Rationale The coordination of vascular smooth muscle cell (VSMC) constriction plays an important role in vascular function such as regulation of blood pressure. However, the mechanism responsible for VSMC communication is not clear in the resistance vasculature. Pannexins (Panx) are purine releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of VSMC constriction. Objective We investigated the role of pannexins in phenylephrine (PE) and KCl mediated constriction of resistance arteries. Methods and Results Western blot, immunohistochemistry and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries (TDA). Functionally, the contractile response of pressurized TDA to PE was significantly decreased by multiple Panx inhibitors (mefloquine, probenecid and 10Panx1), ectonucleotidase (apyrase) and purinergic receptor inhibitors (suramin and reactive-blue-2). Electroporation of TDA with either Panx1-GFP or Panx1 siRNA showed enhanced and decreased constriction respectively in response to PE. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with co-immunoprecipitation experiments from TDA, which suggested an association between Panx1 and α1D-adrenoreceptor. Conclusions Our data demonstrate for the first time a key role for Panx1 in resistance arteries, by contributing to the coordination of VSMC constriction and possibly regulation of blood pressure.

Objective-To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results-Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NO x concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. Key Words: NO Ⅲ GSNO-R Ⅲ connexin Ⅲ myoendothelial junction Ⅲ nitrosylation W ithin the vessel wall of resistance arteries, coordinated vascular smooth muscle cell (SMC) and endothelial cell (EC) function is integrated by complex intercellular signaling to regulate the constriction and dilation of the artery. The anatomic structures that facilitate direct SMC and EC communication within the resistance artery are myoendothelial junctions (MEJs), which are cellular extensions from ECs or SMCs that project through the internal elastic lamina [1][2][3] and link the plasma membranes of the 2 different cell types together. The gap junctions (GJs) at the MEJ provide a conduit for second messenger and electric signaling between the 2 cell types. 2,4,5 For example, phenylephrine (PE) stimulation of SMCs induces inositol 1,4,5-triphosphate (IP 3 ) generation and an increase in [Ca 2ϩ ] i concentrations, constricting the artery. It is thought that the IP 3 progresses to the adjacent EC through GJs at the MEJ, initiating an increase in [Ca 2ϩ ] i and the release of NO to modulate the magnitude of vasoconstriction, thereby regulating the tone of the artery. 6 -8 Elucidation of the mechanisms regulating this process could provide novel insight into blood pressure regulation; however, the process remains uncharacterized. Conclusion-This study provides evidence for compartmentalized S-nitrosylation/denitrosylationGJs are intracellular signaling channels formed by 2 hexameric hemichannels, with each adjacent cell contributing 1 hemichannel. Connexin (Cx) proteins compose the channels, of which 4 different Cxs have been identified in the vasculature, with multiple studies demonstrating a potentially important role for Cx43 at the MEJ. 9 Recent studies have demonstrated that GJ communication and trafficking of Cx43 are mod...

NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.

The functions of caveolae and͞or caveolins in intact animals are beginning to be explored. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in several postnatal vascular paradigms. First, increasing levels of Cav-1 do not increase caveolae number in the endothelium in vivo. Second, despite a lack of quantitative changes in organelle number, endothelial-specific expression of Cav-1 impairs endothelial nitric oxide synthase activation, endothelial barrier function, and angiogenic responses to exogenous VEGF and tissue ischemia. In addition, VEGF-mediated phosphorylation of Akt and its substrate, endothelial nitric oxide synthase, were significantly reduced in VEGF-treated Cav-1 transgenic mice, compared with WT littermates. The inhibitory effect of Cav-1 expression on the Aktendothelial nitric oxide synthase pathway was specific because VEGFstimulated phosphorylation of mitogen-activated protein kinase (ERK1͞2) was elevated in the Cav-1 transgenics, compared with littermates. These data strongly support the idea that, in vivo, Cav-1 may modulate signaling pathways independent of its essential role in caveolae biogenesis.nitric oxide ͉ caveolae ͉ VEGF ͉ signal transduction C aveolae are 50-to 100-nm flask-shaped invaginations of the plasma membrane (1) and are present in most mammalian cells. The putative functions of caveolae include cholesterol transport (2, 3), endocytosis (4), potocytosis (5), and signal transduction (6-9). Recent insights into the physiological roles of caveolae and their primary coat proteins, caveolins, have been dissected in genetically modified mice (10, 11). Caveolin-1 (Cav-1) and Cav-3 are dispensable during vascular and organ development but are essential for caveolae formation in specialized cells including most endothelia, adipocytes, and skeletal͞cardiac myocytes. In vitro data have shown that signaling molecules can potentially interact with Cav-1, and that interactions with Cav-1 can increase or decrease the fidelity or magnitude of signaling (12)(13)(14). The ability of proteins to localize in caveolae, in addition to direct interactions of proteins with caveolins, has led to the hypothesis that caveolae may compartmentalize signaling in the plasma membrane and that the interactions (direct and indirect) between resident proteins and Cav-1 may fine-tune the signaling cascades.Physiologically, the loss of caveolae results in impairment of cholesterol homeostasis (15), insulin sensitivity (16), nitric oxide (NO) (10, 11), calcium signaling (10), and cardiac function (17). These studies validate the in vivo importance of caveolae and caveolins beyond cell-based studies that are largely hampered by operational definitions of biochemical fractions containing caveolins and the lack of specificity inherent in reagents that remove cellular cholesterol. Although caveolin knockout mice are useful to delineate the importance of caveolae in a given response, there are additional questions that are difficult to dis...

Functional hyperemia requires the coordination of smooth muscle cell relaxation along and between branches of the arteriolar network. Vasodilation is conducted from cell to cell along the arteriolar wall through gap junction channels composed of connexin protein subunits. Within skeletal muscle, it is unclear whether arteriolar endothelium, smooth muscle, or both cell layers provide the cellular pathway for conduction. Furthermore, the constitutive profile of connexin expression within the microcirculation is unknown. We tested the hypothesis that conducted vasodilation and connexin expression are intrinsic to the endothelium of arterioles (17 +/- 1 microm diameter) that supply the skeletal muscle fibers in the cremaster of anesthetized C57BL/6 mice. ACh delivered to an arteriole (500 ms, 1-microA pulse; 1-microm micropipette) produced local dilation of 17 +/- 1 microm; conducted vasodilation observed 1 mm upstream was 9 +/- 1 microm (n = 5). After light-dye treatment to selectively disrupt endothelium (250-microm segment centered 500 microm upstream, confirmed by loss of local response to ACh while constriction to phenylephrine and dilation to sodium nitroprusside remained intact), we found that conducted vasodilation was nearly abolished (2 +/- 1 microm; P < 0.05). Whole-mount immunohistochemistry for connexins revealed punctate labeling at borders of arteriolar endothelial cells, with connexin40 and connexin37 in all branches and connexin43 only in the largest branches. Immunoreactivity for connexins was not apparent in smooth muscle or in capillary or venular endothelium, despite robust immunolabeling for alpha-actin and platelet endothelial cell adhesion molecule-1, respectively. We conclude that vasodilation is conducted along the endothelium of mouse skeletal muscle arterioles and that connexin40 and connexin37 are the primary connexins forming gap junction channels between arteriolar endothelial cells.

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Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries,highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels.

To determine whether simulated microgravity in rats is associated with vascular dysfunction, we measured responses of isolated, pressurized mesenteric resistance artery segments (157- to 388-microm ID) to vasoconstrictors, pressure, and shear stress after 28-day hindlimb suspension (HS). Results indicated no differences between HS and control (C) groups in 1) sensitivity or maximal responses to vasoconstrictors (norepinephrine, phenylephrine, serotonin, KCl); 2) ID, external diameter, or ratio of wall thickness to ID; 3) distensibility; or 4) vasodilatory responses to shear stress. Myogenic tone was attenuated (P< 0.05) in HS arteries vs. C, as evidenced by 1) decreased magnitude of tone in larger vessels (second-order branch off superior mesenteric artery, 261- to 388-microm ID) at pressures >/=40 mmHg in the presence of phenylephrine (10(-7) M) and 2) decreased magnitude of tone in smaller vessels (third-order branch off superior mesenteric artery, 157- to 277-microm ID), which exhibited spontaneous tone, at pressures > or =70 mmHg. This attenuation of myogenic tone after HS could contribute to orthostatic intolerance because myogenic tone contributes to the overall tone of resistance arteries.