Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P-3 induces the ac tivation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P-3 recep tors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concen tration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caus ed near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2release. Similar results were obtained using another Ins(1,4,5)P-3-receptor agonist, 2-hydroxyethyl-alpha-D-glucopyranoside 2,3',4'-trisphosphate. Ant i-type-1 Ins(1,4,5)P-3-receptor monoclonal antibody 18A10 inhibited vasopre ssin-stimulated Ca2+ inflow but had no observable effect on vasopressin-ind uced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. How ever, it did not substantially affect Ca2+ inflow and ER Ca2+ release induc ed by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release indu ced by ionomycin or Ins(1,4,5)P3P4(5)-1-(2-nitrophenyl)ethyl eater ['caged' Ins(1,4,5)P-3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)ho mopiperazine: HCl (ML-9), an inhibitor of myosin light-chain kinase, also i nhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow an d ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-in duced Ins(1,4,5)P-3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P-3 requires the participation of a small region of the ER, which is distinguished from other regions of the E R by a different apparent affinity for Ins(1,4,5)P-3 analogues and is assoc iated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation o f SOCs.