti-Cx40 antibody (1:500) overnight at 4°C (ADI, TX), and then with Alexa-568–labeled goat anti-rabbit secondary antibody (Molecular Probes, OR) for 1 hour at room temperature, as described previously.24 The immunoreactivity was examined using an Olympus Fluoview confocal microscope.

Experimental Protocols
Cathodal electrical stimulation of second and third order arterioles was the basic mode of stimulation. Using a micromanipulator (Burleigh TS-5000-I50), the stimulation micropipette tip was positioned below the superficial cremasteric connective tissue and above the arteriole. Voltage and distance of the micropipette tip from the selected arteriole were adjusted to induce a local constriction of about 50%. After recording the local response, the arteriole was stimulated repeatedly at 3-minute intervals, and the changes in diameter were measured at locations 500, 1000, and 2000 µm upstream to evaluate the conduction of vasomotor responses. The upstream locations were selected using a calibrated eyepiece reticule and each measurement corresponded to a separate stimulus. To confirm the reproducibility of conducted responses, at the end of each stimulation series (local, 500, 1000, and 2000 µm), the response at 500 µm was assessed again. In addition, in some experiments, the arterioles were stimulated repeatedly at 3-minute intervals, and the changes in diameter were measured at the local site or at 2000 µm upstream. Maximal diameter was estimated during superfusion of 1 mmol/L adenosine. Variations in diameter were expressed as percentage of resting diameter.

Blockade of Perivascular Nerves
To assess the participation of perivascular nerves in the response to electrical stimulation, arterioles of wild-type (6 vessels, 4 animals) or Cx40-/- (10 vessels, 9 animals) cremaster muscles were stimulated in control conditions and after superfusing 1 µmol/L tetrodotoxin (TTX) or 100 nmol/L prazosin for 15 to 20 minutes.

Long-Pulse Stimulation
To evaluate the involvement of voltage-operated, fast-inactivating ion channels in the propagation of electrically induced vasomotor responses, the pulse duration was varied from 1 to 100 ms to DC (3 vessels, 3 animals) with fixed amplitude of 30 V. In this series of experiments, the responses were observed at the local site, 500, and 1000 µm upstream.

Chemicals
All biochemical reagents and chemicals of analytical grade were purchased from Sigma Chemical Co.

Statistical Analysis
Results are presented as mean±SEM. Two-way ANOVA was used to assess significance of differences as a function of time. Comparisons between groups were made using paired or unpaired Student’s t test or one-way ANOVA plus Newman-Keuls post hoc test as appropriate. The level for establishing a significance of difference was set at P<0.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Maximum diameter of the mouse cremasteric arterioles studied ranged from 27 to 67 µm, and the mean resting diameter was 26.7±1.2 µm (n=33) in wild -type mice and 23.3±1.6 µm in Cx40-/- mice (n=15, P>0.05 by unpaired t test). In both groups of animals, the arterioles presented a similar degree of resting tone (39.9±2.2% in wild-type mice, n=25, and 34.6±2.9% in Cx40-/-, n=14).

Electrically Induced Vasomotor Responses
A representative response of wild-type arterioles to depolarizing electrical stimulation is depicted in Figure 1A. Electrical stimulation resulted in a local vasoconstriction and a conducted vasodilation that spread along the vessel without apparent decay. The local vasoconstriction was restricted to a short vessel segment underneath the stimulation pipette (approximately 50 to 100 µm) and lasted for the entire duration of the stimulus. This reduction in diameter attained a maximum of -48.7±2.2% at {approx}9 seconds of stimulation and returned to control level within 40 to 60 seconds. The vasodilation was propagated along the vessel without a detectable delay and showed a very fast initial phase followed by a slower component, which typically reached a maximum by the end of the stimulation period (n=33; Figures 1A and 1B). The recovery of the conducted responses was very rapid. The arteriolar diameter returned to baseline within 3 to 5 seconds after stimulation and typically showed a small undershoot.



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  		Figure 1. Time course of vasomotor responses induced by electrical stimulation in wild-type animals. Arterioles of 2nd and 3rd order of wild-type mice were stimulated every 3 minutes for 10 seconds with a train of electrical pulses (30 Hz, 1 ms, 30 to 50 V). Vasomotor responses were observed at the local site (pipette site) and at locations 500, 1000, and 2000 µm upstream. A, Changes in vessel diameter of a representative experiment. B, Summary data of changes in vessel diameter expressed as percentage of resting diameter. C, Vasomotor response induced by 5 successive electrical stimulations observed at local site (left) or at 2000 µm upstream (right). Horizontal bars indicate the period of stimulation.

The local constriction showed a striking, progressive reduction in response to successive stimuli (Figure 1C). After a series of stimuli, the arteriolar reactivity recovered over 10 to 15 minutes. In sharp contrast to the local response, the conducted vasodilator response was sustained with multiple stimuli and showed no evidence of tachyphylaxis (Figure 1C).

In Cx40-/- mice, cathodal electrical stimulation consistently induced a local vasoconstriction (Figure 2A), which was similar to that observed in wild-type animals (-55.6±4.0%, n=15, P>0.05 by unpaired t test). The conducted responses in the knockout animals were quite different. The average data for all Cx40-/- arterioles (n=15) showed a small, slow vasoconstriction for the conducted vasomotor responses, in contrast to the abrupt, marked dilation in control animals (compare Figure 1 with Figure 2A). On careful examination of the data, we noted that there were two qualitatively distinct groups of animals based on the responses observed at 500 µm. In 8 arterioles, focal electrical stimulation resulted in a conducted vasoconstriction (Figure 2B), and in seven arterioles, a conducted vasodilation was observed (Figure 2C). Note that in both cases, the magnitude of the local vasoconstriction was similar (-58.9±5.0% conducted constriction group, and -51.9±6.5% conducted dilation group). As shown in Figure 3, electrically induced conducted vasodilation in wild-type animals as well as the conducted vasoconstriction observed in one group of Cx40-/- arterioles did not decay along the vessel (Figure 3 and Table). In contrast, the conducted vasodilation elicited by electrical stimulation in Cx40-/- animals decreased progressively with distance (Figure 3 and Table). In addition, as compared with wild-type mice, the increase in diameter induced by electrical stimulation of arterioles from the Cx40-/- mice was slower (compare Figures 1B and 2C) and smaller at 1000 and 2000 µm (Table).



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  		Figure 2. Time course of vasomotor responses induced by electrical stimulation in Cx40 knockout animals (same parameters were used as in Figure 1). Summary data for all Cx40 knockout arterioles is show in A. Two different groups of animals were identified based on the response observed at 500 µm, which are shown in B and C. Horizontal bars indicate the period of stimulation.



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  		Figure 3. Superposition of the local and the conducted responses of wild-type and Cx40 knockout animals. Time course and maximal responses of data show in Figures 1B, 2B, and 2C are compared. At the left is shown the time course of local and conducted responses merged in one graphic and at the right the maximal responses observed at 500, 1000, and 2000 µm upstream. A, Wild-type animals (see Figure 1B). B, Group 1 of Cx40 knockout mice (see Figure 2B). C, Group 2 of Cx40 knockout mice (see Figure 2C). Horizontal bars indicate the period of stimulation. *P<0.001 vs 500 or 1000 µm by one-way ANOVA plus Newman-Keuls post hoc test.


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  		Table 1. Vasomotor Response Induced by Electrical Stimulation in Wild-Type and Cx40 Knockout Mice

Perivascular Nerves
To assess the participation of perivascular nerves in the response to electrical stimulation, arterioles of wild-type and Cx40-/- mice were stimulated in control conditions and in the presence of 1 µmol/L TTX. In wild-type animals, treatment with TTX did not affect resting microvessel diameter and did not block either the local vasoconstriction or the conducted vasodilation induced by electrical stimulation (Figure 4A). TTX enhanced the vasodilator response observed at upstream sites (Figure 4A), suggesting that TTX may have blocked a constrictor component. Consistent with the activation of a constrictor component, 100-nmol/L prazosin, a {alpha}1-adrenoceptor antagonist, also enhanced the electrically induced conducted vasodilation without affecting the magnitude of the local vasoconstriction (-42.4±6.1% control and -48.2±8.2% prazosin, n=3). At 500 and 1000 µm upstream, the maximal vasodilation was 137.2±8.0% and 136.3±13.4% in control conditions and 144.7±8.1% (P<0.015 by paired t test) and 153.8±21.6% in presence of prazosin.



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  		Figure 4. Effect of perivascular nerve blockade on the electrically induced vasomotor responses in wild-type animals. A, Arterioles were stimulated (30Hz, 1 ms, 30 to 50 V) for 10 seconds in control conditions and after topical application for 15 to 20 minutes of 1 µmol/L tetrodotoxin (TTX). B, Arterioles were stimulated with fixed amplitude of 30 V and the pulse duration was increase from 1 ms (30 Hz) to 100 ms (5 Hz) and to direct current (DC, continuous stimulation). Changes in vessel diameter are shown as percentage of resting diameter. Horizontal bars indicate the period of stimulation. *P<0.001 vs control by Student’s paired t test.

We also attempted to rule out a role for voltage-dependent sodium channels in the propagation of this response by showing that the vasodilator response was not blunted by stimulation with either a train of long duration pulses or DC10 (Figure 4B).

As in wild-type animals, superfusion with TTX did not alter control diameter and did not block the electrically induced local vasoconstriction in Cx40-/- mice (Figure 5). However, TTX abolished the conducted vasoconstriction observed in these animals and revealed a small vasodilation (Figure 5A). The small vasodilation unmasked by TTX decayed along the vessel length and the temporal changes in diameter only reached significance at 500 and 1000 µm upstream (P<0.0001 by 2-way ANOVA). The finding shown in Figure 5A contrasts with the observation that in the Cx40-/- mice in which electrical stimulation elicited a conducted vasodilation, this response was TTX-insensitive (Figure 5B).



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  		Figure 5. Effect of perivascular nerve blockade on the electrically induced vasomotor responses in Cx40 knockout animals. Arterioles were stimulated (30 Hz, 1 ms, 30 to 50 V) for 10 seconds in control conditions and after topical application for 15 to 20 minutes of 1 µmol/L tetrodotoxin (TTX). A, Group 1 of Cx40 knockout mice (see Figure 2B). B, Group 2 of Cx40 knockout mice (see Figure 2C). Changes in vessel diameter are shown as percentage of resting diameter. Horizontal bars indicate the period of stimulation.

Identification of Cx40
The presence and distribution of Cx40 in cremaster muscle microvessels was assessed by immunocytochemical analysis. A clear staining for Cx40 was apparent only in the endothelium of both orders of arterioles (Figure 6A). In arteriolar smooth muscle cells, we detected no positive reaction for this connexin. As expected, the presence of Cx40 staining was not found in cremasteric arterioles from Cx40-/- mice (Figure 6B).



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  		Figure 6. Immunocytochemical localization of Cx40 in mouse cremaster muscle of wild-type and Cx40 knockout mice. A, In wild-type mice, Cx40 is detected as homogenous staining in the endothelial layer of arterioles, but it is absent in smooth muscle cells. B, Lack of immunoreactivity for Cx40 in arterioles of Cx40 knockout mice. Arrows indicate the internal elastic lamina (IEL).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Our results show that depolarizing electrical stimuli cause the activation of perivascular nerves, smooth muscle cells, and/or endothelial cells. The result is a complex vasomotor response composed of a local constriction and a Cx40-dependent conducted vasodilation. The expression of Cx40 was only detected in the endothelial layer and in Cx40-/- mice the propagation of electrically induced vasodilation was dramatically impaired, which highlights the role of endothelial cell communication by Cx40 gap junctions in the control and coordination of microvascular vasomotor responses.

Electrically Induced Vasomotor Response
Focal stimulation of second or third order cremasteric arterioles with a train of cathodal electrical pulses caused a local vasoconstriction limited to a short segment immediately underneath the stimulation micropipette and a very fast vasodilation that was propagated along the entire vessel (Figure 1). Recently, Emerson and Segal9 described a response of similar characteristic in isolated resistance arteries subjected to focal electrical field stimulation using two stimulus micropipettes, the cathode in the adventitia, and the anode directly at the other side of the vessel. However, the transverse stimulation used in that study caused a conducted vasodilation only with a train of pulses of high intensity and after blocking the perivascular nerve. In the current experiments, this response was observed in control conditions, a finding that we attribute to the fact that the stimulation threshold is higher when the electric field is applied in a transverse orientation, because one half of the tissue is depolarized and the other half is hyperpolarized.29 Such transverse stimulation may have favored the activation of perivascular nerves on the cathode side of the isolated resistance arteries. In our experimental set up in vivo, we surrounded the tissue with the reference electrode to allow a more homogenous stimulation across the vessel.

Recent reports have suggested that calcium can diffuse from smooth muscle cells into endothelial cells via myoendothelial gap junctions.R30-126738 30,31 As a result, the increase in intracellular calcium concentration of smooth muscle that is associated with a vasoconstriction, can trigger a subsequent endothelium-dependent vasodilator signal, as calcium diffuses through the myoendothelial junctions.32 However, in the present study, there was no detectable delay between the local vasoconstriction and the vasodilation observed upstream (Figure 3), which suggests that electrical stimulation might directly activate the endothelial cells, as proposed by Emerson and Segal.9 Furthermore, the conducted vasodilator response was sustained with multiple stimuli, although the local constriction showed a progressive tachyphylaxis (Figure 1C), suggesting that the vasodilation elicited by electrical stimulation was independent of the mechanical activation of the smooth muscle cells or a hemodynamic effect.

Perivascular Nerves in the Electrically Induced Vasomotor Response
The vasodilation in response to current stimulation did not show the characteristic exponential decay of electrotonic conduction, suggesting the involvement of a regenerative mechanism as part of the conducted response. The mouse cremaster microcirculation is provided with an extensive network of vasomotor nerves,R33-126738 33,34 but topical application of TTX did not alter the resting arteriolar diameter, excluding a role for TTX-sensitive nerves such as the sympathetics in the control of resting tone. TTX did enhance the electrically elicited conducted vasodilator response (Figure 4A), indicating that current stimulation activates at least two conducted vasomotor components, a nerve-independent vasodilation and a TTX-sensitive vasoconstriction that is overcome by the conducted vasodilation. In all likelihood, the TTX-sensitive vasoconstrictor signal originated from the sympathetic perivascular nerves.R11-126738 11,33 Consistent with this assumption, the blockade of {alpha}1-adrenoceptors with prazosin also enhanced the electrically induced vasodilation.

Cremaster muscle microvessels are also innervated by branches of primary sensory afferent neurons, which possess TTX-resistant sodium channels.R34-126738 R35-126738 34–36 The voltage-sensitive sodium channels rapidly inactivate during depolarization.R29-126738 29,37 Therefore, we stimulated the arterioles with a train of long pulse-duration or DC to preclude repetitive triggering of action potentials and, in consequence, the propagation of a nerve dependent-vasomotor signal. This maneuver did not inhibit the conducted vasodilator response (Figure 4B), supporting the idea that the generation and the propagation mechanism of this response are nerve-independent.

Gap Junctions in the Conduction of Electrically Induced Responses
Deletion of Cx40 had a dramatic effect on the conducted vasodilation evoked by electrical stimulation, converting the marked conducted dilation into a strong, conducted constriction or in some cases a vasodilation that decayed rapidly with distance (Figures 1 through 3). This transformation occurred in spite of the fact that the vasoconstriction observed at the local site was similar in the two mouse lines (Figures 1 through 3). Also it is noteworthy that among the three groups identified (wild-type, constricting Cx40-/-, and dilating Cx40-/-), the degree of resting tone was similar.

The diverse conducted responses observed in the two groups of Cx40-/- animals may correspond to a different balance of the two conducted vasomotor components disclosed in wild-type mice using TTX (Figure 4A). Perhaps there was a higher degree of sympathetic activation with stimulation in the group of vessels shown in Figure 5A, thus making them more sensitive to TTX. In the Cx40-/- arterioles in which electrical stimulation elicited a conducted vasodilation, the TTX-sensitive constrictor component was smaller or absent (Figure 5B). In the Cx40-/- animals that showed a constriction, TTX blocked the conducted vasoconstriction and revealed a small vasodilation that decayed along the vessel length (Figure 5A), making the two groups of animals much more similar.

The endothelium would seem to be the most likely pathway for the conducted vasodilator signal. Although Cx40 gap junctional protein is reported to be present in both endothelial cells and smooth muscle cells of large arteries and in the hamster cheek pouch microcirculation,R20-126738 R21-126738 R22-126738 20–22,38 in mouse cremaster, we found Cx40 expression only in the endothelium (Figure 6), confirming a previous report.25 When taken in the context of the impaired vasodilation observed in Cx40-/- mice, this finding strongly supports the idea that the electrically induced vasodilation is propagated via endothelial cells. The finding also highlights the functional role of Cx40 in the coordination of vasomotor responses.

Impaired propagation of ACh-induced vasodilator signals has also been observed in Cx40-/- mice.25 However, deletion of Cx40 produces a modest reduction in the conducted responses to ACh compared with the effect of electrical stimulation (Figure 2). The absence of Cx40 reduces, but does not eliminate the conduction of either the electrically or ACh-induced vasodilation. This might suggest that other endothelial Cxs participate in the propagation of the vasodilation. Alternatively, vasodilatory signals may spread along the vessel via smooth muscle cells.R5-126738 R8-126738 5,8,39

Although multiple connexins and cell types may be involved in the coordination of vasomotor responses, Cx40 gap junction seems to play a special role because Cx40 knockout mice are hypertensive,25 and the development of hypertension in spontaneously hypertensive rats is associated with a reduction in endothelial cells length and in the density of endothelial gap junction plaques containing Cx40 but not Cx37 or Cx43.40

As mentioned, the rapid and nondecremental nature of the conducted vasodilation observed in wild-type animals points strongly to the activation of a regenerative process. Without an active response, a length constant on the order of 2 mm would have been predicted.R5-126738 5,41 Thus, the longitudinal decay of vasodilation evoked by current stimulation in Cx40-/- compared with the wild-type mice implies that the regenerative process was interrupted. This fact combined with the observation of the localization of Cx40 exclusively in the endothelium (Figure 6), leads us to hypothesize that Cx40 coupling is somehow necessary for an active propagation (regenerative) of the vasodilator signal, but that other connexins can mediate an electrotonic conduction.

Endothelial cells are typically assumed to be electrically unexcitable,13 but they are not insensitive to voltage change.42 Field electrical stimulation has been reported to induce a nonneuronal, endothelium-dependent vasodilation in vitro.R9-126738 R14-126738 R15-126738 R16-126738 9,14–17 Moreover, it has been shown that effluent obtained from the bath of freshly isolated bovine aortic endothelial cells subjected to field electrical stimulation causes relaxation of an endothelium-denuded ring precontracted with phenylephrine.16 These findings show that endothelial cells are endowed with a voltage-sensitive mechanism. In addition, endothelial cells have been reported to possess voltage-sensitive Ca2+ channels,R43-126738 R44-126738 R45-126738 R46-126738 43–46,47 which have been proposed to participate in impulse conduction and pacemaking in the heart.48 The presence of these channels in the endothelial cells could help to explain a regenerative propagation of an endothelium-dependent vasodilator signal.

It is important to note that although voltage-sensitive Ca2+ channels inactivate during depolarization, they continue to exhibit a window current, as manifested by a voltage range where the steady-state activation and inactivation curves overlap. In this voltage range, channels cycle between closed, open, and inactivated states.49 Therefore, in a tonically depolarized tissue, window current of the Ca2+ channels may allow a sustained calcium entry, such as is shown by the maintained vasoconstriction observed with DC stimulation (Figure 4B).

It is also noteworthy that voltage-dependent Ca2+ channels are sometimes found in caveolae, a membrane microdomain specialized in signal transduction.R50-126738 50,51 This location provides these channels with a special proximity to Ca2+-dependent signaling molecules present in caveolae, such as nitric oxide synthase, calmodulin, Ca2+-activated potassium channels, and plasma membrane Ca2+ pumps.R50-126738 R51-126738 50–52 Interestingly, gap junctions can also be targeted to caveolae or other subcellular compartments in a pattern dependent on gap junction type.53 Therefore, we hypothesized that the subcellular location of gap junctions plays a key role in the functional selectivity of this intercellular channels and, thereby, in the regenerative propagation of the electrically induced Cx40-dependent vasodilation.

In summary, we have confirmed a previous report11 that depolarizing electrical stimulation of cremasteric arterioles causes vasoconstriction by the activation of perivascular nerves and smooth muscle cells. In addition, our data indicate that electrical stimulation also initiates a conducted dilation that spreads rapidly along the endothelium without apparent decay, suggesting that a regenerative mechanism is involved in this process. Knockout of Cx40 demonstrates that the gap junctions play a key role in this process, although it remains uncertain whether the gap junctional channels are critical to the conduction process per se or are somehow involved in smooth muscle/endothelial cell coupling.


*    Acknowledgments
 
This research was supported by NIH Grants HL53318 and HL12792.

Received December 4, 2002; revision received February 12, 2003; accepted February 26, 2003.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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