stilbenes 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

Regulation of intracellular pH (pHi) was studied in cultured bovine tracheal epithelial cells using microspectrofluorimetry of the fluorescent indicator 2',7'-biscarboxyethyl- 5(6)-carboxyfluorescein (BCECF). The cells, which were grown on coverslips and superfused in a chamber on the stage of a microscope, were acidified by NH4Cl-prepulses, and pHi recovery was measured (in DeltapH/min) at approximately pHi 6.7. In HCO3-free solutions the recovery rate was 0.14 pH/min, and addition of amiloride or Na-free solution reduced this rate to 0.02-0.03 pH/min. In HCO3/CO2-buffered Ringer's, the rate of recovery was 0.32 pH/min, and amiloride or Na-free reduced the rate to 0.08-0.10 pH/min. This residual Na-independent and HCO3-dependent pHi recovery was studied by using inhibitors of HCO3 and H transporters. Bafilomycin (inhibits H-ATPases) at 100 nM did not significantly affect pHi recovery, while 100 microM SCH28080 (inhibits H,K-ATPase) had a variable inhibitory effect (25-75%), indicating that a gastric-like H, K-ATPase, but not electrogenic H pump, may contribute in a minor way to the recovery from acidification. Cl-free solution and 500 microM H2DIDS (dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, blocks anion exchange and the outwardly rectifying Cl channel, ORCC), both blocked apparent anion exchange activity, but had no effect on the recovery; 100 microM DNDS (4-4''-dinitro-2-2'-stilbenedisulfonate blocks the ORCC but not the cystic fibrosis transmembrane conductance regulator, CFTR) had no effect on pHi recovery; DPC (diphenylamine carboxylate, blocks the CFTR and the ORCC) caused a complete and reversible inhibition of the recovery. When [K] was increased ten fold to depolarize the cell's membrane potential, the magnitude of the pHi recovery (though not the rate) was enhanced. Thus, the HCO3-dependent, Na- and Cl-independent, DPC-blockable pHi recovery may be largely due to an influx of HCO3 via CFTR Cl channels. Under physiological conditions, when the electrochemical gradient for HCO3 is likely to be outwardly rather than inwardly directed, the CFTR (or another HCO3-permeable channel) may mediate HCO3 secretion and contribute to regulation of pH of the periciliary fluid.

Topics: Amiloride; Animals; Antiporters; Bicarbonates; Cattle; Cells, Cultured; Chloride Channels; Chloride-Bicarbonate Antiporters; Culture Media; Diuretics; Epithelial Cells; Epithelium; Fluoresceins; Hydrogen-Ion Concentration; Sodium-Hydrogen Exchangers; Stilbenes; Trachea

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

In order to investigate the membrane-located mechanisms that control intracellular pH in resistance arteries, mesenteric vessels from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) at 5 weeks of age were mounted in a myograph and loaded with 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein (BCECF). Resting intracellular pH was studied over 10 min in the presence of amiloride (1 mmol/l), or 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 0.2 mmol/l). In the presence of DIDS, there was no significant difference in the resulting fall in intracellular pH over 10 min between rat strains. However, in the presence of amiloride there was a significantly greater fall in intracellular pH in SHR (P less than 0.001). These data indicate that in the resting state Na(+)-H+ exchange is increased in SHR resistance arteries at the time when blood pressure is rising and vascular remodelling is taking place.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypertension; In Vitro Techniques; Male; Mesenteric Arteries; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Stilbenes; Vascular Resistance