Decreased Intracellular Calcium Stimulates Renin Release via Calcium-Inhibitable Adenylyl Cyclase

Abstract

Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 μmol/L of the cytosolic calcium chelator 5′5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04±0.92 pM/mL per milligram of protein, and it increased by125±33% ( P <0.01) with BAPTA-AM. Basal renin was 1.28±0.40 μg of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182±62% ( P <0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of ≈135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM–stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.