Heldwein K A, Redick D L, Rittenberg M B, Claycomb W C, Stenzel-Poore M P
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USA.
Endocrinology. 1996 Sep;137(9):3631-9. doi: 10.1210/endo.137.9.8756527.
CRH is the principal mediator of the stress response in mammals. In addition to pituitary and central nervous system effects, peripheral effects of CRH have been observed involving the immune and cardiovascular systems. Two CRH receptor subtypes, CRH-R1 and CRH-R2, have been cloned and show significant amino acid homology (69%), but differ in their tissue distribution. CRH-R1 is expressed predominantly in the brain and pituitary, whereas the CRH-R2 subtype is highly expressed in heart and skeletal muscle. To investigate the role of CRH in cardiac signaling, we analyzed the effect of CRH on freshly isolated neonatal rat cardiomyocytes and murine atrial cardiomyocyte tumor cells, AT-1, which express CRH-R2 messenger RNA. We show that stimulation of these cells with CRH and the CRH-related peptides, sauvagine from frog and urotensin I from fish, elicits large increases in the intracellular level of cAMP. This stimulation is transient, reaching a maximum in 5-15 min in neonatal cardiomyocytes and in 2-4 min in AT-1 cells, followed by a rapid decline. We show that stimulation of AT-1 cells by these peptides is specific for CRH receptors, as the CRH antagonist, alpha-helical CRH-(9-41) inhibits cAMP increases. Furthermore, we show that CRH, sauvagine, and urotensin I stimulations are dose dependent in both neonatal cardiomyocytes and AT-1 cells. Sauvagine and urotensin I are more potent than CRH at stimulating an increase in intracellular cAMP in neonatal cardiomyocytes (EC50 = 1.74, 2.61, 6.42 nM, respectively) and AT-1 cells (EC50 = 16.2, 15.8, and 149 nM, respectively). This rank order is consistent with that previously demonstrated in CRH-R2-transfected HEK293 cells and parallels the in vivo vasodilatory activity of these peptides. In summary, this is the first evidence that CRH, sauvagine, and urotensin I act directly on cardiac myocytes to stimulate increases in intracellular cAMP, presumably through CRH-R2. In addition, these results indicate that cardiac myocytes may be an informative in vitro model to investigate the effects of CRH and its role in the cardiovascular response to stress.
促肾上腺皮质激素释放激素（CRH）是哺乳动物应激反应的主要介质。除了对垂体和中枢神经系统有作用外，还观察到CRH的外周作用涉及免疫系统和心血管系统。已克隆出两种CRH受体亚型，即CRH-R1和CRH-R2，它们显示出显著的氨基酸同源性（69%），但组织分布不同。CRH-R1主要在脑和垂体中表达，而CRH-R2亚型在心脏和骨骼肌中高度表达。为了研究CRH在心脏信号传导中的作用，我们分析了CRH对新鲜分离的新生大鼠心肌细胞和表达CRH-R2信使核糖核酸的小鼠心房心肌肿瘤细胞AT-1的影响。我们发现，用CRH以及与CRH相关的肽（青蛙的 sauvagine和鱼的尾加压素I）刺激这些细胞，会使细胞内cAMP水平大幅升高。这种刺激是短暂的，在新生心肌细胞中5 - 15分钟达到峰值，在AT-1细胞中2 - 4分钟达到峰值，随后迅速下降。我们发现这些肽对AT-1细胞的刺激对CRH受体具有特异性，因为CRH拮抗剂α-螺旋CRH-(9 - 41)可抑制cAMP的升高。此外，我们发现CRH、sauvagine和尾加压素I的刺激在新生心肌细胞和AT-1细胞中均呈剂量依赖性。在刺激新生心肌细胞（EC50分别为1.74、2.61、6.42 nM）和AT-1细胞（EC50分别为16.2、15.8和149 nM）内cAMP升高方面，sauvagine和尾加压素I比CRH更有效。这种排序与先前在转染了CRH-R2的HEK293细胞中所证明的一致，并且与这些肽在体内的血管舒张活性相似。总之，这是首个证据表明CRH、sauvagine和尾加压素I直接作用于心肌细胞以刺激细胞内cAMP升高，推测是通过CRH-R2。此外，这些结果表明心肌细胞可能是研究CRH的作用及其在心血管应激反应中的作用的一个有用的体外模型。
