Clin Biochern, Vol. 24, pp. 241-247, 1991 Printed in Canada. All rights reserved.

0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.

Regulation of Cytoplasmic pH in Phagocytic Cell Function and Dysfunction SERGIO GRINSTEIN, 1 CAROL J. SWALLOW, 2 and ORI D. ROTSTEIN 2 1Division of Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto M5G 1X8 and 2Department of Surgery, Toronto General Hospital and University of Toronto, Toronto, Canada To ensure effective antimicrobial or tumouricidal function, phagocytic cells must maintain their cytoplasmic pH (pHt) at a level conducive to optimal intracellular enzyme activity. The mechanisms by which neutrophils and macrophages regulate their cytoplasmic pH include bicarbonate-independent ion transport systems, most notably the Na+/H + exchanger, and bicarbonate-dependent ion transport systems, which can be subdivided into the cation-independent and Na+-dependent forms of chloride/bicarbonate exchange. In addition, macrophages have been shown to recover from intracellular acid loading by means of an ATP-dependent proton extrusion mechanism, which has the characteristics of a vacuolar-type H÷ ATPase. In the microenvironment typically associated with abscesses, the low extracellular pH and the presence of short chain fatty acid by-products of bacterial metabolism tend to induce cytoplasmic acid loading. In this setting, the ability of the various pH~ regulatory mechanisms to protect pHi may be overcome, leading to cytoplasmic acidification. Several investigators have shown that cytoplasmic acidification impairs the ability of neutrophils to migrate in response to chemotactic stimuli, and also impairs their ability to generate a respiratory burst, thus inhibiting the release of toxic oxygen radicals. This may result in the inability of phagocytes to effect complete abscess resolution.

KEY WORDS: acid-base; phagocytic cells; cytoplasmic pH; Na+/H + antiport; H ÷ ATPase. Introduction

Regulation of

pH i

in p h a g o c y t i c cells

The regulatory movement of proton equivalents across the plasma membrane of phagocytes appears to be, to a large extent, tightly coupled to the movement of other inorganic ions. For the most part, these coupled fluxes are passive, driven by the electrochemical gradient of the inorganic ions. These, in turn, can be passively distributed across the plasma membrane, or driven across the membrane by an active, energy-consuming process. The proton equivalent transporting systems of mammalian cells can be arbitrarily divided into two groups, those that are independent of the presence of bicarbonate and those that are not. B I C A R B O N A T E - I N D E P E N D E N T TRANSPORT SYSTEMS

p

hagocytic cells are frequently required to function under conditions which threaten their cytoplasmic pH (pHi), such as the acidic microenvironment typically prevailing in tumours and abscesses. At low extracellular pH, the cytoplasm of phagocytic cells tends to become acidic due to inward leakage of proton equivalents, a process that is facilitated by the presence of such permeable weak organic acids as succinic, butyric and propionic acids, which are by-products of bacterial metabolism. Yet, maintenance of pH i within a narrow range is essential since departures from the normal pH i of - 7.2 can impair phagocytic cell chemotaxis and bactericidal or tumouricidal activity. Thus, ac-

Correspondence: Sergio Grinstein, Division of Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8 Canada. Manuscript received June 18, 1990; revised September 20, 1990; accepted November 1, 1990. CLINICAL BIOCHEMISTRY,

tive mechanisms must exist to protect pHi, counteracting the spontaneous tendency of the cells to become acidotic and enabling them to function in acidic environments. The object of this paper is to briefly review the mechanisms whereby pH i is regulated in neutrophils and macrophages and the importance of phi homeostasis to phagocytic cell physiology and pathophysiology.

VOLUME

24, J U N E

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The most prominent of the bicarbonate-independent transporters is the Na+/H + exchanger or antiport (1,2). In its physiological or "forward" mode, this system exchanges extracellular Na + for internal H ÷ (Figure 1, I). The direction of transport under physiological conditions is dictated by the combined gradients of Na + and H +, Because the Na+/K + pump induces the formation of a large inward electrochemical Na + gradient, the potential energy inherent in this gradient can be utilized to power the ejection of protons by the antiport. The antiport is extremely cation selective, transporting Na + and also to a lesser extent Li ÷, but not K ÷, Rb +, Cs +, monovalent organic cations or divalent cations. Because the stoichiometry of the exchange is one Na ÷ for one H ÷, the transport cycle is electroneutral. The rate of Na+/H + exchange is a saturable function of the external Na ÷ concentration, following Michaelis-Menten kinetics. This behavior is consistent with translocation of a single Na + ion per transport cycle. In view of the one-to-one stoichi241

GRINSTEIN, SWALLOW, AND ROTSTEIN EXTRACELLULAR

H*

cl-

*,

I

V

III Ngr~l~ .~. . .' \ ,

He03

...

~

+P~

.co; CYTOPLASM ,~f' " , ~



Figure 1--Mechanisms of cytoplasmic pH regulation in phagocytic cells. This diagram illustrates the best-characterized plasma membrane ion-transport systems which serve to regulate cytoplasmic pH in neutrophils and/or macrophages. (I) Na+/H + antiport; (H) Na+-dependent HCO3-/Cl- exchanger; (HI) cation-independent HCO3-/ C1- exchanger; (IV) H + ATPase.

ometry of the exchange process, a single proton equivalent must be ejected per cycle. Whereas forward transport is stimulated by elevating the external Na + concentration, it is inhibited by increasing external [H+]. This is thought to result from competition between Na ÷ and H ÷. Inhibition is also observed in the presence of any one of a family of pyrazine derivatives, typified by amiloride (3). In most reported instances, amiloride inhibits exchange competitively, interacting externally with the Na + binding site. Finally, inhibition of net forward Na+/H + exchange has also been observed when the internal Na + concentration is elevated, as expected from competition with intracellular H ÷ for the inwardfacing transport site. In contrast to the inhibitory effect of extracellular protons, a pronounced stimulation of forward Na+/H + exchange is observed when the cytoplasm is acidified. The relationship between internal proton concentration and the rate of exchange suggests a cooperative process. This effect has been attributed to the existence of a cytoplasmic allosteric site which, when protonated, stimulates the rate of transport (1,2). This putative allosteric site is thought to be different from the site that binds the protons to be transported across the membrane. By modifying the rate of Na+/H + exchange, the allosteric site is thought to confer on the system its pH i regulatory property. Thus, Na+/H + exchange will take place at significant rates only if the cytoplasm is acidified, when the allosteric controlling site is protonated. Conversely, transport will essentially cease once the normal resting pH i has been attained, due to deprotonation of the allosteric modifier. Under certain conditions, the "set point" of the allosteric site of the antiport can be reset upward,

242

rendering the cytoplasmic pH more alkaline. This has been reported to occur in neutrophils (4-6) and macrophages (7) stimulated with chemotactic agents or phorbol esters. The molecular mechanism underlying this shift is not understood. However, as discussed in detail below, the resulting cytosolic alkalinization can stimulate chemotaxis and oxygen radical production, potentially enhancing the bactericidal capability of the cells. BICARBONATE-DEPENDENT TRANSPORT SYSTEMS

In a variety of cell types, two widely distributed bicarbonate-transporting systems are known to participate in pH i regulation. Both systems countertransport chloride for bicarbonate, but they differ in their requirement for Na +. One type of exchanger is independent of cations, while the second type requires the presence of Na +. In contrast to the Na+/H + antiport which is ubiquitous, only the cation-independent anion exchanger has been reported to exist in h u m a n neutrophils (8). In U937 cells, a monocytic cell line, the Na+-dependent but not the cation-independent system was found to be present (9).

Cation-independent chloride~bicarbonate exchange The cation-independent chloride/bicarbonate exchanger (Figure 1, III) translocates anions across the plasma membrane with a stoichiometry of oneto-one and, like the Na+/H + antiport, is therefore electroneutral. This anion exchanger also transports other halides and larger, polyvalent anions such as sulfate and phosphate, in addition to its main physiological substrates, chloride and bicarbonate. In most cell types, chloride/bicarbonate exchange is inhibited by externally added stilbene derivatives such as 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) (10). However, neutrophils seem to be particularly insensitive to these agents (8). Instead, cation-independent exchange in these cells can be inhibited by cyanohydroxycynnamic acid derivatives at millimolar concentrations (8). In h u m a n red cells, where it has been studied most extensively, chloride/bicarbonate exchange is mediated by a polypeptide of molecular weight 95,000100,000, known as capnophorin or '%and three." Its abundance and large transport capacity in red cells have facilitated the thorough kinetic, biochemical and pharmacological characterization of the anion exchanger, culminating in the elucidation of its primary sequence in erythroid cells (see 10, 11 for reviews). The putative role of the cation-independent chloride/bicarbonate exchanger in pH i regulation has been investigated in several m a m m a l i a n tissues and cells, including phagocytes (8). Because the in-

CLINICAL BIOCHEMISTRY, VOLUME 24, JUNE 1991

CYTOPLASMIC PH IN PHAGOCYTES

ward gradient for chloride exceeds that for bicarbonate in alkalotic cells, the direction of net transport predicted for an electroneutral one-to-one exchange influx of chloride and effiux of bicarbonate, leading to intracellular acidification. Accordingly, recent studies have suggested that chloride/bicarbonate exchange may play a role in acidifying alkaline-loaded cells, operating at pHi values where the Na+/H + antiports are largely quiescent (8). Under normal resting conditions, the combined chloride and bicarbonate gradients will also drive the uptake of acid equivalents into the cells. This phenomenon would compound the spontaneous tendency of the cells to acidify due to metabolic proton generation and electrophoretic accumulation of H ÷ equivalents. Recent evidence indicates that the chloride/ bicarbonate exchanger of nucleated mammalian cells, unlike that of red cells, is greatly inhibited at pH i levels of - 7.2 or below (12). Thus, the cation-independent exchanger can function effectively in the defense of intracellular pH in the alkaline range, without contributing significantly to spontaneous acidification under physiological conditions.

Na+-dependent chloride~bicarbonate exchange Whereas cation-independent chloride/bicarbonate exchange has long been known to operate in mammalian cells, the Na÷-dependent anion exchanger was, for a long time, believed to exist only in invertebrates. More recently, however, a Na+-cou pled chloride/bicarbonate exchange system (Figure 1, H) has been demonstrated in several mammalian cells. This transporter normally exchanges extracellular Na ÷ and bicarbonate for intracellular chloride and possibly one proton, with a stoichiometry of 1 Na+: 1 CI-: 2 acid/base equivalents. Several modes of transport have been proposed, including the exchange of one Na ÷ and one bicarbonate for one chloride and one proton. An alternative model has envisaged exchange of the NaCO3- ion pair for one C1-. In mammalian cells, the stoichiometry has not been defined. As is the case for the cation-independent exchanger, the Na+-dependent chloride/bicarbonate transporter of m a m m a l i a n cells, including the U937 line, is inhibited by stilbene derivatives such as DIDS and SITS (9,12). In contrast, despite being Na ÷ dependent, this system is insensitive to amiloride and thus distinct from the Na+/H + antiport. Yet, like the latter, the Na+-dependent chloride/bicarbonate exchanger is quiescent at alkaline pHi but becomes activated as pH i falls below a certain threshold, which is close to the physiological pH i of ~ 7.2. Due mainly to the inward Na + gradient, the Na +dependent anion exchanger is thermodynamically poised to extrude acid from resting as well as from acid-loaded cells. For these reasons, it is considered to be a functional acid secretion mechanism, which may be important in the maintenance of the rest-

CLINICAL BIOCHEMISTRY, VOLUME 24, JUNE 1991

ing pH i and in preventing acidosis during metabolic activation. Indeed, under conditions where Na+/H + exchange is impaired (e.g., in the presence of amiloride or its analogs), cells can maintain pH i within the physiological range provided bicarbonate is present. This most likely reflects the operation of Na +-dependent chloride/bicarbonate exchange. Despite their similar pharmacology (i.e., susceptibility to stilbene derivatives), it is generally believed that the Na÷-dependent and cation-independent modes of chloride/bicarbonate exchange are mediated by separate and distinct molecular entities. This is suggested by the fact that some cell types seem to display only one of the transport modalities (e.g., Na+-dependent exchange in U937 cells). Moreover, in cells possessing both transport systems, differential sensitivity to certain inhibitors has been demonstrated. Thus, ethacrynic acid preferentially inhibits cation-independent chloride/ bicarbonate exchange, with little effect on the Na +dependent system, whereas picrysulfonate has the opposite effect (13).

Electrogenic bicarbonate transport A third type of bicarbonate transporting system has been described in the basolateral membrane of some epithelial cells. Unlike the mechanisms described above, bicarbonate transport by this epithelial system is independent of the presence of chloride and is not electroneutral. It is thought to involve the co-transport of one Na ÷ with 2 or most likely 3 bicarbonate ions. The negative charge of the transported complex renders the process electrogenic and therefore susceptible to changes in membrane potential. It is not clear whether this system is present in neutrophils or macrophages, or whether the electrochemical gradient of Na + and bicarbonate is suited to the net uptake of bicarbonate, which would be required to regulate pHi in resting or metabolically activated cells. PROTON EXTRUSION PUMPS

Conceivably, acid extrusion from the cytoplasm could also be accomplished by primary active, ATPdriven processes. Indeed, proton-pumping ATPases have been described in a variety of intracellular membranes. However, with the exception of specialized, acid-secreting cells in the renal and gastric epithelia, proton pumps have not been detected in the plasma membranes of other mammalian cells. Recently, however, we have obtained evidence which is consistent with the existence of proton pumps in the plasma membrane of murine peritoneal macrophages (Ref. 14, Figure 1, IV). We found that, following acid-loading, these cells were able to regain near normal pHi in the absence of Na ÷ and in the nominal absence of bicarbonate

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GRINSTEIN, SWALLOW,AND ROTSTEIN (Figure 2B). Therefore, a process other than Na+/ H + exchange or Na+-coupled chloride/bicarbonate exchange must be implicated. This conclusion was strengthened by the observation that pH i recovery proceeded in the presence of amiloride (Figure 2A) or its analogs and even after the cells were treated with disulfonic stilbenes (Figure 2C). Instead, recovery was blocked by N-ethylmaleimide and by N,N'-dicyclohexylcarbodiimide, which are powerful inhibitors of proton-pumping ATPases in other systems (15). Moreover, the Na +- and bicarbonate-independent p h i recovery was virtually eliminated in ATP-depleted cells, strongly suggesting the involvement of a primary active proton extrusion process. Additional evidence indicated that the cytoplasmic alkalinization was paralleled by an increased rate of acid extrusion into the medium, demonstrating that trans-membrane flux of proton equivalents occurred. The existence of a plasma membrane H ÷ pump could account for most, if not all of the above observations. Recent studies have shown that this ATP-dependent proton extrusion process is markedly sensitive to the highly selective inhibitor of vacuolar-type H + ATPases, bafilomycin A1, implying that it is mediated by H ÷ ATPases of the vacuolar type located in the macrophage plasma membrane (16). The substantial pH i recovery displayed by macrophages in the nominal absence of Na + and bicarbonate has not been reported in other non-epithelial animal cells, such as fibroblasts and lymphocytes. It is conceivable that an additional pHi regulatory mechanism was developed by macrophages and perhaps also neutrophils to ensure their survival in the acidic milieu of turnouts and abscesses. The low extracellular pH that prevails in these environments would tend to inhibit Na÷/H + exchange by competition between Na + and H ÷ at the external transport site and would also reduce the concentration of bicarbonate, which is required by the bicarbonate-dependent pH regulatory systems. Under these conditions, proton pumping might be essential for the maintenance of pHi.

Roleof pHiin r e g u l a t i n g

A

Na +

~MILORIDE

PHi

I 2rain I

6.4 7.3

- -

B

NIGERICIN

K+

PHi

NMG +

6.47.3C

NIGERICIN

PH i

6.4-

p h a g o c y t i c cell

function

Having defined the mechanisms of pH i regulation in phagocytic cells, consideration should be given to the effects of changes in cytoplasmic pH on their function. Because relatively little functional information is presently available regarding macrophages, the following discussion is devoted mainly to neutrophils. In broad terms, neutrophil functions can be divided into two general areas: 1) migration and 2) microbicidal activity. MIGRATION Alterations in cytoplasmic pH appear to have two distinct effects on neutrophil migration. Sev244

7.3-

Figure 2--Na ÷- and HCO3--independent cytoplasmic pH recovery in acid-loaded macrophages. Cytoplasmic pH was measured fluorimetrically using the pH-sensitive cytoplasmic dye, 2', 7' -biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Macrophages were acid loaded by preincubation with 40 mM NH4C1 for 15 rain at 37 °C, followed by resuspension in NH4+-free medium. Traces start upon resuspension of cells in 2 mL of the indicated medium. All media were nominally HCOs--free. (A) cells resuspended in 140 mM NaCl medium with or without 800 ~M amiloride; (B) cells resuspended in Na÷-free medium with 140 mM of either KC1 or N-methyl-D-glucamine (NMG÷) C1; (C) cells resuspended in Na+-free, 140 mM KC1 medium containing 4, acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 1 raM). The K+/H + ionophore nigericin (1 }zM) was added where indicated by arrows, for purposes of calibration. Traces are representative of at least three experiments. The pH of all media was 7.35. Temperature: 37 °C. (From Ref. 14.) CLINICAL BIOCHEMISTRY,VOLUME 24, JUNE 1991

C Y T O P L A S M I C PH IN P H A G O C Y T E S

eral lines of evidence suggest that the initial acidification phase associated with exposure to chemotactic factors such as formyl-methionyl-leucylphenylalanine (f-met-leu-phe) plays a role in initiating cell migration (17,18). This conclusion is derived from studies which accept the premise that changes in cytoskeletal structure, as evidenced by actin polymerization, signify an early event in cell migration (19). These include: 1) cell acidification, whether induced by agonist or by weak acids, initiates polymerization of actin. Like the agonist-stimulated response, the induction of actin polymerization by acidification appears to be, at least in part, mediated through G proteins, as evidenced by its sensitivity to pertussis toxin (20); 2) prevention of agonistinduced or weak acid-induced acidification with protonophores obviates actin polymerization; 3) the initiation of acidification and actin polymerization are coincidental. In spite of these correlations, the question of whether acidification is necessary for the initiation of actin polymerization and subsequent cell migration requires further study. The finding that neutrophils from patients with chronic granulomatous disease exhibit normal migration, despite their failure to acidify following stimulation with an agonist (21), makes it unlikely that acidification alone is the critical signal for migration. Regardless of whether acidification signals actin polymerization, it is clear that alterations in cytoplasmic pH appear to modulate the chemotactic response to various stimuli. Simchowitz and Cragoe (22) demonstrated a linear correlation between net migration (the difference between directed and random motility) and pH i over an intracellular pH range from 6.8 to 8.6. In general, cytoplasmic acidification impaired migration, whether acidification was achieved through inhibition of the Na+/H + antiport or through acid loading of the cell. These results are supported by other studies in which pHi was not measured directly, but was effectively reduced by manoeuvers such as reducing extracellular pH or substituting extracellular Na + with other cations (23-25). At elevated pHi levels, Simchowitz and Cragoe (22) found that migration was stimulated. These authors postulated a major role for pH i in the regulation of actin polymerization as a mechanism for its modulatory effect on cell migration. In keeping with the demonstrated stimulation of neutrophil locomotion at alkaline pH i levels, alkalinization has been associated with an increased polymerization of actin in several other cell systems (26). RESPIRATORY BURST

Toxic oxygen radicals generated following stimulation of the neutrophil respiratory burst are an integral part of the neutrophil's microbicidal armamentarium. Reduction of pH i impairs activation of the respiratory burst in response to both f-met-leuphe (27) and the phorbol ester 12-O-tetradecanoylCLINICAL BIOCHEMISTRY, VOLUME 24, JUNE 1991

phorbol-13-acetate (TPA) (28). This latter effect suggests that cellular acidification causes inhibition via an effect on protein kinase C or on a later stage of activation. In this regard, it has been reported that the NADPH oxidase system displays a pH optimum in the 6.8 to 7.9 range in vitro (29). Thus, inhibition of this enzyme system could account for the observed results. By contrast, an alternate explanation must be cited to account for the stimulation of superoxide production at pHi levels >7.22, as reported by Simchowitz (27). While the mechanism underlying this enhancement was not specifically studied, it did not appear to be due to increased receptor-ligand interactions at elevated pH.

Potential implications of altered p h a g o c y t e pH i in vivo The pH in the purulent centre of clinical abscesses has been reported to be as low as 5.7 (30). This average pH value likely underestimates the true degree of acidification in some localized areas of the abscess where metabolic activity may be accelerated. Nevertheless, such low extracellular pH levels are capable of reducing neutrophil cytoplasmic pH to levels at which both cell migration and microbicidal activity are markedly reduced. In addition, in abscesses containing anaerobic bacteria, the short chain fatty acid by-products of bacterial metabolism may further exaggerate pHi reduction and thus result in further inhibition of neutrophil function. Support for this hypothesis is derived from studies in which the short chain fatty acid, succinate, a by-product of anaerobic bacterial metabolism, was examined for its effect on human neutrophil pHi and oxygen consumption in response to stimulation with the phorbol ester, TPA, at several different extracellular pH levels (31). At extracellular pH 6.5 or less, the presence of 30 mM succinate in the incubation medium significantly reduced neutrophil pH i when compared to succinate-free medium. Presumably, the weak acid operated as a protonophore, shuttling protons across the plasma membrane. The augmentation of cytoplasmic acidification effected by succinate at low pH accounted for the virtually complete abolition of the respiratory burst, as measured by oxygen consumption. These studies have been extended by investigation of crude Bacteroides fragilis culture filtrates. This bacterial species, which is the anaerobe most frequently recovered from intra-abdominal infections, was shown to secrete a factor(s) during its growth in vitro which impaired both neutrophil migration (32) and killing functions. Subsequent studies demonstrated that short chain organic acids generated by this species aider it reached the stationary phase of growth (as would occur in abscesses) were entirely responsible for the inhibition, and also that the inhibitory effect was due to the ability of these metabolic acids to reduce neutrophil pHi. In this regard, it is noteworthy that both low pH (30) 245

GRINSTEIN, SWALLOW,AND ROTSTEIN and high fatty acid concentrations have been measured in clinical abscesses containing Bacteroides species (33). This suggests a possible role for alterations in the cytoplasmic pH of phagocytic cells as a pathogenic mechanism contributing to the formation and persistence of abscesses in vivo.

Acknowledgments Research in the authors' laboratories is supported by the Medical Research Council of Canada, the Canadian Cystic Fibrosis Foundation and the National Cancer Institute. S. G. is the recipient of a Medical Research Council Scientist Award. C. J. S. is the recipient of an Ethicon-Society of University Surgeons Surgical Research Fellowship and a Medical Research Council of Canada Fellowship.

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ride antiport. J Biol Chem 1987; 262: 7486-91. 14. Swallow CJ, Grinstein S, Rotstein OD. Cytoplasmic pH regulation in macrophages by an ATP-dependent and N,N'-dicyclohexylcarbodiimide-sensitive mechanism. Possible involvement of a plasma membrane proton pump. J Biol Chem 1988; 263: 19558-63. 15. A1-Awqati Q. Proton-translocating ATPases. A n n u Rev Cell Biol 1986; 2: 179-200. 16. Swallow CJ, Grinstein S, Rotstein OD. A vacuolar type H+-ATPase regulates cytoplasmic pH in murine macrophages. J Biol Chem 1990; 265: 7645-54. 17. Naccache PH. Signals for actin polymerization in neutrophils. Biomed Pharmacother 1987; 41: 297304. 18. Yuli I, Oplatka A. Cytosolic acidification as an early transductory signal of human neutrophil chemotaxis. Science 1987; 235: 340-2. 19. White JR, Naccache PH, Sha'afi RI. Stimulation by chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils: effects of calcium and cytochalasin B. J Biol Chem 1983; 258: 14041-7. 20. Molski TFP, Sha'afi RI. Intracellular acidification, guanine nucleotide binding proteins and cytoskeletal actin. Cell Motility 1987; 8: 1-6. 21. Grinstein S, Furuya W, Biggar WD. Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na+/I-I÷ exchange. J Biol Chem 1986; 261: S12-S14. 22. Simchowitz L, Cragoe EJ. Regulation of human neutrophil chemotaxis by intracellular pH. J Biol Chem 1986; 261: 6492-500. 23. Retstein OD, Fiegel VD, Simmons RL, Knighton DL. The deleterious effect of reduced pH and hypoxia on neutrophil migration in vitro. J Surg Res 1988; 45: 298-303. 24. Rabinovitch M, DeStefano MJ, Dziezanowski MA. Neutrophil migration under agarose: stimulation by lowered medium pH and osmolarity. J Reticuloendothel Soc 1980; 27: 189-200. 25. Showell HJ, Becker EL. The effects of external K ÷ and Na ÷ on the chemotaxis of rabbit peritoneal neutrophils. J Immunol 1976; 116: 99-105. 26. Begg DA, Rebhun LI. pH regulates the polymerization of actin in the sea urchin egg cortex. J Cell Biol 1979; 83: 241-8. 27. Simchowitz L. Intracellular pH modulates the generation of superoxide radicals by human neutrophils. J Clin Invest 1985: 76: 1079-89. 28. Nasmith PE, Grinstein S. Impairment of Na+/H ÷ exchange underlies inhibitory effect of Na+-free media on leukocyte function. F E B S Lett 1986; 202: 7985. 29. Tauber AI, Goetzl EJ. Structural and catalytic properties of the solubilized superoxide-generating activity of human polymorphonuclear leukocytes. Solubilization, stabilization in solution and partial characterization. Biochemistry 1979; 18: 5576-84. 30. Bryant RE, Rashad AL, Mazza JA, Hammond D. Beta-lactamase activity in human pus. J Infect Dis 1980; 142: 594-601. 31. Retstein OD, Nasmith PE, Grinstein S. The Bacteroides by-product, succinic acid, inhibits neutrophil respiratory burst by reducing intracellular pH. Infect I m m u n 1987; 55: 864-70. 32. Rotstein OD, Wells CL, Pruett TL, Sorenson JJ, Simmons RL. Succinic acid production by Bacteroi-

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Regulation of cytoplasmic pH in phagocytic cell function and dysfunction.

To ensure effective antimicrobial or tumouricidal function, phagocytic cells must maintain their cytoplasmic pH (pHi) at a level conductive to optimal...
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