Store-operated Ca2+ inflow in Reuber hepatoma cells is inhibited by voltage-operated Ca2+ channel antagonists and, in contrast to freshly isolated hepatocytes, does not require a pertussis toxin-sensitive trimeric GTP-binding protein

AbstractThe treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca2+ inflow (assayed using intracellular fluo-3 and a Ca2+ add-back protocol). No stimulation of Mn2+ inflow by thapsigargin was detected. Thapsigargin-stimulated Ca2+ inflow was inhibited by Gd3+ (maximal inhibition at 2 μM Gd3+), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca2+ channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca2+ inflow and release of Ca2+ from intracellular stores. No inhibition of either Ca2+ inflow or Ca2+ release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca2+ inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca2+ inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of Gi2α with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of Gi2α and substantially less total Gi2α than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca2+ channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca2+ channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require Gi2α for activation. Possible explanations for differences in the requirement for Gi2α in the activation of Ca2+ inflow are briefly discussed