Regulation of nitric oxide-responsive recombinant soluble guanylyl cyclaseby calcium

Calcium (Ca2+) and cyclic GMP (cGMP) subserve antagonistic functions that a re reflected in their coordinated reciprocal regulation in physiological sy stems. However, molecular mechanisms by which Ca2+ regulates cGMP-dependent signaling remain incompletely defined. In this study, the inhibition of re combinant nitric oxide (NO)-stimulated soluble guanylyl cyclase (SGC) by Ca 2+ was demonstrated. The alpha- and beta-subunits of recombinant rat SGC we re heterologously coexpressed in HEK 293 cells which do not express NO synt hase, whose Ca2+-stimulated activity can confound the effects of that catio n on SGC. Ca2+ inhibited basal and NO-stimulated SGC in a concentration- an d guanine nucleotide-dependent fashion. This cation inhibited SGC in crude cell extracts and immunopurified preparations. Ca2+ lowered both the V-max and K-m of SGC via an uncompetitive mechanism through direct interaction wi th the enzyme. In intact HEK 293 cells, increases in the intracellular Ca2 concentration induced by ionomycin, a Ca2+ ionophore, and thapsigargin, wh ich releases intracellular stores of that cation, inhibited NO-stimulated i ntracellular cGMP accumulation. Similarly, carbachol-induced elevation of t he intracellular Ca2+ concentration inhibited NO-stimulated intracellular c GMP accumulation in HEK 293 cells. These data demonstrate that SGC behaves as a sensitive Ca2+ detector that may play a central role in coordinating t he reciprocal regulation of Ca2+- and cGMP-dependent signaling mechanisms.