Properties and subcellular distribution of guanylate cyclase activity in rat renal medulla: correlation with tissue content of guanosine 3',5'-monophosphate

Abstract

The outer and inner medulla of rat kidney were examined and compared with those of the renal cortex. A gradation in steady-state cyclic[c]GMP levels was observed in incubated slices of these tissues (inner medulla > outer medulla > cortex). This correlated with the proportion of total guanylate cyclase [GC] activity in the 100,000 g particulate fraction of each tissue, but was discordant with the relative activities of GC (highest in cortex) and of cGMP-phosphodiesterase (lowest in cortex) in whole tissue homogenates. Soluble GC of cortex and inner medulla exhibited typical Michaelis-Menten kinetics with an apparent Km for MnGTP of 0.11 mM, while the particulate enzyme from inner medulla exhibited apparent positive cooperative behavior and a decreased dependence on Mn2+. The particulate enzyme played a key role in regulating cGMP levels in the intact cell where Mn2+ concentrations are low. The soluble and particulate enzymes from the inner medulla were further distinguished by their responses to several test agents. The soluble enzyme was activated by Ca2+, NaN3, NaNO2 and phenylhydrazine, whereas particulate activity was inhibited by Ca2+ and was unresponsive to the latter agents. In the presence of NaNO2, Mn2+ requirement of the soluble enzyme was reduced and equivalent to that of the particulate preparation. The relative responsiveness of the soluble enzyme to NaNO2 was potentiated when Mg2+ replaced Mn2+ as the sole divalent cation. These changes in metal requirements were involved in the action of NaNO2 to increase cGMP in intact kidney. Soluble GC of cortex was clearly more responsive to stimulation by NaN3, NaNO2 and phenylhydrazine than was soluble activity from either medullary tissue. The effectiveness of the agonists on soluble activity from outer and inner medulla also was distinguished. Regulation and properties of soluble GC and subcellular enzyme distribution are distinct in the 3 regions of the kidney.