Hypotonicity-Induced Renin Exocytosis from Juxtaglomerular Cells Requires Aquaporin-1 and Cyclooxygenase-2

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C_m) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C_m significantly and enhanced outward current. Indomethacin, PLA₂ inhibition, and an antagonist of prostaglandin transport impaired the C_m and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2^{−/ −} and AQP1^{−/ −} mice, hypotonicity increased neither C_m nor outward current, but 0.1-μM PGE₂ increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C_m and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA₂/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.Juxtaglomerular (JG) granular cells in the terminal part of the renal afferent glomerular arterioles are the only cells in the organism that synthesize preprorenin, process it to active renin, and store active renin in mature secretory granules. The rate of renin granule exocytosis determines the level of activation of the renin-angiotensin-aldosterone system. Renin secretion from most^1,2 but not all^3,4 in vitro preparations displays a uniquely high sensitivity to changes in extracellular osmolality such that a moderate reduction in the extracellular osmolality leads to rapid increases in renin secretion. The sensing and transduction events for renin release in response to osmotic perturbations are not known. At the glomerular tuft, the extracellular osmolality may vary depending on sodium chloride (NaCl)transport rate by the adjacent macula densa and thick ascending limb cells, which are relatively water impermeable.^5,6 JG cell capacitance (C_m), an index of cell surface area, increased when extracellular osmolality was decreased by 5 to 10% at the single cell level.⁷ This observation shows exocytotic release of renin in response to decreases in extracellular osmolality.⁷ Introduction of a pipette solution with slightly increased osmolality to the cell cytoplasm is sufficient to initiate exocytosis of renin in JG cells.⁷ This indicates that cell swelling, and not granule swelling, is involved in the response and shows that sensing and transduction of the initial change in osmolality is not dependent on an extracellular receptor for the agent used (e.g., sucrose or mannitol), as shown recently to be the case for succinate.⁴ The estimated number of granules recruited for exocytosis by a hypotonic extracellular challenge corresponded closely to the number that fused after receptor-dependent activation of cAMP formation.⁷ The existing data predict an involvement of water fluxes across the JG cell membrane, but aquaporin water channels have not been demonstrated in JG cells. Marked cell swelling normally initiates a regulatory volume decrease response whereby the cell, through coordinated activation of ion and organic osmolyte efflux, regains cell volume.⁸ A distinct role for phospholipase A2 (PLA₂) and prostaglandin E2(PGE₂) EP2 receptors in swelling-induced activation of regulatory processes in single cells has been demonstrated.⁹ Prostaglandin E₂ and PGI₂ enhance outward current and renin secretion from JG cells.¹⁰ In the study presented here, we hypothesized that JG cells respond to a decrease in extracellular osmolality by water uptake, Cyclooxygenase (COX)-dependent prostaglandin formation and renin release. The hypotheses were tested using single JG cells subjected to whole-cell patch-clamp analysis and primary cultures enriched in JG cells from rats, wild-type mice, and mice with targeted deletions of COX-2 and aquaporin-1 (AQP1).