| Abstract: | The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ ( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of Ca2+]i. |
