VECTORIAL CA2-SPACE TO THE ENDOPLASMIC-RETICULUM VIA A RESTRICTED CYTOPLASMIC COMPARTMENT REGULATES INOSITOL 1,4,5-TRISPHOSPHATE-STIMULATEDCA2+ RELEASE FROM INTERNAL STORES IN VASCULAR ENDOTHELIAL-CELLS( FLUXFROM THE EXTRACELLULAR)

Depletion of the Ins(1,4,5)P3-sensitive intracellular Ca2+ store of va scular endothelial cells after selective inhibition of the endoplasmic -reticulum (ER) Ca2+ pump by thapsigargin or 2,5-di-t-butylhydroquinon e (BHQ) increases Ca2+ influx from the extracellular space in the abse nce of phosphoinositide hydrolysis. One model to account for these res ults suggests a close association between the internal store and the p lasmalemma, allowing for the vectorial movement of Ca2+ from the extra cellular space to the ER. Furthermore, recent evidence suggests that I ns(1,4,5)P3-induced Ca2+ release from intracellular stores is regulate d by the free cytosolic Ca2+ concentration ([Ca2+]i). Thus agonist-ind uced Ca2+ entry may directly regulate Ca2+ release from internal store s. To test these hypotheses, we examined the effect of xyphenyl)propox y]-4-methoxyphenethyl}-1H-imidazole (SKF 96365), an inhibitor of Ca2influx, on unidirectional Ca-45(2+) efflux (i.e. retrograde radioisoto pe flux via the influx pathway) and on [Ca2+]i as measured by fura-2. Bradykinin produced a transient increase in [Ca2+]i, reflecting releas e 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-4 5(2+) efflux; 50-60 % of the Ca-45(2+) associated with the cell monola yer was released within 2 min after addition of bradykinin. Both the b radykinin-induced change in [Ca2+], and the stimulation of Ca-45(2+) w as completely blocked by loading the cells with the Ca2+ chelator BAPT A. At a supermaximal concentration of bradykinin (50 nM). SKF 96365 (5 0 muM) inhibited the rise in [Ca2+]i attributed to influx without affe cting release from internal stores. At a threshold concentration of br adykinin (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 BAPT A. These results suggest the existence of a restricted sub-plasmalemma l 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 inc reases Ca2+ release from internal stores, and suggests that a rise in [Ca2+]i within the restricted space as a direct result of influx inhib its 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 ve ctorial Ca2+ flux through the cell.