title: Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells.
abstract: Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: N R
authors.initials: NR
authors.lastname: Danthuluri
authors.forename: D
authors.initials: D
authors.lastname: Kim
authors.forename: T A
authors.initials: TA
authors.lastname: Brock
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008744:Methylamines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D010716:Phosphatidylinositols / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D008744:Methylamines; D010716:Phosphatidylinositols; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D000255:Adenosine Triphosphate; C027451:methylamine; D002118:Calcium