Properties of the guanylate cyclase-guanosine 3':5'-monophosphate system of rat renal cortex. Activation of guanylate cyclase and calcium-independent modulation of tissue guanosine 3':5'-monophosphate by sodium azide.

The effects of sodium azide on guanylate cyclase activity of homogenates of rat renal cortex and on the guanosine 3':5'-monophosphate (cGMP) content of cortical slices were examined and compared to those of carbamylcholine and NaF. In complete Krebs-Ringer bicarbonate buffer containing 10 mM theophylline, tissue cGMP content was increased 5- to 6-fold by 0.05 mM carbamylcholine or 10 mM NaN3, and 3-fold by 10 mM NaF. Increases in cGMP were maximal in response to these concentrations of the agonists and occurred within 2 min. Exclusion of Ca2+ from the incubation media reduced basal cGMP by 50% in 20 min and abolished responses to carbamylcholine and NaF, while exclusion of Mg2+ was without effect. Analogous reductions in cGMP were observed in complete buffer containing 1 mM tetracaine, an agent which blocks movement of Ca2+ across and binding to biologic membranes. By contrast, exclusion of Ca2+ or addition of tetracaine did not alter relative cGMP responses to NaN3 (6-fold increase over basal), although levels were reduced in slices exposed to these buffers for 20 min. When slices were incubated without Ca2+ or with tetracaine for only 2 min prior to addition of agonists, basal cGMP did not decline. Under these conditions, both absolute and relative increases in cGMP in response to NaN3 were comparable to those of slices incubated throughout in complete buffer, while carbamylcholine and NaF effects on cGMP were abolished. NaN3 increased guanylate cyclase activity of whole homogenates (10- to 20-fold), and of the 100,000 X g soluble (20-fold) and particulate (4-fold) fractions of cortex. Prior incubation of slices with NaN3 in the presence or absence of Ca2+ or with Ca2+ plus tetracaine also markedly enhanced enzyme activity in homogenates and subcellular fractions subsequently prepared from these slices. In the presence of 3 mM excess MnCl2, NaN3 raised the apparent Km for MnGTP of soluble guanylate cyclase from 0.11 mM to 0.20 mM, and reduced enzyme dependence on Mn2+. Thus, when Mg2+ was employed as the sole divalent cation in the enzyme reaction mixture basal and NaN3-responsive activities were 7% and 30% of those seen with optimal concentrations of Mn2+, respectively. Under a variety of assay conditions where responses to NaN3 were readily detectable, alterations in guanylate cyclase activities could not be demonstrated in response to carbamylcholine or NaF. By contrast Ca2+ increased the guanylate cyclase activity 6- to 7-fold over basal under conditions of reduced Mn2+ (0.75 mM Mn2+/1 mM GTP). This latter effect of Ca2+ was shared by Mg2+ and not blocked by tetracaine. Carbamylcholine, NaF, Ca2+, and NaN3 all failed to alter cGMP phosphodiesterase activity in cortex. Thus, while carbamylcholine and NaF enhance renal cortical cGMP accumulation through actions which are dependent upon the presence of extracellular Ca2+, NaN3 stimulates cGMP generation in this tissue through an apparently distinct Ca2+-independent mechanism.