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 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.

Two pathways for choline transport in eel erythrocytes: a saturable carrier and a volume-activated channel

1994 ◽  
Vol 267 (3) ◽  
pp. R773-R779 ◽  
Author(s):  
S. E. Joyner ◽  
K. Kirk
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Anguilla Anguilla ◽  
Anion Transport ◽  
Response Curves ◽  
Choline Transport ◽  
Human Red Blood Cells ◽  
Selective Channel ◽  
Transport Inhibitors ◽  
In Cells

Choline transport in eel (Anguilla anguilla) erythrocytes was investigated in cells suspended in isotonic and hypotonic media. In cells in isosmotic solution choline transport was mediated by a saturable system with a Michaelis constant (Km; 62 +/- 6 microM) similar to that of the choline carrier of human erythrocytes but a maximal transport rate (Vmax; 4.5 +/- 0.4 mmol.1 red blood cells-1.h-1) almost two orders of magnitude higher than that in human red blood cells. This pathway was inhibited by hemicholinium-3 and dodecyltrimethylammonium, but not by any of a range of anion transport inhibitors tested. Swelling the cells by suspending them in hyposmotic media activated a second choline transport component that was kinetically and pharmacologically distinct from the saturable system. The volume-activated component was nonsaturable (up to 50 mM choline). It was not inhibited by hemicholinium-3 or dodecyltrimethylammonium but was inhibited by anion transport inhibitors, the most potent of which was 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; half-maximal inhibitory concentration = 14 microM). Dose-response curves for the effect of NPPB on swelling-activated choline transport and the swelling-activated transport of taurine, a sulfonic amino acid, were superimposable. It is postulated that the transport of choline and taurine (as well as that of other small organic solutes) in osmotically swollen fish erythrocytes is mediated by a volume-activated, anion-selective channel.

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.

Na-Ca exchange in ferret red blood cells

1989 ◽  
Vol 256 (2) ◽  
pp. C390-C398 ◽  
Author(s):  
M. A. Milanick
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Red Cells ◽  
Ionophore A23187 ◽  
Maximal Velocity ◽  
Free Solution ◽  
Exchange System ◽  
Ca Pump ◽  
Na Efflux ◽  
Intracellular Ca

Ferrets have high Na (140 mmol/l) red blood cells. To determine whether ferret red cells had a Na-Ca exchange system, Na effluxes via the Na + K + 2Cl cotransrpoter and Ca effluxes via the Ca pump had to be inhibited. This was accomplished by replacing cell chloride with nitrate and by loading the cells with vanadate that inhibits the Ca pump. Under these conditions, extracellular Na (Naout) inhibited Ca influx. Intracellular Na (Nain) was required for the large Ca influx as replacement of Na with Li reduced Ca influx to less than one-tenth of the original rate. Caout stimulated Na efflux by about twofold. The Ca efflux from cells depleted of Na was increased from 0.8 to 3.2 mmol.l packed cells-1.h-1 by the presence of Naout. Cells placed in a Na-free solution accumulated Ca: total intracellular Ca was 20-fold higher than free Caout. Most of this Ca was released on addition of the Ca ionophore, A23187. Because the Na gradient had driven net Ca uphill, the fluxes of Na and Ca are coupled. In a Na-free solution, the K1/2 for Ca influx was usually approximately 10 microM (occasionally approximately 100 microM), and the maximal velocity (Vmax was 1.5-4.4 mmol.l packed cells-1.h-1. At Naout = 150 mM, K1/2 increased 5- to 150-fold. In some cells, Naout decreased Vmax by approximately fourfold, suggesting that Naout does not always compete with Caout.(ABSTRACT TRUNCATED AT 250 WORDS)

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-.

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