The control of ion movements in human red blood cells.

The thesis describes studies on the control of ion movements across membranes of human red blood cells. The first part deals with whether active and passive movements of sodium and potassium are influenced by changing the potential difference (p.d.) across the membrane. Replacement of external chloride with non-penetrating anions changed the chloride gradient and, as chloride was passively distributed, the p.d. was calculated. With low external chloride the intracellular chloride concentration was greater than that outside, indicating reversal of the sign of the membrane potential from -9 to +30 mV. Ouabain-sensitive (pump) sodium and potassium fluxes were little affected. Potassium efflux and ouabain-insensitive sodium efflux were increased ten- and three-fold respectively, whilst ouabain-insensitive potassium influx was halved and sodium influx unaffected. Thus, the main effect of reversing the chloride gradient was a marked increase in passive outward movements of sodium and potassium ions. Part Two describes a study of chemical reactions of the sodium pump enzyme in relation to the regulation of active sodium and potassium movements. The main aspect was to see whether p-nitrophenylphosphate (pNPP) hydrolysis by human red blood cells, ghosts and fragmented membranes was mediated by the sodium pump. In cells, pNPP uptake was inversely proportional to external chloride and the uptake mechanism was similar to that for inorganic phosphate. Hydrolysis of pNPP by both cells and ghosts was insensitive to external potassium and ouabain. Hydrolysis by fragmented membranes was partly sensitive to potassium and ouabain. The main finding was that there was a marked decrease in enzymic activity on raising the ionic strength of the medium, whereas there was no effect on ATPase activity. It is this effect, as well as the presence of a large amount of intracellular phosphatase, which accounts for the absence of a ouabain-sensitive component of pNPP hydrolysis when cells or ghosts are incubated in physiological electrolyte media.