Abstract
The effects of extracellular Na+ on store-dependent Ca2+ influx were compared for transfected Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (CK1.4 cells) and vector-transfected control cells. Store-dependent Ca2+ influx was elicited by depletion of intracellular Ca2+ stores with ionomycin, thapsigargin, or extracellular ATP, a purinergic agonist. In each case, the rise in [Ca2+]i upon the addition of extracellular Ca2+ was reduced in CK1.4 cells compared with control cells at physiological [Na+]o. When Li+ or NMDG was substituted for Na+, the CK1.4 cells showed a greater rise in [Ca2+]i than control cells over the subsequent 3 min after the addition of Ca2+o. Under Na+-free conditions, SK&F 96365 (50 microM), a blocker of store-operated Ca2+ channels, nearly abolished the thapsigargin-induced rise in [Ca2+]i in the control cells but only partially inhibited this response in the CK1.4 cells. We conclude that in the CK1.4 cells, Ca2+ entry through store-operated channels was counteracted by Na+o-dependent Ca2+ efflux at physiological [Na+]o, whereas Ca2+ entry was enhanced through Na+i-dependent Ca2+ influx in the Na+-free medium. We examined the effects of thapsigargin on Ba2+ entry in the CK1.4 cells because Ba2+ is transported by the Na+-Ca2+ exchanger, but it enters these cells only poorly through store-operated channels, and it is not sequestered by intracellular organelles. Thapsigargin treatment stimulated Ba2+ influx in a Na+-free medium, consistent with an acceleration of Ba2+ entry through the Na+-Ca2+ exchanger. We conclude that organellar Ca2+ release induces a regulatory activation of Na+-Ca2+ exchange activity.
Highlights
Agents that promote the production of 1,4,5-inositol trisphosphate (InsP3)1 give rise to a biphasic increase in cytosolic Ca2ϩ
Ca2ϩ Efflux from CK1.4 Cells—The results presented above suggest that Naϩo-dependent Ca2ϩ efflux via the Naϩ-Ca2ϩ exchanger is responsible for the attenuation of Tg-induced Ca2ϩ entry in the presence of Naϩ
In the CK1.4 cells, physiological concentrations of extracellular Naϩ retard store refilling and attenuate Tg-induced Ca2ϩ entry compared with control cells
Summary
InsP3, inositol 1,4,5-trisphosphate; CHO, Chinese hamster ovary; NMDG, N-methyl-D-glucamine; PSS, physiological salts solution; SDCI, store-dependent Ca2ϩ influx; Tg, thapsigargin; Mops, 3-(N-morpholino)propanesulfonic acid. The Ca2ϩ influx pathway involves low conductance Ca2ϩ channels and is activated, through a poorly understood mechanism, by the loss of Ca2ϩ from the InsP3sensitive stores [1,2,3,4]. This process is designated as capacitative Ca2ϩ entry [3] or store-dependent Ca2ϩ influx (SDCI) [4]. Comparing the effects of Naϩ on Ca2ϩ mobilization between transfected and vectortransfected control cells provides a means of identifying functional roles of exchange activity in relation to other cellular mechanisms for intracellular Ca2ϩ handling.
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