Cyclic GMP induces oscillatory calcium signals in rat hepatocytes

Abstract

The ability of guanosine-3',5'-cyclic monophosphate (cGMP) to induce increases in the intracellular free calcium ion concentration ([Ca2+](i)) was studied at the single cell level in fura-2-loaded rat hepatocytes. Both 8- bromo-cGMP (Br-cGMP) and dibutyryl cGMP (db-cGMP) produced oscillatory [Ca2+](i) increases in hepatocytes. In addition, Br-cGMP increased the frequency of agonist-induced spiking or converted [Ca2+](i) oscillations into sustained nonoscillatory [Ca2+](i) responses. Addition of the nitric oxide donor sodium nitroprusside also produced oscillatory [Ca2+](i) increases similar to those generated by cGMP analogues. In the absence of extracellular Ca2+, cGMP-induced [Ca2+ responses were significantly reduced and mainly appeared as single transient [Ca2+](i) increases. The effects of cGMP analogues do not appear to be mediated by a secondary increase in cAMP or activation of cAMP-dependent protein kinase (PKA), since [Ca2+](i) responses to cGMP analogues were inhibited by the G-kinase inhibitor 8-bromoguanosine- 3',5'-cyclic monophosphorothioate (Rp-Br-cGMP[S]). Both Br-cGMP and db-cGMP also increased [Ca2+](i) in the presence of the PKA inhibitor 8-bromoadenosine- 3',5'-cyclic monophosphorothioate (Rp-Br-cAMP[S]) and when the cGMP-inhibitable cAMP phosphodiesterase activity was inhibited by pretreatment with siguazodan. Br-cGMP stimulated the Mn2+induced quench of compartmentalized fura-2 in intact hepatocytes, indicating a site of action at the level of the Ca2+ stores. This locus was further supported by the finding that pretreatment of hepatocytes with Br-cGMP potentiated submaximal inositol 1,4,5-trisphosphate (InsP3)-induced Mn2+ quench in subsequently permeabilized hepatocytes. db- cGMP also decreased PKA-mediated back phosphorylation of the hepatic type-1 InsP3 receptor, indicating that G-kinase phosphorylates the InsP3 receptor at sites targeted by PKA. These data indicate that phosphorylation of the hepatic InsP3 receptor by G-kinase increases the sensitivity to InsP3 for [Ca2+](i) release and is associated with the production of [Ca2+](i) oscillations in single rat hepatocytes.