The role of extracellular Ca²⁺ in the response of the hepatocyte to Ca²⁺-dependent hormones

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 orginial 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).