Abstract 1133: Endothelium-Derived Hyperpolarizing Factor Signaling Is Critically Regulated By Caveolin-1 In Endothelial Cells.
In endothelial cells, caveolin-1 (cav-1), the structural protein of caveolae, acts as scaffolding protein to cluster lipids and signaling molecules within caveolae and in some instances, to regulate the activity of protein targeted to caveolae. Here, we specifically determined whether cav-1 modulates the signaling pathways responsible for the EDHF relaxation. Using contractility and electrophysiologic measurements in vessels from caveolin-1 knock-out mice (cav-1 KO) and their wild type littermate, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from cav-1 KO mice (n=3–7, P<0.01 versus Wild type) and of the smooth muscle cell membrane hyperpolarization evoked by acetylcholine (Wild type: from −51.8±2.0mV, n=6 to −60.3±0.9mV, n=4 versus cav-1 KO: from −49.9±1.3mV, n=11 to −51.7±1.6mV, n=6). The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, specifically a decreased activity of Ca2+ permeable TRPV4 cation channels that participate in the NO- and EDHF-mediated relaxation. Accordingly, pretreatment of endothelial cells with a cav-1-directed siRNA blocked the increase in fura-2 Ca2+ signal evoked by the TRPV4 specific activator, 4αPDD (a.u.:0.48±0.03. in cav-1-targeted siRNA treated cells vs 1.2±0.23 in control endothelial cells, P<0.05, n=6 each). Furthermore, the morphological characterisation of cav-1 KO arteries by electron microscopy showed a decreased expression of Connexins 40, 43 and 37 and an altered cell-cell communication via the gap-junctions, as assessed by a decreased transfer of calcein from the endothelium to the media in cav-1 KO arteries compared with Wild type. Finally, using cellular fractionation and isopycnic centrifugation on endothelial cells extracts, we demonstrated that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolae-enriched fragments of the plasma membrane. In conclusion, the expression of caveolin-1 is required for the EDHF-related relaxation by modulating membrane location and activity of the TRPV4 channels and connexins, both implicated at different steps in the EDHF-signaling pathway.