scholarly journals Direct estimate of 1:1 stoichiometry of K+-Cl−cotransport in rabbit erythrocytes

2001 ◽  
Vol 281 (3) ◽  
pp. C825-C832 ◽  
Author(s):  
Michael L. Jennings ◽  
Mark F. Adame
Keyword(s):  
Membrane Potential ◽  
Red Blood Cells ◽  
Okadaic Acid ◽  
Blood Cells ◽  
Model System ◽  
Disulfonic Acid ◽  
Calyculin A ◽  
Direct Measure ◽  
Direct Estimate ◽  
Exchange Flux

This work was undertaken to obtain a direct measure of the stoichiometry of Na+-independent K+-Cl−cotransport (KCC), with rabbit red blood cells as a model system. To determine whether86Rb+can be used quantitatively as a tracer for KCC,86Rb+and K+effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated K+efflux into isosmotic NaCl was smaller than that for86Rb+by a factor of 0.68 ± 0.11 (SD, n = 5). This correction factor was used in all other experiments to calculate the K+efflux from the measured86Rb+efflux. To minimize interference from the anion exchanger, extracellular Cl−was replaced with SO[Formula: see text], and 4,4′-diisothiocyanothiocyanatodihydrostilbene-2,2′-disulfonic acid was present in the flux media. The membrane potential was clamped near 0 mV with the protonophore 2,4-dinitrophenol. The Cl−efflux at 25°C under these conditions is ∼100,000-fold smaller than the uninhibited Cl−/Cl−exchange flux and is stimulated ∼2-fold by NEM. The NEM-stimulated36Cl−flux is inhibited by okadaic acid and calyculin A, as expected for KCC. The ratio of the NEM-stimulated K+to Cl−efflux is 1.12 ± 0.26 (SD, n = 5). We conclude that K+-Cl−cotransport in rabbit red blood cells has a stoichiometry of 1:1.

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).

Mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles

Biochemistry ◽  
1974 ◽  
Vol 13 (16) ◽  
pp. 3315-3330 ◽  
Author(s):  
Peter J. Sims ◽  
Alan S. Waggoner ◽  
Chao-Huei Wang ◽  
Joseph F. Hoffman
Keyword(s):  
Membrane Potential ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Cyanine Dyes ◽  
Measure Membrane Potential

scholarly journals A Hundred-Year Researching History on the Low Ionic Strength in Red Blood Cells: Literature Review

2021 ◽  
Vol 2 (3) ◽  
pp. 139-168
Author(s):  
GF Fuhrmann ◽  
KJ Netter
Keyword(s):  
Membrane Potential ◽  
Ionic Strength ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Sucrose Solution ◽  
Critical Survey ◽  
Unequal Distribution ◽  
Donnan Equilibrium ◽  
Disulfonic Stilbene ◽  
Low Ionic Strength

This review article provides a critical survey of work from 1904 to 2003 on the effects of low ionic strength in Red Blood Cells (RBCs) incubated in media with impermeable sugars such as sucrose. In 1904 Gürber A washed RBCs of different species with isotonic sucrose solution to eliminate the outside ions in order to better analyse their intracellular ionic composition; however, this approach was not feasible because of a substantial salt efflux from the cells. A prominent feature of the salt loss is the shrinking of the RBCs. A central role in the understanding of the ionic movements is thereby the new Donnan equilibrium of the anions. Experimental evidence has been given by Jacobs MH and Parpart AK in 1933. In the sucrose medium two phases could be predicted: 1) a very rapid anionic shift resulting in an unequal distribution of chloride and hydroxyl anions on both sides of the membrane and 2) a leakage of salts from the RBCs. In 1940 Wilbrandt W assumed that a positive membrane potential is in line with the salt loss at low ionic strength in RBCs. In 1977 Knauf PA, Fuhrmann GF, Rothstein S and Rothstein A observed in RBCs an inhibition of both, anion exchange and also of net anion efflux, by incubation with disulfonic stilbene derivates. At low ionic strength the Donnan equilibrium is immediately obtained by the Anion Exchanger Protein (AEP). The resulting positive membrane potential opens at least two new types of cation pores or channels. Thereby is the conductivity pathway for the anions, namely the AEP, in charge of the net anion loss at low ionic strength. The AEP pathway is extensively blocked by disulfonic stilbene compounds. The permeability ways for cations through these pores or channels are not yet explored.

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.

Urea alters set point volume for K-Cl cotransport, Na-H exchange, and Ca-Na exchange in dog red blood cells

1993 ◽  
Vol 265 (2) ◽  
pp. C447-C452 ◽  
Author(s):  
J. C. Parker
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Okadaic Acid ◽  
Blood Cells ◽  
Regulatory System ◽  
Set Point ◽  
Volume Sensor

Urea equilibrates rapidly across the red blood cells of mammals. It was speculated that urea might affect the cell volume sensor by virtue of its properties as a protein perturbant. At concentrations of 0.1-0.6 M, urea caused a decrease in the set points for shrinkage-induced Na-H exchange, swelling-induced K-Cl cotransport, and swelling-induced Ca-Na exchange of dog red blood cells. Okadaic acid opposed the action of urea on all three pathways. The effects were reversible and not due to cyanate. Formamide and acetamide had actions similar to urea but not as potent. Equimolar concentrations of methanol had no effect. The coordinated influence of urea on three separate volume-activated transporters suggests that it acts on a mutual regulatory system that senses and transduces volume stimuli.

scholarly journals The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway.

1993 ◽  
Vol 102 (3) ◽  
pp. 551-573 ◽  
Author(s):  
J R Sachs ◽  
D W Martin
Keyword(s):  
Signal Transduction ◽  
Red Blood Cells ◽  
Okadaic Acid ◽  
Blood Cells ◽  
Kinase Inhibitor ◽  
Signal Transduction Pathway ◽  
Maximal Activity ◽  
Cell Protein ◽  
Intact Cells ◽  
Human Red Blood Cells

Volume-sensitive K-Cl cotransport occurs in red blood cells of many species. In intact cells, activation of K-Cl cotransport by swelling requires dephosphorylation of some cell protein, but maximal activity requires the presence of intracellular ATP. We have examined the relation between K-Cl cotransport activity and ATP in ghosts prepared from human red blood cells. K-Cl cotransport activity in swollen ghosts increased by ATP, and the increase requires Mg so that it almost certainly results from the phosphorylation of some membrane component. However, even in ATP-free ghosts residual volume-sensitive K-Cl cotransport can be demonstrated. This residual cotransport in ATP-free ghosts is greater in the presence of vanadate, a tyrosyl phosphatase inhibitor, and in ghosts that contain ATP cotransport is reduced by genistein, a tyrosyl kinase inhibitor. Okadaic acid, an inhibitor of serine and threonine phosphatases, inhibits K-Cl cotransport in ghosts as it does in intact cells. Experiments in which ghosts were preexposed to okadaic acid showed that the protein dephosphorylation that permits K-Cl cotransport can proceed to completion before the ghosts are swollen and K transport measured and therefore dephosphorylation is not a response to ghost swelling. In experiments with ATP-free ghosts we found that phosphorylation is not necessary to increase the cotransport rate when shrunken ghosts are swollen, nor is rephosphorylation necessary to decrease the cotransport rate when swollen ghosts are shrunken. Cotransport is greater in swollen than in shrunken ghosts even when the swollen and shrunken ghosts have the same concentration of cytoplasmic solutes. We conclude that, although phosphorylation and dephosphorylation modify the activity of the cotransporter in swollen and in shrunken ghosts, neither of these processes nor any other known messenger is involved in signal transduction between the cell volume sensor and the cotransporter as originally proposed by Jennings and Al-Rohil (Jennings, M. L., and N. Al-Rohil. 1990. Journal of General Physiology. 95: 1021-1040).

Chloride and taurine effluxes occur by different pathways in skate erythrocytes

1996 ◽  
Vol 271 (6) ◽  
pp. R1544-R1549 ◽  
Author(s):  
E. M. Davis-Amaral ◽  
M. W. Musch ◽  
L. Goldstein
Keyword(s):  
Epithelial Cells ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Disulfonic Acid ◽  
Raja Erinacea ◽  
Western Blots ◽  
Cl Channel ◽  
Cl Channels

The aim of this study was to determine whether volume-activated taurine and Cl- effluxes occur via the same system in skate (Raja erinacea) red blood cells (RBC). The effluxes were measured in isotonic and hypotonic elasmobranch Ringer solutions, in which NaCl was replaced by mannitol and the remaining exchangeable anions with gluconate. Methazolamide (0.1 mM) was added to minimize HCO3- formation. RBC Cl- content fell approximately 50%/h in both isotonic and hypotonic media, with no detectable K- loss in either medium. The observed Cl- loss was accompanied by an increase in pH. Both the Cl- loss and pH rise were inhibited by 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM), suggesting that Cl- efflux was due to H(+)-Cl- cotransport. 36Cl- effluxes in isotonic and hypotonic media were (means +/- SE, n = 11) 2.8 +/- 0.6 and 3.5 +/- 0.9 mumol.g dry wt RBC-1.min-1, respectively, whereas [3H]taurine effluxes in the same media were 0.045 +/- 0.02 and 2.1 +/- 0.05 mumol.g dry wt RBC-1.min-1, respectively (n = 6). These results indicate that taurine and Cl- effluxes occur via different pathways in skate RBC. In addition, the swelling-activated Cl- channel reported in epithelial cells does not appear to be present in skate RBC. This conclusion was confirmed by Western blots with an antibody to swelling-activated Cl- channels. Taurine and Cl- fluxes are apparently under different pathway influences in these RBC: taurine diffuses via a channel, whereas Cl- is transported by cotransporters.

scholarly journals Activation of electroneutral K flux in Amphiuma red blood cells by N-ethylmaleimide. Distinction between K/H exchange and KCl cotransport.

1987 ◽  
Vol 90 (2) ◽  
pp. 209-227 ◽  
Author(s):  
J S Adorante ◽  
P M Cala
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Driving Force ◽  
Flow Analysis ◽  
Cell Swelling ◽  
Direct Effects ◽  
Kcl Cotransport ◽  
Thermodynamic Forces ◽  
Exchange Flux ◽  
Stimulation Of

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.

scholarly journals Volume-responsive sodium and proton movements in dog red blood cells.

1984 ◽  
Vol 84 (3) ◽  
pp. 379-401 ◽  
Author(s):  
J C Parker ◽  
V Castranova
Keyword(s):  
Membrane Potential ◽  
Red Blood Cells ◽  
Fluorescent Probe ◽  
Blood Cells ◽  
Indirect Method ◽  
Cell Types ◽  
The Other ◽  
Na Transport ◽  
Transport Pathway ◽  
Transport Pathways

Shrinkage of dog red blood cells (RBC) activates a Na transport pathway that is Cl dependent, amiloride sensitive, and capable of conducting Na-proton counterflow. It is possible to establish transmembrane gradients for either Na or protons and to demonstrate that each cation species can drive reciprocal movements of the other. The nature of the coupling between Na and proton movements was investigated using the fluorescent probe diS-C3(5) and also by an indirect method in which K movements through valinomycin channels were used to draw inferences about the membrane potential. No evidence was found to suggest that the Na-proton pathway activated by shrinkage of dog RBC is a conductive one. By exclusion, it is presumed that the coupling between the counterflow of Na and protons is electroneutral. The volume-activated Na-proton fluxes in dog RBC have certain properties that distinguish them from similar transport pathways in other cell types.

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