Store-operated Ca(2+) inflow in Reuber hepatoma cells is inhibited by voltage-operated Ca(2+) channel antagonists and, in contrast to freshly isolated hepatocytes, does not require a pertussis toxin-sensitive trimeric GTP-binding protein.
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.
Auld
A
A
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, SA, Adelaide, Australia.
Chen
J
J
Brereton
H M
HM
Wang
Y J
YJ
Gregory
R B
RB
Barritt
G J
GJ
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005682:Gadolinium / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006873:Hydroquinones / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D037342:Pertussis Toxin; D013449:Sulfonamides / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002147:Calmodulin; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D007093:Imidazoles; D007211:Indoles; D013449:Sulfonamides; D010566:Virulence Factors, Bordetella; D020126:Brefeldin A; C019359:2,5-di-tert-butylhydroquinone; C031938:calmidazolium; D004533:Egtazic Acid; C017967:W 7; D019284:Thapsigargin; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D005682:Gadolinium; D037342:Pertussis Toxin; D020962:Heterotrimeric GTP-Binding Proteins; D000252:Calcium-Transporting ATPases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; C000543:cyclopiazonic acid