Evidence obtained using single hepatocytes for inhibition by the phospholipase C inhibitor U73122 of store-operated Ca 2+ inflow

Abstract

The ability of 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipase C (Smith et al., J Pharmacol Exp Ther 253: 688–697, 1992), to inhibit agonist-stimulated and store-operated Ca 2+ inflow in single hepatocytes was investigated with the aim of testing whether the activation of phospholipase C is a necessary step in the process of agonist-stimulated Ca 2+ inflow in this cell type. U73122 inhibited the release of Ca 2+ from intracellular stores and plasma membrane Ca 2+ inflow induced by vasopressin. An inactive analogue of U73122, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidone-dione (U73433), did not inhibit vasopressin-induced release of Ca 2+ from intracellular stores, but did partially inhibit Ca 2+ inflow. Neither U73122 nor ‘inactive’ analogue U73433 inhibited the release of Ca 2+ from intracellular stores when this was initiated by the photolysis of ‘caged’ guanosine (5′-[γ-thio]triphosphate (GTPγS) introduced to the cytoplasmic space by microinjection. However, both compounds inhibited GTPγS-stimulated Ca 2+ inflow. U73122 also inhibited the actions of glycerophosphoryl- myo-inositol-4,5-diphosphate (GPIP 2), a slowly-hydrolysed analogue of inositol 1,4,5-triphosphate (InsP 3) which is released by photolysis of ‘caged’ 1-(α-glycerophosphoryl)- myo-inositol-4,5-diphosphate, P 4(5)-1-(2-nitrophenyl) ethyl ester, and thapsigargin in stimulating Ca 2+ inflow. U73122 did not inhibit GPIP 2-stimulated release of Ca 2+ from intracellular stores, but did partially inhibit the ability of thapsigargin to induce Ca 2+ release. It is concluded that, while U73122 does inhibit phospholipase C β in hepatocytes, complete inhibition of this enzyme in situ requires an intracellular concentration of U73122 higher than that achieved in the present experiments. Moreover, both U73122 and ‘inactive’ analogue U73433 have one or possibly two additional sites of action. These are likely to be the hepatocyte plasma membrane Ca 2+ inflow channel protein (or a protein involved in the activation of this channel by the InsP 3-sensitive intracellular Ca 2+ store), and a protein involved in thapsigargin action.