REGULATION OF ATRIAL NATRIURETIC PEPTIDE-STIMULATED CGMP PRODUCTION IN THE INNER MEDULLA

Studies were performed to examine the regulation of atrial natriuretic peptide- (ANP) stimulated guanylate cyclase in the the inner medulla. Primary cultures of rat inner medullary collecting tubular cells exposed to 10(-7) m ANP increased cGMP formation to 31.2 +/- 1.8 compared to the basal production of 2.1 +/- 0.6 fm/mu-g protein. This response did not appear to be transduced via a G(i) protein, as preincubation with pertussis toxin did not alter the response to 10(-7) m ANP, and saponized cells exposed to 10-mu-M GTP-gamma-S did not enhance the response to ANP (77.3 +/- 5.9 vs. 86.7 +/- 6.3 g/mu-g). Likewise, changes in extracellular Ca2+ from 0.5 to 3.0 mM, decrements in intracellular Ca2+ with EGTA or increments in intracellular Ca2+ with ionmycin 5-mu-M) did not significantly alter the response to ANP. Neither activation of protein kinase A with forskolin (36.5 +/- 5.1) nor of protein kinase C with s,n-1,2-dioctanolyglycerol (33.2 +/- 2.5) altered the response to 10(-7) M ANP (32.2 +/- 3.3, NS). As the inner medullary environment was hypertonic, the effect of altering tonicity was studied. Cells grown for 48 hours in hypertonic media (600 mOsm/kg H2O) displayed enhanced response to 10(-8) and 10(-7) M ANP when osmolality was raised by either Na+ alone or in combination with urea, but not by urea alone. Our studies demonstrate that ANP-stimulated guanylate cyclase is insensitive to alterations in either intra- or extracellular Ca2+, is not subject to inhibition by protein kinase, and does not involve a pertussis-sensitive G protein. The response is highly sensitive to elevations in tonicity, with Na+ as the mediator of this effect.