the of biological chemistry vol. 266, no. 36, issue of pp. … · 2001-06-12 · the journal of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 36, Issue of December 25, pp. 24540-24548, 1991 Printed in U.S.A.

Determinants of the Phagosornal pH in Macrophages IN SZTU ASSESSMENT OF VACUOLAR H+-ATPase ACTIVITY, COUNTERION CONDUCTANCE, AND H’ “LEAK”*

(Received for publication, June 25, 1991)

Gergely L. Lukacs$§V, Ori D. Rotstein§, and Sergio GrinsteinSIl From the $Division of Cell Biology, Research Institute, Hospital for Sick Children, M5G 1x8, the §Department of Surgery, Toronto General Hospital, 200 Elizabeth St., M5G 2C4, and The Institute of Medical Science, University o/ Toronto, Toronto, M5S 229, Canada

We studied the factors that determine the intraphagosomal pH (pHp) in elicited murine peritoneal macrophages. pHp was measured in situ by recording the fluorescence of covalently fluoresceinated Staphylo- coccus aureus ingested by the macrophages. Following spontaneous acidification of the phagosomes, passive (leak) H+ permeability was determined measuring the rate of change of pHp upon complete inhibition of the H+ pump with bafilomycin AI. A significant, but comparatively low passive H+ permeability was detected. The existence of a passive H+ leak implies that continuous energy expenditure is required for the maintenance of an acidic pH,. In combination with ionophores, bafilomycin was also used to estimate the counterion permeability. The counterion conductance was found to be severalfold higher than the H+ leak. Ion substitution experiments in electropermeabilized cells and the inhibitory effects of quinine and 5-nitro-2-(3- phenylpropy1amino)benzoic acid suggest that both monovalent anions and cations permeate the phagosomal membrane. The activity of the H+ pump was measured at various pH,, levels. In the steady state, the rate of H+ pumping was considerably lower than counterion permeation. These findings suggest that the phagosomal membrane potential is insignificant. Consistent with this notion, increasing phagosomal conductance with ionophores failed to accelerate the rate of H+ pumping. Thus, the transmembrane ApH is the predominant component of the proton-motive force across the phagosomal membrane in the steady state. The rate of H+ pumping was found to decrease steeply as the phagosomal lumen became acidified. Therefore, the pH sensitivity of the H+ pump, which possibly reflects a kinetic or allosteric effect, is the primary determinant of pHp.

Vacuolar (V)-type H+-ATPases are integral components of the limiting membrane of many intracellular organelles of

* This work was supported by the Canadian Cystic Fibrosis Foun- dation, the Medical Research Council of Canada, and by an Interna- tional Scholars Award from the Howard Hughes Medical Institute (to S. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Recipient of a Canadian Cystic Fibrosis Foundation Postdoctoral Fellowship. Permanent address: Dept. of Physiology, Semmelweis Medical School, Budapest, H1444, POB 259, Hungary.

(1 To whom correspondence should be addressed Div. of Cell Bi- ology, Hospital for Sick Children, 555 University Ave., Toronto, M5G 1x8, Canada.

both animal and plant cells. The acidic pH generated in the interior of these compartments by the V-type H+-ATPases appears to play a crucial role in the regulation of several important functions, including receptor-ligand dissociation, vesicular and protein trafficking, transport of solutes, and intracellular hydrolysis of macromolecules (see Refs. 1-6, for reviews). While similar, if not identical pumps are present in different compartments, the pH of their luminal space can range from -7.0 to 4.5 (1, 7, 8). The mechanism underlying this differential regulation of organellar acidification remains poorly understood.

Since V-type H+-ATPases are electrogenic ion pumps, a transmembrane potential (lumen positive) tends to develop when H’ are translocated into the organelles. Together with the pH gradient, this electrical potential determines the magnitude of the proton motive force generated across the organellar membrane, which is limited by the free energy derived from the hydrolysis of ATP by the pump. Thus, the degree of organellar acidification attained in the steady state is in- versely proportional to the magnitude of the membrane potential generated by the ATPase (4, 6, 9, 10). The fractional contribution of the membrane potential and pH gradient to the proton motive force is dictated largely by the following two parameters: the buffering power of the luminal compartment and the availability of permeable counterions. While little is known about the determinants of the organellar buffering capacity, counterion permeability has been studied in some detail. A central role for C1- has been established using isolated endomembrane preparations (9-19). Parallel migration of this anion with H’ is thought to be the primary mechanism for preservation of electroneutrality, allowing the generation of a sizable pH gradient. For this reason, regulation of the electrical component of the proton motive force by variations in C1- conductance has been proposed to account for the heterogeneity of the intraorganellar pH (4, 5, 20). Direct modulation of the activity of the pump by anions or by boundary lipids has also been postulated, and differential subunit composition of the pumps has similarly been invoked (1, 4,5).

The vast majority of studies examining the regulation of the pH of acidic compartments have been performed using either isolated organelles (e.g. endosomes, lysosomes, coated vesicles, synaptic vesicles, chromaffin granules, and ghosts) or purified V-type H’-ATPases reconstituted into phospho- lipid vesicles (7, 12-14, 17, 18, 21-24). By contrast, little information is available concerning the regulation of the internal pH of acidic organelles in intact cells. In principle, the latter approach is preferable, inasmuch as subcellular fractionation can result in loss of soluble regulatory factors and might induce changes in the permeability properties of

24540

Determinants of Phagosomal p H 24541

the membrane. The purpose of the present experiments was to analyze the determinants of the intraphagosomal pH in intact murine macrophages. We recently reported that acidification of the phagosomal lumen is mediated by a V-type H+- ATPase (25). In that study, H+ fluxes across the phagosomal membrane were monitored fluorimetrically in intact cells following phagocytosis of fluorescein-labeled Staphylococcus aureus. A similar experimental protocol was used in the present study in conjunction with ionophores, transport inhibitors, and ionic substitution experiments to define the relative contribution of the pumps, counterions, and H' "leak" fluxes to the maintenance of the phagosomal pH in intact macrophages.

EXPERIMENTAL PROCEDURES

Materials and Solutions-Medium RPMI 1640 (with L-glutamine, bicarbonate-free), Ca2+ and Me-f ree Hanks' balanced salt solution and phosphate-buffered saline were from GIBCO. Heparin sodium (1000 USP units/ml) was from Organon, Canada and powdered Brewer's thioglycolate from Difco. HEPES,' nonactin, gramicidin D, nigericin, CCCP, oligomycin, ouabain, and FITC were obtained from Sigma. The acetoxymethyl ester of BCECF, antifluorescein antibody, and bis-(l,3-diethylthiobarbituric)trimethine oxonol (bis-oxonol) were purchased from Molecular Probes, Eugene, OR. Bafilomycin AI was the kind gift of Prof. K. Altendorf (Universitat Osnabruck, Germany). 5-Nitro-2-(3-phenylpropylamino)-benzoic acid was the kind gift of Dr. R. Greger (Universitat Freiburg, Germany). IAA-94 was synthesized by Dr. D. Landry and was the kind gift of Drs. A. Edelman and Q. AI-Awqati (Columbia University).

HEPES-RPMI was prepared by titrating HCOi-free RPMI with 20 mM HEPES-Na to pH 7.35 at 37 "C. Na-medium contained (in mM) 140 NaCI, 5 KCI, 10 glucose, 10 HEPES, pH 7.35 at 37 "C. The permeabilization medium contained 140 KC1 or K-gluconate, 5 NaCI, 10 glucose or 2-deoxy-D-glucose (as indicated), 10 HEPES-K and 0.5 m M EGTA to give a final free [Ca'+] of <30 nM. The osmolarity of the media was adjusted to 290 2 5 moSM with the major solute. Phosphate-buffered saline contained 144 mM NaCl and 10 mM sodium phosphate at pH 7.4. Thioglycolate was solubilized in water, auto- claved at 12.5 "C for 45 min, and stored in the dark until uniformly green.

Macrophage Isolation and Characterization-Six- to 8-week-old female Swiss Webster mice (Charles River) were injected intraperi- toneally with 2 ml of thioglycolate medium, and the elicited macrophages were isolated as described (25). The cells were washed twice with Hanks' solution (9 min, 600 X g at room temperature), counted using a Coulter Counter model ZF, and resuspended in HEPES- RPMI. The proportion of peritoneal cells identified as macrophages by nonspecific esterase staining and Wright's staining was SO-90%. Viability was >95%, as assessed by trypan blue exclusion.

Phagosomal p H Measurement-The phagosomal pH (pH,) was determined by measuring alterations in the fluorescence of ingested S. aureus, which had been previously labeled covalently with FITC. Bacterial culture and labeling were performed exactly as described (25). To synchronize phagocytosis, labeled and opsonized S. aureus were adhered to the surface of macrophages by incubating the cells with bacteria for 20 min at 4 "C. The bacteria to cell ratio was =50:1. Bacterial ingestion was initiated by transiently raising the temperature to 37 "C. After 10 min, phagocytosis was stopped and excess bacteria removed by washing the suspension twice with ice cold Hanks' solution (9 min, 600 X g). Any residual adherent, extracellular 5'. aureus were further removed by specific enzymatic lysis of the bacterial cell wall. For this purpose, cells were resuspended in HEPES-RPMI (2 x lo' cells/ml) and incubated with 10 units/ml lysostaphin at room temperature for 5 min. The cells were again washed twice with ice-cold Hanks' solution as above, resuspended in

' The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid pH,, pH in the phagosomal compartment; FITC, fluorescein isothiocyanate; BCECF, 2',7'-biscarboxyethyl-

RPMI 1640 buffered with 20 mM HEPES-NaOH; NPPB, 5-nitro-2- 5(6)-carboxyfluorescein; HEPES-RPMI, bicarbonate-free medium

(3-phenylpropy1amino)benzoic acid, IAA-94, 2-[(2-cyclopentyl-6, 7- d~chloro-2,3-dihydro-2-methyl-l-oxo-1H-inden-5-yl)oxyl]acetic acid CCCP, carbonyl cyanide p-chlorophenylhydrazone; EGTA, [etheyle- nebis(oxyethylenenitri1o))t.etraacetic acid.

HEPES-RPMI medium, and stored on ice. To determine the pH,, 0.5-1 X lo6 cells containing phagocytosed FITC-labeled S. aureus were added to a thermostatted cuvette containing 1.2 ml of the indicated medium at 37 "C. Antifluorescein antibody was present in the medium to quench contaminating extracellular fluorescence. Flu- orescence intensity was monitored with excitation at 490 nm and emission at 525 nm, using 5 and 10-nm slits, respectively, on a Perkin- Elmer Cetus 650-40 spectrofluorimeter. Phagosomal pH calibration was carried out in each experiment by equilibrating the intraphago- soma1 pH with the extracellular pH by addition of monensin and nigericin, as described (25). These alkali cation/proton exchangers collapse the sodium and potassium gradients between the extracellular and intracellular (including the subcellular) compartments and consequently establish a uniform pH inside and outside the cells (25- 27). Thus, by titrating the external milieu, pH, could be adjusted to desired levels.

Electropermeabilization of Macrophages-Macrophages were allowed to phagocytose FITC-labeled S. aureus and permeabilized immediately before use for pH, determinations. To achieve permeabilization, 1-2 X lo6 cells were resuspended in 0.8 ml ice-cold permeabilization medium and subjected to two or three 2.3 kV/cm discharges from a 25-pF capacitor, using the Bio-Rad Gene Pulser. The suspension was gently stirred between pulses. The cells were utilized immediately after permeabilization, or stored on ice for up to 7 min. Previous studies demonstrated that electroporation under these conditions selectively permeabilized the plasma membrane without affecting the integrity of the phagosomal membrane. In such electroporated cells, the development of an acidic pH, required the addition of exogenous ATP-Mg to the incubation medium (25).

Cytosolic p H Determination-Cytosolic pH (pH;) was measured fluorimetrically in cells loaded with the pH-sensitive dye BCECF. Cell suspensions (10' cells/ml in HEPES-RPMI) were incubated with 2 wg/ml of the acetoxymethylester form of BCECF for 12 min at 37 "C, washed, and then allowed to phagocytose unlabeled S. aureus. Free bacteria were eliminated by washing three times with Hanks' solution at 4 "C. Fluorescence of a suspension containing lo6 cells/ ml was measured with excitation at 495 nm and emission at 525 nm, using 5- and 10-nm slits, respectively. Calibration was performed using monensin and nigericin in Na-medium as previously described (25).

Membrane Potential Measurements-The anionic fluorescent probe bis-oxonol was used to estimate the membrane potential (28). Fluorescence was measured with excitation at 540 nm (4-nm slit) and emission at 580 nm (10-nm slit). After addition of 100 nM bis-oxonol to the cell suspension, the fluorescence was allowed to stabilize before further additions were made. Calibration was made by adding gramicidin in isotonic media containing varying ratios of Na+ and N- methyl-D-glucammonium+ or K' and choline+. Neither N-methyl-D- glucammonium' nor choline+ permeate through the gramicidin channel. E, was therefore calculated as

Em = 60 1% ([Nal, + [KId/(INal, + IKlJ

assuming that Na+ and K+ permeation through gramicidin are comparable, and/or that the intra- and extracellular concentrations of permeant monovalent cations are equal at equilibrium. Calibration curves obtained in Na' or K+ media were identical (not shown). Transference numbers for specific ions were calculated as described (28). Unless otherwise specified, all experiments were carried out at 37 "C. Data are presented as the mean 2 1 S.E. of the number of experiments indicated in parenthesis.

RESULTS

Passive H" Permeability of Phagosomes The fluorescence emission of macrophages containingphag-

ocytosed, FITC-labeled S. aurew spontaneously and rapidly decreased upon resuspension of the cells at 37 "C (Fig. U). Previous studies demonstrated that this reduction of fluorescence intensity reflects an acidification of the phagosomal compartment (25). This acidification reached a steady state at pHp 5.7-6.0 in 6-8 min and was completely obliterated by 200 nM bafilomycin AI, a specific inhibitor of V-type H'- ATPases (29).

The acidification of pHp is determined by the difference between the rate of H+ accumulation catalyzed by the

24542 Determinants of Phagosomal pH 6.

i I

f r t I 6ot J I

" ~ I I

0

bafilomycin A1 (pM) 0.01 0.1 1.0 10.0

FIG. 1. Effect of bafilomycin A1 and CCCP on the phagosomal pH (pH,). Phagosomal pH was determined in suspended macrophages measuring the fluorescence of internalized, FITC-labeled S. aureu.s as described under "Experimental Procedures." Panel A, following spontaneous phagosomal acidification upon rewarming, rapid H' efflux was induced by sequential addition of 500 nM bafilomycin A, (BAF) and the indicated concentration of CCCP (in p ~ ) . Panel E , sequential addition of 80 p M CCCP, 500 nM bafilomycin AI, and 33 mM NHICl. Traces in panels A and B are representative of three experiments. Panel C, dose dependence of the inhibitory effect of bafilomycin AI, determined indirectly from the rate of dissipation of pH,. To detect small alterations in the proton pump activity, efflux was enhanced by the protonophore CCCP (20 p ~ ) , and the rate of H+ efflux was measured as a function of bafilomycin A1 concentration. Results are means f S.E. of three experiments and are expressed as percent of the proton efflux at 10 p~ bafilomycin AI. Where absent, the error bars were smaller than the symbol.

ATPases and the ostensibly passive H' efflux (leak). In the experiments described below, these two components were studied separately. To estimate the passive H' permeability of the phagosomal membrane, we analyzed the rate of dissipation of the phagosomal acidification following inhibition of the ATPases. As shown in Fig. L4, pH, was first allowed to attain near steady state. Bafilomycin AI was added next, while continuously monitoring pH,,. The pump inhibitor initiated a slow net efflux of acid equivalents, manifested as a phagosomal alkalinization averaging 0.09 f 0.01 pH/min (n = 14). This observation implies that the phagosomal membrane has a measurable passive H' permeability and that continuous activity of the ATPases is required for the steady state maintenance of an acidic pH,,. Several explanations could account for the seemingly low H' permeability of the phagosome. First, the reduced rate of net H' efflux may result from incomplete inhibition of the V-type ATPases by bafilomycin AI. In this instance, residual pump activity could partially compensate for rapid H' efflux. To ensure that complete inhibition of the pump was attained, H' efflux was measured at increasing bafilomycin AI concentrations (Fig. 1C). The rate of phagosomal alkalinization reached a maximum at 50 nM and remained constant as the concentration of the inhibitor was increased up to 10 pM. Half-maximal effects were obtained at a concentration of bafilomycin AI that was slightly higher than the reported K, for inhibition of V-type ATPases in cell-free systems (29,30). Because a supramaximal concentration of bafilomycin (500 nM) was present in the experiment of Fig. lA, the slow rate of H' efflux cannot be attributed to incomplete inhibition of the phagosomal H'-ATPases. This concentration of the inhibitor was used in all subsequent experiments.

The low rate of phagosomal alkalinization following inhibition of the pumps could also result from restricted movement of counterions. In this case, the development of a luminal-negative diffusion potential would limit the exit of H' from the phagosomes. To estimate the counterion permeability of the phagosomal membrane, we used the conductive protonophore CCCP. If the movement of counterions restricts the rate of H' efflux from phagosomes, further increasing H' conductance by means of the ionophore is expected to have little effect on the rate of alkalinization. Contrary to this prediction, when added after bafilomycin AI, CCCP (20-80 PM) promoted a large increase in the rate of net H' efflux (Fig. lA). Using 20 p~ CCCP this rate averaged 0.48 f 0.03 pH/min ( n = 13). The effect of CCCP was not the result of nonspecific disruption of the phagosomal membrane, as it required the simultaneous presence of bafilomycin (Fig. 1B). These observations suggest that counterion conductance is not the factor limiting the rate of net H' efflux from phagosomes. This conclusion was further substantiated by studies using valinomycin, a conductive K'-selective ionophore. In the presence of bafilomycin AI, addition of valinomycin had only a marginal effect on the rate of H' efflux (107.5 f 12% of control rate, n = 4; e.g. Fig. 2B). However, the K+ ionophore enhanced the H' efflux induced by subsequent addition of CCCP greatly (10-13-fold; range of three experiments), implying that valinomycin was able to reach the phagosomal membrane (cf. Fig. 2, A and B; notice the change in time scale following addition of CCCP). Like valinomycin, the conductive ionophores gramicidin and nonactin had little effect on the H' leakage promoted by bafilomycin but effectively stim- ulated the CCCP-induced efflux (Fig. 2, C and D). When considered together, these data indicate that the slow dissipation of the phagosomal acidification is not due to limited counterion conductance and suggest instead that the endogenous H' permeability is low.

In the experiments of Figs. 1 and 2, passive H' permeability was estimated following spontaneous phagosomal acidification. Because a variety of ion channels are exquisitely sensitive to changes in pH (e.g. 31, 32), it is conceivable that the low H+ permeability of the phagosomal membrane is a con- sequence of the luminal acidification. To consider this possi-

I \

FIG. 2. Effects of alkali cation ionophores on the steady state pH, and on the rate of H+ efflux evoked by bafilomycin AI and CCCP. pH,, measured as in Fig. 1, was allowed to attain steady state, and then net H' efflux was induced by sequential addition of 500 nM bafilomycin AI (BAF) and 20 p M CCCP. The following additions were made prior to dissipation of pH,: A, none; B, 10 p M valinomycin (VAL); C, 10 pM gramicidin (GRA); D, 10 pM nonactin (NON). Note that the recording speed was doubled in every instance after addition of CCCP, to facilitate comparison of the rates of pH change. Traces are representative of two to four experiments.

Determinants of Phagosomal pH 24543

0.25 -

0.20 -

0.15 .

0.0 0.5 1.0 1.5 2.0 2.5 3.0

FIG. 3. Relationship between the rate of passive (leak) H+ efflux and the intraphagosomal H+ concentration. The spontaneous acidification of the phagosomal compartment was inhibited at different pH, levels by addition 500 nM bafilomycin AI (BAF) and the rate of net H’ efflux was measured ( n = 3). A typical experiment is shown in the inset. Data from three similar experiments are summarized in the main panel. The line was fitted by linear regres- sion.

bility, the rate of bafilomycin-induced alkalinization was measured as a function of pH, (Fig. 3). This was accomplished by adding the H+-ATPase inhibitor at increasing times after the initiation of the spontaneous acidification (see inset in Fig. 3) and measuring the rate of change of pH,. Since cytoplasmic pH (measured in parallel experiments using BCECF)’ was essentially invariant during the course of phagosomal acidification, the pH gradient across the phagosomal membrane varies along with pH,. As shown in the main panel of Fig. 3, the rate of net H+ efflux was a linear function of the intraphagosomal H’ concentration, consistent with a diffu- sional pathway. Thus, no evidence was found for a modulatory role of pHp on the passive H’ permeability of the phagosomal membrane.

Effect of Membrane Potential on pH,

In some cells, the plasmalemma1 Na,K-ATPase is known to be incorporated into the early endosomal membrane (33, 34). The enzyme remains active following endocytosis and, due to its rheogenic mode of action, generates an electrical potential (inside positive) across the endosomal membrane. As a result, the rate of electrogenic H’ pumping into the endosome is partially inhibited (33, 34). Since phagosomes are also generated by invagination of the plasmalemma, we considered the possibility that phagosomal acidification is similarly affected by the Na,K-ATPase. For this purpose, ouabain (1 mM), an impermeant specific inhibitor of the Na,K-ATPase, was included in the medium during phagocytosis of the labeled bacteria. As shown in Table I, the glycoside had no effect on the steady state pH,. This contrasts with the behavior of early endosomes, which attain more acidic pH levels in the presence of ouabain (33, 34). Failure of the glycoside to alter the final pHp could result from inactivation or exclusion of the Na,K pumps from the phagosomal membrane. However, this is unlikely since the rate of phagosomal acidification was in fact significantly increased in the presence of ouabain (Table I). Because phagosomal acidification in fact begins during the phagocytosis and wash periods, the “initial” pHp recorded upon resuspension at 37 “C was also significantly lower in ouabain-treated samples (Table I), reflecting the accelerated rate of acidification. The apparent discrepancy

Macrophages were first loaded with BCECF and then allowed to phagocytose unlabeled S. aureus, to avoid interference between the fluorescence of FITC and that of BCECF. The cytoplasmic pH was determined as in Ref. 25.

TABLE I Effect of ouabain on phagosornal acidification

Phagosomal pH (pH,) was determined fluorimetrically, as described under “Experimental Procedures.” The data are means f S.E. of 8-10 determinations (four separate experiments) of the initial or steady state pHp and of the maximal rate of phagosomal acidification. Initial pHp was measured following a 10-min phagocytosis period followed by removal of contaminating extracellular bacteria. Statis- tical significance was calculated using the paired t test. NS = not significant ( p > 0.05).

Steady state Rate of phagosomal RH, acidification

pHfmin Control 7.00 f 0.05 5.95 f 0.10 0.16 +. 0.03 Ouabain-treated 6.75 f 0.06 5.90 f 0.06 0.22 +. 0.03

~<0.001 NS D < 0.01

between the effects of the glycoside on the initial and final pH, can be explained by: (a) inactivation of the Na,K-ATPase at acidic intraphagosomal pH; (b) removal of the internalized ATPases from the membrane during the transition from early to late phagosomes, as has been postulated for endosomes (33, 34); or (c) by the large difference in the rates of initial and steady state H’ pumping (see below). During the initial phase of spontaneous acidification, substantial movement of counterions is required to neutralize the high rate of H+ translocation (0.2-0.25 pH/min). Under these conditions, passive counterion permeability can become limiting to the acidification, particularly when the Na,K-ATPase contributes to net cation translocation into the phagosome. In contrast, counterion permeability exceeds the much lower rates of H’ pumping required to maintain the steady state acidification (see “Discussion”).

These results imply that functional Na,K-ATPases are present in the phagosomal membrane, at least during the early stages of acidification. In addition, the data indicate that the endogenous counterion permeability of the phagosomal membrane is relatively high, suggesting that in the steady state the fractional contribution of the membrane potential to the proton-motive force is small. To confirm the latter prediction, we evaluated the effect of dissipating any residual membrane potential on the steady state pHp. Na+-conducting ionophores were added to cells that were allowed to phagocytose bacteria in Na+-rich medium. Neither nonactin nor gramicidin potentiated phagosomal acidification under these conditions (Fig. 2, C and D), suggesting that the membrane potential component of the proton motive force is too small to measurably suppress H+-pump activity. As discussed above, potentiation of the alkalinizing effects of CCCP (Fig. 2) confirms the ability of the alkali cation ionophores to reach the phagosomal membrane. The steady state pH, was similar when phagocytosis was performed in the standard Na’ medium and in K+-rich medium without or with valinomycin (not shown). The latter conditions are known to depolarize the plasma membrane (see below) and are anticipated to have the same effect on the phagosomal membrane. These findings are also consistent with the minor role of phagosomal membrane potential in the determination of the rate of H+ pumping in the steady state.

Components of the Phagosomal Membrane Ion Permeability

Anion Permeability-This section describes experiments designed to define the nature of the comparatively high counterion conductance of the phagosomal membrane. For this purpose, we studied the properties of the rapid H+ efflux induced by CCCP in the presence of bafilomycin AI. As demonstrated above by means of conductive ionophores, this

24544 Determinants of Phagosomal p H

pHp A. 7.0 ,- control

J

5.9

J NPPB

L~ I, I CCCP 2 min

0 25 50 75 100 125 NPPE (pM)

FIG. 4. Inhibition of net H+ efflux by NPPB. Panel A, macrophages were allowed to attain steady state pHp in the absence (control; left trace) or presence of 100 PM NPPB (right trace). The traces start upon addition of 500 nM bafilomycin A1 (BAF) , followed by 20 PM CCCP. Note that the recording speed was doubled when CCCP was added in both traces. Panel B, dose dependence of the inhibitory effect of NPPB (circles) on the rate of net phagosomal H' efflux, measured as in panel A. The rate of H' efflux was determined at a comparable pHp in control and experimental samples. Data are means f S.E. of three experiments and are expressed as percent of H' efflux in the absence of NPPB. The inhibitory effect of IAA-94 (100 pM; n = 5) is also shown.

dissipation is limited by and can be used as an indicator of the counterion conductance. This experimental protocol en- abled us to test the effects of potential inhibitors of phagosomal counterion permeability in situ. To assess the contribution of C1- to phagosomal conductance, the effect of NPPB and IAA-94 was examined. These drugs are potent inhibitors of C1- channels in a variety of tissues (35, 36). When 100 p~ NPPB was added, H' efflux induced by bafilomycin AI and CCCP was inhibited by 66% (Fig. 4A): A detailed concentration dependence of the effect of NPPB is presented in Fig. 4B. Inhibition was incomplete at the highest concentration tested (120 pM) and was half-maximal at 25 pM. The struc- turally unrelated C1- channel inhibitor IAA-94 (an indanylox- yacetic acid derivate) also inhibited the CCCP and bafilomycin Al-induced dissipation of the ApH,. At 100 p~ IAA-94 inhibition was 39 f 3% (n = 5; Fig. 4B). Control experiments using bacterial suspensions showed that neither NPPB nor IAA-94 interfered with the fluorescence or pH sensitivity of the FITC-labeled S. aureus . A direct effect of the channel blockers on CCCP was also ruled out (not shown). Together, these findings suggest that C1- conductance contributes to the phagosomal counterion permeability.

The incomplete block of the net H' efflux by NPPB and IAA-94 may indicate the presence of C1- channels insensitive to the concentrations of the inhibitors used. Alternatively, cation influx may compensate the charge of the exiting H'. The following observations are consistent with the latter possibility. First, phagosomal acidification was detected in cells that were C1- depleted by electropermeabilization in a Cl--free, K-glutamate medium. Under these conditions, residual cytosolic C1-, determined isotopically by preloading with 36Cl-, was 51 mM, yet near normal rates of phagosomal

The steady state pHp attained in the presence of NPPB was slightly higher than that observed in its absence. This is possibly due to the moderate protonophoric activity of NPPB (48).

acidification were observed upon addition of ATP and Mg2' (data not shown; see Ref. 25). Second, preliminary experiments showed that an acidification developed in phagosomes isolated by nitrogen cavitation and suspended in C1--free medium in the presence of ATP and M e . This acidification was blocked by bafilomycin A1 (not illustrated). Assuming that large organic anions such as glutamate are impermeant, these observations are most readily explained by neutraliza- tion of the charges associated with H' translocation by cation counterflow.

Cation Permeability-The phagosomal membrane is generated by the rapid internalization of the plasma membrane. We therefore hypothesized that, at the early stages of phagosomal development, the ionic permeabilities of the phagosomal and plasma membranes would be similar. To determine the relative conductance of the plasma membrane to the major monovalent ions, the effects of extracellular ionic substitution on the membrane potential were determined. The plasma membrane potential was estimated in freshly isolated cells using the fluorescent potential-sensitive dye bis-oxonol. As illustrated in Fig. 5A varying extracellular [CI-] or [Na+] from t 1 0 mM to 145 mM while maintaining osmolarity with an impermeant substituent caused a minimal hyperpolarization or depolarization, respectively. In contrast, varying extracellular [K'] markedly altered the plasma membrane potential (Fig. 5A). The transference number for K' (calculated between 10 and 140 mM K') was 0.76, similar to the value reported for human monocytes (37). If the relative ionic permeabilities of the phagosome resemble those of the plasmalemma, transport of H' could therefore be neutralized by counterflow of alkali cations, particularly K'.

Important biochemical and morphological changes occur in the plasma membrane of macrophages during phagocytosis. It is therefore conceivable that the ionic permeability is similarly affected during the phagocytic process. To assess this possibility, experiments like those described in Fig. 5A were repeated using cells following the ingestion of unlabeled S. aureus. As shown by the results in Fig. 523, the membrane conductance was also dominated by the permeability to K' under these conditions. In this case, the calculated transference number for K+ was 0.86.

To assess the contribution of K' to phagosomal counterion permeability, a variety of K' channel blockers were tested. Charybdotoxin (60 nM) and tetraethylammonium (10 mM) had negligible effects on the plasma membrane potential and were therefore not investigated further. Barium (6 mM), which is impermeant and must be added together with the bacteria to reach the phagosomal lumen, was found to inhibit phagocytosis. In contrast, quinine was found to effectively inhibit K' conductance (38) and was able to traverse the plasmalemma to reach the phagosomal membrane in intact cells. Addition of quinine led to a spontaneous depolarization of the plasma membrane (inset, Fig. 6). Half-maximal effects were observed at 20 PM. As illustrated by the main panel of Fig. 6, the depolarization was attributable to diminution of the conductance to K+. The K' transference number was reduced from 0.86 to 0.45 by 50 p M quinine.

If quinine-sensitive K+ channels are incorporated into the phagosomal membrane, K' ions might serve as counterions during H+ translocation. This hypothesis was tested by measuring the quinine sensitivity of the H+ efflux induced by the combination of CCCP and bafilomycin A1. Addition of 100 PM quinine to the suspension had only a modest (50.15 pH) effect on the steady state pH,. The small magnitude of this weak base effect reflects the comparatively high buffering power of the phagosomal lumen or a compensatory action of

Determinants of Phagosomal p H 24545

P"P

A. A.

u- ICCCP 2 min

-90 I I 1 10 100

concentration (mM)

quinine (pM)

FIG. 7. Inhibition of net H+ efflux from phagosomes by quinine. Panel A , macrophages were allowed to attain steady state pH, in the absence (control; left trace) or presence of 83 p~ quinine (right trace). The traces start upon addition of 500 nM bafilomycin A, (BAF) , followed by 20 ~ L M CCCP. Note that the recording speed was doubled when CCCP was added in both traces. Panel B , dose dependence of the inhibitory effect of quinine on the rate of net phagosomal H+ efflux, measured as in panel A . The rate of H+ efflux was determined at a comparable pH, in control and experimental samples. Data are means f S.E. of three experiments and are expressed as percent of H+ efflux in the absence of quinine. Where absent, error bars were smaller than the symbol.

-90 1 I 1 10 100

concentration (mM)

FIG. 5. The plasma membrane potential of macrophages (ordinate) as a function of external Na+, K+, and Cl- concentration (abscissa). Cells were suspended in isoosmotic media containing the indicated concentrations of K+, Na+, or C1- and the membrane potential was measured using bis-oxonol as described under "Experimental Procedures." Osmolarity was maintained by replacing cations with choline or N-methyl-D-ghcammonium and C1- with gluconate. Panel A , freshly isolated macrophages (prior to phagocytosis); panel B, macrophages were allowed to phagocytose unlabeled S. aureus before their membrane potential was determined. Curves were fitted by eye. Data are means f S.E. of three to four experiments.

r r 0

c- > -30 E E

v

W -60 ICCCP I I

U U

FIG. 8. Additivity of the effects of quinine and NPPB. Mac- rophages were allowed to attain steady state pH, in the absence (control; kftmost trace) or presence of 83 p M quinine (second trace), 100 p~ NPPB (third trace) or both quinine plus NPPB (rightmost trace). The traces start upon addition of 500 nM bafilomycin AI (BAF, downward arrow), followed by 20 pM CCCP ( f i r s t upward arrow). Where indicated, 40 mM NH,Cl was added to collapse the residual pH,. In three similar experiments, quinine inhibited the rate of H+ efflux by 47.6 f 4.4%, NPPB inhibited 63.6 f 4.0%, and the combination of inhibitors by 79.8 f 2.0%. The rates of H+ efflux were determined at the same pH,. Note that the recording speed was doubled in all cases simultaneously with the addition of CCCP.

2 min

-90 L 1

FIG. 6. Effect of quinine on the plasma membrane potential of macrophages. Main panel, the extracellular K+ concentration dependence of the membrane potential was measured in the absence (open syrnbok) or presence (solid syrnbok) of 50 pM quinine. Extra- cellular K+ was isosmotically replaced by choline. Membrane potential was determined as in Fig. 5. Inset, concentration dependence of the depolarization induced by quinine in macrophages suspended in low (3 mM) K+ medium. Data are means f S.E. of three experiments.

the H+ pump. As shown in Fig. 7A, quinine caused a 50% reduction in the rate of pH, dissipation induced by CCCP and bafilomycin AI. This effect is opposite to that expected for a weak base and likely reflects blockade of counterion (K+) permeability. Analysis of the concentration dependence (Fig. 7 B ) demonstrated that, in the range studied, only about 60% of the flux is susceptible to inhibition by quinine. Half- maximal effects were attained at ~ 2 0 NM quinine. It is note- worthy that a comparable concentration was required for half- maximal depolarization of the plasma membrane potential, suggesting the involvement of a similar pathway. Quinine did not interfere with the pH sensitivity of FITC-labeled bacteria in suspension, nor did it interact with CCCP (not shown).

The results presented above suggest that both anion comi-

gration and cation counterflow can serve to maintain electroneutrality during H+ translocation across the phagosomal membrane. The multiplicity of counterion permeation path- ways can be illustrated by the simultaneous use of quinine and NPPB (Fig. 8). Whereas NPPB and quinine added separately produce intermediate levels of inhibition, nearly 80% of the H+ flux is obliterated when the channel blockers are used jointly (see legend to Fig. 8). This inhibition can be bypassed by addition of NH4C1 or valinomycin (not illustrated). Together with the ion substitution experiments described above, these findings indicate that monovalent cations as well as anions can serve as H' counterions. Shunting of the phagosomal membrane potential by the combined monovalent ion permeability reduces the contribution of the elec-

24546 Determinants of Phagosomal pH

trical component to the proton motive force, allowing pH, to attain very acidic levels.

Role of Phagosomal pH in the Regulation of Proton Pumping

In view of the high counterion conductance, the pH gradient across the phagosomal membrane is expected to be the primary determinant of the rate of H' pumping. This prediction was tested by measuring the rate of pumping as a function of pH,, at constant cytosolic pH. The activity of the pump was estimated based on the notion that, in the steady state, the net proton flux is zero, implying that the rates of H' pumping and leakage are identical. Thus, the initial rate of net H' efflux recorded upon addition of maximally inhibitory doses of bafilomycin A1 provides a measure of the activity of the pump. Varying steady levels of pH, were obtained by addition of moderate amounts of the electroneutral alkali cation/H+ exchanger monensin. As shown in Fig. 9A, this ionophore readily reaches the phagosomal membrane and can largely dissipate ApH, when the pump is inhibited. When the pump remains active, however, intermediate steady pH, levels can be reached after a transient increase, when moderate doses of monensin are used (e.g. Fig. 9B). Under these conditions, the subsequent addition of bafilomycin A, triggers an alkalinization that is proportional to the activity of the pump in the preceding steady state. Fig. 9C summarizes the results of five such experiments. Elevation of pH, from the original steady state (5.7-6.0) by 0.4 units caused a nearly 10-fold increase in the rate of pumping. The same relationship between pH, and H' pump activity was found when small doses of nigericin were used to partially collapse the phagosomal acidification (empty squares in Fig. 9C). A similar behavior of the pump was observed when the pH gradient was partially dissipated using CCCP (e.g. Fig. 1B). These data indicate that pH, or

I 0.0 0.1 0.2 0.3 0.4 0.5

ApHp

FIG. 9. pH, dependence of phagosomal H+ pump activity. Panel A, phagosomes were allowed to acidify spontaneously and where indicated, bafilomycin A, (500 nM) was added. Subsequent addition of 10 JIM monensin ( M O N ) rapidly collapsed the phagosomal pH gradient. Panel B, phagosomal acidification was allowed to develop, and then the indicated concentrations of monensin (in pM) were added. After a new steady state pH, was attained, the H+ pump was blocked with bafilomycin A, (500 nM). Panel C, the activity of the H' pump was estimated at different pHp levels from the bafilomycin- induced rate of net H' efflux in experiments like that illustrated in panel B (see text for details). pHp = 0 refers to cells allowed to acidify without addition of ionophores (average steady state pH, ~ 5 . 8 ) Data from five experiments using either monensin (solid squares) or nigericin (open squares) to vary steady state pH, are presented.

the pH gradient across the phagosomal membrane are major determinants of the rate of H+ pumping.

DISCUSSION

The purpose of the present experiments was to study the determinants of the phagosomal pH in situ. In the steady state, pH, in murine peritoneal macrophages averaged ~ 5 . 8 . Several factors seem to contribute to the attainment and maintenance of this acidic pH. First, the passive H' permeability of the phagosomal membrane was found to be comparatively low. Following inhibition of the H+-ATPase, the spontaneous rate of alkalinization was only 0.09 pH/min. Dissi- pation of the pH gradient was not limited by counterion permeability, as demonstrated using cation ionophores (Fig. 2). Thus, the relative impermeability of phagosomes to H' (equivalents) compares favorably with that reported for other endomembrane compartments, studied after isolation (39,40). This relative "tightness" may be an intrinsic property of phagosomes but may also reflect the fact that the measurements were carried out in intact cells. Preliminary experiments indicate that pH, dissipation rates after inhibition of the ATPase are considerably faster in phagosomes isolated by cavitation and even in phagosomes within permeabilized cells, suggesting that soluble factors may control H' permeability. Even in situ, however, a finite H+ leak is detectable, implying that continuous expenditure of metabolic energy by the ATPase is required for the maintenance of the acidic pH,,.

Addition of conductive cation ionophores, expected to collapse the electrical potential across the phagosomal membrane, failed to lower the steady state pH,. This finding is indicative of a low phagosomal membrane potential, and suggests a high intrinsic conductance to ions other than H'. In agreement with this notion, the intraphagosomal acidity dissipated rapidly upon addition of bafilomycin Al and CCCP (e.g. Fig. l), revealing the presence of a sizable counterion permeability. Both cations and anions seem to contribute to this permeability. A component of the phagosomal counterion conductance is inhibited by quinine and is likely mediated by K' channels. A predominant K' conductance is indeed detectable in the plasma membrane (Fig. 5), which invaginates to form the phagosomes. Accordingly, patch clamping experiments have demonstrated the presence of at least three types of K' channels in the surface membrane of macrophages: inward rectifying, calcium-activated, and delayed (outward) rectifying channels (38; see Ref. 41, for review). The plasma- lemma1 K' conductance is inhibited by quinine, as evident from the reduction in the K+ transference number and from the spontaneous depolarization caused by micromolar concentrations of the alkaloid (Fig. 6). Importantly, comparable concentrations of quinine reduced the counterion permeability of the phagosomal membrane, suggesting the involvement of similar K+ channels. The presence of cation channels in other acidic endomembrane compartments has been suggested (19, 39, 42).

A second component of the phagosomal counterion permeability was inhibited by NPPB, a well established blocker of anion (primarily C1-) channels in a variety of tissues (see Ref. 35, for review). We were unable to determine the effects of NPPB on the plasmalemma1 conductance, due to interference of the channel blocker with the fluorescence assay. However, the ionic substitution data in Fig. 5 indicate that, as in other leukocytes (28, 37, 41), the C1- conductance of the plasma membrane of macrophages is comparatively low. Electrophys- iological experiments have revealed the presence of anion- selective channels in macrophages, but these are mainly detectable after depolarization or patch excision (38, 41). In

Determinants of Phagosomal pH 24547

preliminary experiments using thioglycolate-elicited macrophages, 2 out of 12 patches were found to display anion- selective channel activity and one of these was reversibly inhibited by NPPB.' Therefore, while C1- channels are readily apparent in isolated endomembrane vesicles (10, 11, 15, 19, 20, 39, 43, 4 4 , they are not very active in the plasma membrane. It is therefore possible that anion channels are inserted into the phagosomal membrane upon fusion with endosomes or lysosomes, a process that is detectable within minutes of phagocytosis (45). Alternatively, previously quiescent plas- malemmal channels may become activated during the phagocytic process. In this regard, exposure of macrophages to zymosan has been reported to activate anion-selective channels on the plasma membrane (46).

The joint rate of phagosomal anion and cation permeation exceeds the rate of H' pumping in the steady state (at least 5-fold; e.g. Fig. 1), implying that the extent of acidification is not limited by the membrane potential. In agreement with this, further increases in monovalent cation conductance by addition of ionophores failed to accentuate the acidification (Fig. 2). Hence, the transmembrane pH appears to be the predominant determinant of the protonmotive force. Despite the tightness of the membrane to H' leakage, however, the ApH attained in the steady state falls far short of the value predicted thermodynamically, based on the free energy of hydrolysis of ATP. Under standard (cytosolic) conditions, and considering a maximum stoichiometry of 3H'/ATP (from Ref. 4), the 58 kJ liberated/mol of nucleotide hydrolyzed could theoretically drive the intraphagosomal pH to below 4, assuming that the electrical potential is negligible. An even more acidic pH would be attainable if a H+/ATP stoichiometry of less than 3 is considered (e.g. Ref. 6). The source of the discrepancy between the calculated minimal pH, and the recorded pHp (5.7-5.9) remains unclear. However, a possible explanation is suggested by the data in Fig. 9. These results show a steep correlation between pHp and the rate of H+ pumping. If extrapolated, the linear relationship in Fig. 9C suggests that complete inactivation of the pump would occur at a pHp 0.1-0.2 units below the steady state (i.e. at or above pHp 5.5). In view of the calculations discussed above, cessation of net pumping at this pH cannot be attributed to thermo- dynamic equilibration and must instead be determined kinet- ically. The deprotonation of the substrate site on the luminal side of the membrane may become rate limiting to the transport cycle or, alternatively, allosteric inactivation of the pump may occur. Validation of the linear extrapolation will require the development of methods for artificially acidifying phagosomes in situ below the steady state pH,.

The steep pH dependence of the rate of pumping (Fig. 9) also indicates that the activity of the ATPase is very high at more alkaline levels, e.g. during the early stages of phagosomal acidification. Under these conditions, counterion permeability may become limiting and the development of a membrane potential can restrict the activity of the pump. Consistent with this, inhibition of the electrogenic Na,K-ATPase by ouabain accelerated the initial rate of phagosomal acidification, yet had little effect on the steady state pH,,. Moreover, inclusion of quinine and NPPB in the medium reduced the initial rate of phagosomal acidification by 35% (data not shown).

With the exception of lysosomes, which are very acidic, other organelles have been postulated to have higher luminal pH levels because of a limited permeability to chloride, the primary counterion (4,20). This proposed mechanism differs from the one postulated above for phagosomes, which appear

G. L. Lukacs and P. Pahapill, unpublished observations.

to have an elevated counterion permeability. The differential behavior could be attributed to the predominantly plasmalem- mal origin of the phagosomal membrane, to an effect of the phagocytosed bacteria on the phagosomal components or to the fact that the measurements were performed in situ. Fur- ther experiments will be required to define the determinants of the internal pH of other acidic organelles within intact cells.

In summary, the membrane of the phagosome in murine macrophages is comparatively impermeable to H' (equivalents), yet quite permeable to counterions, which in all like- lihood include both monovalent anions and cations. As a result, the transmembrane ApH is the main component of the protonmotive force in this organelle. In the steady state, pH,, (or the ApH across the phagosomal membrane) is the primary determinant of the rate of H' pumping, possibly due to an allosteric effect on the ATPase. Inactivation of the pump by intraphagosomal acid could result from increased slippage (47), altered stoichiometry, or direct inhibition of the ATPase activity. Differential sensitivity of the H+ pumps to the luminal pH could underlie the reported differences in the internal pH of various organelles.

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