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

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.     

Sodium and calcium movements in dog red blood cells

Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed.     

Glutaraldehyde fixation of sodium transport in dog red blood cells

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.     

Heterogeneity in dog red blood cells: sodium and potassium transport

After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Heterogeneity among dog red blood cells

A phthalate density-separation technique has been used to study the heterogeneity of dog red blood cells that becomes manifest when they are suspended in KCl media. It is demonstrated that the proportions of cells that separate into light and dense fractions can be varied by altering the tonicity of the KCl medium. This results from the fact that the Na and K permeabilities of each cell are continuous functions of cell volume. It was found that quinidine inhibits selectively the volume dependence of Na permeability. In the presence of this drug, the heterogeneity demonstrated by KCl incubation disappears. The notion that dog red blood cells are heterogeneous in their permeabilities to Na and K is thus upheld, but the heterogeneity is not an abruptly discontinuous one, as has been claimed. A sample of dog blood does not contain two discrete populations of red cells.     

Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways

To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.     

Volume-activated transport systems in dog red blood cells

1. When dog red blood cells are shrunken or swollen, transport pathways are activated that do not function discernibly when the cell is at normal volume. Swelling the cells turns on two pathways, a Ca-Na exchanger and a Cl-dependent K pathway. 2. Shrinking the cells activates a Na-H antiporter. 3. The passive net flow of ions through these transporters is in such a direction as to correct the perturbation of cell volume: when the cell water content has returned to normal, the transporters turn off. 4. Recently we have investigated agents that can lock or fix the volume-responsive transporters in the activated state. Na-H exchange, for example, can be fixed in the on position with either glutaraldehyde or N-phenylmaleimide. 5. Ca-Na exchange can be locked on by the sulfhydryl-oxidizing agent, diamide. We have used these effects to investigate the relationships between cell volume and the transport mechanisms. 6. It is possible, for instance, to distinguish whether certain inhibitors act on the transporters per se or on the apparatus that perceives cell volume and communicates with the transporters. 7. Furthermore, in the case of the Ca-Na exchanger some indication of the membrane polypeptides involved in volume regulation has been possible, using radioactive compounds that bind covalently to sulfhydryl groups.     

Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

Role of calcium in volume regulation by dog red blood cells

Dog red blood cells (RBC) are shown to regulate their volume in anisosmotic media. Extrusion of water from osmotically swollen cells requires external calcium and is associated with net outward sodium movement. Accumulation of water by osmotically shrunken cells is not calcium dependent and is associated with net sodium uptake. Net movements of calcium are influenced by several variables including cell volume, pH, medium sodium concentration, and cellular sodium concentration. Osmotic swelling of cells increases calcium permeability, and this effect is diminished at acid pH. Net calcium flux in either direction between cells and medium is facilitated when the sodium concentrations is low in the compartment from which calcium moves and/or high in the compartment to which calcium moves. The hypothesis is advanced that energy for active sodium extrusion in dog RBC comes from passive, inward flow of calcium through a countertransport mechanism.