Jackson W F, Boerman E M, Lange E J, Lundback S S, Cohen K D
Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824, USA.
Br J Pharmacol. 2008 Oct;155(4):514-24. doi: 10.1038/bjp.2008.276. Epub 2008 Jul 7.
alpha(1)-Adrenoceptor agonists induce Ca(2+)-transients in endothelial cells (ECs) of arterioles. However, the presence of alpha(1)-adrenoceptors on arteriolar ECs has not been excluded, and the identity of alpha(1)-adrenoceptor subtypes in arterioles only has been inferred from pharmacology. Therefore, we determined which subtypes were expressed by vascular smooth muscle cells (VSMCs) and ECs, and which subtype mediated alpha(1)-adrenoceptor-induced constriction.
EC Ca(2+)-transients in isolated, cannulated hamster cremasteric arterioles or freshly isolated ECs were studied using Fura 2. Arteriolar diameter was measured by video microscopy. alpha(1)-Adrenoceptor expression was assessed by western blot of whole-arteriolar homogenates and real-time RT-PCR on enzymatically isolated VSMCs and ECs.
Phenylephrine-induced constriction and EC Ca(2+)-transients were abolished by the alpha(1)-adrenoceptor antagonist prazosin (30 nM) in arterioles. Phenylephrine-induced constriction was inhibited by the alpha(1D)-adrenoceptor antagonist BMY 7378 (K(B)=2.96 nM) and the alpha(1A)-adrenoceptor antagonist 5-methylurapidil (K(B)=4.08 nM), suggesting a significant role for alpha(1D)-adrenoceptors. Western blots confirmed alpha(1D)-adrenoceptor expression, but did not detect alpha(1A)-adrenoceptors. VSMCs expressed alpha(1D)- and alpha(1A)-, but not alpha(1B)-, adrenoceptor transcripts. No alpha(1)-adrenoceptor transcripts were detected in ECs. Neither phenylephrine (10 microM) nor noradrenaline (0.1-1 microM) elicited Ca(2+)-transients in freshly isolated ECs, whereas the endothelium-dependent vasodilators methacholine (1 microM) and substance P (100 nM) consistently increased Ca(2+).
We reject the hypothesis that hamster cremasteric arteriolar ECs express alpha(1)-adrenoceptors and conclude that alpha(1)-adrenoceptor agonists predominantly act on VSMC alpha(1D)-adrenoceptors to cause vasoconstriction and a subsequent rise in EC Ca(2+).
α1肾上腺素受体激动剂可诱导微动脉内皮细胞（ECs）产生Ca2+瞬变。然而，微动脉ECs上α1肾上腺素受体的存在尚未被排除，微动脉中α1肾上腺素受体亚型的身份仅从药理学推断而来。因此，我们确定了血管平滑肌细胞（VSMCs）和ECs表达哪些亚型，以及哪种亚型介导α1肾上腺素受体诱导的收缩。
使用Fura 2研究分离并插管的仓鼠提睾肌微动脉或新鲜分离的ECs中的EC Ca2+瞬变。通过视频显微镜测量微动脉直径。通过全微动脉匀浆的蛋白质印迹法以及对酶解分离的VSMCs和ECs进行实时RT-PCR来评估α1肾上腺素受体的表达。
在微动脉中，α1肾上腺素受体拮抗剂哌唑嗪（30 nM）消除了去氧肾上腺素诱导的收缩和EC Ca2+瞬变。去氧肾上腺素诱导的收缩受到α1D肾上腺素受体拮抗剂BMY 7378（K B = 2.96 nM）和α1A肾上腺素受体拮抗剂5-甲基尿嘧啶（K B = 4.08 nM）的抑制，表明α1D肾上腺素受体起重要作用。蛋白质印迹证实了α1D肾上腺素受体的表达，但未检测到α1A肾上腺素受体。VSMCs表达α1D-和α1A-肾上腺素受体转录本，但不表达α1B-肾上腺素受体转录本。在新鲜分离的ECs中，去氧肾上腺素（10 μM）和去甲肾上腺素（0.1 - 1 μM）均未引发Ca2+瞬变，而内皮依赖性血管舒张剂乙酰甲胆碱（acholine，1 μM）和P物质（100 nM）持续增加Ca2+。
我们否定仓鼠提睾肌微动脉ECs表达α1肾上腺素受体这一假说，并得出结论：α1肾上腺素受体激动剂主要作用于VSMC的α1D肾上腺素受体，导致血管收缩并随后使EC Ca2+升高。
