Dependence of Soluble Guanylyl Cyclase Activity on Calcium Signaling in Pituitary Cells

Silvana A Andric,Tatjana S Kostic,Melanija Tomić,Taka-Aki Koshimizu,Stanko S Stojilkovic

doi:10.1074/jbc.m004406200

Abstract

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca(2+) in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K(+) and Bay K 8644, an L-type Ca(2+) channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca(2+)-deficient medium. This inhibition was independent from the pattern of Ca(2+) signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca(2+). These results indicate that inactivation of sGC by intracellular Ca(2+) serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.

Highlights

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized
Depolarization-induced down-regulation of soluble guanylyl cyclase (sGC) activity was observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca2؉deficient medium
The Characterization of NO-derived Enzymes in Pituitary Cells—Western blot analysis confirmed the presence of detectable levels of neuronal NO synthase (nNOS) in pituitary tissue (Fig. 1A, line 2), cultured cells, and control tissue. endothelial NOS (eNOS) was detectable in pituitary tissue, dispersed pituitary cells, and control tissue. iNOS was not detectable in pituitary tissue and dispersed cells, but was expressed in pituitary cells stimulated with LPSϩIFN-␥ for 16 h

Summary

Introduction

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. Depolarization-induced down-regulation of sGC activity was observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca2؉deficient medium This inhibition was independent from the pattern of Ca2؉ signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca2؉. It is reasonable to propose that the negative feedback effect of Ca2ϩ on sGC would provide the necessary mechanism to coordinate the regulation of intracellular cGMP and Ca2ϩ concentrations To test this hypothesis, we chose rat anterior pituitary cells, the majority of which exhibit spontaneous [Ca2ϩ]i transients of high amplitude that are sufficient to trigger hormone secretion [10]. We further characterized the role of calcium in the control of sGC activity in intact pituitary cells

Methods

Results

Conclusion