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SUMMARY1. In the large single muscle fibres from the barnacle Balanus nubilus, the total fibre Mg concentration was estimated as 1541 m-mole/kg wet wt., of which about 3-3'5 m-mole/kg wet wt. was extracellular. The diffusible Mg, measured by internal sampling, was 11P5 m-mole/kg wet wt., of which at least half may be completed to larger diffusible molecules. The free ionized Mg level was estimated as < 5 m-mole/kg wet wt.2. The loss of [28Mg]MgCl2 from both Maia and Balanus muscle fibres following axial micro-injection approximated to first-order kinetics. The maximum rate constant for the loss was 1-51 + 0-20 (S.E.) X 10-5 sec-1 for Balanus (sixty-seven fibres) and 106 + 0-46 (s.E.) x 10-5 sec-1 for Maia (seven fibres) at 20-25°C.3. The calculated Mg efflux was in the range 6-12 p-mole/cm2. sec based on this rate constant, assuming isotopic equilibration internally and that the surface area of the fibres approximated to that of a simple cylinder. If account was taken of the area of the cleft system the efflux was reduced by about fifteen times.4. The diffusion coefficient for injected 28Mg was estimated as 2-3 x 10-6 cm2 sec-1, about half the value in free solution. Injections of 2 M-MgCl2 or 200 mM-EDTA subsequent to the injection of the isotope caused about a 30 % reduction in the tracer efflux.5. External application of salines containing 100 mM-Ca or Mg caused a rapid but reversible inhibition of the magnesium efflux. Similar effects were observed with salines containing 32 mM-Co or Mn chlorides or 1-2 mM-La or Gd chlorides. Polyarginine (200 jug/ml.) had no effect.6. The Mg efflux had a Q10 of 3-4 over the temperature range of about 5-20' C. It was irreversibly inhibited by the sulphydryl reagent NEM (1 mM), but PCMBS (0-2-2 mM) had no effect. Contractile agents (5 mM caffeine or 200 mM-K salines) and a variety of inhibitors of ion movement C. C. ASHLEY AND J. C. ELLORY or active transport had no appreciable effect on the Mg efflux. Lowering the pH of the saline from 7 to 5 produced a 70 % reduction in the efflux which was reversible over short periods of application.7. Replacement of external Na, but not Ca or Mg, with Li, choline or sucrose caused a rapid and partially reversible reduction of the Mg efflux, but increasing the internal Na by micro-injection in zero Na salines had no consistent effect. It is suggested that the extrusion of Mg from these muscle cells is largely dependent upon the inward movement of Na.

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.

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.

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