Role of caveolin-1 in endothelial BKCachannel regulation of vasoreactivity

Abstract

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca2+-activated K+(BKCa) channels is present following in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. We tested the hypothesis that CH removes an inhibitory effect of the scaffolding domain of caveolin-1 (Cav-1) on EC BKCachannels to permit activation, thereby affecting vasoreactivity. Experiments were performed on gracilis resistance arteries and ECs from control and CH-exposed (380 mmHg barometric pressure for 48 h) rats. EC membrane potential was hyperpolarized in arteries from CH-exposed rats and arteries treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD) compared with controls. Hyperpolarization was reversed by the BKCachannel antagonist iberiotoxin (IBTX) or by a scaffolding domain peptide of Cav-1 (AP-CAV). Patch-clamp experiments documented an IBTX-sensitive current in ECs from CH-exposed rats and in MBCD-treated cells that was not present in controls. This current was enhanced by the BKCachannel activator NS-1619 and blocked by AP-CAV or cholesterol supplementation. EC BKCachannels displayed similar unitary conductance but greater Ca2+sensitivity than BKCachannels from vascular smooth muscle. Immunofluorescence imaging demonstrated greater association of BKCaα-subunits with Cav-1 in control arteries than in arteries from CH-exposed rats, although fluorescence intensity for each protein did not differ between groups. Finally, AP-CAV restored myogenic and phenylephrine-induced constriction in arteries from CH-exposed rats without affecting controls. AP-CAV similarly restored diminished reactivity to phenylephrine in control arteries pretreated with MBCD. We conclude that CH unmasks EC BKCachannel activity by removing an inhibitory action of the Cav-1 scaffolding domain that may depend on cellular cholesterol levels.