Relationship between inositol polyphosphate production and the increase of cytosolic free Ca²⁺ induced by vasopressin in isolated hepatocytes

Addition of vasopressin to rat hepatocytes prelabeled with myo-[2-³H]inositol resulted in a very rapid decrease [³H]phosphatidylinositol 4,5-bisphosphate (Ptd-Ins-4,5-P₂) which was paralleled by increases of up to 3-fold in the levels of [³H]inositol trisphosphate (Ins-P₃) and [³H]inositol bisphosphate (Ins-P₂). Increases of [³H]inositol phosphate (Ins-P) were not detected until about 5 min after hormone addition. These data indicate that the major pathway for hormone-induced lipid breakdown in liver is through a phosphodiesterase for PtdIns-4,5-P₂ and that decreases of phosphatidylinositol are a secondary result of increased PtdIns-4,5-P₂ resynthesis. Using the fluorescent Ca²⁺ indicator Quin 2, cytosolic free Ca²⁺ increased from 160 nM to about 400 nM after vasopressin addition to hepatocytes and preceded the conversion of phosphorylase b to a. Half-maximal and maximal increases of cytosolic free Ca²⁺ and phosphorylase a activity were observed at 0.2 and 1 nM vasopressin, respectively. The dose-response curve for the initial rate of cytosolic free Ca²⁺ increase was very similar to those obtained for the initial rates of Ins-P₃ production and PtdIns-4,5-P₂ breakdown. Pretreatment of hepatocytes with Li⁺ caused a 3-4-fold potentiation of vasopressin-induced elevations of Ins-P, Ins-P₂, and Ins-P₃, with half-maximal effects at 0.5, 1, and 5 mM, respectively. The calculated maximal concentrations of Ins-P₃ in cells treated with 20 nM vasopressin were 10 and 30 μM, respectively, without and with Li⁺. Lithium did not affect the initial rate of inositol polyphosphate production or Ca²⁺ mobilization. The increase of Ins-P₃ which correlated with peak cytosolic free Ca²⁺ elevation was about 0.6 μM. In a saponin-permeabilized hepatocyte preparation, Ins-P₃ (1 μM) caused Ca²⁺ release from a vesicular, ATP-dependent Ca²⁺ pool. The data presented here suggest that Ins-P₃ may be a second messenger for the mobilization of intracellular Ca²⁺ by hormones in liver.