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).

scholarly journals PIEZO1 and the mechanism of the long circulatory longevity of human red blood cells

2021 ◽  
Vol 17 (3) ◽  
pp. e1008496
Author(s):  
Simon Rogers ◽  
Virgilio L. Lew
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Time Course ◽  
Clinical Manifestations ◽  
Cell Subpopulation ◽  
Unexpected Finding ◽  
Human Red Blood Cells ◽  
Acceptable Model ◽  
New Finding ◽  
Extended Longevity

Human red blood cells (RBCs) have a circulatory lifespan of about four months. Under constant oxidative and mechanical stress, but devoid of organelles and deprived of biosynthetic capacity for protein renewal, RBCs undergo substantial homeostatic changes, progressive densification followed by late density reversal among others, changes assumed to have been harnessed by evolution to sustain the rheological competence of the RBCs for as long as possible. The unknown mechanisms by which this is achieved are the subject of this investigation. Each RBC traverses capillaries between 1000 and 2000 times per day, roughly one transit per minute. A dedicated Lifespan model of RBC homeostasis was developed as an extension of the RCM introduced in the previous paper to explore the cumulative patterns predicted for repetitive capillary transits over a standardized lifespan period of 120 days, using experimental data to constrain the range of acceptable model outcomes. Capillary transits were simulated by periods of elevated cell/medium volume ratios and by transient deformation-induced permeability changes attributed to PIEZO1 channel mediation as outlined in the previous paper. The first unexpected finding was that quantal density changes generated during single capillary transits cease accumulating after a few days and cannot account for the observed progressive densification of RBCs on their own, thus ruling out the quantal hypothesis. The second unexpected finding was that the documented patterns of RBC densification and late reversal could only be emulated by the implementation of a strict time-course of decay in the activities of the calcium and Na/K pumps, suggestive of a selective mechanism enabling the extended longevity of RBCs. The densification pattern over most of the circulatory lifespan was determined by calcium pump decay whereas late density reversal was shaped by the pattern of Na/K pump decay. A third finding was that both quantal changes and pump-decay regimes were necessary to account for the documented lifespan pattern, neither sufficient on their own. A fourth new finding revealed that RBCs exposed to levels of PIEZO1-medited calcium permeation above certain thresholds in the circulation could develop a pattern of early or late hyperdense collapse followed by delayed density reversal. When tested over much reduced lifespan periods the results reproduced the known circulatory fate of irreversible sickle cells, the cell subpopulation responsible for vaso-occlusion and for most of the clinical manifestations of sickle cell disease. Analysis of the results provided an insightful new understanding of the mechanisms driving the changes in RBC homeostasis during circulatory aging in health and disease.

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.

Cell volume and metabolic dependence of NEM-activated K+-Cl- flux in human red blood cells

1985 ◽  
Vol 249 (1) ◽  
pp. C124-C128 ◽  
Author(s):  
P. K. Lauf ◽  
C. M. Perkins ◽  
N. C. Adragna
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Time Course ◽  
Transport Activity ◽  
Human Red Blood Cells ◽  
Atp Breakdown ◽  
The Difference ◽  
Metabolic Dependence ◽  
K Transport

The effects of incubation in anisosmotic media and of metabolic depletion on ouabain-resistant (OR) Cl--dependent K+ influxes stimulated by N-ethylmaleimide (NEM) were studied in human red blood cells using Rb+ as K+ analogue. The NEM-stimulated but not the basal Rb+-Cl- influx measured in phosphate-buffered anisosmotic media was found to be cell volume dependent. When cellular ATP, [ATP]c, was lowered to less than 0.10 of its initial level by exposure to nonmetabolizable 2-deoxy-D-glucose, the NEM-stimulated but not the basal Cl--dependent Rb+ influxes were abolished. Metabolically depleted red blood cells subsequently repleted by incubation in glucose plus inosine regained the NEM-inducible Rb+ (K+) transport activity. The difference in the time course of ATP breakdown and Rb+ influx inhibition suggests that energization of the NEM-stimulated Rb+ flux by metabolism may involve factors additional to ATP.

scholarly journals The circulatory physiopathology of human red blood cells investigated with a multiplatform model of cellular homeostasis. III. Senescence changes during the full circulatory lifespan

2020 ◽  
Author(s):  
Simon Rogers ◽  
Virgilio L. Lew
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Time Course ◽  
Clinical Manifestations ◽  
Cell Subpopulation ◽  
Unexpected Finding ◽  
Sickle Cells ◽  
Human Red Blood Cells ◽  
Decay Sequence ◽  
Health And Disease

AbstractHuman red blood cells (RBCs) have a circulatory lifespan of about four months. Under constant oxidative and mechanical stress, but devoid of organelles and deprived of biosynthetic capacity for protein renewal, RBCs undergo substantial homeostatic changes, progressive densification followed by late density reversal among others, changes assumed to have been harnessed by evolution to sustain the rheological competence of the RBCs for as long as possible. The unknown mechanisms by which this is achieved are the subject of this investigation. Each RBC traverses capillaries between 1000 and 2000 times per day, roughly one transit per minute, a total of about 2•105 transits during their lifespan. A dedicated Lifespan model of RBC homeostasis was developed as an extension of the RCM introduced in the first paper of this series to explore the cumulative patterns predicted for repetitive capillary transits over a standardized lifespan period of 120 days, using experimental data to constrain the parameter space. Capillary transits were simulated by periods of elevated cell/medium volume ratios and by transient deformation-induced permeability changes attributed to PIEZO1 channel mediation as outlined in the second paper of this series. The first unexpected finding was that quantal changes generated during single capillary transits cease accumulating after a few days and cannot account for the observed progressive densification of RBCs on their own, thus ruling out the quantal hypothesis. The second unexpected finding was that the documented patterns of RBC densification and late reversal could only be emulated by the implementation of a strict time-course of decay in the activities of the calcium and Na/K pumps, but only in addition to the quantal changes. These results showed that both quantal changes and pump-decay regimes were necessary to account for the documented lifespan pattern, neither sufficient on their own. They also suggested a strong selective component in the pump decay sequence. A third finding was that RBCs exposed to levels of calcium permeation above certain thresholds in the circulation could develop a pattern of late or early hyperdense collapse followed by delayed density reversal. When tested over much reduced lifespan periods the results emulated the known circulatory fate of irreversible sickle cells, the cell subpopulation responsible for vaso-occlusion and for most of the clinical manifestations of sickle cell disease. Analysis of the results provided an insightful new understanding of the mechanisms driving the changes in RBC homeostasis during circulatory aging in health and disease.

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.

Proton-sulfate cotransport: external proton activation of sulfate influx into human red blood cells

1984 ◽  
Vol 247 (3) ◽  
pp. C247-C259 ◽  
Author(s):  
M. A. Milanick ◽  
R. B. Gunn
Keyword(s):  
Membrane Potential ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Half Cycle ◽  
Ph Values ◽  
Human Red Blood Cells ◽  
Internal Ph ◽  
Ph Range ◽  
External Ph ◽  
Chloride Concentrations

Sulfate influx into human red blood cells was measured at 0 and 22 degrees C at several fixed external pH values between 3 and 10. These cells had normal internal pH and chloride concentrations so that sulfate influx was not limited by the efflux half-cycle reactions. The flux was a Michaelis-Menten function of sulfate concentration at each pH with K1/2SO4 = 4-10 mM. External protons activated influx 100-fold at a single site with a pK = 5.9 at 22 degrees C and 5.5 at 0 degrees C. This pK is similar to the value 5.99 +/- 0.3 for external proton binding to the sulfate-loaded transporter at 0 degrees C (J. Gen. Physiol. 79: 87-114, 1982). The flux was stilbene sensitive even in valinomycin-treated cells and was independent of membrane potential. This proton-activated influx appears to be proton-sulfate cotransport. At high pH there was a proton-independent flux that was membrane potential and stilbene sensitive. This proton-insensitive flux appears to be SO4(2-)/Cl- exchange or net sulfate influx. The sulfate influx over the entire pH range may be described in terms of an equation for the sum of the influxes through these two pathways on band 3.

scholarly journals South american rattlesnake venom: its hemolytic power

1989 ◽  
Vol 22 (4) ◽  
pp. 171-175 ◽  
Author(s):  
Édimo Garcia de Lima ◽  
Paulo Inácio da Costa ◽  
Carlos Julio Laure
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
South American ◽  
Experimental Conditions ◽  
Crotalus Durissus Terrificus ◽  
Sheep Red Blood Cells ◽  
Human Red Blood Cells ◽  
Rattlesnake Venom ◽  
Positive Plate ◽  
Crotalus Durissus

The hemolytic power of rattlesnake venom (Crotalus durissus terrificus) was Studied. A high percentage of sample with negative hemolytic power was detected when sheep red blood cells were used. A large number of venoms with hemolytic power, though with a low hemolysis percentage, were detected when liquid, recently extracted venom was used. When crystallized venom was used under the same experimental conditions, a higher percentage ofpositivityfor hemolysis was obtained. When the results obtained on agar plates were compared to those obtained in test tubes, a large number of animals with a higher percentage of hemolysis were detected, though this value was not proportional to the number of animals showing positive plate hemolysis. When the hemolytic power of these venoms was tested on human red blood cells, a large percentage of animals with venoms having a low hemolytic power was also detected. Hemolytic power was much greater when human red blood cells were tested with crystallized venom. The preparation of red blood cells also had an important effect and the use of red blood cells from defibrinated blood is recommended. We conclude that rattlesnake venom has hemolytic power that increases when the venom is crystallized. Red blood cells should be properly preparedfor the lysis reactions. We suggest that the lytic power of the venom is related to venom concentration and to the purity of its fractions.

scholarly journals Quantitative time-course metabolomics in human red blood cells reveal the temperature dependence of human metabolic networks

2017 ◽  
Vol 292 (48) ◽  
pp. 19556-19564 ◽  
Author(s):  
James T. Yurkovich ◽  
Daniel C. Zielinski ◽  
Laurence Yang ◽  
Giuseppe Paglia ◽  
Ottar Rolfsson ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Temperature Dependence ◽  
Blood Cells ◽  
Time Course ◽  
Metabolic Networks ◽  
Human Red Blood Cells

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.

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