title: Actions of vasopressin and the Ca(2+)-ATPase inhibitor, thapsigargin, on Ca2+ signaling in hepatocytes.
abstract: When hepatocytes were loaded with fura-2 by incubation with the acetoxymethyl ester (fura-2/AM), addition of Mn2+ resulted in a rapid quench of a fraction of cellular fura-2 fluorescence. Addition of vasopressin caused a second, rapid quench of cellular fura-2, whereas the addition of thapsigargin had no effect. When hepatocytes were loaded by microinjection of fura-2 acid, addition of Mn2+ caused a slower, sustained rate of quench, and both vasopressin and thapsigargin increased this rate of quench. When Mn2+ was removed from the medium of fura-2/AM-loaded cells after preincubation with Mn2+, vasopressin still caused quench of cellular fura-2. In contrast, neither vasopressin nor thapsigargin increased fura-2 quench when Mn2+ was removed from fura-2-injected cells. When fura-2/AM-loaded cells were permeabilized with saponin, only a fraction of the cell-associated fura-2 was quenched by addition of Mn2+. A second fraction was then quenched by addition of inositol 1,4,5-trisphosphate. These results indicate that in hepatocytes loaded with the acetoxymethyl ester of fura-2, the increased quench of cellular fura-2 seen with phospholipase C-linked agonists is not due to effects of the agonist on Mn2+ entry across the plasma membrane, but rather is due to agonist activation of Mn2+ penetration into an intracellular organelle, presumably through inositol 1,4,5-trisphosphate-regulated channels. Thus, it appears that compartmentalization of fura-2 accounts for previously reported anomalies in Ca2+ signaling in hepatocytes, such as the apparent failure of Ca(2+)-ATPase inhibition to increase divalent cation entry, as well as the apparent ability of phospholipase C-linked agonists to stimulate efflux of Ca2+.
authors.affiliation: Calcium Regulation Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: M C
authors.initials: MC
authors.lastname: Glennon
authors.forename: G S
authors.initials: GS
authors.lastname: Bird
authors.forename: C Y
authors.initials: CY
authors.lastname: Kwan
authors.forename: J W
authors.initials: JW
authors.lastname: Putney
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D005453:Fluorescence; D016257:Fura-2; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014667:Vasopressins / Q000494:pharmacology*
chemical_list: D013729:Terpenes; D014667:Vasopressins; D008345:Manganese; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2