CGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle.

Abstract

Recent studies on the role of nitric oxide (NO) in gastrointestinal smooth muscle have raised the possibility that NO-stimulated cGMP could, in the absence of cGMP-dependent protein kinase (PKG) activity, act as a Ca(2+)-mobilizing messenger [K. S. Murthy, K.-M. Zhang, J.-G. f1p4 J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G660-G671, 1993]. This notion was examined in dispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) and with NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 microM), NO (1 microM), and VIP (1 microM) stimulated 45Ca2+ release (21 +/- 3 to 30 +/- 1% decrease in 45Ca2+ cell content); Ca2+ release stimulated by 8-BrcGMP was concentration dependent with an EC50 of 0.4 +/- 0.1 microM and a threshold of 10 nM. 8-BrcGMP and NO increased cytosolic free Ca2+ concentration ([Ca2+]i) and induced contraction; both responses were abolished after Ca2+ stores were depleted with thapsigargin. With VIP, which normally increases [Ca2+]i by stimulating Ca2+ influx, treatment with PKA and PKG inhibitors caused a further increase in [Ca2+]i that reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release nor contraction induced by cGMP and NO in permeabilized muscle cells was affected by heparin or ruthenium red. Ca2+ release induced by maximally effective concentrations of cGMP and inositol 1,4,5-trisphosphate (IP3) was additive, independent of which agent was applied first. We conclude that, in the absence of PKA and PKG activity, cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.