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 20 mM K+ and 200 nM Bay K 8644 (20 K/Bay K) or with one containing 80 mM K+(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.
Highlights
Caveolae are flask shaped invaginations, 50–100 nm in diameter, present in the plasma membrane of endothelial cells (ECs) and smooth muscle cells (SMCs) (Cohen et al, 2004)
Cholesterol depletion by M-β-CD disrupts caveolae in smooth muscle and endothelial cells of rat femoral artery M-β-CD depletes membrane cholesterol, which is essential for caveolar stability (Zidovetzki & Levitan, 2007)
After treatment with M-β-CD, caveolae were seen less frequently, and those that were present showed an opened-out, cup-like profile. These images establish that treatment with M-β-CD disrupts caveolae of rat femoral ECs and SMCs
Summary
Caveolae are flask shaped invaginations, 50–100 nm in diameter, present in the plasma membrane of endothelial cells (ECs) and smooth muscle cells (SMCs) (Cohen et al, 2004). Caveolin-1 has a tonic inhibitory effect on endothelial NO synthase (eNOS) activity, and it is thought that on cholesterol-depletion caveolin-1 re-distributes from caveolar to non-caveolar membranes (Xu et al, 2008) This results in a reduction in eNOS activation in response to endothelium-dependent vasodilators, possibly due to disrupted organisation of signalling complexes normally located in caveolae (Xu et al, 2008). 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. 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
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