Spontaneous lung dysfunction and fibrosis in mice lacking connexin 40 and endothelial cell connexin 43.

Gap junction proteins (connexins) facilitate intercellular communication and serve several roles in regulation of tissue function and remodeling. To examine the physiologic effects of depleting two prominent endothelial connexins, Cx40 and Cx43, transgenic mice were generated by breeding Cx40-deficient mice (Cx40(-/-)) with a vascular endothelial cell (VEC)-specific Cx43-deficient mouse strain (VEC Cx43(-/-)) to produce double-connexin knockout mice (VEC Cx43(-/-)/Cx40(-/-)). The life span in VEC Cx43(-/-)/Cx40(-/-) mice was dramatically shortened, which correlated with severe spontaneous lung abnormalities as the mice aged including increased fibrosis, aberrant alveolar remodeling, and increased lung fibroblast content. Moreover, VEC Cx43(-/-)/Cx40(-/-) mice exhibited cardiac hypertrophy and hypertension. Because VEC Cx43(-/-)/Cx40(-/-) mice demonstrated phenotypic hallmarks that were remarkably similar to those in mice deficient in caveolin-1, pulmonary caveolin expression was examined. Lungs from VEC Cx43(-/-)/Cx40(-/-) mice demonstrated significantly decreased expression of caveolin-1 and caveolin-2. This suggests that expression of caveolin-1 may be linked to expression of Cx40 and endothelial Cx43. Moreover, the phenotype of caveolin-1(-/-) mice and VEC Cx43(-/-)/Cx40(-/-) mice may arise via a common mechanism.

Gap junctions in the control of vascular function.

Direct intercellular communication via gap junctions is critical in the control and coordination of vascular function. In the cardiovascular system, gap junctions are made up of one or more of four connexin proteins: Cx37, Cx40, Cx43, and Cx45. The expression of more than one gap-junction protein in the vasculature is not redundant. Rather, vascular connexins work in concert, first during the development of the cardiovascular system, and then in integrating smooth muscle and endothelial cell function, and in coordinating cell function along the length of the vessel wall. In addition, connexin-based channels have emerged as an important signaling pathway in the astrocyte-mediated neurovascular coupling. Direct electrical communication between endothelial cells and vascular smooth muscle cells via gap junctions is thought to play a relevant role in the control of vasomotor tone, providing the signaling pathway known as endothelium-derived hyperpolarizing factor (EDHF). Consistent with the importance of gap junctions in the regulation of vasomotor tone and arterial blood pressure, the expression of connexins is altered in diseases associated with vascular complications. In this review, we discuss the participation of connexin-based channels in the control of vascular function in physiologic and pathologic conditions, with a special emphasis on hypertension and diabetes.

Dissection of two Cx37-independent conducted vasodilator mechanisms by deletion of Cx40: electrotonic versus regenerative conduction.

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 microm 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 mechanism of regenerative conduction of vasodilator responses. Connexin40 is essential for the ACh-activated regenerative vasodilator mechanism. However, neither connexin40 nor connexin37 is indispensable for the electrotonic spread of hyperpolarizing signals.

Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo.

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.

Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.

Although much physiology in resistance vessels has been attributed to the cytoplasmic connection between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), little is known of the protein expression between the two cell types. In an attempt to identify the proteins between ECs and VSMCs, mouse cremaster arterioles were stained with phalloidin-Alexa 594 and viewed on a confocal microscope that resolved "actin bridges" within the internal elastic lamina between ECs and VSMCs. To determine the incidence of protein, the pixel intensity from the antibodies on actin bridges were compared with the pixel intensity from antibodies within ECs or VSMCs. N-cadherin, desmin, connexin (Cx)40, and Cx43 and phosphorylated Cx43 at serine-368 were identified on actin bridges, but NG2, CD31, and Cx45 were not evident. Cx37 expression was more variable than the other connexins examined. Using this method on rat mesentery, we confirm the previously published predominance of Cx37 and Cx40 at the myoendothelial junction that was determined using electron microscopy. We conclude that this new method represents an important screening mechanism in which to rapidly test for protein expression between ECs and VSMCs and possibly a first-step in quantifying protein expression at the myoendothelial junction.

Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.

Smooth muscle-targeted knockout of connexin43 enhances neointimal formation in response to vascular injury.

OBJECTIVE: Vascular disease alters and reduces connexin expression and a reduction in connexin 43 (Cx43) expression diminishes the extent of atherosclerosis observed in a high-cholesterol diet murine model. We hypothesized that connexins might play a role in the smooth muscle cell response to vascular injury. METHODS AND RESULTS: We therefore studied a line of smooth muscle cell-specific, Cx43 gene knockout mice (SM Cx43 KO) in which the carotid arteries were injured, either by vascular occlusion or by a wire injury. In the SM Cx43 KO mice both types of injury manifested accelerated growth of the neointima and of the adventitia. Isolated vascular smooth muscle cells from the SM Cx43 KO mice grew at a slightly faster rate in culture, and to marginally higher saturation densities than those of control mice, but these changes were not adequate to explain the large changes in the injured vessels. CONCLUSIONS: These observations provide direct evidence that smooth muscle Cx43 gap junctions play a multi-faceted role in modulating the in vivo growth response of vascular smooth muscle cells to vascular injury.

Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction.

Second messenger signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is poorly understood, but intracellular Ca2+ concentrations ([Ca2+]i) in the 2 cells are coordinated, possibly through gap junctions at the myoendothelial junction. To study heterocellular calcium signaling, we used a vascular cell coculture model composed of monolayers of ECs and VSMCs. Stimulation of either cell type leads to an increase in [Ca2+]i in the stimulated cell and a secondary increase in [Ca2+]i in the other cell type that was blocked by gap junction inhibitors. To determine which second messengers are involved, we initially depleted Ca2+ stores in the endoplasmic reticulum Ca2+ with thapsigargin in ECs or VSMCs, but this had no effect on heterocellular calcium signaling. Alternatively, we loaded ECs or VSMCs with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to buffer changes in [Ca2+]i. BAPTA loading of ECs inhibited agonist-induced increases in intracellular calcium concentration ([Ca2+]i), in both ECs and VSMCs. In contrast, BAPTA loading of the VSMCs blunted the VSMC response but did not alter the secondary increase in EC [Ca2+]i. Xestospongin C (an inositol 1,4,5-trisphosphate receptor inhibitor) had no effect on the secondary Ca2+ response, but when xestospongin C or thapsigargin was loaded into ECs and BAPTA into VSMCs, intercellular Ca2+ signaling was completely blocked. We conclude that 1,4,5-trisphosphate and Ca2+ originating in the VSMCs induces the secondary increase in EC [Ca2+]i but stimulation of the ECs generates a Ca2+ dependent response in the VSMCs.

Connexin43 is required for production of the aqueous humor in the murine eye.

Connexin43 is a major component of the gap junctions between pigmented and non-pigmented cells of the double-layered epithelium in the ciliary body of the eye. We directly tested the hypothesis that gap junctions play a crucial role in the production of the aqueous humor by inactivating the GJA1 (connexin43) gene in the pigmented epithelium with cre-loxP technology. To accomplish this, we crossed a line expressing cre recombinase driven by the nestin promoter and a line with floxed connexin43 alleles. Resultant lines exhibited loss of connexin43 from the pigmented epithelium, iris, retinal pigment epithelium and the lens. We observed plasma proteins in the aqueous humor and pathological changes consistent with a loss of intraocular pressure. As the ciliary body is responsible for aqueous humor production, these data support the hypothesis that the gap junctions between pigmented and non-pigmented epithelium are necessary for production of the aqueous humor that is in turn required for the generation of normal intraocular pressure and nourishment of the postnatal lens. The loss of connexin43 expression in the iris correlated with a separation of the posterior pigmented epithelium from the anterior myoepithelium and with meiosis, possibly resulting from a loss of function of the dilator pupillae.

Oxidized phospholipids alter vascular connexin expression, phosphorylation, and heterocellular communication.

OBJECTIVE: In endothelial cells (EC) and vascular smooth muscle cells (VSMC) from atherosclerotic mice, connexin (Cx) expression becomes distorted. Lipoprotein-derived phospholipid oxidation products (OxPAPC) play a critical role in atherosclerosis, and we hypothesized that they may act as trigger molecules causing the changes in connexin expression. METHODS AND RESULTS: We applied OxPAPC to murine carotid arteries in vivo and vascular cell cocultures. OxPAPC applied to carotids induced an upregulation of both Cx37 and Cx43 in the VSMC. In EC, Cx43 was upregulated and Cx37 was downregulated, whereas Cx40 in EC remained constant. In the vascular cell coculture, OxPAPC caused similar changes in Cx37 and Cx43 but caused a decrease in Cx40 in EC and an elevation of Cx40 in VSMC. In the coculture model, OxPAPC treatment led to the selective disappearance of Cx40 at the myoendothelial junction. Biocytin dye transfer between EC and VSMC coupling was dramatically reduced by OxPAPC. The decrease in dye transfer after OxPAPC treatment was correlated with an increase in tyrosine 265 phosphorylation of Cx43, especially at the in vitro myoendothelial junction. CONCLUSIONS: We conclude that OxPAPC could be responsible for the changes in connexin expression previously reported in atherosclerosis.

Reactive oxygen species mediate modification of glycocalyx during ischemia-reperfusion injury.

The glycocalyx (Gcx) is a complex and poorly understood structure covering the luminal surface of endothelial cells. It is known to be a determinant of vascular rheology and permeability and may be a key control site for the vascular injuries caused by ischemia-reperfusion (I/R). We used intravital-microscopy to evaluate the effects of I/R injury on two properties of Gcx in mouse cremasteric microvessels: exclusion of macromolecules (anionic-dextrans) and intracapillary distribution of red blood cells (RBC). In this model, the Gcx is rapidly modified by I/R injury with an increase in 70-kDa anionic-dextran penetration without measurable effect on the penetration of 580-kDa anionic-dextran or on RBC exclusion. The effects of I/R injury appear to be mediated by the rapid production of reactive oxygen species (ROS) because they are ameliorated by the addition of exogenous superoxide dismutase-catalase. Intravenous application of allopurinol or heparin also inhibited the effects of I/R injury, and we interpret efficacy of allopurinol as evidence for a role for xanthine-oxidoreductase (XOR) in the response to I/R injury. Heparin, which is hypothesized to displace XOR from a heparin-binding domain in the Gcx, reduced the effects of I/R. The effects of I/R injury were also partially prevented or fully reversed by the intravascular infusion of exogenous hyaluronan. These data demonstrate: 1) the liability of Gcx during I/R injury; 2) the importance of locally produced ROS in the injury to Gcx; and 3) the potential importance of heparin-binding sites in modulating the ROS production. Our findings further highlight the relations between glycosaminoglycans and the pathophysiology of Gcx in vivo.

Connexin40 and connexin43 in mouse aortic endothelium: evidence for coordinated regulation.

In the vessel wall, endothelial cells are metabolically and electrically coupled to each other and to the adjacent smooth muscle cells by gap junctions composed of connexins. Gap junctions may be formed from combinations of several different connexin proteins, and deletion of one connexin can lead to modification of the expression of another. To reveal a possible interaction between connexin40 (Cx40) and connexin43 (Cx43) in endothelium, we studied their distribution in vessels from C57Bl/6 and Cx40 knockout mice (Cx40-/-) using immunoblots and immunocytochemistry on aortic cross sections and en face whole mounts. En face preparations from C57Bl/6 mice revealed two distinct pools of Cx43, one pericellular and the other intracellular. Cx40 was largely restricted to the periphery of the cells, and in Cx40-/- mice it was, as expected, undetectable. In the Cx40-/- mice, total Cx43 protein was also modestly reduced (immunoblots), but there was a major redistribution of the protein within the cell. The pericellular component of Cx43 was rendered virtually undetectable, and the intracellular compartments were normal or even slightly elevated. Smooth muscle Cx43 was also reduced in the Cx40-/- animals. These findings indicate that the cellular distribution of Cx43 is dependent on the presence of Cx40, and in view of the profound effects on the pericellular pool of the Cx43, the findings suggest that interactions between Cx40 and Cx43 regulate communication between endothelial cells and perhaps between smooth muscle and endothelial cells as well.

Heterocellular contact at the myoendothelial junction influences gap junction organization.

Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated Cx37 and Cx43 in both cell types, whereas Cx40 was found only in EC. Cx37 was excluded from the MEJ in both EC and VSMC. Connexin composition as well as functionality of the gap junctions at the MEJ was assessed by measuring diffusional transfer of biocytin and Cy3. Using connexin-specific blockers and manipulations of expression of individual connexin proteins, we confirmed that Cx37 is not a part of EC-VSMC coupling, and we demonstrated that heterotypic gap junctions are functional at the MEJ. We speculate that specific gap junction organization may be a vital component of EC-VSMC contact at the MEJ.

Connexins: gaps in our knowledge of vascular function.

Gap junctions are common features in the vasculature, long thought to provide a pathway for cell-cell signaling. Emerging understanding of the gap-junctional proteins (connexins) and new tools for their investigation now offer the opportunity to explore the vital role that the gap junctions may play in cardiovascular homeostasis and pathophysiology.

Adenosine A3 receptor activation modulates the capillary endothelial glycocalyx.

The endothelial glycocalyx is a dynamic extracellular matrix composed of cell surface proteoglycans, glycoproteins, and adsorbed serum proteins that has been implicated in the regulation and modulation of capillary tube hematocrit, permeability, and hemostasis. High tissue adenosine levels have been shown to adversely affect microvascular function and tissue survival after an ischemic episode, and previous work in this laboratory has shown that adenosine causes arteriolar constriction and degranulation of mast cells via the A3 receptor (A3AR). We hypothesized that adenosine exerts at least part of its effect through modification of the glycocalyx via the A3AR. We used an in vivo cremaster model (hamster and mouse) in which circulating plasma was labeled with a 70-kDa FITC-dextran, and the capillaries were examined before and after superfusion with varying concentrations of adenosine (or other vasoactive molecules). Measurements of the dextran exclusion from an endothelial cell surface layer and red cell separation from the endothelial cell surface were made for up to 30 minutes. Our data indicate that adenosine causes a rapid and profound decrease in the ability of the glycocalyx to exclude dextran but only affects red blood cell exclusion at pharmacological levels. Knockout mice deficient in the A3AR were completely protected from glycocalyx changes attributable to adenosine. These data show a potential link between a known vasoactive tissue metabolite, adenosine, and regulation of the glycocalyx, which may be important during (patho)physiological changes in microvascular function during inflammatory insults.

Endothelial cell signaling during conducted vasomotor responses.

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.

Participation of intracellular Ca2+ stores in arteriolar conducted responses.

We examined the role played by intracellular Ca2+ stores in conducted vasomotor responses induced by phenylephrine (PE) in isolated hamster cremasteric arterioles. When applied briefly ( approximately 1 s) to isolated, cannulated arterioles by using pressure-pulse ejection from a micropipette, PE produced a strong local vasoconstriction and a very small biphasic conducted response (a small constriction followed by a dilation) that propagated several hundred micrometers along the vessel length. The conducted vasomotion was associated with a monophasic elevation of the endothelial cell intracellular Ca2+ concentration ([Ca2+]i) at the site of stimulation, as measured with the Ca2+ indicator fura 2. The Ca2+ pump inhibitor thapsigargin was used to limit filling of Ca2+ stores in smooth muscle and endothelial cells. Thapsigargin reduced baseline diameter and elicited a strong dilator component at the local site while enhancing both the constrictor and dilator components of the PE-induced conducted response. The enhanced conducted constrictor component induced by thapsigargin was mimicked by extraluminal application of tetraethylammonium or charybdotoxin but not by iberiotoxin, apamin, glibenclamide, barium, or 4-aminopirydine. Thapsigargin increased the estimated basal endothelial cell [Ca2+]i by approximately 60 nM and converted the PE-induced change in [Ca2+]i from monotonic to biphasic with a late elevation of [Ca2+]i above baseline that coincided with the increased dilatory component of the conducted response. Luminal application of charybdotoxin plus apamin significantly reduced the dilatory component of the conducted response. These results indicate that intracellular Ca2+ stores play a dynamic role in regulating conducted vasomotor responses apparently through modulation of KCa channels in both cell types.

Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo.

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 microm 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 micromol/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.