scholarly journals Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.

1994 ◽  
Vol 104 (5) ◽  
pp. 961-983 ◽  
Author(s):  
J C Freedman ◽  
T S Novak ◽  
J D Bisognano ◽  
P R Pratap
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Disulfonic Acid ◽  
Mean Cell Volume ◽  
Partial Inhibition ◽  
Human Red Blood Cells ◽  
Specific Effects ◽  
Voltage Curve ◽  
Current Voltage Curve ◽  
Cl Conductance

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.

Proton fluxes associated with the Ca pump in human red blood cells

1990 ◽  
Vol 258 (3) ◽  
pp. C552-C562 ◽  
Author(s):  
M. A. Milanick
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Disulfonic Acid ◽  
Surprising Result ◽  
Exchange System ◽  
Response Curves ◽  
Ca Pump ◽  
Human Red Blood Cells ◽  
Pump Activity ◽  
Ph Stat

Ca fluxes and H fluxes were measured in human red blood cells at 37 degrees C to characterize the effects of extracellular protons (Hout) on the Ca pump and to determine the stoichiometry of Ca-H exchange. A pH-stat technique was used to measure the rate of H influx, and 45Ca was used to determine the rate of Ca efflux. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was used to reduce proton permeability. A La-sensitive H influx was observed in Ca-loaded cells (Ca = 2 mmol/l packed cells) and was not observed in the cells loaded with vanadate as well as Ca. Similar results were obtained in Ca-loaded ghosts. The La dose-response curves for H influx and for Ca efflux were similar [50% inhibitory concentration (IC50) = approximately 5 microM] in intact red blood cells. The stoichiometry of the La-sensitive fluxes among different experiments ranged from 1.7 to 2.1 H/Ca when extracellular pH (pHout) = 6.3. Thus the Ca pump in intact red blood cells mediates Ca-2H exchange at pHout = 6.3. A 100-fold decrease in Hout [from pH 6.5 to 8.5; intracellular pH (pHin) approximately 7.4] only decreased Ca efflux 1.5- to 3-fold, hence Hout had little effect on the overall rate under the conditions studied. The small effect of Hout was a surprising result for a Ca-H exchange system, since one would have expected a steep dependence of Ca pump on Hout at Hout less than the Michaelis constant (Km). However, no La-sensitive H influx was observed when pHout = 8. On the basis of these data, it is suggested that the Ca pump also mediates Ca efflux uncoupled from H influx (Ca2+/phi H+). Ca efflux in the presence of 11 mM extracellular Ca (Caout) was one-fifth the value obtained in the absence of Caout at pHout = 8.5; this inhibition was reversed by increasing Hout (to pH 6.1). These results are consistent with a model in which 1) the Ca pump mediates Ca2+/2H+ exchange at high Hout; 2) the Ca pump mediates Ca2+/phi H+ exchange at low pHout; 3) the rates of the two processes are less than or equal to 4-fold different; 4) Caout inhibits pump activity at low Hout; and 5) Caout competes with Hout for binding.

scholarly journals Kinetics of residual chloride transport in human red blood cells after maximum covalent 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid binding.

1993 ◽  
Vol 101 (5) ◽  
pp. 715-732 ◽  
Author(s):  
P K Gasbjerg ◽  
J Funder ◽  
J Brahm
Keyword(s):  
Red Blood Cells ◽  
Lipid Bilayers ◽  
Blood Cells ◽  
Chloride Transport ◽  
Covalent Binding ◽  
Chloride Concentration ◽  
Disulfonic Acid ◽  
Human Red Blood Cells ◽  
O 19 ◽  
Kinetics Of

Irreversible inhibition, 99.8% of control values for chloride transport in human red blood cells, was obtained by well-established methods of maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The kinetics of the residual chloride transport (0.2%, 106 pmol.cm-2 x s-1) at 38 degrees C, pH 7.2) was studied by means of 36Cl- efflux. The outside apparent affinity, expressed by Ko1/2,c, was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Borders, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemistry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of the residual chloride flux depended on the extracellular chloride concentration, but was not purely competitive. The half-inhibition concentrations at [Cl(o)] = 150 mM in control cells (Ki,o) and covalently DIDS-treated cells (Ki,c) were: DIDS, Ki,c = 73 nM; DNDS, Ki,o = 6.3 microM, Ki,c = 22 microM; phloretin, Ki,o = 19 microM, Ki,c = 17 microM; salicylate, Ki,o = 4 mM, Ki,c = 8 mM; Killer III, Ki,o = 10 microM, Ki,c = 10 microM.

scholarly journals Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

1997 ◽  
Vol 109 (2) ◽  
pp. 201-216 ◽  
Author(s):  
Jeffrey C. Freedman ◽  
Terri S. Novak
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Constant Field ◽  
Cell Shrinkage ◽  
Experimental Conditions ◽  
Maximal Conductance ◽  
Voltage Gated ◽  
Human Red Blood Cells ◽  
Current Voltage ◽  
Cl Conductance

Current-voltage curves for DIDS-insensitive Cl− conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl− conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl− were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl− effluxes, induced by valinomycin or gramicidin at varied [K+]o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl− currents yields PCl = 0.13 h−1 with DIDS, compared to 0.49 h−1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl− current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.

Glycine transport by human red blood cells and ghosts: evidence for glycine anion and proton cotransport by band 3

1991 ◽  
Vol 261 (5) ◽  
pp. C814-C821 ◽  
Author(s):  
P. A. King ◽  
R. B. Gunn
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Band 3 ◽  
Kinetic Constants ◽  
Disulfonic Acid ◽  
Human Red Blood Cells ◽  
Proton Cotransport ◽  
Glycine Transport ◽  
Ph Range ◽  
Resealed Ghosts

Stilbene-sensitive glycine transport was investigated in human red blood cells and ghosts. We have found that this component of glycine transport was inhibited by the stilbene derivatives 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS); the apparent constant for inhibition by DNDS was 4 microM in the presence of 150 mM chloride. DNDS-sensitive glycine influx was modulated by pH such that as pH was increased from 5.9 to 9.2, transport increased from 2.5 to 140 mumol.kg Hb-1.h-1 at 37 degrees C and 100 microM glycine. The increased transport was correlated with an increase in the amount of glycine present as the anion over this pH range (0.03-40 microM glycine anion), but, in addition, pH had a direct effect on transport. Glycine influx was studied as a function of glycine anion concentration with anion varied by changing pH at a constant total glycine concentration and by changing total glycine at a constant pH. A comparison of these data demonstrated that the stilbene-sensitive glycine anion flux is stimulated by protons with half-maximal stimulation below pH 6.5 and suggests that the glycine anion and a proton are cotransported. Inorganic anions transported by band 3, including Cl, NO3, and SO4, inhibited glycine transport. Glycine flux into resealed ghosts was inhibited by Cl with an inhibition constant of 25 mM. The similarities between the kinetic constants for transport inhibition by Cl and DNDS and the kinetic constants for Cl and DNDS binding to band 3 suggest that the DNDS-sensitive glycine anion and proton cotransport is via band 3.

scholarly journals Proton (or hydroxide) fluxes and the biphasic osmotic response of human red blood cells.

1993 ◽  
Vol 102 (1) ◽  
pp. 99-123 ◽  
Author(s):  
J D Bisognano ◽  
J A Dix ◽  
P R Pratap ◽  
T S Novak ◽  
J C Freedman
Keyword(s):  
Membrane Potential ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Time Course ◽  
Rapid Development ◽  
Disulfonic Acid ◽  
Proton Efflux ◽  
Experimental Conditions ◽  
Human Red Blood Cells ◽  
Hydroxide Fluxes

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).

Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells

1990 ◽  
Vol 259 (4) ◽  
pp. C570-C576 ◽  
Author(s):  
J. O. Alda ◽  
R. Garay
Keyword(s):  
Red Blood Cells ◽  
Dissociation Constant ◽  
Anion Exchanger ◽  
Blood Cells ◽  
Initial Rate ◽  
Disulfonic Acid ◽  
Copper Uptake ◽  
Selective Electrode ◽  
Apparent Dissociation Constant ◽  
Human Red Blood Cells

The initial rate of Cu2+ uptake in human red blood cells was measured by atomic absorption. About 80% of Cu2+ uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) concentrations greater than 5-10 microM. DIDS-sensitive Cu2+ uptake required the presence of external HCO3- or external Cl-. Cl- strongly stimulated Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (KCl) of 72 +/- 9.4 (SD) mM (n = 6 experiments). HCO3- stimulated DIDS-sensitive Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (Kbic) of 10 +/- 1.9 (SD) mM (n = 4 experiments). Maximal rates (of Cl(-)- or HCO3(-)-stimulated Cu2+ uptake) were nonadditive. DIDS-sensitive Cu2+ uptake was not modified by physiological concentrations of phosphate or sulfate. Conversely, it was strongly inhibited by physiological concentrations of L-histidine and cysteine (at a Cu2+ concentration of 100 microM, these physiological ligands exhibited KHis and KCys of 50 and 80 microM, respectively). By using a copper-selective electrode, we found that at pH 7-7.4 copper is associated with OH-, particularly in the form of Cu(OH)2 complexes. In conclusion, the anion exchanger is the major transport mechanism for red blood cell Cu2+ uptake. The translocating species can be the monovalent anion complexes of copper with OH-, Cl-, and/or HCO3-.

The Influence Of Red Cell Size On Platelet-Vessel Wall Interaction

1981 ◽  
Author(s):  
P A M M Aarts ◽  
J J Sixma ◽  
K S Sakariassen ◽  
P A Bolhuis ◽  
R M Heethaar
Keyword(s):  
Red Blood Cells ◽  
Cell Size ◽  
Blood Cells ◽  
Vessel Wall ◽  
Human Platelets ◽  
Red Cell ◽  
Mean Cell Volume ◽  
Human Red Blood Cells ◽  
Platelet Adherence ◽  
Order Of Magnitude

The hematocrit is one of the main factors influencing platelet adherence to the vessel wall. Raising the hematocrit causes an increase on platelet accumulation of about one order of magnitude.These studies disregard the influence of the red cell size. We have studied this effect using an annular perfusion chamber according to Baumgartner, with human umbilical arteries and a steady flow system. Perfusions were performed with reconstituted blood consisting of the appropriate washed red cells, aspirin treated and 51Cr labelled human platelets and human plasma. Perfusions were at a calculated vessel wall shear rate of 800 sec-1 for 5 min at 37°C. Platelet adherence was measured by 51Cr counting and controlled by morphometry. Human, goat and rabbit red blood cells were utilized. Human red blood cells (mean cell volume 100 f1) increase platelet adherence sevenfold, as the hematocrit increases from 0 to 60%. Human erythrocyte ghosts had a similar effect. Goat erythrocytes (m.c.v. 25 f1) caused no increment in adherence in the same hematocrit range. Rabbit erythrocytes (m.c.v. 70 f1) caused an intermediate increase in adherence.These data indicate that the red cell size is of major importance for platelet adherence. They may be explained by postulating that red cell rotation is responsible for diffusivity.

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