Cholesterol Depletion Disrupts Caveolae and Differentially Impairs Agonist-Induced Arterial Contraction

Abstract

Objective-This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results-Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-␤-cyclodextrin, and the effects on force and Ca 2ϩ handling were evaluated. In cholesterol-depleted preparations, the force responses to ␣ 1 -adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 mol/L AlCl 3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by Ͼ50%. The rise in global intracellular free Ca 2ϩ concentration in response to 5-HT was attenuated, as was the generation of Ca 2ϩ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT 2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT 2A receptors, but not ␣ 1 -adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. Conclusions-These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae. Key Words: smooth muscle Ⅲ caveolae Ⅲ 5-hydroxytryptamine Ⅲ endothelin Ⅲ intracellular calcium C ellular cholesterol, of which most (up to 90%) 1 resides in the plasma membrane, is crucial for normal membrane permeability and fluidity and also plays a role in cellular signaling, via several proposed mechanisms that fall into at least 4 categories. First, cellular cholesterol may influence gene transcription in the nucleus through sterol regulatory element binding proteins. 2 Second, the activity of membrane receptors, ion channels, and transporters may depend on the membrane fluidity, per se. 3 Third, membrane protein function may be regulated through specific cholesterol-protein interactions. 3,4 Fourth, cholesterol stabilizes the structure of caveolae and lipid rafts.Caveolae, which are 50-to 100-nm membrane invaginations that are abundant in vascular endothelium and smooth muscle cells, are defined by their characteristic morphology and contents of caveolin and cav-p60. 5,6 No definitive definition of rafts has appeared because they do not exhibit a characteristic structure, but the term is used for planar aggregations of specific lipids and proteins. Caveolae and lipid rafts are envisaged to serve as platforms for a dynamic association of signaling proteins and for the initiation or modulation of signaling. 5,7,8 Some agonists causing contraction of vascular smooth muscle act on receptors that are believed to be located in caveo...

Smooth muscle caveolae differentially regulate specific agonist induced bladder contractions

“…Restoration of membrane caveolae following cholesterol administration largely re-established normal contractile responses to these agonists, thus demonstrating that the functional e¡ects of cyclodextrin treatment are e¡ectively reversible and cholesterol-speci¢c. These caveolae mediated contractile responses are consistent with previous functional studies performed in the denudated rat caudal artery in which disruption of caveolae was found to impair serotonin-induced contractions [Dreja et al, 2002] and in cultured glioma cells in which Cav-1 knockdown diminished 5-HT 2A mediated calcium signaling [Bhatnagar et al, 2004]. Several G-protein-coupled receptors are thought to be regulated by caveolae in certain types of cells, Neurourology and Urodynamics DOI 10.1002/nau Fig.…”

“…A similar lack of e¡ect of cyclodextrin on KCl-induced contractions has been demonstrated in the aorta and tail artery [Dreja et al, 2002;Je et al, 2003]. In addition, our ultrastructure results provide evidence that, while the number of caveolae are markedly reduced by this experimental approach, the morphology and integrity of smooth muscle cells remained unaltered after cyclodextrin treatment.…”

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

“…We observed in this study that methyl-␤-cyclodextrin did not change smooth muscle contractile responses to phenylephrine, indicating that this ␣-adrenergic agonist does not require signaling molecules in cholesterol-rich domains to cause contraction. A similar observation was also reported in tail artery (Dreja et al, 2002).…”

“…Cyclodextrin, a water-soluble cyclic oligosaccharide formed of seven glucopyranose units able to accept one molecule of cholesterol in its hydrophobic core, is a membrane-impermeable molecule that depletes cellular cholesterol content through solubilization of the plasmalemmal cholesterol (Kilsdonk et al, 1995). This cholesterol-binding agent has been efficiently used as a pharmacological tool to study the role of caveolae in vascular reactivity (Dreja et al, 2002;Kaiser et al, 2002;Je et al, 2004). Indeed, in our experiments we observed by electron microscopy that endothelial caveolae were disassembled after exposure of aortic rings to methyl-␤-cyclodextrin.…”

Pathogenesis of pulmonary hypertension: a case for caveolin-1 and cell membrane integrity

“…Disruption of caveolin-1 has been shown to reduce myogenic tone and impair contractile responses to several agonists (3,28). In addition, caveolin-1 keeps proliferative pathways including extracellular signal-regulated kinases inactive in caveolae.…”