PROTON (OR HYDROXIDE) FLUXES AND THE BIPHASIC OSMOTIC RESPONSE OF HUMAN RED-BLOOD-CELLS

Upon exposure of human red blood cells to hypertonic sucrose, the fluo rescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyani ne iodide, denoted diS-C3(5), displays a biphasic time course indicati ng the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stil bene)-sensitive decline of the voltage. In addition to monitoring memb rane potential, proton (or hydroxide) fluxes were measured by a pH sta t method, cell volume was monitored by light scattering, and cell elec trolytes were measured directly when red cells were shrunken either wi th hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mueq/g Hb.min and a Cl shift of 21-31 mueq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the ce lls are initially close to Donnan equilibrium and exhibit no detectabl e shift of Cl or protons. Experiments with the carbonic anhydrase inhi bitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs -Stewart cycle contribute to dissipation of the increased outward Cl c oncentration gradient. With maximally inhibitory concentrations of eth oxzolamide, a residual proton efflux of 2 mueq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is co mpletely inhibited by DIDS. The ethoxzolamide-insensitive apparent pro ton efflux may be driven against the electrochemical gradient, and is thus consistent with HCI cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibrat ion after shrinkage of human red blood cells by hypertonic sucrose occ urs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induc ed apparent proton efflux is mediated by the Jacobs-Stewart cycle, wit h the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).