title: 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.
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)
authors.affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030.
authors.forename: O A
authors.initials: OA
authors.lastname: Cabello
authors.forename: W P
authors.initials: WP
authors.lastname: Schilling
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002417:Cattle; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
chemical_list: D006873:Hydroquinones; D007093:Imidazoles; D013729:Terpenes; C019359:2,5-di-tert-butylhydroquinone; D004533:Egtazic Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D001920:Bradykinin; D002118:Calcium