4-4--dinitro-2-2--stilbenedisulfonic-acid and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K(+) efflux and Cl(-) loss. When human erythrocytes were suspended in a physiological NaCl solution (pH(o) = 7.4), the measured pH(i) was 7.19 + or - 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH(i) to 7.70 + or - 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl(-) content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H(+) influx with the measured unidirectional K(+) efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H(+) influx and a K(+) efflux in LIS solution of 108.2 + or - 20.4 mmol (l(cells) hr)(-1) and 98.7 + or - 19.3 mmol (l(cells) hr)(-1), respectively. For bovine and porcine erythrocytes, in LIS media, H(+) influx and K(+) efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K(+)(Na(+))/H(+) exchanger, inhibited the K(+) efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K(+)(Na(+))/H(+) exchanger in the human erythrocyte membrane.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anion Exchange Protein 1, Erythrocyte; Anions; Bumetanide; Cattle; Chlorides; Egtazic Acid; Erythrocyte Membrane; Extracellular Space; Female; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen; Hydrogen-Ion Concentration; Intracellular Fluid; Ion Channel Gating; Ion Transport; Male; Mitochondria; Niflumic Acid; Nigericin; Osmolar Concentration; Ouabain; Potassium; Quinacrine; Sodium; Sodium-Hydrogen Exchangers; Species Specificity; Stilbenes; Swine

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the substrate and inhibitor specificity of monocarboxylate transport into isolated rat heart cells. Further evidence was obtained for the presence of two lactate carriers present in heart cells (Wang et al., Biochem. J. 290: 249-258, 1993) both distinct from the recently cloned monocarboxylate transporter isoform 1 (MCT-1) found in many other cell types. Only one isoform was potently inhibited by alpha-cyano-4-hydroxycinnamate [CHC; inhibitor constant (Ki) 190 microM] and the stilbene disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki 79 microM) and 4,4'-dinitrostilbene-2,2'-disulfonate (Ki of cis- and trans-isomers 38 and 171 microM, respectively; neither isomer inhibits MCT-1). The second carrier had a Ki of approximately 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues, in rat heart cells these inhibitors are not effective at blocking lactate transport totally unless used at very high concentrations. Both carriers were inhibited by 3-isobutyl-1-methylxanthine (Ki 340 microM) and neither by 5-nitro-2-(3-phenylpropylamino)benzoate (a potent inhibitor of MCT-1). The overall Michaelis constant (Km) and maximum reaction rate (Vmax) for transport of a variety of substituted monocarboxylates (C2-C5) were determined, although it was not possible to elucidate the kinetic parameters of the two isoforms. Of physiological interest, the ketone bodies D-beta-hydroxybutyrate and acetoacetate had K(m) values of 10 and 5.4 mM, respectively. Vmax values were similar to those of L-lactate and pyruvate and indicate that transport could limit rates of utilization of ketone bodies. No stereoselectivity for L-over D-isomers of 2-chloro or 2-hydroxy acids was observed.

Topics: 1-Methyl-3-isobutylxanthine; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Carrier Proteins; Cell Separation; Cinnamates; Coumaric Acids; Fluoresceins; Fluorescent Dyes; Isomerism; Lactic Acid; Monocarboxylic Acid Transporters; Myocardium; Rats; Stilbenes; Substrate Specificity