Activation of the renal cortical and hepatic guanylate cyclase-guanosine 3′,5′-monophosphate systems by nitrosoureas divalent cation requirements and relationship to thiol reactivity

Abstract

Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca 2+-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca 2+. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn 2+-dependent and decreased 85% when either Mg 2+ or Ca 2+ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn 2+ or Mg 2+, but not with Ca 2+, as sole divalent cation. Improved utilization of Mg 2+ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg 2+ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg 2+ or Mn 2+ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity. Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone. Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca 2+. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.