Caveolar disruption causes contraction of rat femoral arteries via reduced basal NO release and subsequent closure of BKCa channels

Abstract

Background and Purpose: Caveolae act as signalling hubs in endothelial and smooth muscle cells. Caveolar disruption by the membrane cholesterol depleting agent methyl-β-cyclodextrin (M-β-CD) has various functional effects on arteries including (i) impairment of endothelium-dependent relaxation, and (ii) alteration of smooth muscle cell (SMC) contraction independently of the endothelium. The aim of this study was to explore the effects of M-β-CD on rat femoral arteries. Methods: Isometric force was measured in rat femoral arteries stimulated to contract with a solution containing 20mMK+ and 200 nM Bay K 8644 (20 K/Bay K) or with one containing 80mMK+ (80 K). Results: Incubation of arteries with M-β-CD (5 mM, 60 min) increased force in response to 20 K/Bay K but not that induced by 80 K. Application of cholesterol saturated M-β-CD (Ch-MCD, 5 mM, 50 min) reversed the effects of M-β-CD. After mechanical removal of endothelial cells M-β-CD caused only a small enhancement of contractions to 20 K/Bay K. This result suggests M-β-CD acts via altering release of an endothelial-derived vasodilator or vasoconstrictor.When nitric oxide synthase was blocked by pre-incubation of arteries with L-NAME (250 μM) the contraction of arteries to 20 K/Bay K was enhanced, and this effect was abolished by pre-treatment with M-β-CD. This suggests M-β-CD is inhibiting endothelial NO release. Inhibition of large conductance voltage- and Ca2+-activated (BKCa) channels with 2 mM TEA+ or 100 nM Iberiotoxin (IbTX) enhanced 20 K/Bay K contractions. L-NAME attenuated the contractile effect of IbTX, as did endothelial removal. Conclusions: Our results suggest caveolar disruption results in decreased release of endothelial-derived nitric oxide in rat femoral artery, resulting in a reduced contribution of BKCa channels to the smooth muscle cell membrane potential, causing depolarisation and contraction.