Cyclic GMP induces oscillatory calcium signals in rat hepatocytes.
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+]i 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.
Rooney
T A
TA
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Joseph
S K
SK
Queen
C
C
Thomas
A P
AP
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D006152:Cyclic GMP / Q000502:physiology*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000378:metabolism*; D008297:Male; D008345:Manganese / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010507:Periodicity; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D008345:Manganese; D017868:Cyclic AMP-Dependent Protein Kinases; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D002118:Calcium