Angiotensin II and cAMP regulate AT 1-mRNA expression in rat cardiomyocytes by transcriptional mechanism

Abstract

Mechanisms of angiotensin II and cAMP regulating the expression of angiotensin II type 1 (AT 1) receptor mRNA were studied in neonatal rat cardiomyocytes. Angiotensin II induced a transient decrease of AT 1-mRNA expression in time- and dose-dependent manner. Maximal decrease (49.2±9.5% of control) occurred at 6 h of angiotensin II (10 nmol/l) treatment. AT 1 receptor antagonists 4-ethyl-2- n-propyl-1-[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-5-carboxylic acid (DMP811) and losartan as well as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) reversed the down-regulation of AT 1-mRNA expression. 6 h of phorbol 12-myristate 13-acetate (PMA) stimulation caused a decrease of AT 1-mRNA level. Treatment by angiotensin II plus actinomycin D for 6 h produced the same effect as actinomycin D alone. These results suggest that angiotensin II down-regulates AT 1-mRNA level of rat cardiomyocytes by inhibiting the transcription of AT 1 gene, which is mediated by AT 1 receptor and related to the activation of protein kinase C. Stimulation by forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) decreased the expression of AT 1-mRNA to 68.1±21.5% of control at 6 h treatment; while increased to 207.9±27.1% of control at 48 h treatment. A series of 5′-upstream deletion mutants of AT 1A promoter were produced and then were recombined with pGL 3 basic vector utilizing luciferase as reporter gene. Among all the constructors, p(−201/+74)Luc was of the highest luciferase activity (5.9 times higher than control) after stimulation by forskolin for 48 h. Further deletion from −201 to −61 resulted in a large decrease of activity. These results indicate that cAMP induces a time-dependent bi-directional regulation of AT 1-mRNA expression. The cAMP responsible element (CRE) cis-element located in the region −201/−61 of rat AT 1A promoter is forskolin inducible, which may mediate the up-regulation of AT 1-mRNA expression induced by cAMP long-lasting stimulation.