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

Abstract

The 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 Ca(2+) inflow (assayed using intracellular fluo-3 and a Ca(2+) add-back protocol). No stimulation of Mn(2+) inflow by thapsigargin was detected. Thapsigargin-stimulated Ca(2+) inflow was inhibited by Gd(3+) (maximal inhibition at 2 microM Gd(3+)), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca(2+) channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca(2+) inflow and release of Ca(2+) from intracellular stores. No inhibition of either Ca(2+) inflow or Ca(2+) release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca(2+) 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 Ca(2+) 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 G(i2(alpha)) with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of G(i2(alpha)) and substantially less total G(i2(alpha)) than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca(2+) 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 Ca(2+) channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require G(i2(alpha)) for activation. Possible explanations for differences in the requirement for G(i2(alpha)) in the activation of Ca(2+) inflow are briefly discussed.

Plasma Membrane Ca2+ Release-Activated Ca2+ Channels With A High Selectivity for Ca2+ Identified by Patch–Clamp Recording in Rat Liver Cells

“…[7][8][9][10] Moreover, other studies with H4-IIE cells have shown that Ca 2ϩ inflow through SOCs (measured using the fluorescent Ca 2ϩ sensor, fura-2) is almost completely inhibited by 2 mol/L Gd 3ϩ or 100 mol/L W7. 25 Taken together with the present results, which show that I SOC in H4-IIE cells can be completely inhibited by 2 mol/L La 3ϩ or 150 mol/L W7, these observations suggest that the Ca 2ϩ -specific SOCs characterized here play a major role in replenishing the Ca 2ϩ that is lost from intracellular stores during hormone-induced Ca 2ϩ signaling in H4-IIE cells. Because a current similar to I SOC has also been observed in rat hepatocytes (Rychkov G, Brereton HM, Barritt GJ, unpublished results, April, 2000), it is likely that rat hepatocytes also possess Ca 2ϩ -specific SOCs (CRAC-like channels) that replenish Ca 2ϩ lost from the endoplasmic reticulum during the generation of hormone-induced Ca 2ϩ signals.…”

“…H4-IIE cells (ATCC CRL 1548) were cultured at 37°C in 5% (vol/vol) CO 2 in air in Dulbecco's modified Eagle medium (DMEM) supplemented with penicillin (100 units/mL), streptomycin (100 g/mL), 10 mmol/L HEPES (pH 7.4), and 10% (vol/vol) fetal bovine serum (complete DMEM). [23][24][25] Flasks (25-cm 2 ) were seeded with approximately 0.5 ϫ 10 6 cells. The incubation medium was changed every 2 days, and cultures were confluent in 1 week.…”

Calcium Influx Factor from Cytochrome P-450 Metabolism and Secretion-like Coupling Mechanisms for Capacitative Calcium Entry in Corneal Endothelial Cells

“…BFA was reported to inhibit CCE in hepatocytes (66), platelets (59), and HEK293 cells (67). But it did not affect CCE in Xenopus oocytes (20) and Reuber hepatoma cells (68). In corneal endothelial cells, treatment with BFA (100 M) for 12 h inhibited CCE in both vehicle-and BN-treated cells (Fig.…”

Role of Calcium and Calcium-activated Proteases in CYP2E1-dependent Toxicity in HEPG2 Cells

“…Nifedipine, verapamil, and diltiazem (inhibitors of L-type calcium channels in excitable cells and store-operated channels at higher concentration in liver cells) (34) did not prevent the toxicity. SKF 96365 (an inhibitor of receptor-operated calcium channels) and benzamil (an inhibitor of Na ϩ /Ca 2ϩ exchange) (34) at 1 and 10 M were also not protective.…”