Active and passive Ca movements in dog red blood cells and resealed ghosts

1979 ◽  
Vol 237 (1) ◽  
pp. C10-C16 ◽  
Author(s):  
J. C. Parker
Keyword(s):  
Body Temperature ◽  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Volume Regulation ◽  
Cell Volume Regulation ◽  
Low Ph ◽  
Coupling Ratio ◽  
Cell Shrinkage ◽  
Resealed Ghosts

Dog red blood cells accumulate Ca rapidly when deprived of substrate or cooled to 5 degrees C. The latter effect is reversible as the cells are rewarmed to body temperature. Resealed ghosts extrude Ca, provided ATP is incorporated in them. Passive fluxes of Ca are stimulated by Na on the opposite side of the membrane, consistent with a model for Ca-Na countertransport. Quinidine, cell shrinkage, and low pH--all known to suppress net Ca influx--have no accelerating effect on Ca efflux, thus validating earlier conclusions about the variability of the coupling ratio for Ca-Na exchange. The significance of these findings for cell volume regulation is discussed.

scholarly journals Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways.

1980 ◽  
Vol 76 (6) ◽  
pp. 683-708 ◽  
Author(s):  
P M Cala
Keyword(s):  
Membrane Potential ◽  
Alkali Metal ◽  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Ion Flux ◽  
Regulatory Response

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.

scholarly journals Evidence of Coordinated and Adjustable Osmolytes Movements Following Hyposmotic Swelling in Rainbow Trout Red Blood Cells

2021 ◽  
Vol 55 (S1) ◽  
pp. 185-195
Author(s):  
Valérie Maxime ◽  
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Volume Regulation ◽  
Rapid Change ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Gravimetric Method ◽  
Ph Control ◽  
Na Uptake

BACKGROUND/AIMS: The osmolytes involved in the volume regulation of hyposmotically-swollen fish cells are well identified. However, if a coordination and adjustments of their fluxes are obvious, few studies have clearly illustrated these aspects. METHODS: Trout red blood cells volume variations were estimated from water contents obtained by a gravimetric method. Intracellular K+ and Na+ contents, and Cl- content of haemolysed cells were determined by photometry and colorimetry, respectively. The taurine contribution to cell volume regulation was calculated from the net changes of water, K+, Cl- and Na+ contents. The intracellular pH was calculated from the chloride distribution across the cells membranes according to the Donnan equilibrium. RESULTS: Cells responses to a rapid change (from 296 to 176 mOsm.kg-1)
of the saline osmolality were examined in three conditions designed to not impact (Hypo. I)
or to reduce the K+ (Hypo. II) and Cl- (Hypo. III) contributions to the volume regulation. Hypo. I condition caused an immediate increase in water content, followed by a 90 min. full regulation, concomitant with gradual lowering of K+ and Cl- contents and a surprising increase in Na+ content. Hypo. II and III conditions showed a partial and complete volume regulation, respectively. This was made possible by an increase in the taurine involvement. These experiments allowed to confirm that K+ and Cl- were released via KCl cotransport and by separate channels. The comparison of Hypo. I and III conditions led to the observation that the partially amiloride-sensitive Na+ influx is proportional to the taurine efflux; the latter being sustained mainly by a Na+/taurine cotransport. The Hypo. II condition was suitable for the (Na+/K+)ATPase activity inhibition. This effect could explain the observed lack of Na+ uptake, the consecutive depletion of intracellular taurine stock and the incomplete volume regulation. Finally, the results support the importance of taurine in pH control under Hypo. I (physiologic) condition. The alkalosis observed in Hypo. II and III conditions were the consequences of changes in the salines compositions, not of physiologic adjustments. CONCLUSION: The regulatory volume decrease process of trout RBCs is complex and adjustable through coordinated osmolytes movements. The obliged decrease in K+ and/or Cl- contributions stimulates taurine and Na+ pathways. This study highlights the importance of taurine as a compensatory variable in cell volume regulation and explains for the first time the significance of the Na+ uptake during this process

scholarly journals Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange.

1983 ◽  
Vol 82 (6) ◽  
pp. 761-784 ◽  
Author(s):  
P M Cala
Keyword(s):  
Alkali Metal ◽  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Volume Regulation ◽  
Time Course ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Membrane Conductance ◽  
Ionophore A23187

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.

scholarly journals Volume regulation by flounder red blood cells in anisotonic media

1977 ◽  
Vol 69 (5) ◽  
pp. 537-552 ◽  
Author(s):  
PM Cala
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Water Flow ◽  
Blood Cells ◽  
Volume Regulation ◽  
Control Cell ◽  
Regulatory Volume Decrease ◽  
Electrochemical Gradient ◽  
Osmotic Swelling ◽  
Inorganic Cation

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.

Passive calcium movements in dog red blood cells: anion effects

1983 ◽  
Vol 244 (5) ◽  
pp. C318-C323 ◽  
Author(s):  
J. C. Parker
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Calcium Transport ◽  
Calcium Influx ◽  
Low Ph ◽  
Irreversible Change ◽  
Cell Shrinkage ◽  
Sodium Efflux ◽  
Sodium Dependent ◽  
Stimulation Of

Calcium influx in dog red blood cells was stimulated by replacing chloride in the medium with nitrate or thiocyanate. These anion effects were due to stimulation of a sodium-dependent calcium pathway, because calcium influx in the presence of nitrate or thiocyanate was 1) inhibited by external sodium, 2) dependent on internal sodium, 3) inhibited by cell shrinkage and low pH, and 4) inhibited by quinidine. All these characteristics had previously been shown to hold for calcium movements in the presence of chloride. Neither nitrate nor thiocyanate caused an irreversible change in calcium transport in the concentrations studied. Calcium-stimulated sodium efflux is stimulated when chloride is replaced by thiocyanate but not by nitrate. Several limiting features of the system are discussed, which preclude a conclusive interpretation of the data. The possibility is considered that the rates of sodium-dependent calcium transport in the presence of chloride, nitrate, and thiocyanate are a function of the conductance of these anions.

Extracellular Na+ inhibits Na+/H+ exchange: cell shrinkage reduces the inhibition

2004 ◽  
Vol 287 (2) ◽  
pp. C336-C344 ◽  
Author(s):  
Philip B. Dunham ◽  
Scott J. Kelley ◽  
Paul J. Logue
Keyword(s):  
Growth Factors ◽  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Hyperbolic Function ◽  
Maximal Velocity ◽  
Cell Shrinkage ◽  
Inhibitory Site ◽  
Kinetics Of ◽  
In Cells

Na+/H+ exchangers (NHE) are ubiquitous transporters participating in regulation of cell volume and pH. Cell shrinkage, acidification, and growth factors activate NHE by increasing its sensitivity to intracellular H+ concentration. In this study, the kinetics were studied in dog red blood cells of Na+ influx through NHE as a function of external Na+ concentration ([Na+]o). In cells in isotonic media, [Na+]o inhibited Na+ influx >40 mM. Osmotic shrinkage activated NHE by reducing this inhibition. In cells in isotonic media + 120 mM sucrose, there was no inhibition, and influx was a hyperbolic function of [Na+]o. The kinetics of Na+-inhibited Na+ influx were analyzed at various extents of osmotic shrinkage. The curves for inhibited Na+ fluxes were sigmoid, indicating more than one Na+ inhibitory site associated with each transporter. Shrinkage significantly increased the Na+ concentration at half-maximal velocity of Na+-inhibited Na+ influx, the mechanism by which shrinkage activates NHE.

scholarly journals Glutaraldehyde fixation of sodium transport in dog red blood cells.

1984 ◽  
Vol 84 (5) ◽  
pp. 789-803 ◽  
Author(s):  
J C Parker
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Cross Linking ◽  
Glutaraldehyde Fixation ◽  
Cell Shrinkage ◽  
Transport Pathway ◽  
Turn On ◽  
Two Phases ◽  
Chemical Cross Linking

The large increase in passive Na flux that occurs when dog red blood cells are caused to shrink is amiloride sensitive and inhibited when Cl is replaced by nitrate or thiocyanate. Activation and deactivation of this transport pathway by manipulation of cell volume is reversible. Brief treatment of the cells with 0.01-0.03% glutaraldehyde can cause the shrinkage-activated transporter to become irreversibly activated or inactivated, depending on the volume of the cells at the time of glutaraldehyde exposure. Thus, if glutaraldehyde is applied when the cells are shrunken, the amiloride-sensitive Na transporter is activated and remains so regardless of subsequent alterations in cell volume. If the fixative is applied to swollen cells, no amount of subsequent shrinkage will turn on the Na pathway. In its fixed state, the activated transporter is fully amiloride sensitive, but it is no longer inhibited when Cl is replaced by thiocyanate. The action of glutaraldehyde thus allows one to dissect the response to cell shrinkage into two phases. Activation of the pathway is affected by anions and is not prevented by amiloride. Once activated and fixed, the anion requirement disappears. Amiloride inhibits movement of Na through the activated transporter. These experiments demonstrate how a chemical cross-linking agent may be used to study the functional properties of a regulable transport pathway.

scholarly journals Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells

2010 ◽  
Vol 298 (3) ◽  
pp. C510-C520 ◽  
Author(s):  
Alejandro Ortiz-Acevedo ◽  
Robert R. Rigor ◽  
Hector M. Maldonado ◽  
Peter M. Cala
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Coordinated Control ◽  
Cell Swelling ◽  
Cell Shrinkage ◽  
Relaxation Kinetic ◽  
Shrinkage And Swelling ◽  
Final Steady State ◽  
Single Exponential Function

The Na+/H+ and K+/H+ exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H+ exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na+/H+ and K+/H+ exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na+/H+ and K+/H+ exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na+/H+ exchange activation increases with cell shrinkage, whereas the rate of Na+/H+ exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K+/H+ exchange activation increases with cell swelling, whereas the rate of K+/H+ exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K+/H+ exchange are reciprocally related to those controlling Na+/H+ exchange.

Interactions of sodium-proton exchange mechanism in dog red blood cells with N-phenylmaleimide

1987 ◽  
Vol 253 (1) ◽  
pp. C60-C65 ◽  
Author(s):  
J. C. Parker ◽  
P. S. Glosson
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Functional Groups ◽  
Blood Cells ◽  
Proton Exchange ◽  
Cell Swelling ◽  
Cell Shrinkage ◽  
Activated State ◽  
Sodium Proton Exchange ◽  
Number Of Cells

Dog red blood cells (RBC) have a Na-H exchanger that is reversibly activated by cell shrinkage. The Na-H exchanger can be fixed in the on or off mode by treating the cells with N-phenylmaleimide. This action depends on the volume of the cells at the time of exposure to N-phenylmaleimide and also on the concentration of the reagent per number of cells. If the cells are swollen in hypotonic media during N-phenylmaleimide exposure, the Na-H exchanger becomes irreversibly inactivated, so that on subsequent shrinkage of the cells, no amiloride-sensitive Na flux is seen. This effect is maximal at N-phenylmaleimide concentrations of greater than 20 mumol/g hemoglobin. If the cells are shrunken in hypertonic media during N-phenylmaleimide exposure, the response of the Na-H exchanger depends critically on the concentration of the reagent. At N-phenylmaleimide concentrations of less than 20 mumol/g hemoglobin, the Na-H exchanger is fixed in the activated state, so that even when the volume stimulus is removed by subsequent cell swelling, an amiloride-sensitive flux is seen. Higher concentrations of N-phenylmaleimide applied to shrunken cells inhibit the Na-H exchanger. The results are accounted for in a model that envisions a volume-responsive switching mechanism for Na-H exchange that has two functional groups capable of reacting with N-phenylmaleimide. The accessibility of these groups is determined by cell volume.

Volume regulation by Amphiuma red blood cells: cytosolic free Ca and alkali metal-H exchange

1986 ◽  
Vol 250 (3) ◽  
pp. C423-C429 ◽  
Author(s):  
P. M. Cala ◽  
L. J. Mandel ◽  
E. Murphy
Keyword(s):  
Alkali Metal ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Volume Regulation ◽  
Null Point ◽  
Cell Swelling ◽  
Maximal Concentration ◽  
Cell Shrinkage ◽  
Osmotic Swelling ◽  
Intracellular Ca

Osmotic swelling of Amphiuma red blood cells results in activation of electroneutral K-H exchange, whereas cell shrinkage activates an electroneutral Na-H exchange. These K-H and Na-H exchangers function to restore cell volume to normal after cell swelling and shrinkage, respectively. Our previous studies have suggested that Ca plays a role in volume-dependent activation of K-H exchange. In the present studies, intracellular free Ca levels were measured employing the Ca-sensitive extracellular dye arsenazo III and a previously described null-point method. Control values for intracellular free Ca averaged 0.46 +/- 0.15 microM. Cell shrinkage caused this value to decrease to 0.16 +/- 0.11 microM, whereas either cell swelling or addition of 5 microM A23187 resulted in saturation of intracellular Ca buffers, suggesting that both treatments caused an increase in intracellular free Ca. In the presence of 7 microM A23187, the rate of K-H exchange displayed a hyperbolic relationship as a function of extracellular Ca (Cao). The apparent half-maximal concentration for Cao (in the presence of 7 microM A23187) was 0.27 mM for osmotically swollen cells and 1.9 mM for cells in isotonic medium, suggesting that the Ca affinity of a modulating site is increased in swollen cells. Inhibitors of Ca-mediated processes, such as quinidine and the phenothiazines, inhibited K-H exchange. In contrast, the phenothiazines chlorpromazine and trifluoperazine stimulated Na-H exchange by osmotically shrunken cells. These results suggest that increases in intracellular free Ca are involved in stimulating K-H exchange while repressing Na-H exchange in Amphiuma red blood cells.

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