Myoendothelial feedback in mouse mesenteric resistance arteries is similar between the sexes, dependent on nitric oxide synthase, and independent of TPRV4.

Myoendothelial feedback (MEF), the endothelium-dependent vasodilation following sympathetic vasoconstriction (mediated by smooth muscle to endothelium gap junction communication), has been well studied in resistance arteries of males, but not females. We hypothesized that MEF responses would be similar between the sexes, but different in the relative contribution of the underlying nitric oxide and hyperpolarization mechanisms, given that these mechanisms differ between the sexes in agonist-induced endothelium-dependent dilation. We measured MEF responses (diameter changes) of male and female first- to second-order mouse mesenteric arteries to phenylephrine (10 µM) over 30 min using isolated pressure myography ± blinded inhibition of nitric oxide synthase (NOS) using Nω-nitro-l-arginine methyl ester (l-NAME; 0.1-1.0 mM), hyperpolarization using 35 mM KCl, or transient receptor potential vanilloid 4 (TRPV4) channels using GSK219 (0.1-1.0 µM) or RN-1734 (30 µM). MEF was similar [%dilation (means ± SE): males = 26.7 ± 2.0 and females = 26.1 ± 1.9 at 15 min] and significantly inhibited by l-NAME (1.0 mM) at 15 min [%dilation (means ± SE): males = 8.2 ± 3.3, P < 0.01; females = 6.8 ± 1.9, P < 0.001] and over time (P < 0.01) in both sexes. l-NAME (0.1 mM) + 35 mM KCl nearly eliminated MEF in both sexes (P < 0.001-0.0001). Activation of TRPV4 with GSK101 (0.1-10 µM) induced similar dilation between the sexes. Inhibition of TRPV4, which is reportedly involved in the hyperpolarization mechanism, did not inhibit MEF in either sex. Similar expression of eNOS was found between the sexes with Western blot. Thus, MEF is prominent and similar in murine first- and second-order mesenteric resistance arteries of both sexes, and reliant primarily on NOS and secondarily on hyperpolarization, but not TRPV4.NEW & NOTEWORTHY We found that female mesenteric resistance arteries have similar postconstriction dilatory responses (i.e., myoendothelial feedback) to a sympathetic neurotransmitter analog as male arteries. Both sexes use nitric oxide synthase (NOS) and hyperpolarization, but not TRPV4, in this response. Moreover, the key protein involved in this pathway (eNOS) is similarly expressed in these arteries between the sexes. These similarities are surprising given that agonist-induced endothelium-dependent dilatory mechanisms differ in these arteries between the sexes.

Shear Stress Attenuates Inward Remodeling in Cultured Mouse Thoracodorsal Arteries in an eNOS-Dependent, but Not Hemodynamic Manner, and Increases Cx37 Expression.

BACKGROUND: Arteries chronically constricted in culture remodel to smaller diameters. Conversely, elevated luminal shear stress (SS) promotes outward remodeling of arteries in vivo and prevents inward remodeling in culture in a nitric oxide synthase (NOS)-dependent manner. OBJECTIVES: To determine whether SS-induced prevention of inward remodeling in cultured arteries is specifically eNOS-dependent and requires dilation, and whether SS alters the expression of eNOS and other genes potentially involved in remodeling. METHODS: Female mouse thoracodorsal arteries were cannulated, pressurized to 80 mm Hg, and cultured for 2 days with low SS (<7 dyn/cm2), high SS (≥15 dyn/cm2), high SS + L-NAME (NOS inhibitor, 10-4 M), or high SS in arteries from eNOS-/- mice. In separate arteries cultured 1 day with low or high SS, eNOS and connexin (Cx) 37, Cx40, and Cx43 mRNA were assessed with real-time PCR. RESULTS: High SS caused little change in passive diameters after culture (-4.7 ± 2.0%), which was less than low SS (-18.9 ± 1.4%; p < 0.0001), high SS eNOS-/- (-18.0 ± 1.5; p < 0.001), or high SS + L-NAME (-12.0 ± 0.6%; nonsignificant) despite similar constriction during culture. Cx37 mRNA expression was increased (p < 0.05) with high SS, but other gene levels were not different. CONCLUSIONS: eNOS is involved in SS-induced prevention of inward remodeling in cultured small arteries. This effect does not require NO-mediated dilation. SS increased Cx37.

Flow does not alter eNOS phosphoryation at Ser1179 or Thr495 in preconstricted mouse mesenteric arteries.

In arteries, endothelium-dependent vasodilatory agonists and flow-induced shear stress cause vasodilation largely by activation of the endothelial enzyme eNOS, which generates nitric oxide that relaxes vascular smooth muscle. Agonists activate eNOS in part through increased phosphorylation at Ser1179 and decreased phosphorylation at Thr495. We previously found that preconstriction of intact, isolated mouse mesenteric arteries with phenylephrine also caused increased Ser1179 and decreased Thr495 eNOS phosphorylation, and sequential treatment with the vasodilatory agonist acetylcholine did not cause any further change in phosphorylation at these sites, despite producing vasodilation. The present study tests the hypothesis that luminal flow in these arteries preconstricted with phenylephrine also produces vasodilation without phosphorylation changes at these sites. First-order mesenteric arteries, isolated from male C57/BL6 mice (7-20 weeks of age) anesthetized with pentobarbital (50 mg/kg, i.p.), were cannulated, pressurized, and treated with stepped increases in luminal flow (15-120 µL/min). Flow resulted in dilation that plateaued at ~60 µL/min (31.3 ± 3.0% dilation) and was significantly (P < 0.001) NOS-dependent at all flow rates (determined by 10-4 mol/L L-NAME treatment). In separate arteries, preconstriction with phenylephrine (10-5 mol/L) resulted in increased eNOS phosphorylation at Ser1179 (P < 0.05) and decreased phosphorylation at Thr495, but subsequent flow at 60 µL/min for 5 or 15 min did not cause further changes in phosphorylation, despite causing dilation. Thus, flow-induced dilation does not require changes in these eNOS phosphorylation sites beyond those induced by alpha1-adrenergic stimulation with phenylephrine, indicating that eNOS is activated by other mechanisms during acute flow-induced dilation of preconstricted arteries.

Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function.

OBJECTIVE: In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. APPROACH AND RESULTS: We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. CONCLUSIONS: Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.

Increased myoendothelial feedback is associated with increased connexin37 and IK1 channel expression in mesenteric arteries of diet-induced hyperhomocysteinemic mice.

OBJECTIVE: Previously, we found that diet-induced HHcy in mice caused decreased eNOS expression and signaling in mesenteric arteries, but greatly enhanced non-NOS, non-prostacyclin-dependent vasodilation, which involves MEJ communication. To further assess whether HHcy enhances MEJ communication, this study examined endothelium-dependent attenuation of phenylephrine-induced vasoconstriction (myoendothelial feedback) and key molecules involved. METHODS: Myoendothelial feedback was examined in isolated mouse mesenteric arteries, after 6-weeks diet-induced HHcy, using pressure myography. Gap junction (Cx37, Cx40, Cx43), NOS (eNOS, nNOS, iNOS), and potassium channel (IK1) protein expression were measured with immunoblots, and connexin mRNAs with real-time PCR. Contribution of nNOS + iNOS to vasomotor responses was assessed using the drug TRIM. RESULTS: Myoendothelial feedback was significantly (P < .05) enhanced in HHcy arteries compared to control, coincident with significantly greater Cx37 and IK1 protein and Cx37 mRNA. Cx43 protein, but not mRNA, was significantly less in HHcy, and Cx40 was not different. eNOS protein was significantly less in HHcy. nNOS and iNOS were not different. TRIM had little effect on vasomotor function. CONCLUSIONS: Diet-induced HHcy enhanced myoendothelial feedback, and increased Cx37 and IK1 expression may contribute. nNOS or iNOS did not upregulate to compensate for decreased eNOS, and they had little involvement in vasomotor function.

Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries.

Activation of arterial smooth muscle alpha(1)-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurized (75 mm Hg) and treated either by 1) 5 min of phenylephrine superfusion (10(-5)M) (PE5), 2) 15 min of phenylephrine (PE15), 3) 15 min phenylephrine followed by acetylcholine (10(-4)M) (PE+ACh), or 4) 20 min time control with no treatment (NT) [4-5 arteries pooled per treatment per blot; 5 blots performed]. These treatments allowed correlation between vasomotor changes, namely maximal constriction (PE5), slow vasodilation (PE15), and maximal dilation (PE+ACh), and relative phosphorylation changes. Phosphorylation of eNOS at Ser1179 was increased relative to NT by more than 2-fold at PE5 and remained similarly increased at PE15 and PE+ACh. Phosphorylation of eNOS at Thr495 was less in all treatments relative to NT, but not significantly. Treatment with L-NAME (10(-4)M) or endothelial denudation indicated that the slow dilation in response to phenylephrine was completely due to nitric oxide synthase and was endothelial dependent. These results indicate that eNOS phosphorylation at Ser1179 occurs before the slow dilation and is not actively involved in this vasodilation or dilation to acetylcholine, but may play a permissive role in eNOS activation by other mechanisms. It is not yet known what mechanism is responsible for Ser1179 phosphorylation with phenylephrine stimulation.

Pannexin1 regulates α1-adrenergic receptor- mediated vasoconstriction.

RATIONALE: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell 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 vascular smooth muscle cell constriction. OBJECTIVE: We investigated the role of pannexins in phenylephrine- 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. Functionally, the contractile response of pressurized thoracodorsal resistance arteries to phenylephrine was decreased significantly by multiple Panx inhibitors (mefloquine, probenecid, and (10)Panx1), ectonucleotidase (apyrase), and purinergic receptor inhibitors (suramin and reactive blue-2). Electroporation of thoracodorsal resistance arteries with either Panx1-green fluorescent protein or Panx1 small interfering RNA showed enhanced and decreased constriction, respectively, in response to phenylephrine. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with coimmunoprecipitation experiments from thoracodorsal resistance arteries, which suggested an association between Panx1 and α1D-adrenergic receptor. CONCLUSIONS: Our data demonstrate for the first time a key role for Panx1 in resistance arteries by contributing to the coordination of vascular smooth muscle cell constriction and possibly to the regulation of blood pressure.

Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.

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. CONCLUSIONS: This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.

Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.

RATIONALE: Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined. OBJECTIVE: Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation. METHODS AND RESULTS: MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells. Using a vascular cell coculture we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the vascular cell coculture, recombinant PAI-1 added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1(-/-) mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1(-/-) coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the vascular cell coculture was temporally enhanced when recombinant PAI-1 was present, and prolonged when PAI-1 was absent. CONCLUSION: Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (eg, diabetes mellitus).

Chronic diet-induced hyperhomocysteinemia impairs eNOS regulation in mouse mesenteric arteries.

Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species, thereby reducing nitric oxide (NO.) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO., endothelial nitric oxide synthase (eNOS), also contributes. It is also unclear whether resistance vessels that utilize both NO.and non-NO.vasodilatory mechanisms, undergo alteration of non-NO.mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hcy: 89.2 +/- 49.0 microM) compared with age-matched controls (Hcy: 6.6 +/- 1.9 microM), using first-order mesenteric arteries. Dilation to ACh (10(-9)-10(-4) M) was measured in isolated, cannulated, and pressurized (75 mmHg) arteries with and without N(G)-nitro-l-arginine methyl ester (l-NAME) (10(-4) M) and/or indomethacin (10(-5) M) to test endothelium-dependent dilation and non-NO.-dependent dilation, respectively. The time course of dilation to ACh (10(-4) M) was examined to compare the initial transient dilation due to non-NO., non-prostacyclin mechanism and the sustained dilation due to NO.. These experiments indicated that endothelium-dependent dilation was attenuated (P < 0.05) in HHcy arteries due to downregulation of only NO.-dependent dilation. Western blot analysis indicated significantly less (P < 0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no difference in phospho-T495-eNOS/eNOS. S1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic diet-induced HHcy in mice impairs eNOS protein expression and phosphorylation at S1179, coincident with impaired NO.-dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation and microvascular disease that is common with HHcy.

Age-related changes in conducted vasodilation: effects of exercise training and role in functional hyperemia.

Conducted vasodilation may coordinate blood flow in microvascular networks during skeletal muscle contraction. We tested the hypotheses that 1) exercise training enhances conducted vasodilation and 2) age-related changes in the capacity for conduction affect muscle perfusion during contractions. To address hypothesis 1, young (4-5 mo), adult (12-14 mo), and old (19-21 mo) C57BL6 male mice were sedentary or given access to running wheels for 8 wk. Voluntary running distances were significantly different (in km/day): young = 5.8 +/- 0.1, adult = 3.9 +/- 0.1, old = 2.2 +/- 0.1 (P < 0.05). In gluteus maximus muscles, conducted vasodilation was greater in adult than in young or old mice (P < 0.05) and greater in young sedentary than in old sedentary mice but was not affected by exercise training. Citrate synthase activity was greater with exercise training at all ages (P < 0.05). mRNA for endothelial nitric oxide synthase did not differ among ages, but endothelial nitric oxide synthase protein expression was greater in adult and old mice with exercise training (P < 0.05). Connexin 37, connexin 40, and connexin 43 mRNA were not affected by exercise training and did not differ by age. To address hypothesis 2, perfusion of the gluteus maximus muscle during light to severe workloads was assessed by Doppler microprobe at 3-26 mo of age. Maximum perfusion decreased linearly across the lifespan. Perfusion at the highest workload, absolute and relative to maximum, decreased across the lifespan, with a steeper decline beyond approximately 20 mo of age. In this model, 1) exercise training does not alter conducted vasodilation and 2) muscle perfusion is maintained up to near maximum workloads despite age-related changes in conducted vasodilation.

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo.

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.

Endothelial-specific expression of caveolin-1 impairs microvascular permeability and angiogenesis.

The functions of caveolae and/or 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 Akt-endothelial nitric oxide synthase pathway was specific because VEGF-stimulated phosphorylation of mitogen-activated protein kinase (ERK1/2) 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.

Independence of connexin expression and vasomotor conduction from sympathetic innervation in hamster feed arteries.

OBJECTIVE: Vasomotor responses can travel along the wall of resistance microvessels by two distinct mechanisms: cell-to-cell conduction through gap junctions or the release of neurotransmitter along perivascular nerves. It is unknown whether vascular innervation influences the expression of connexin molecules which comprise gap junctions, or the conduction of vasomotor responses. In feed arteries of the hamster retractor muscle (RFA), the authors tested whether sympathetic denervation would alter the expression of connexin isoforms and the conduction of vasomotor responses. METHODS: Using intact vessels with sympathetic innervation and those 7-8 days following denervation surgery, mRNA expression was quantified using real-time PCR, cellular localization of Cx protein was characterized using immunohistochemistry, and vasomotor responses to dilator and constrictor stimuli were evaluated in isolated pressurized RFA. RESULTS: Connexin protein localization and mRNA expression were similar between innervated and denervated vessels. mRNA levels were Cx43 = Cx37 > Cx45 >> Cx40. Vasodilation to acetylcholine conducted >/=2000 microm along innervated and denervated vessels, as did the biphasic conduction of vasoconstriction and vasodilation in response to KCl. Vasoconstriction to phenylephrine conducted <500 microm and was attenuated (p <.05) in denervated vessels. CONCLUSIONS: The profile of connexin expression and the conduction of vasomotor responses are largely independent of sympathetic innervation in feed arteries of the hamster retractor muscle (RFA).

Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.

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.

Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries.

OBJECTIVES: Conduction of vasoconstrictor and vasodilator responses in the microcirculation involves electrical coupling through gap junction channels among cells of the vascular wall. The present study determined whether reported differences in the properties of conduction along the arterioles of the epithelial hamster cheek pouch (CPA) and feed arteries of its retractor skeletal muscle (RFA) result from differences in the expression profile of specific connexin (Cx) isoforms and the gap junctions they comprise. METHODS: Real-time PCR, immunohistochemistry and serial section electron microscopy were used to compare wall morphology and the distribution of gap junctions between respective vessels. RESULTS: Expression of mRNA for Cx37, 40, 43 and 45 was similar between CPA and RFA. In the endothelium, Cx37, 40 and 43 proteins were expressed abundantly between adjacent cells while Cx37 was present in the smooth muscle. In both vessels, endothelial and smooth muscle cell (SMC) layers were well connected by myoendothelial gap junctions (MEGJs), which were found near endothelial cell (EC) gap junctions. CONCLUSIONS: The absence of differential gap junctional expression between CPA and RFA, in spite of documented differences in cellular conduction pathways, supports the hypothesis that conductance of vascular gap junction channels can be differentially modulated in resistance microvessels.

Peripheral vascular responses to heat stress after hindlimb suspension.

PURPOSE: The purpose of this study was to determine whether hindlimb suspension (which simulates the effects of microgravity) results in impaired hemodynamic responses to heat stress or alterations in mesenteric small artery sympathetic nerve innervation. METHODS: Over 28 d, 16 male Sprague-Dawley rats were hindlimb-suspended, and 13 control rats were housed in the same type of cage. After the treatment, mean arterial pressure (MAP), colonic temperature (Tcol), and superior mesenteric and iliac artery resistances (using Doppler flowmetry) were measured during heat stress [exposure to 42 degrees C until the endpoint of 80 mm Hg blood pressure was reached (75 +/- 9 min); endpoint Tcore = 43.6 +/- 0.2] while rats were anesthetized (sodium pentobarbital, 50 mg x kg(-1) BW). RESULTS: Hindlimb-suspended and control rats exhibited similar increases in Tcol, MAP, and superior mesenteric artery resistance, and similar decreases in iliac resistance during heat stress (endpoint was a fall in MAP below 80 mm Hg). Tyrosine hydroxylase immunostaining indicated similar sympathetic nerve innervation in small mesenteric arteries from both groups. CONCLUSION: Hindlimb suspension does not alter the hemodynamic or thermoregulatory responses to heat stress in the anesthetized rat or mesenteric sympathetic nerve innervation, suggesting that this sympathetic pathway is intact.