EVIDENCE OBTAINED USING SINGLE HEPATOCYTES FOR INHIBITION BY THE PHOSPHOLIPASE-C INHIBITOR U73122 OF STORE-OPERATED CA2+ INFLOW

The ability of 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] am ino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipas e C (Smith et al., J Pharmacol Exp Ther 253: 688-697, 1992), to inhibi t agonist-stimulated and store-operated Ca2+ inflow in single hepatocy tes was investigated with the aim of testing whether the activation of phospholipase C is a necessary step in the process of agonist-stimula ted Ca2+ inflow in this cell type. U73122 inhibited the release of Ca2 + from intracellular stores and plasma membrane Ca2+ inflow induced by vasopressin. An inactive analogue of U73122, 1-[6-[[17 10)-trien-17-y l]amino]hexyl]-2,5-pyrrolidone-dione (U73433), did not inhibit vasopre ssin-induced release of Ca2+ from intracellular stores, but did partia lly inhibit Ca2+ inflow. Neither U73122 nor 'inactive' analogue U73433 inhibited the release of Ca2+ from intracellular stores when this was initiated by the photolysis of 'caged' guanosine (5'-[gamma-thio]trip hosphate (GTP gamma S) introduced to the cytoplasmic space by microinj ection. However, both compounds inhibited GTP gamma S-stimulated Ca2inflow. U73122 also inhibited the actions of glycerophosphoryl-myo-ino sitol-4,5-diphosphate (GPIP(2)), a slowly-hydrolysed analogue of inosi tol 1,4,5-triphosphate (InsP(3)) which is released by photolysis of 'c aged' a-glycerophosphoryl)-myo-inositol-4,5-diphosphate, P-4(5)-1-(2-n itrophenyl)ethyl ester, and thapsigargin in stimulating Ca2+ inflow. U 73122 did not inhibit GPIP(2)-stimulated release of Ca2+ from intracel lular stores, but did partially inhibit the ability of thapsigargin to induce Ca2+ release. It is concluded that, while U73122 does inhibit phospholipase C-beta in hepatocytes, complete inhibition of this enzym e in situ requires an intracellular concentration of U73122 higher tha n that achieved in the present experiments. Moreover, both U73122 and 'inactive' analogue U73433 have one or possibly two additional sites o f action. These are likely to be the hepatocyte plasma membrane Ca2+ i nflow channel protein (or a protein involved in the activation of this channel by the InsP(3)-sensitive intracellular Ca2+ store), and a pro tein involved in thapsigargin action.