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

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 Ca-45(2+) 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, Ca-45(2+) efflux was slow and monoexponential with time. Addition of BK dramatically increased Ca-45(2+) efflux; 50-60 % of the Ca-45(2+) associated with the cell monolayer was released within 2 min after addition of bradykinin. Both the bradykinin-induced change in [Ca2+], and the stimulation of Ca-45(2+) was completely blocked by loading the cells with the Ca2+ chelator BAPTA. At a supermaximal concentration of bradykinin (50 nM). SKF 96365 (50 muM) 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 Ca-45(2+) efflux. Thapsigargin (200 nM) and BHQ (10 muM) produced an increase in Ca-45(2+) 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 Ca2+-influx pathway but is devoid of Ca2+ pumps, and by a section of ER that is rich in thapsigargin-sensitive Ca2+-pump units. Blockade of Ca2+ influx through the agonist-activated pathway by SKF 96365 increases Ca2+ release from internal stores, and suggests that a rise in [Ca2+]i within the restricted space as a direct result of influx inhibits either Ins(1,4,5)P3 binding or effect at the Ca2+-release channel. A model is proposed in which these structural features provide for vectorial Ca2+ flux through the cell.