Regulation of alpha 1-adrenergic receptor-coupled calcium flux in cultured vascular smooth muscle cells.
Regulation of alpha 1-adrenergic receptor-coupled calcium flux in cultured vascular smooth muscle cells.
复制标题
培养血管平滑肌细胞中α1-肾上腺素受体耦合钙通量的调节。
DOI：
10.1161/01.hyp.6.2_pt_2.i19
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Alexander,RW
中科院分区：
文献类型：
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作者：
Colucci,WS;Brock,TA;GimbroneJr,MA;Alexander,RW
为了研究α-肾上腺素能受体(AAR)通路在儿茶酚胺诱导的血管收缩中的作用，我们建立了直接测定AAR及其偶联钙离子外流的方法。α1和α2选择性放射性配基[3 H]-哌唑嗪和[3 H]-育亨宾分别与AAR结合。用去甲肾上腺素刺激的45Ca外流作为AAR介导的钙流出的一个指标。[~3H]-哌唑嗪结合具有高亲和力(Kd=0.15 nM)、可饱和性(Bmax=75-125fmol/mg蛋白)，并可被激动剂和拮抗剂按α1受体的预期顺序竞争。[~3H]-育亨宾无特异性结合。去甲肾上腺素刺激的~(45)Ca外流呈浓度依赖性(EC_(50)为100 nM)，可被哌唑嗪(IC_(50)为0.1 nM)所阻断，但育亨宾(IC_(50)>100 nM)不能阻断。结合结合数据，表明去甲肾上腺素刺激的~(45)Ca外流是由α1介导的。1-去甲肾上腺素(0.1 nM)处理细胞24小时后，AAR密度和去甲肾上腺素刺激的45Ca最大流出量分别减少72%+/-14%和91%+/-9%。这种培养的细胞系统为研究血管平滑肌中α-肾上腺素能受体的偶联和调节机制提供了几个有利条件。
To study the role of the alpha-adrenergic receptor (AAR) pathway in the regulation of catecholamine-induced vascular contraction, we developed methods for the direct evaluation of AAR and AAR-coupled calcium efflux in cultured vascular smooth muscle cells derived from the rabbit aorta by enzymatic dissociation. AAR were characterized by the binding of the alpha 1- and alpha 2-selective radioligands [3H]-prazosin and [3H]-yohimbine, respectively. Norepinephrine-stimulated efflux of 45calcium from preloaded cells was measured as an index of AAR-mediated calcium flux. The [3H]-prazosin binding was of high affinity (Kd = 0.15 nM), saturable (Bmax = 75-125 fmol/mg protein), and competed for by agonists and antagonists in the order expected for an alpha 1 receptor. There was no specific binding of [3H]-yohimbine. Norepinephrine-stimulated 45Ca efflux was concentration-dependent (EC50 congruent to 100 nM), and potently blocked by prazosin (IC50 congruent to 0.1 nM), but not yohimbine (IC50 greater than 100 nM), indicating, together with the binding data, that norepinephrine-stimulated 45Ca efflux in this system is alpha 1 mediated. Following treatment of cells with 1-norepinephrine (0.1 nM) for 24 hours, the density of AAR and maximal norepinephrine-stimulated 45Ca efflux were decreased by 72% +/- 14% and 91% +/- 9%, respectively. This cultured cell system offers several advantages for study of the mechanisms of coupling and regulation of alpha-adrenergic receptors in vascular smooth muscle.