title: Store-operated Ca2+ entry and coupling to Ca2+ pool depletion in thapsigargin-resistant cells.
abstract: The release of Ca2+ from intracellular Ca2+ pumping pools and the entry of extracellular Ca2+ are tightly coupled events. The potent and specific intracellular Ca2+ pump inhibitor, thapsigargin, blocks Ca2+ accumulation and allows Ca2+ release from pools within mammalian cells, inducing major changes in endoplasmic reticulum function and cell growth. Recent studies characterized the pools of Ca2+ within permeabilized DC-3F/TG2 cells (a thapsigargin-resistant variant form of the DC-3F Chinese hamster lung fibroblast line, able to grow in 2 microM thapsigargin), revealing highly thapsigargin-resistant intracellular Ca2+ pumping activity capable of accumulating Ca2+ within an inositol 1,4,5-trisphosphate-releasable Ca2+ pool (Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961). Using intact fura-2-loaded thapsigargin-resistant DC-3F/TG2 cells, the present study investigated the role of this unusual Ca2+ pumping activity in maintaining cytosolic Ca2+, generating Ca2+ signals, and mediating Ca2+ entry. The thapsigargin-resistant Ca2+ pumping pool was capable of generating rapid cytosolic Ca2+ signals in response to the phospholipase C-coupled agonist, oleoyl lysophosphatidic acid. The resting level of cytosolic Ca2+ in DC-3F/TG2 cells was 2-fold elevated compared with control cells (the parent DC-3F line), and transient extracellular Ca2+ removal induced a large "overshoot" in cytosolic Ca2+. The overshoot response was blocked by the Ca2+ influx inhibitor, SKF96365, and was kinetically identical to that induced in parent DC-3F cells after thapsigargin-induced Ca2+ pool emptying, indicating that the thapsigargin-resistant DC-3F/TG2 cells had "constitutively" opened Ca2+ entry channels coupled to an emptied or partially emptied thapsigargin-sensitive Ca2+ pumping pool. Even though oleoyl lysophosphatidic acid-mediated Ca2+ release induced little Ca2+ entry, complete ionomycin-activated emptying of the thapsigargin-resistant Ca2+ pool in DC-3F/TG2 cells induced a large, sustained entry of Ca2+ that was also completely blocked by SKF96365. The results revealed that the thapsigargin-resistant Ca2+ pump does maintain physiological Ca2+ levels, is able to fill an agonist-responsive Ca2+ pool in DC-3F/TG2 cells, and is likely responsible for the ability of these cells to function and grow in the presence of thapsigargin. In addition, Ca2+ influx in the resistant DC-3F/TG2 cells reflects emptying of pools that accumulate Ca2+ by both thapsigargin-sensitive and -resistant Ca2+ pumps; since these pumps accumulate Ca2+ in distinct pools in parent DC-3F cells, it is possible that more than one pool is coupled to Ca2+ influx in the resistant DC-3F/TG2 cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Waldron
authors.forename: A D
authors.initials: AD
authors.lastname: Short
authors.forename: D L
authors.initials: DL
authors.lastname: Gill
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002451:Cell Compartmentation / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D004351:Drug Resistance; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007476:Ionophores; D008246:Lysophospholipids; D015759:Ionomycin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium