Kinetics of empty store-activated Ca2+ influx in HeLa cells.
The intracellular Ca2+ indicator Indo-1 was used to monitor changes in cytosolic [Ca2+] ([Ca2+]i) in single HeLa cells upon readmission of external Ca2+ after a short incubation in Ca(2+)-free solution. HeLa cells were responsive to histamine but not to caffeine, and their histamine-sensitive store was totally depleted by a 60-min exposure to 2 microM thapsigargin. The resting [Ca2+]i in thapsigargin-treated cells was higher than in control cells and low amplitude [Ca2+]i oscillations were observed in about 20% of the cells. Readmission of external Ca2+ after a brief withdrawal of extracellular Ca2+ resulted in a transient [Ca2+]i rise, which then decayed to the same elevated [Ca2+]i measured before the Ca2+ withdrawal period. The [Ca2+]i rise was associated with an increased rate of Mn2+ entry, measured as the rate of quenching of intracellular Fura-2. The same procedure did not affect the [Ca2+]i in control cells not pretreated with thapsigargin. The amplitude of this [Ca2+]i transient in thapsigargin pretreated cells depended on the duration of prior incubation in Ca(2+)-free medium. The [Ca2+]i rise induced by elevating the extracellular [Ca2+] from 1.5 to 10 mM was more pronounced if the [Ca2+]i during the initial incubation in 1.5 mM Ca2+ was first lowered by depolarizing the cells. We conclude that an empty store stimulates a Ca2+ entry pathway consisting of two components: a continuously elevated basal leak and a second component that is transient due to the high [Ca2+]i-induced inhibition of the Ca2+ entry pathway. This inhibition and the subsequent recovery from it as the [Ca2+]i is brought to resting levels could cause the oscillatory Ca2+ entry that we recorded in a fraction of the thapsigargin-treated cells.
Missiaen
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Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Belgium.
De Smedt
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H
Pary
J B
JB
Oike
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M
Casteels
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D001692:Biological Transport; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D013729:Terpenes; D002110:Caffeine; D019284:Thapsigargin; D006632:Histamine; D002118:Calcium