title: Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria.
abstract: The effects of membrane potential on resting and bradykinin-stimulated changes in [Ca2+]i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic [Ca2+]i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient [Ca2+]i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in [Ca2+]i was determined by varying [K+]o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular [K+] (52 mV change/10-fold change in [K+]o). The observed decrease in resting and bradykinin-stimulated changes in [Ca2+]i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in [Ca2+]i in confluent endothelial cell monolayers. These [Ca2+]i oscillations between the plateau and resting [Ca2+]i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying [Ca2+]i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.
authors.affiliation: Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
authors.forename: R E
authors.initials: RE
authors.lastname: Laskey
authors.forename: D J
authors.initials: DJ
authors.lastname: Adams
authors.forename: A
authors.initials: A
authors.lastname: Johns
authors.forename: G M
authors.initials: GM
authors.lastname: Rubanyi
authors.forename: C
authors.initials: C
authors.lastname: van Breemen
mesh_terms: D000818:Animals; D001572:Benzofurans; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005456:Fluorescent Dyes; D016257:Fura-2; D006325:Heart Atria; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001572:Benzofurans; D005456:Fluorescent Dyes; D009569:Nitric Oxide; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2