Coordinated regulation of Na/H exchange and [K-Cl] cotransport in dog red cells

Swelling-activated [K-Cl] cotransport and shrinkage-activated Na/H exchange were studied in dog red cells with altered internal Mg or Li content. The two pathways responded in a coordinated fashion. When cells were depleted of Mg, [K-Cl] cotransport was stimulated and Na/H exchange was inhibited. Raising internal Mg had the opposite effect: [K-Cl] cotransport was inhibited and Na/H exchange was stimulated. Li loading, previously shown to stimulate Na/H exchange, inhibited [K-Cl] cotransport. From these reciprocal effects and from other evidence, we surmise that the regulation of Na/H exchange and [K-Cl] cotransport is conducted and coordinated by a discrete mechanism that responds to changes in cell volume and is sensitive to cytoplasmic Mg and Li concentrations.     

Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells

Dog red cell membranes contain two distinct volume-sensitive transporters: swelling-activated K-Cl cotransport and shrinkage- activated Na/H exchange. Cells were prepared with intracellular salt concentration and weight percentage of cell water (%cw) varied independently by transient permeabilization of the cell membrane to cations. The dependence of transporter-mediated Na and K influxes upon %cw and upon extracellular salt concentration (c(ext)) was measured in cells so prepared. It was found that the critical value of %cw at which transporters are activated, called the set point, is similar for the two transporters, and that the set points for the two transporters decrease similarly with increasing extracellular salt concentration. These findings suggest a common mechanism of regulation of these two transporters. Cellular Na, K, and Cl concentrations were measured as functions of %cw and c(ext). Using these data together with data from the literature for other solute concentrations, empirical expressions were developed to describe the dependence of the intracellular concentrations of all significant small molecule electrolytes, and therefore the intracellular ionic strength, upon %cw and c(ext). A mechanistic model for the dependence of the set point of an individual transporter upon intracellular ionic strength is proposed. According to this model, the set point represents a critical extent of association between the transporter and a postulated soluble regulatory protein, called regulator. Model functions are presented for the calculation of the thermodynamic activity of regulator, and hence extent of regulator- transporter association, as a function of total intracellular protein concentration (or %cw) and ionic strength. The experimentally observed dependence of set point %cw on c(ext) are simulated using these functions and the empirical expressions described above, together with reasonable but not uniquely determined values of model parameters.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

K-Cl cotransport in red blood cells from patients with KCC3 isoform mutants.

Red blood cells (RBCs) possess the K-Cl cotransport (KCC) isoforms 1, 3, and 4. Mutations within a given isoform may affect overall KCC activity. In a double-blind study, we analyzed, with Rb as a K congener, K fluxes (total flux, ouabain-sensitive Na+/K+ pump, and bumetanide-sensitive Na-K-2Cl cotransport, Cl-dependent, and ouabain- and bumetanide-insensitive KCC with or without stimulation by N-ethylmaleimide (NEM) and staurosporine or Mg removal, and basal channel-mediated fluxes, osmotic fragility, and ions and water in the RBCs of 8 controls, and of 8 patients with hereditary motor and sensory neuropathy with agenesis of corpus callosum (HMSN-ACC) with defined KCC3 mutations (813FsX813 and Phe529FsX532) involving the truncations of 338 and 619 C-terminal amino acids, respectively. Water and ion content and, with one exception, mean osmotic fragility, as well as K fluxes without stimulating agents, were similar in controls and HMSN-ACC RBCs. However, the NEM-stimulated KCC was reduced 5-fold (p < 0.0005) in HMSN-ACC vs control RBCs, as a result of a lower Vmax (p < 0.05) rather than a lower Km (p = 0.109), accompanied by corresponding differences in Cl activation. Low intracellular Mg activated KCC in 6 out of 7 controls vs 1 out of 6 HMSN-ACC RBCs, suggesting that regulation is compromised. The lack of differences in staurosporine-activated KCC indicates different action mechanisms. Thus, in HMSN-ACC patients with KCC3 mutants, RBC KCC activity, although indistinguishable from that of the control group, responded differently to biochemical stressors, such as thiol alkylation or Mg removal, thereby indirectly indicating an important contribution of KCC3 to overall KCC function and regulation.     

Extracellular ATP stimulates volume decrease in Necturus red blood cells

This study examined whether extracellular ATP stimulatesregulatory volume decrease (RVD) in Necturusmaculosus (mudpuppy) red blood cells (RBCs). Thehemolytic index (a measure of osmotic fragility) decreased withextracellular ATP (50 µM). In contrast, the ATP scavenger hexokinase(2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, theATP-dependent K⁺ channelantagonist glibenclamide (100 µM) increased the hemolytic index, andthis inhibition was reversed with ATP (50 µM). We also measured cellvolume recovery in response to hypotonic shock electronically with aCoulter counter. Extracellular ATP (50 µM) enhanced cell volumedecrease in a hypotonic (0.5×) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml)inhibited cell volume recovery. The inhibitory effect of hexokinase wasreversed with the Ca²⁺ ionophoreA-23187 (1 µM); it also was reversed with the cationophore gramicidin(5 µM in a choline-Ringer solution), indicating that ATP was linkedto K⁺ efflux. In addition,glibenclamide (100 µM) and gadolinium (10 µM) inhibited cell volumedecrease, and the effect of these agents was reversed with ATP (50 µM) and A-23187 (1 µM). Using the whole cell patch-clamp technique,we found that ATP (50 µM) stimulated a whole cell current underisosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide(100 µM), and gadolinium (10 µM) inhibited whole cell currents thatwere activated during hypotonic swelling. The inhibitory effect ofapyrase was reversed with the nonhydrolyzable analog adenosine5'-O-(3-thiotriphosphate) (50 µM), and the effect of glibenclamide or gadolinium was reversed withATP (50 µM). Finally, anionic whole cell currents were activated withhypotonic swelling when ATP was the only significant charge carrier,suggesting that increases in cell volume led to ATP efflux through aconductive pathway. Taken together, these results indicate thatextracellular ATP stimulated cell volume decrease via aCa²⁺-dependent step that led toK⁺ efflux.

     

Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride

Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl(-) transport. Reductions in either cell volume or cytosolic Cl(-) concentration ([Cl(-)](i)) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl(-)](i) was evaluated in model duck red blood cells. Replacement of [Cl(-)](i) with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl(-)](i) at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl(-)](i) is associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl(-)](i) accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl(-)] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl(-)]. The major effect of Cl(-) on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein.     

Comparison of K-Cl cotransport expression in high and low K dog erythrocytes.

K-Cl cotransport plays a crucial role in regulatory volume decrease of erythrocytes. K-Cl cotransport activities in dog erythrocytes with an inherited high Na-K pump activity (HK) and normal erythrocytes (LK) were compared. Nitrite (NO(2)) stimulated K-Cl cotransport activity in HK cells around 14-fold at 2.4 mM, and it also increased the Km value of this cotransporter. Real-time PCR and western blot analysis revealed that K-Cl cotransporter 1 was dominant, and that the quantity of K-Cl cotransporter 1 protein was comparable between HK and LK erythrocytes. These results suggest that the difference in cotransport activity was not caused by the amount of K-Cl cotransport protein but by a difference in the regulation system, which is susceptible to oxidant.     

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.     

Coordinated regulation of shrinkage-induced Na/H exchange and swelling- induced [K-Cl] cotransport in dog red cells. Further evidence from activation kinetics and phosphatase inhibition

Hypertonic shrinkage of dog red cells caused rapid activation of Na/H exchange and rapid deactivation of [K-Cl] cotransport. Hypotonic swelling caused delayed deactivation of Na/H exchange and delayed activation of [K-Cl] cotransport. Okadaic acid stimulated shrinkage-induced Na/H exchange and inhibited swelling-induced [K-Cl] cotransport. The data are compatible with the kinetic model of Jennings and Al-Rohil (1990. J. Gen. Physiol. 95:1021-1040) for volume regulation of [K-Cl] cotransport in rabbit red cells and suggest that in dog red cells Na/H exchange and [K-Cl] cotransport are controlled by a common regulatory system. The proposal of Jennings and Schulz (1991. J. Gen. Physiol. 96:799-817) that activation/deactivation of volume-sensitive transport involves phosphorylation/dephosphorylation of a regulatory protein is supported by these observations.