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

K. Dreja, M. Voldstedlund, J. Vinten, J. Tranum-Jensen, P. Hellstrand, K. Swärd
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引用次数: 182

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-&bgr;-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to &agr;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 &mgr;mol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ 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-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not &agr;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.
胆固醇消耗破坏小窝和差异损害激动剂诱导的动脉收缩
目的:本研究评估富含胆固醇的膜区(包括小泡)在动脉收缩性调节中的作用。方法与结果:采用胆固醇受体甲基-环糊精处理内皮缺失大鼠尾动脉,观察其对力和Ca2+处理的影响。在低胆固醇制剂中,对1-肾上腺素能受体、膜去极化、肌球蛋白轻链磷酸酶的抑制以及用20 mmol/L NaF和60 mmol/L AlCl3的混合物激活G蛋白的力反应不受影响。相比之下,5-羟色胺(5-HT)、血管加压素和内皮素的反应降低了50%以上。响应5-HT的全球细胞内游离Ca2+浓度的上升被减弱,细胞水平上Ca2+波的产生也被减弱。通过电子显微镜,发现胆固醇消耗会破坏小泡。外源性胆固醇可以恢复5-羟色胺反应,也可以恢复小窝。Western blot结果显示,5-HT2A受体和小窝蛋白-1的水平不受胆固醇提取的影响。蔗糖梯度离心显示,在含有小窝蛋白-1的组分中,富集了5-HT2A受体,但没有富集&agr;1-肾上腺素能受体,提示前者定位于小窝。结论:这些结果表明,调节平滑肌收缩的信号通路的子集特别依赖于胆固醇。此外,5 -羟色胺能信号通路中的胆固醇依赖性步骤发生在通路的早期,并依赖于小泡的完整性。
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