The ability to regulate cell volume in the face of perturbation is a commonly observed property of cells (Parker, 1993;Hoffmann & Dunham, 1995). Shrinkage in response to an initial increase in volume is termed regulatory volume decrease (RVD); swelling in response to shrinkage is termed regulatory volume increase (RVI). In the short term, RVI and RVD are carried out by a variety of membrane transport systems, which use pre-existing electrochemical gradients for the dissipative movement of a range of solutes (or osmolytes). Water then passively follows these solutes by osmosis. Volume regulatory capacities, together with the identity of the transport systems, vary with cell type and species. This is well illustrated by examining the pattern of volume regulatory responses found in vertebrate red cells (Cossins & Gibson, 1997), whose ease of procurement and homogeneity have made them a popular choice for the study of such processes. Avian red cells possess a powerful Na¤-K¤-2Cl¦ cotransport system (

SUMMARYPotassium (using 86Rb+ as a tracer), amino acid and taurine fluxes were measured in horse red blood cells (RBCs). No volume-sensitive component of alanine and glycine transport was observed, and although volume-sensitive taurine fluxes were observed in most animals, their absolute magnitudes were small. K+ fluxes, however, were shown to be particularly volume sensitive; they were stimulated by cell swelling and inhibited by cell shrinkage. Sizeable fluxes were present at normal cell volumes. The volume-sensitive K+ flux was Cl-dependent and was abolished by Cl-replacement with methylsulphate. The Cl--dependent K+ fluxes in horse red blood cells were stimulated by lowering in external pH to 6-9 and by treatment with the sulphydryl-reacting agent, N-ethylmaleimide. They were inhibited by the potent K+-Cl cotransport inhibitor, DIOA, ([(dihydroindenyl)oxy]alkanoic acid) but were insensitive to the Na+-K+-Cl-co-transport inhibitors, frusemide and bumetanide. A Cl-channel inhibitor, 5-nitro-2-(phenylpropyl-amino)-benzoate (NPPB), produced partial inhibition. These results suggest that regulatory volume decrease in horse red blood cells is achieved predominantly by volume-sensitive K+ efflux mediated via a K+-Cl-co-transport system with similar properties to those observed in the red blood cells of other species. The significance of these findings and their rheological consequences are discussed.

The effect of urea and its interactions with oxygen tension (PO2), cell volume and inhibitors of protein phosphatases/kinases (PP/PK) on the K influx into equine red blood cells were studied. K influx was measured using 86Rb as a radioactive tracer for K. As in other species, Cl-dependent K influxes were stimulated by urea, with peak fluxes occurring at about 750 mM. This effect was not mediated via changes in cell volume or following formation of cyanate, the hydrolysis product of urea. Stimulation by urea was prevented by pre-treatment with calyculin A (100 nM) at all urea concentrations tested. At low concentrations, urea-stimulated influx was O2 dependent, and sensitive to changes in cell volume and subsequent treatment with calyculin A. By contrast, at high concentrations, urea-stimulated influxes were largely unaffected by these manipulations. Like pharmacological manipulations, e.g. by N-ethylmaleimide, staurosporine and depletion of intracellular Mg by A23187, but unlike cell swelling per se, urea was able to affect transport regardless of PO2. K-Cl cotransport in cells treated with N-ethylmaleimide (1 mM) alone, or with combinations of N-ethymaleimide and calyculin A, was no longer stimulated by addition of urea, rather it was inhibited. Results are consistent with urea acting predominantly as a direct inhibitor of the regulatory PK, with a smaller inhibitory effect downstream of this phosphorylation step possibly on the transporter itself.

SUMMARYPotassium transport was measured in equine red blood cells, using 8fRb+ influx as a convenient assay. A significant component of volume-and pH-sensitive K+-Cl-cotransport to the overall K+ flux was observed in all blood samples studied, although fluxes were variable between animals, and within individuals when measured at intervals over a period of weeks. The aryloxyacetic acid [(dihydroindenyl)oxy]alkanoic acid (DIOA), at a final concentration of 100jUM, inhibited most (>95 %) of the Cl--dependent K+ flux, and DIOA sensitivity was therefore used to define the activity of the K+-Cl-cotransporter. K+-Cl-cotransport was also sensitive to protein phosphatase inhibition with calyculin A or okadaic acid, with inhibition constants of 9 + 1 nm for calyculin and about 100 nm for okadaic acid. Peak fluxes were observed at an extemal pH of 6-7-7 0, with inhibition at higher and lower values. Volumesensitive K+ fluxes assayed in autologous plasma, controlled for osmolality, pH and potassium concentration, were significantly lower (28 + 8 % of control values, n = 6) than those measured in saline. This inhibition was mimicked by the culture medium RPMI, but disappeared following dialysis of the plasma. Phosphate (5 6 mM) inhibited volume-sensitive K+ fluxes by 48 + 2 %, n = 3; no significant effect was observed by increasing external magnesium concentrations to 0.5 or 2 mm. Thus, inhibition by RPMI, but not that by plasma, may be due to phosphate. Finally, volume-and pH-sensitive K+ fluxes were sensitive to oxygen tension and were abolished reversibly by equilibrating solutions with nitrogen, as opposed to air. Use of solutions equilibrated with different values of PO, may account for some of the variability in equine red blood cell KCI fluxes. The importance of these observations to equine red blood cell homeostasis and haemodynamics is discussed.

The effect of oxidants on K(+)-Cl(-) cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O(2). The substituted benzaldehyde, 12C79 (5 mM), markedly increased O(2) affinity. In N(2), however, O(2) saturation was low (<10%) but KCC remained active. Nitrite (NO(2)(-)) oxidized heme to methemoglobin (metHb). High concentrations of NO(2)(-) (1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O(2) independent but remained sensitive to other stimuli. 1-Chloro-2, 4-dinitrobenzene (1-3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O(2)-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.