CORTICOTROPIN-RELEASING HORMONE-RECEPTOR EXPRESSION AND FUNCTIONAL COUPLING IN NEONATAL CARDIAC MYOCYTES AND AT-1 CELLS

CRH is the principal mediator of the stress response in mammals. In ad dition to pituitary and central nervous system effects, peripheral eff ects of CRH have been observed involving the immune and cardiovascular systems. Two CRH receptor subtypes, CRH-R1 and CRH-R2, have been clon ed and show significant amino acid homology (69%), but differ in their tissue distribution. CRH-R1 is expressed predominantly in the brain a nd pituitary, whereas the CRH-R2 subtype is highly expressed in heart and skeletal muscle. To investigate the role of CRH in cardiac signali ng, we analyzed the effect of CRH on freshly isolated neonatal rat car diomyocytes and murine atrial cardiomyocyte tumor cells AT-1, which ex press CRH-RB messenger RNA. We show that stimulation of these cells wi th CRH and the CRH-related peptides, sauvagine from frog and urotensin I from fish, elicits large increases in the intracellular level of cA MP. This stimulation is transient, reaching a maximum in 5-15 min in n eonatal cardiomyocytes and in 2-4 min in AT-1 cells, followed by a rap id decline. We show that stimulation of AT-1 cells by these peptides i s specific for CRH receptors, as the CRH antagonist, alpha-helical CRH -(9-41) inhibits cAMP increases. Furthermore, we show that CRH, sauvag ine,and urotensin I stimulations are dose dependent in both neonatal c ardiomyocytes and AT-1 cells. Sauvagine and urotensin I are more poten t than CRH at stimulating an increase in intracellular cAMP in neonata l cardiomyocytes (EC(50) = 1.74, 2.61, 6.42 mM, respectively) and AT-1 cells (EC(50) = 16.2, 15.8, and 149 mu M, respectively). This rank or der is consistent with that previously demonstrated in CRH-Ra-transfec ted HEX 293 cells and parallels the in vivo vasodilatory activity of t hese peptides. In summary, this is the first evidence that CRH, sauvag ine, and urotensin I act directly on cardiac myocytes to stimulate inc reases 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 card iovascular response to stress.