Chotani Maqsood A, Mitra Srabani, Eid Ali H, Han Seon A, Flavahan Nicholas A
Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA.
Am J Physiol Heart Circ Physiol. 2005 Jan;288(1):H69-76. doi: 10.1152/ajpheart.01223.2003. Epub 2004 Sep 2.
The physiological role of alpha(2)-adrenoceptors (alpha(2)-ARs) in cutaneous, arteriolar, vascular smooth muscle cells (VSMs) is to mediate cold-induced constriction. In VSMs cultured from human cutaneous arterioles, there is a selective increase in alpha(2C)-AR expression after serum stimulation. In the present study, we examined the cellular mechanisms contributing to this response. Serum induction of alpha(2C)-ARs was paralleled by increased expression of cyclooxygenase-2 (COX-2), increased release of prostaglandins, and increased intracellular concentration of cAMP. Inhibition of COX-2 by acetyl salicylic acid (1 mM), NS-398 (5 microM), or celecoxib (3 microM) abolished the increase in cAMP and markedly reduced alpha(2C)-AR induction in response to serum stimulation. The cAMP agonists, forskolin (10 microM), isoproterenol (10 microM), and cholera toxin (0.1 microg/ml) each dramatically increased expression of alpha(2C)-ARs in human cutaneous VSMs. The A-kinase inhibitor H-89 (2 microM) inhibited phosphorylation of cAMP response element binding protein, but not the increase in alpha(2C)-AR expression in response to these agonists. cAMP-dependent but A-kinase independent signaling can involve activation of guanine nucleotide exchange factors for the GTP-binding protein, Rap. Indeed, pull-down assays demonstrated Rap1 activation by serum and forskolin in VSM. Transient transfections using alpha(2C)-AR promoter-luciferase reporter construct demonstrated that Rap1 increased reporter activity, whereas the A-kinase catalytic subunit decreased reporter activity. These results indicate that cAMP signaling can have dual effects in cutaneous VSMs:activation of alpha(2C)-AR transcription mediated by Rap1 GTPase and suppression mediated by A-kinase. The former effect predominates in serum-stimulated VSMs leading to a COX-2, cAMP, and Rap 1-dependent increase in alpha(2C)-AR expression. Such increased expression of alpha(2C)-ARs may contribute to enhanced cold-induced vasoconstriction and Raynaud's phenomenon.
α₂ - 肾上腺素能受体（α₂ - ARs）在皮肤、小动脉血管平滑肌细胞（VSMs）中的生理作用是介导冷诱导的血管收缩。在从人皮肤小动脉培养的VSMs中，血清刺激后α₂C - AR的表达有选择性增加。在本研究中，我们研究了促成这种反应的细胞机制。血清诱导α₂C - ARs的同时伴随着环氧合酶 - 2（COX - 2）表达增加、前列腺素释放增加以及细胞内cAMP浓度升高。乙酰水杨酸（1 mM）、NS - 398（5 μM）或塞来昔布（3 μM）抑制COX - 2可消除cAMP的增加，并显著降低血清刺激后α₂C - AR的诱导。cAMP激动剂福斯可林（10 μM）、异丙肾上腺素（10 μM）和霍乱毒素（0.1 μg/ml）均可显著增加人皮肤VSMs中α₂C - ARs的表达。蛋白激酶A抑制剂H - 89（2 μM）抑制了cAMP反应元件结合蛋白的磷酸化，但不抑制这些激动剂诱导的α₂C - AR表达增加。cAMP依赖性但蛋白激酶A非依赖性信号传导可能涉及对GTP结合蛋白Rap的鸟嘌呤核苷酸交换因子的激活。事实上，下拉试验证明血清和福斯可林可在VSM中激活Rap1。使用α₂C - AR启动子 - 荧光素酶报告构建体的瞬时转染表明，Rap1增加报告基因活性，而蛋白激酶A催化亚基降低报告基因活性。这些结果表明，cAMP信号传导在皮肤VSMs中可产生双重作用：由Rap1 GTP酶介导的α₂C - AR转录激活和由蛋白激酶A介导的抑制作用。前一种作用在血清刺激的VSMs中占主导，导致α₂C - AR表达呈COX - 2、cAMP和Rap 1依赖性增加。这种α₂C - ARs表达的增加可能有助于增强冷诱导的血管收缩和雷诺现象。
