(706.1) EC Membrane Cholesterol Content Regulates the Contribution of TRPV4 Channels in ACh-induced vasodilation in Rat Gracilis Arteries

Poster Board Number: E1

Sophia Salbato (University of New Mexico Health Science Center), Emily Morin (University of New Mexico Health Science Center), Benjimen Walker (University of New Mexico Health Science Center), Jay Naik (University of New Mexico Health Science Center)

Numerous studies show that ion channel activity is sensitive to the level of membrane cholesterol, with cholesterol most often suppressing channel activity. Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48-hr of exposure to chronic hypoxia (CH). Moreover, CH exposure also couples endothelial Transient Receptor Potential Subfamily V Member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium-dependent hyperpolarization (EDH)-pathway not present in control animals. TRPV4 appears to provide activator Ca2+ for endothelial large-conductance Ca2+-activation K+ channels in this setting. Taken together, this work suggests that changes in endothelial cell membrane cholesterol mediate this CH-induced increase in ion channel functionality.  Interestingly, both BK and TRPV4 channels contain Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs that bind cholesterol through protein-sterol interactions. It is essential to separate the effects of CH from the influence of EC membrane cholesterol changes related to hypoxic exposure. Thus, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelial-dependent vasodilation. To test this hypothesis, vasodilation responses to acetylcholine (ACh) were determined in gracilis arteries from male Sprague-Dawley rats using standard pressure myography. Rats were exposed to either ambient atmospheric (atm.) pressure or 48-hrs of hypoxia (0.5 atm). Arteries were pretreated with N-nitro-L-arginine and indomethacin.  EC membrane cholesterol was depleted using MβCD and supplemented with MβCD-cholesterol. In arteries from normoxic animals (a replete cholesterol state), inhibition of TRPV4 did not affect ACh-induced vasodilation. However, TRPV4 channels contribute to ACh-induced vasodilation in these arteries when EC membrane cholesterol is depleted with MβCD. In contrast, inhibition of TRPV4 channels attenuated ACh-induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion with MβCD-cholesterol in arteries from CH animals abolished the contribution of TRPV4 channels to ACh-induced vasodilation. In conclusion, our results demonstrate that EC membrane cholesterol impedes the contribution of TRPV4 channels in EDH-mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.

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