Aquaporin-2 Promoter Is Synergistically Regulated by Nitric Oxide and Nuclear Factor of Activated T Cells

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Role of Cholesterol in the Regulation of Hydrogen Sulfide Signaling within the Vascular Endothelium

H2S is a gaseous signaling molecule enzymatically produced in mammals and H2S-producing enzymes are expressed throughout the vascular wall. We previously reported that H2S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BKCa) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BKCa potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H2S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H2S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-β-cyclodextrin (MBCD, 100 µM) increased H2S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BKCa channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BKCa are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H2S-induced vasodilation through effects on EC TRPV4 and BKCa channels

Novel role of endothelial BKCa channels in altered vasoreactivity following hypoxia

The systemic vasculature exhibits attenuated vasoconstriction following hypobaric chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased activity of endothelial cell (EC) large-conductance, calcium-activated potassium (BKCa) channels contributes to this response. Gracilis resistance arteries from hypobaric CH (barometric pressure = 380 mmHg for 48 h) rats demonstrated reduced myogenic reactivity and hyperpolarized VSM membrane potential (Em) compared with controls under normoxic ex vivo conditions. These differences were eliminated by endothelial disruption. In the presence of cyclooxygenase and nitric oxide synthase inhibition, combined intraluminal administration of the intermediate and small-conductance, calcium-activated K+ channel blockers TRAM-34 and apamin was without effect on myogenic responsiveness and VSM Em in both groups; however, these variables were normalized in CH arteries by intraluminal administration of the BKCa inhibitor iberiotoxin (IBTX). Basal EC Em was hyperpolarized in arteries from CH rats compared with controls and was restored by IBTX, but not by TRAM-34/apamin. K+ channel blockers were without effect on EC basal Em in controls. Similarly, IBTX blocked acetylcholine-induced dilation in arteries from CH rats, but was without effect in controls, whereas TRAM-34/apamin eliminated dilation in controls. Acetylcholine-induced EC hyperpolarization and calcium responses were inhibited by IBTX in CH arteries and by TRAM-34/apamin in controls. Patch-clamp experiments on freshly isolated ECs demonstrated greater K+ current in cells from CH rats that was normalized by IBTX. IBTX was without effect on K+ current in controls. We conclude that hypobaric CH induces increased endothelial BKCa channel activity that contributes to reduced myogenic responsiveness and EC and VSM cell hyperpolarization