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

Abstract

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 mol/L of the cytosolic calcium chelator 5Ј5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,NЈ,NЈ-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Key Words: renin Ⅲ adenylyl cyclase Ⅲ calcium Ⅲ juxtaglomerular cell Ⅲ cAMP R enin is the rate-limiting enzymatic step in the formation of angiotensin (Ang); thus, control of renin secretion by the kidney is a critical element in regulating systemic blood pressure and renal function. The common element in all of the renin-stimulating pathways is the second messenger cAMP, 1 the product of adenylyl cyclase activity. 2 However, it is also well established that renin secretion by the JG cells, unlike almost all secretory cells, is inversely related to intracellular calcium concentration such that paradoxically elevated intracellular calcium is a potent inhibitor of renin release. [3][4][5] Increased calcium in the juxtaglomerular (JG) cells suppresses basal renin release and blunts stimulation of renin secretion. 6 -9 Decreased JG cell intracellular calcium increases basal renin secretion and amplifies stimulated renin levels. 3,6,9 -11 Although the influence of these 2 "second messenger" regulatory signals, cAMP and calcium, has been established for years, 1,5,6,10,12 the precise nature of their interactions is unresolved and an area of considerable interest and debate.There are at least 9 isoforms of adenylyl cyclase, 2 including 2 (types V and VI) that are inhibited by increased intracellular calcium. 13 Because renin release is inhibited by increased intracellular calcium and stimulated by cAMP, we hypothesized that reducing intracellular calcium stimulates renin release by activating a calcium-inhibitable adenylyl cyclase, types V and/or VI in JG cells, enhancing cAMP levels, and thereby stimulating renin release. To test this hypothesis, we used primary cultures of isolated mouse JG cells, which exhibit the classic phenotypic character of the JG cell but are unencumbered by the many extracellular signaling pathways that can influence renin secretion in vivo or in less homogeneous in vitro preparations. Our results provide a unique answer to this longstanding question of how these 2 classic second messengers interact to control the release of renin from the JG cell.

Adenylyl Cyclase Isoform V Mediates Renin Release From Juxtaglomerular Cells

“…Although we observed a provocative colocalization of our antibody with renin on what appeared to be granule-like foci within the cytoplasm, 6 we were unable to distinguish between the 2 isoforms to answer the critical question of which calciuminhibitable isoform of adenylyl cyclase is in the JG cell and mediates this stimulation of renin secretion. Although either or both isoforms might exist within the JG cell, based on reports in some other cell types that AC-VI seems to localize in the cell membrane 11 contrary to the cytoplasmic localization we observed, 6 we hypothesized that it would be the AC-V isoform in the JG cells, and this isoform would be the enzymatic mediator of cAMP stimulation of renin secretion in response to decreased [Ca ϩ2 ] i .…”

“…Although we observed a provocative colocalization of our antibody with renin on what appeared to be granule-like foci within the cytoplasm, 6 we were unable to distinguish between the 2 isoforms to answer the critical question of which calciuminhibitable isoform of adenylyl cyclase is in the JG cell and…”

“…C57/BL6 mice were given free access to food and water and were euthanized by cervical dislocation to avoid adverse effects of anesthesia on the harvested cells. Using a sterile technique, the kidneys were removed, and the renal cortex was dissected, minced, and then incubated in a digestion buffer 6 containing 0.25% trypsin (activity: 15 500 U/mg; Sigma-Aldrich) and 0.1% collagenase (activity: 0.17 U/mg, type A; Roche) at 37°C for 75 minutes. The tissue was passed through 74-m and then 22-m sieves and then resuspended in 1-mL HEPES buffer.…”

“…[3][4][5] The question of how [Ca ϩ2 ] i and adenylyl cyclase might act or interact to regulate renin has been a longstanding subject of debate and investigation. 1,5 Recently, using a preparation of primary cultures of isolated mouse JG cells that seem to completely maintain their phenotype and release renin, 6 we found that a calcium-inhibitable form of adenylyl cyclase existed within the JG cell, suggesting that changes in [Ca ϩ2 ] i regulate adenylyl cyclase activity, and this could account for this paradoxical relationship between [Ca ϩ2 ] i and renin. We found that decreased [Ca ϩ2 ] i leads to an increase in cAMP and, subsequently, renin release from isolated JG cells.…”

“…Previously, 6 using an antibody that does not discriminate between the 2 isoforms, we found that either 1 or both of these calcium-inhibitable adenylyl cyclases existed within the JG cell, and their presence could explain the paradoxical effect of how decreasing [Ca ϩ2 ] i could result in a cAMPmediated stimulation of renin secretion. Although we observed a provocative colocalization of our antibody with renin on what appeared to be granule-like foci within the cytoplasm, 6 we were unable to distinguish between the 2 isoforms to answer the critical question of which calciuminhibitable isoform of adenylyl cyclase is in the JG cell and mediates this stimulation of renin secretion.…”