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(Hypertension. 2000;36:1093.)
© 2000 American Heart Association, Inc.

Colin Johnston - A Celebration

Capacity for Purinergic Control of Renin Promoter via P2Y₁₁ Receptor and cAMP Pathways

Louise van der Weyden; David J. Adams; Brian J. Morris

From the Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, The University of Sydney, Sydney, Australia.

Correspondence to Brian J. Morris, DSc, Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, Building F13, The University of Sydney, NSW 2006, Australia. E-mail: brianm{at}physiol.usyd.edu.au

Abstract—Renin secretion can be stimulated by ATP via purinergic P2Y receptors. ATP is a cotransmitter with norepinephrine and is released from the cytosol during cell damage. Such release could account for the de novo renin expression seen in the proximal tubule in renal disease and in myocardial infarct borders. Whereas most P2Y purinoceptor subtypes utilize phosphoinositide signal-transduction pathways, the effector mechanisms of the subtype P2Y₁₁ also involve increases in cAMP, a well-known renin secretagogue and stimulus to renin production. The present study tested the effect of ATP on human renin gene (REN) promoter activity and the role of P2Y₁₁. By means of reverse transcriptase–polymerase chain reaction, we found that renin-expressing Calu-6 cells express P2Y₁₁ mRNA. Expression was also detected in the brain, kidney, testis, muscle, liver, and spleen. We made a novel cell line (Calu-6/P2Y11) in which P2Y₁₁ cDNA, under the control of a strong promoter, was stably integrated into genomic DNA. These cells produced P2Y₁₁ mRNA during culture. Treatment of Calu-6/P2Y11 cells with 1 mmol/L ATP caused a 3-fold increase in renin mRNA and protein over 36 hours. Transient transfection of Calu-6/P2Y11 cells with constructs containing 896 bp of human REN 5'-flanking DNA linked to the luciferase reporter gene led to a 5.8±0.6-fold increase (mean±SEM) in reporter activity in response to ATP (P=0.0015). In contrast, UTP produced only a 1.4±0.1-fold increase (P=0.016). For ADP, it was 1.7±0.1-fold (P=0.011). The response profile was ATP>ADP>AMP=adenosine=0, consistent with a P2Y₁₁ effect. Mutation of the cAMP response element (CRE) located at –222 in the REN promoter DNA abolished the effect of ATP. Furthermore, ATP induced a rapid, time-dependent increase in the phosphorylation of CRE binding protein (CREB) and activating transcription factor-1. These data implicate a cAMP pathway in mediation of the P2Y₁₁ effect. In conclusion, we have made a novel cell line that overexpresses the P2Y₁₁ purinoceptor. Stimulation of these cells by ATP activates a cAMP signal-transduction pathway that phosphorylates CREB and stimulates renin promoter activity via the CRE at –222. The data raise the possibility of a contribution of ATP/P2Y₁₁ effects to sympathetic stimulation of renin, as well as to responses in renin seen after tissue damage, such as in kidney disease and myocardial infarction.

Key Words: renin gene • human • purinergic receptor P2Y₁₁ • ATP • Calu-6 cells • cAMP • cAMP response element • CREB • ATF-1

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