An increase in the free intracellular calcium concentration promotes exocytosis in most secretory cells. In contrast, renin release from juxtaglomerular (JG) cells is suppressed by calcium. The further downstream signaling cascades of this so called "calcium paradoxon" of renin secretion have been incompletely defined. Because cAMP is the main intracellular stimulator of renin release, we hypothesized that calcium might exert its suppressive effects on renin secretion via the inhibition of the calcium-regulated adenylate cyclases AC5 and AC6. In primary cultures of JG cells, calcium-dependent inhibitors of renin release (angiotensin II, endothelin-1, thapsigargin) suppressed renin secretion, which was paralleled by decreases in intracellular cAMP levels [cAMP]. When [cAMP] was clamped by membrane permeable cAMP derivates, renin release was not suppressed by any of the calcium liberators. Additionally, both endothelin and thapsigargin suppressed cAMP levels and renin release in isoproterenol or forskolin-pretreated As4.1 cells, a renin-producing cell line that expresses AC5 and AC6. The calcium-dependent inhibition of intracellular cAMP levels and renin release was prevented by small interfering RNA-mediated knockdown of AC5 and/or AC6 expression, underlining the functional significance of these AC isoforms in renin-producing cells. Finally, in isolated perfused mouse kidneys, angiotensin II completely inhibited the stimulation of renin secretion induced by adenylate cyclase activation (isoproterenol) but not by membrane permeable cAMP analogs, supporting the conclusion that the suppressive effect of calcium liberators on renin release is mediated by inhibition of adenylate cyclase activity.

Abstract-The aim of this study was to investigate the role of cytosolic calcium for renin gene expression in juxtaglomerular cells. For this purpose, we used the immortalized juxtaglomerular mouse cell line As4.1. To increase cytosolic calcium concentration, we treated the cells with thapsigargin and cyclopiazonic acid, inhibitors of the endoplasmatic reticulum CaϪ ATPase. Thapsigargin and cyclopiazonic acid inhibited renin gene expression in a characteristic time and concentration-dependent manner. This effect was concentration-dependently blocked by BAPTA-AM, an intracellular Ca 2ϩ chelator. Pharmacological blocking of protein kinase C activity by calphostin, Gö6976, and Gö6983 did not change the effect of thapsigargin on renin gene expression. Experiments with renin1 C -promoter-reporter constructs revealed that thapsigargin inhibited renin gene transcription. Analysis of deletion constructs of the renin1 C promoter indicated that regulatory elements involved in the calcium-mediated inhibition of renin gene transcription are located in the enhancer region of the renin gene and that Ն3 transcription factor-binding sites are involved in this process. In addition, thapsigargin reduced the renin mRNA half-life from 10 hours (control conditions) to 4 hours. Knockdown studies with small interfering RNA directed to dynamin-1 mRNA revealed that dynamin-1 is likely to be involved in the calcium-mediated destabilization of renin mRNA. These data suggest that calcium inhibits renin gene expression in juxtaglomerular cells via a concerted action of inhibition of renin gene transcription and destabilization of renin mRNA. Key Words: calcium Ⅲ renin-angiotensin-aldosterone system Ⅲ thapsigargin Ⅲ mRNA stability T he renin-angiotensin-aldosterone system is a major regulatory system controlling extracellular fluid volume and blood pressure. The activity of the renin-angiotensin-aldosterone system is rate limited by the activity of the protease renin. 1 Renin itself is also regulated by a variety of factors. In the past few years, our knowledge has increased substantially about the regulation of renin gene expression at the cellular level. [2][3][4] For the transcription of the mouse renin gene, 2 critical regions, a proximal promoter region at Ϫ197 to Ϫ50 bp and an enhancer element at Ϫ2866 to Ϫ2625 bp, have been identified. 4 Also, the regulation of the renin mRNA stability is influenced by various factors, among these, interacting partner proteins like MINT, dynamin, or nucleolin have been found to be involved in the stabilization process of the renin mRNA. 5 The focus of our study was to investigate the role of calcium in the regulation of renin gene expression. This is of particular interest, because vasoactive peptides like angiotensin II or endothelin-1, which are known to be direct regulators of renin gene expression, activate the phospholipase C via a G protein-coupled receptor and, in turn, increase inositol triphosphate and diacylglycerol (DAG). 3 Whereas DAG is known to activate protein kinase C, inositol triphosp...

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