title: Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes.
abstract: Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.
authors.affiliation: Institute of General Pathology, University of Padova, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Dell'Antone
mesh_terms: D000165:Acridine Orange / Q000378:metabolism; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002478:Cells, Cultured; D006859:Hydrogen / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009550:Nigericin / Q000494:pharmacology; D011522:Protons; D051381:Rats; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism*
chemical_list: D011522:Protons; D000001:Calcimycin; D006859:Hydrogen; D000165:Acridine Orange; D009550:Nigericin; D000645:Ammonium Sulfate