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. 1993 Oct 15;295(Pt 2):357–366. doi: 10.1042/bj2950357

Vectorial Ca2+ flux from the extracellular space to the endoplasmic reticulum via a restricted cytoplasmic compartment regulates inositol 1,4,5-trisphosphate-stimulated Ca2+ release from internal stores in vascular endothelial cells.

O A Cabello 1, W P Schilling 1
PMCID: PMC1134890  PMID: 8240234

Abstract

Depletion of the Ins(1,4,5)P3-sensitive intracellular Ca2+ store of vascular endothelial cells after selective inhibition of the endoplasmic-reticulum (ER) Ca2+ pump by thapsigargin or 2,5-di-t-butylhydroquinone (BHQ) increases Ca2+ influx from the extracellular space in the absence of phosphoinositide hydrolysis. One model to account for these results suggests a close association between the internal store and the plasmalemma, allowing for the vectorial movement of Ca2+ from the extracellular space to the ER. Furthermore, recent evidence suggests that Ins(1,4,5)P3-induced Ca2+ release from intracellular stores is regulated by the free cytosolic Ca2+ concentration ([Ca2+]i). Thus agonist-induced Ca2+ entry may directly regulate Ca2+ release from internal stores. To test these hypotheses, we examined the effect of 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF 96365), an inhibitor of Ca2+ influx, on unidirectional 45Ca2+ efflux (i.e. retrograde radioisotope flux via the influx pathway) and on [Ca2+]i as measured by fura-2. Bradykinin produced a transient increase in [Ca2+]i, reflecting release of Ca2+ from internal stores, and a sustained increase indicative of Ca2+ influx. In the absence of agonist, 45Ca2+ efflux was slow and monoexponential with time. Addition of BK dramatically increased 45Ca2+ efflux; 50-60% of the 45Ca2+ associated with the cell monolayer was released within 2 min after addition of bradykinin. Both the bradykinin-induced change in [Ca2+]i and the stimulation of 45Ca2+ efflux was completely blocked by loading the cells with the Ca2+ chelator BAPTA. At a supermaximal concentration of bradykinin (50 nM), SKF 96365 (50 microM) inhibited the rise in [Ca2+]i attributed to influx without affecting release from internal stores. At a threshold concentration of bradykinin (2 nM), SKF 96365 blocked influx, but stimulated Ca2+ release from internal stores, as indicated by increases in both the transient component of the fura-2 response and 45Ca2+ efflux. Thapsigargin (200 nM) and BHQ (10 microM) produced an increase in 45Ca2+ efflux that was completely blocked by SKF 96365 or by cytosolic loading with BAPTA. These results suggest the existence of a restricted sub-plasmalemmal space that is defined by an area of surface membrane which contains the Ca(2+)-influx pathway but is devoid of Ca2+ pumps, and by a section of ER that is rich in thapsigargin-sensitive Ca(2+)-pump units.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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