The intracellular pH (pH;) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of ["C]5,5-dimethyloxazolidine-2,4-dione (DMO). At an extracellular pH (pH.) of 7 .40 in nominally C0 2-free medium, the steady state pHi using either indicator was -7 .25. When pH. was suddenly raised from 7.40 to 8.40 in the nominal absence of C02, pH i slowly rose by -0 .35 during the subsequent hour . A change of similar magnitude in the opposite direction occurred when pH. was reduced to 6 .40. Both changes were reversible . Intrinsic intracellular buffering power, determined by using graded pulses of C02 or NH 4CI, was^"50 mM/pH over the pH i range of 6.8-7 .9 . The course of pH; obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% C02 or by prepulsing with 30 mM NH 4CI, while pH . was maintained at 7.40. In both instances, pHi (6 .80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min . These rates were reduced by -90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-Dglucamine. Recovery was not affected by 1 mM SITS or by 40 mM a-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils . Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4CI or by prepulsing with 18% C0 2, both at a constant pH . 7 .40. In both instances, pHi, which was 7 .65 and 7.76, respectively, recovered to the control value. The recovery rates (0 .033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0 .1 mM) were ineffective . The rate of recovery from NH4CI-induced alkalinization was enhanced twofold by adding 1 mM HC03/0 .2% C02 to the medium (pHo 7.40) . When the membrane was depolarized from -53 to 0 mV in 115 mM K, the recovery during NH 4CI exposure was reduced by -30%, whereas the inward driving force on NH4 was reduced by -80% . Apparently, the entry of NH4 by electrodiffusion plays only a minor role in the recovery . These results make it

The relationship of intracellular pH (pH1) to superoxide radical (02) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pHo 7.40), led to a rise in pH1 from 7.22 to 7.80. This pH, change was sensitive to amiloride (apparent K1 78 MM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated 02 production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pH, transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, 02-release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for 02-generation. By independently varying either pH0, pH1, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pH, transients could be altered. In each instance, the amount of 02-release correlated directly with pHi and was enhanced by intracellular alkalinization.In the absence of FMLP, a rise in pH, to 7.7-7.8 by exposure of cells to 30 mM NH4CI, 10 MM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in 02 generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHj 1 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of 02 under normal incubation conditions. However, these cells could be induced to generate 0°when the degree of alka tion was enhanced by internal Na depletion or by pretreatment with 18% Co2. Together, these results indicate a modulating effect of pH3 on 02 production and suggest that other functional responses of neutrophils may be regulated by their pH1.

Human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction in response to activation by the synthetic chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine.

Chloride content and fluxes were measured in isolated resting human peripheral polymorphonuclear leukocytes. The intracellular Cl concentration of cells kept at 37°C in 148 mM Cl media was -80 meq/liter cell water, fourfold higher than expected for passive distribution at the cell's estimated membrane potential (approximately -53 mV). All intracellular Cl was rapidly exchangeable with external "Cl. Cells lost CI exponentially into Cl-free media, and reaccumulated it when Cl was restored to the bath ; this reuptake was dependent on metabolism . One-way s1CI fluxes in steady state cells were -1 .4 meq/liter min. The bulk (-70%) of these represented electrically silent CI/CI exchange mediated by a carrier insensitive to disulfonic stilbenes but blocked by the anion carrier inhibitor a-cyano-4-hydroxycinnamate (CHC). The remaining fluxes were characterized in some detail . About 20% of "CI influx behaved as active transport: it moved thermodynamically uphill and was absent in cells treated with 2-deoxy-D-glucose, displayed Michaelis-Menten kinetics with K,n(Cl) = 5 mM, V... = 0.25 meq/liter min, and was inhibited by CHC (K ; = 1 .7 mM), ethacrynate (Ki = 50 wM), and furosemide (K, = 50 1M). About 30% of Cl efflux and^-8% of Cl influx behaved as electrodiffusion through a low-permeability pathway (Pc, = 4 x 10 -s cm/s ; gC, = 1 'US/cm 2; PK/PNa/PC, = 10 :1 :1); these fluxes were linear with concentration and strongly voltage sensitive . The putative Cl channel does not appear to be voltage gated, and gives evidence of single filing.

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured . In cells kept at 37°C, [Na]i was 25 mM and [K]i was 120 mM ; both ions were completely exchangeable with extracellular isotopes . One-way Na and K fluxes, measured with 22 Na and 42 K, were all -0 .9 meq/liter cell water -min . Ouabain had no effect on Na influx or K efflux, but inhibited 95 t 7% of Na efflux and 63% of K influx . Cells kept at 0°C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h) ; upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced . External K stimulated Na efflux (Km^-1 .5 mM in 140-mM Na medium) . The PNa/PK permeability ratio, estimated from ouabaininsensitive fluxes, was 0.10 . Valinomycin (1 AM) approximately doubled PR .Membrane potential (V.) was estimated using the potentiometric indicator diS-C3(5) ; calibration was based on the assumption of constant-field behavior .External K, but not Cl, affected Vm . Ouabain caused a depolarization whose magnitude depended on [Na]i . Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin ; this hyperpolarization was abolished by ouabain . We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3 .7-4 .0 /IS/ cm 2) computed from the individual K and Na conductances .