©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Cytoplasmic pH in Osteoclasts
CONTRIBUTION OF PROTON PUMPS AND A PROTON-SELECTIVE CONDUCTANCE (*)

(Received for publication, July 21, 1994; and in revised form, October 20, 1994)

Tommy Nordström (1) (2) Ori D. Rotstein (2) Robert Romanek (1) Satish Asotra (3) Johannes N. M. Heersche (3) Morris F. Manolson (1)(§)(¶) Guy F. Brisseau (2) Sergio Grinstein (1)(¶)(**)

From the  (1)Division of Cell Biology, The Hospital for Sick Children, M5G 1X8 Toronto, the (2)Department of Surgery, Toronto Hospital and University of Toronto, M5G 2C4 Toronto, and the (3)Faculty of Dentistry, University of Toronto, M5G 1G6 Toronto, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pH) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3)-free solutions. When suspended in Na-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na/H antiporter. However, rapid recovery was also observed in Na-free medium when K was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K/H exchange. In contrast, pH recovery in high K medium was largely attributed to a Zn-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H (equivalent) translocation across the membrane. The H current was greatly stimulated by reducing pH, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.


INTRODUCTION

Osteoclasts are multinucleated cells which are primarily responsible for bone resorption. These cells are located on the bone surface and effect resorption of both the inorganic and organic components of bone in a localized area underlying their attachment site. Several lines of evidence suggest that acidification of the osteoclast-bone interface plays a critical role in the resorptive process. First, the pH of the localized extracellular compartment has been determined to be acidic by fluorescence measurements as well as by studies using microelectrodes (Baron et al., 1985; Silver et al., 1988). Second, low pH is capable of dissolving bone mineral (hydroxyapatite) by a process whereby 2H exchange for every Ca ion released from the crystal (Neuman and Neuman, 1958). Finally, pharmacological agents known to impair H efflux from osteoclasts are able to inhibit bone resorption (see below).

The primary cellular mechanism responsible for the localized acidification of the osteoclast-bone interface is a plasmalemmal vacuolar-type HATPase (V-ATPase), (^1)which actively extrudes protons into the extracellular space. Following cell activation, osteoclasts attached to the bone matrix create an underlying resorption zone characterized by a highly folded membrane called the ruffled border, surrounded by an area rich in actin (Kallio et al., 1971). Immunohistochemical studies have documented the accumulation of V-ATPases within this subdomain of the plasma membrane, allowing vectorial proton secretion to the site of bone resorption (Blair et al., 1989; Väänänen et al., 1990). Functional studies support this concept. Bafilomycin A(1), a specific V-ATPase blocker, inhibits bone resorption (Bowman et al., 1988; Sundquist et al., 1990), as does acetazolamide, an inhibitor of carbonic anhydrase (Hall and Kenny, 1987; Hunter et al., 1991). The latter enzyme facilitates the hydration of carbon dioxide, thereby generating carbonic acid which is the primary source of protons pumped into the resorption lacuna by the V-ATPase (Laitala and Väänänen, 1993; Asotra et al., 1994).

Metabolic acidosis is known to be associated with negative calcium balance and bone loss. Indeed, acidification of the extracellular culture medium has been shown to increase the bone resorption activity of osteoclasts cultured on bone (Walsh et al., 1990; Goldhaber and Rabadija, 1987; Shibutani and Heersche, 1993). While the mechanism underlying this effect is not clear, such observations highlight the need to maintain cytoplasmic pH (pH) homeostasis, since conditions leading to a pH imbalance impair normal osteoclast function. Bicarbonate/chloride (HCO(3)/Cl) exchange has been shown to counteract the intracellular alkalinization that tends to develop during proton secretion into the resorption lacuna (Teti et al., 1989). A Na/H antiporter has also been reported to contribute to pHregulation in these cells (Redhead and Baker, 1988; Hall and Chambers, 1990). To our knowledge, these are the only pH regulatory systems reported to operate in osteoclasts.

In certain cell types, mechanisms other than the cation and anion exchangers have been recently found to contribute importantly to pH homeostasis. Cotransporters, pumps and conductive pathways can also translocate H equivalents and can thereby modulate pH. The purpose of the present studies was to investigate whether additional mechanisms of pH regulation exist in mammalian osteoclasts. Given the problems inherent to acquiring pure populations of osteoclasts, we used single cell fluorescence techniques to examine pH recovery in individual osteoclasts, identified morphologically and using histochemistry. These studies confirmed the role of the Na/H antiporter as a major pH regulatory mechanism in acid-loaded osteoclasts but also indicated that the plasmalemmal V-ATPase, while crucial to bone resorption, plays a minor role in pHhomeostasis. Additionally, combined single-cell fluorescence and patch-clamp techniques revealed the presence of a proton-conductive pathway, which contributed significantly to pH regulation, particularly when the Na/H antiporter was rendered inoperative.


EXPERIMENTAL PROCEDURES

Materials

HEPES, PIPES, MES, nigericin, zinc chloride, ammonium chloride, amiloride and medium RPMI 1640 (bicarbonate-free) were obtained from Sigma. Both the free acid and the acetoxymethyl ester of 2`,7`-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were purchased from Molecular Probes Inc. (Eugene, OR). Bafilomycin A(1) was from Kamiya Biomedical Co. (Thousand Oaks, CA). All other reagents were of analytical grade and obtained from Sigma, Aldrich, Fisher, or BDH.

Solutions

The K medium used during single-cell fluorescence experiments contained 140 mM KCl, 1 mM CaCl(2), 1 mM MgCl(2), 10 mM glucose, and 20 mM HEPES (pH 7.4). The Na and NMG media were made by isosmotic replacement of K with the indicated cation. The composition of the buffers used for patch-clamp experiments is shown in Table 1. All of these solutions contained 1 mM MgCl(2) and 1 mM EGTA, except in the case when Zn was used. For the latter experiments, EGTA was omitted and ZnCl(2) was added to a final nominal concentration (^2)of 0.5 mM. A low concentration of chloride was always present to avoid electrode polarization. When one of the above solutions was used to fill the patch pipette, MgATP was added to a final concentration of 1 mM.



Cell Isolation, Characterization, and Acid Loading

Rabbit osteoclasts were isolated as described previously (Kanehisa and Heersche, 1988). Briefly, 1-day-old New Zealand White rabbits were sacrificed by decapitation and the long bones removed. Cell suspensions containing osteoclasts were obtained from the inside of the bony shafts by scraping out the cancellous bone, suspending these particles in culture medium, and releasing the cells by pipetting. Medium 199 (Life Technologies, Inc.) supplemented with 15% fetal bovine serum and antibiotics was used as the plating medium. Osteoclasts were identified visually for use during single-cell fluorescence and patch-clamping studies. Identifying features included cell size and the presence of multiple nuclei. Only cells containing at least 3 clearly discernible nuclei were studied. At the conclusion of the experiment, this visual identification was confirmed by staining for TRAP. Briefly, cells were fixed with 10% neutral buffered formalin for 10 min and washed with phosphate-buffered saline. The cells were then treated with a cold solution containing 98.5% methanol, 1% N-methyl morpholine, and 0.5% cyanuric chloride for 6 h, rinsed with 0.2 M sodium acetate buffer for 10 min, and subsequently stained for TRAP using Naphtol AS-MX phosphate as the substrate. The cells were visualized with a Nikon microscope at 40times magnification and photographed. A grid that had been previously etched onto the coverslip allowed identification of those cells used during the experiment as staining positively for TRAP.

To load the osteoclasts with acid, adherent cells were incubated in Hepes-RPMI containing 40 mM NH(4)Cl for 12 min at 37 °C and then rapidly transferred to a NH(4)-free Na- or K-containing medium. Where indicated, this technique was used repeatedly to examine pH(i) recovery under different conditions in a single cell.

Fluorescence Microscopy of Acridine Orange-stained Osteoclasts

For acridine orange staining of intracellular acidic compartments, osteoclasts cultured on glass coverslips were loaded with 5 µM dye in HBSS for 15 min at room temperature. Next, the coverslips were washed three times with HBSS and the fluorescence was monitored using a Nikon TMD-Diaphot fluorescence microscope with a 470-490-nm excitation filter, a 510-nm dichroic mirror, and a 520-nm long-pass emission filter. A neutral density filter (neutral density of 16, transmittance = 6.25%) was used to reduce the excitation intensity and minimize dye bleaching. The cells were photographed at 1/4-s shutter speed using Kodak Ektachrome 1600 slide film. The photographed images were digitized and separated into their orange and green components. These images were then converted to a 256 gray scale, representative of dye intensity, for illustration.

Single Cell Measurements of pH(i)

For microfluorimetric studies, osteoclasts were analyzed essentially as described earlier for macrophages (Kapus et al., 1993). The cells were plated for 48 h on acid-washed glass coverslips, placed into a Leiden coverslip dish, and maintained at 37 °C. They were then loaded with BCECF by incubation with 1 µM of the parent acetoxymethyl ester for 15 min at 37 °C. The osteoclasts were next washed with RPMI and incubated in the indicated bathing medium. Single cell fluorescence was monitored using a Nikon TMD-Diaphot microscope attached to an M Series dual wavelength illumination system from Photon Technologies Inc. (Romanek et al., 1994). Illumination was on for 2 s and off 20 s, and the photometric data were recorded at a rate of 5 points/s. Mean values for each 2-s illumination period were plotted against time. Calibration of the fluorescence ratio versus pH was performed using the K/H ionophore nigericin. Cells were equilibrated in K medium (140 mM) of varying pH in the presence of 5 µM nigericin and calibration curves were constructed by plotting the extracellular pH (which is assumed to be identical to the internal pH; Thomas et al., 1979), against the corresponding fluorescence ratio. The resulting curve was sigmoidal with an inflection point approx7.0, as expected from the reported pK(a) of BCECF.

Cellular buffering power was measured by pulsing control or acid-loaded cells with ammonium and calculated as described (Roos and Boron, 1981).

Video Imaging of Cells Loaded with BCECF

Video images of osteoclasts, adhered to glass and loaded into a Leiden holder on a Nikon TMD-Diaphot microscope, were collected and analyzed using Metafluor Imaging System software from Universal Images Corporation. An Empix Imaging filter wheel was used to position the two excitation filters (500 ± 10 nm and 440 ± 10 nm) in front of a Nikon xenon lamp. A neutral density filter (neutral density of 2) was used to reduce the intensity of the light exciting the cells in order to minimize dye bleaching and photodynamic damage. The excitation light was directed to the cells via a 510-nm dichroic mirror. Fluorescence emission collected by a Nikon Fluor 40times oil-immersion objective traversed a 542 ± 64 nm band-pass filter. Data were collected every 20 s by irradiating the cells for 1 s at each of the excitation wavelengths after which the irradiating light was off and data collection suspended. The fluorescence image was captured on a Star 1 liquid-cooled CCD camera (Photometrics Ltd.), then downloaded to a Dell 486 computer where the Metafluor software was used to make a ratio of the two images created by dual excitation. Calibration of the fluorescence ratio versus pH was performed using the K/H ionophore nigericin as described above.

Patch-clamping

The whole-cell configuration of the patch-clamp technique was used to record ionic currents in osteoclasts (Hamill et al., 1981; Kapus et al. 1993). Patch electrodes had resistances ranging from 5 to 7 M and junction potentials were neutralized using the appropriate circuitry of the Axopatch-1D amplifier (Axon Instruments Inc., Foster City, CA). Successful pipette-to-cell attachments resulted in seal resistances varying from 10 to 50 G. Under the conditions used, input resistance in the whole-cell mode was found to range from approx5 to 50 G at normal pH(i). Bath perfusion with the appropriate solutions was initiated only after successful establishment of a patch. To reversibly change the internal pH of the cells during the course of patch-clamping experiments, an NH(4)Cl concentration gradient was applied between the bath perfusion medium and the pipette solution (Grinstein et al., 1994). Briefly, the pipette solution contained 50 mM NH(4) and cells were patched initially in medium RPMI containing 50 mM NH(4). Once the whole-cell mode was established, the concentration of NH(4)Cl in the bath perfusion medium was varied while keeping pH(o) at 7.5. Changes in the ratio of intracellular to extracellular NH(4) resulted in predictable changes in the pH(i).

Currents in response to voltage steps were filtered at 100 Hz with a 4-pole Bessel filter and digitized on line at 3-ms intervals (unless indicated otherwise) using pClamp Clampex software (Axon Instruments Inc.). Data analysis was carried out using pClamp Clampfit and Clampan (Axon Instruments Inc.) and Sigma Plot (Jandel Scientific) software. Where indicated, traces were normalized relative to the maximal current generated by depolarizing the cell to +75 mV. These relative current values facilitate comparison between experiments. Leak current was determined by stepping the voltage from the holding potential of -60 mV to -90 mV in two 15-mV steps. When present, significant linear leak was subtracted from the current traces prior to analysis. All current records are shown with the convention that an upward deflection indicates outward current flow. ``Steady state'' currents were quantified over the last 100 ms of the 2.9-s pulse. Tail current amplitudes were determined by curve fitting and back-extrapolating to the first time point of the test pulse. All patch clamping experiments were carried out at room temperature (approx22 °C). Data are presented as representative traces of at least three similar experiments with cells from different animals or as means ± standard error (S.E.) of the number of experiments indicated. The capacitance of the cells was found to range from 35 to 80 pF (mean ± S.E. were 55.9 ± 5.1 pF).

When electrophysiological and fluorescence data were collected simultaneously, osteoclasts were loaded with BCECF by adding the free acid of BCECF (300 µM) directly to the pipette solution. These experiments were carried out at room temperature (approx22 °C).


RESULTS

Osteoclast Identification

Cultures of mammalian osteoclasts are by necessity impure, containing other cell types as well. It is therefore essential to ensure the identity of osteoclasts during the course of single-cell studies. Osteoclasts are generally characterized as being multinucleated cells that stain positively for TRAP. In the experiments discussed below, nuclearity was assessed morphologically in live cells by Hoffmann optics. The visualization of TRAP activity was performed a posteriori in fixed cells identified on a grid etched on the coverslip, using naphthyl phosphate as substrate. The intensity of red staining generated by the enzyme is directly proportional to its activity. Fig. 1(upper panels) shows light micrographs of adherent rabbit osteoclasts stained for TRAP. Several distinct phenotypes were observed. In panel A, an osteoclast showing the ``flat'' phenotype is shown. In panelsB and C, migrating osteoclasts displaying the ``spread'' phenotype are shown. Finally, in panelsD and E, cells with the ``rounded and compact'' phenotype are illustrated. The number of nuclei in the latter cells ranged from 3 to 5, and their diameter averaged 28 µm. The round and compact phenotype comprised approx10% of the total osteoclast population in our cultures. Such morphology is comparable to that most frequently observed when cells are cultured on bone slices (Arkett et al., 1992) and is known to be the active phenotype in terms of bone resorption (Kanehisa and Heersche, 1988). Accordingly, the fraction of compact cells has been noted to increase in proportion to the resorptive activity of an osteoclast population (Shibata et al., 1993). Based on these criteria, compact cells have the most physiological relevance and were therefore used in the majority of studies reported in this paper.


Figure 1: Phenotypic characterization and pHrecovery of osteoclasts cultured on glass coverslips. Upperpanel, light micrographs of TRAP-stained osteoclasts illustrating the different morphological types: A, giant spread osteoclast; B and C, motile osteoclasts of intermediate size; D and E, compact osteoclasts. The bar (panel A) represents 40 µm and applies to A-E. Lower panel, pHrecovery of acid-loaded osteoclasts. Osteoclasts were loaded with BCECF-AM and incubated with 40 mM NH(4)Cl for 20 min at 37 °C. Next, the cells were washed and resuspended in K medium at 37 °C and fluorescence ratio images were collected as described under ``Experimental Procedures'' at 0, 20, 140, and 260 s after the solution change (top row). The same cell was then reloaded with NH(4)Cl and recovery in Na medium monitored. Reversing the order of the recovery solutions (i.e. Na solutions after the first acid load and K after the second) yielded comparable results. Upon completion of the experiment, the pH of the cell was set to 6.3, 6.8, and 7.3 using nigericin/K and the resulting images along with the pH pseudocolor scale are illustrated to the left of the main panel. Data are representative of 3 similar experiments.



Imaging of Intracellular pH

Fluorescence ratio imaging was used to ascertain the cytosolic distribution of the pH-sensitive dye and to assess the existence of intracellular pH gradients. The latter possibility was of interest given the unusually large size of osteoclasts and the polar distribution of H-transporting systems in these cells (see Introduction). Fig. 1(lowerpanels) shows a typical series of images demonstrating pH(i) recovery from an acid-load in a cell perfused with either K-rich (toprow) or Na-rich (bottomrow) medium. Prior to acid loading, the dye appeared to be uniformly distributed in the cytoplasmic compartment and the pH(i) was similar throughout the cytosol. Small inhomogeneities in the fluorescence ratio likely result from optical artifacts, since they persisted after clamping pH(i) with nigericin and were observable at different pH levels (see insets beside calibration scale). In six determinations, the pH(i) of resting osteoclasts in medium RPMI averaged 7.1.

As illustrated in Fig. 1(lowerpanel), the cytosol of osteoclasts acidified uniformly following an NH prepulse. In cells bathed in Na-rich medium a rapid recovery of pH ensued, with restoration of the base-line level within approx4 min. A more gradual recovery was noted in Na-free, K-rich solution (Fig. 1). No subcellular pH gradients were found to develop during the recovery from acid loading in either solution at any of the times tested. The mechanisms underlying H extrusion in Na and K-rich media are analyzed in detail below. A photomultiplier-based fluorescence ratio system, rather than imaging, was used for the subsequent experiments to improve the temporal resolution while minimizing photodynamic damage of the cells.

Na-dependent pH(i) Recovery

To determine the contribution of Na/H exchange to pH(i) homeostasis in acid-loaded osteoclasts, the rate of recovery was compared in Na medium with or without amiloride (1 mM), a potent inhibitor of the antiporter. As shown in Fig. 2A, a rapid acidification to pH(i) approx6.5 developed upon superfusion of NH(4)-loaded cells with Na medium free of NH(4) and HCO(3). After the initial drop, pH(i) recovered rapidly toward the resting level. Under these conditions, the initial rate of alkalinization (measured over the first min) averaged 0.40 ± 0.12 pH/min (n = 4). Over the ensuing 4-min period, the cytosolic pH recovered to 7.2 ± 0.15, a level comparable to the resting pH(i) of these cells. Two differences were observed in the presence of amiloride. First, removal of NH(4)Cl was followed by a much more profound acidification of the cytosol. Second, the initial pH(i) recovery rate was markedly slower, averaging 0.01 ± 0.03 pH/min (n = 5). Therefore, under the conditions tested, amiloride reduced the initial recovery by 97.5%. These studies suggest an important role for an amiloride-sensitive component, in all likelihood the Na/H antiport, in pH(i) regulation by acid-loaded osteoclasts.


Figure 2: pH recovery of acid-loaded osteoclasts. Osteoclasts were loaded with BCECF-AM and incubated with 40 mM NH(4)Cl for 20 min at 37 °C. The pH of single cells was measured photometrically. A, data collection was started upon superfusion with Na medium (without NH(4)Cl) at 37 °C (open circles; n = 4). The cell was then reloaded with NH(4)Cl and recovery from the acid load was monitored in Na medium containing 1 mM amiloride (solidcircles; n = 5). Data are means ± S.E. of the indicated number of experiments. B, acid-loaded osteoclasts were superfused in K medium (opencircles) or NMG medium (solidcircles) at 37 °C. Data are means ± S.E. of geq5 similar experiments.



As an alternative test of this hypothesis, recovery from an acid load was studied in media devoid of Na. As shown in Fig. 2B, replacement of Na by the purportedly impermeant NMG yielded results that were comparable to those obtained with amiloride, i.e. a profound acidification followed by an insignificant recovery. Unexpectedly, a sizable recovery (0.35 ± 0.13 pH min; n = 6) was noted in K-rich medium devoid of Na (opencircles). Because osteoclasts are known to display a prominent K conductance (Arkett et al., 1992), it is likely that the cells depolarized in K solution. These data suggest that the membrane potential may be an important determinant of the pH(i) recovery observed in Na-free medium. The Na-independent pH(i) regulatory processes were studied in detail below.

Role of V-ATPases in pH(i) Regulation

As noted earlier, osteoclasts are endowed with plasmalemmal V-ATPases which play a central role in bone resorption by virtue of their ability to extrude protons into the extracellular medium. Conceivably, these pumps might also contribute to pH(i) regulation as they do in macrophages, cells known to be of similar lineage to osteoclasts. The enhanced Na-independent pH(i) recovery recorded in depolarizing (K-rich) medium is consistent with the electrogenic nature of these pumps. To determine the role of V-ATPases in pH(i) regulation in osteoclasts, acid-loaded cells were permitted to recover in K medium containing the specific V-ATPase inhibitor, bafilomycin A(1) (Bowman et al., 1988). The inhibitor was added to the cells during the final 5 min of the NH(4) pulse and was also present during the assay. As shown in Fig. 3(leftpanel), the addition of bafilomycin A(1) had only a marginal, statistically insignificant inhibitory effect on the pH(i) recovery (cf.Fig. 2B and Fig. 3). To ensure that the concentration of bafilomycin A(1)used in these studies (200 nM) was sufficient to inhibit V-ATPases in osteoclasts, the ability of the macrolide to dissipate V-ATPase-dependent acidification of intracellular organelles was examined using acridine orange. Sequestration of this weak base in acidic endomembrane compartments results in a shift in its fluorescence emission (from green to orange), thereby permitting qualitative evaluation of the intravesicular pH under various conditions (Tsien, 1989). A punctate orange staining was observed upon incubation of otherwise untreated osteoclasts with acridine orange (Fig. 3, upperleftimage), consistent with accumulation of the dye in acidic intracellular organelles. The addition of 200 nM bafilomycin A(1) for 5 min caused almost complete disappearance of the orange fluorescence (Fig. 3, lowerleftimage), with a corresponding rise in green fluorescence (cf.upper right versuslower right images, Fig. 3), findings in keeping with dissipation of the acidic pH of these organelles. Binding of acridine to DNA resulted in green staining of the nuclei (Fig. 3, rightpanels), confirming the multinuclearity of the osteoclasts used in this study. Dissipation of the orange punctate pattern was also observed when the protonophore carbonyl cyanide m-chlorophenylhydrazine (1 µM) was added to the cells (data not shown). Considered together, these data suggest that the concentration of bafilomycin A(1) used in the pH(i) recovery studies was sufficient to inhibit V-ATPases in these cells. (^3)On this basis, we tentatively conclude that active H-pumping through V-ATPases plays only a minor role in pH(i) regulation in cultured osteoclasts.


Figure 3: Effects of bafilomycin A(1) and of Znon pH recovery in K medium. Left panel, osteoclasts were loaded with BCECF-AM and then incubated with 40 mM NH(4)Cl for 20 min at 37 °C. The cells were next superfused with K medium containing 100 µM Zn (solid circles). Alternatively, the cells were pretreated with 200 nM bafilomycin A(1) during the final 5 min of the acid-loading step and then exposed to K medium containing the same concentration of bafilomycin (open circles). Traces are means ± S.E. of geq5 similar experiments. Right panel, acridine orange staining of a control cell (upper images) or a cell treated for 5 min with 200 nM bafilomycin A(1) (lowerimages). The orange and green components of the fluorescent emission from the same cell were separated by use of appropriate filters and are displayed separately. Orange fluorescence, indicative of acidic organelles, is shown in the leftimages, and green fluorescence, reflective of dye distributed at near-neutral pH, is shown to the right.



Role of K/HExchange in pH Regulation

Rapid dissipation of the acid load in high K medium is compatible with the occurrence of K/H exchange. Two types of experiments were designed to test this possibility. First, BCECF-loaded but otherwise untreated cells were bathed in K-rich solution and pH(i) was determined. Rapid replacement of external K by NMG failed to induce the acidification predicted from the exchange of internal K for external H (not illustrated). Second, cells were dialyzed with a patch pipette containing BCECF and lightly buffered, K-free solution at pH 6.0 (Cs-Asp 6.0 with 5 mM MES; Table 1). These cells were then superfused with either NMG- or K-rich solutions at pH 7.5, while holding the voltage at -60 mV. No significant alkalinization was recorded in K solution, as would be expected from active K/H exchange. That the setup was sufficiently sensitive to detect pH(i) changes was demonstrated by depolarizing the cells to +75 mV for 10 s. In three experiments, this maneuver induced an alkalinization of 0.64 ± 0.06 pH units. Together, these experiments argue against the presence of an active K/H antiporter.

Contribution of a Proton-conductive Pathway to pH(i) Recovery

Further studies were performed to define the mechanism(s) responsible for the residual pH(i) recovery in Na-free media. Recent studies have reported the existence of proton-conductive pathways in macrophages, neutrophils, and HL-60 cells (Demaurex et al., 1993; Kapus et al., 1993; DeCoursey and Cherny, 1993). Proton (equivalent) movement via this mechanism was inhibited by Zn, a potent inhibitor of H-selective currents (Mahaut-Smith, 1989; Lukacs et al., 1993), and augmented by depolarizing conditions. Given the common lineage of osteoclasts and macrophages and the observation that Na-independent pH(i) recovery occurred preferentially in K medium, we hypothesized that a H-conductive pathway might also exist in osteoclasts. To examine this possibility, recovery from an acid-load was studied in K medium in the presence of Zn (100 µM). As shown in Fig. 3(left panel), the rate of pH(i) recovery was negligible in the presence of the divalent cation. The effect of Zn was reversible; near normal rates of recovery were recorded after the cation was removed (not illustrated).

Evidence for a Zn-sensitive Outward-rectifying Current in Osteoclasts

Since the fluorescence studies of pH(i) suggested the existence of conductive Hfluxes, electrophysiological studies of single, adherent osteoclasts were undertaken to determine whether H-selective currents could be detected. Osteoclasts were initially patch-clamped in the whole-cell configuration with a pipette containing NMG-Asp solution pH 6.0, while the bath was perfused with NMG-Asp pH 7.5. These ionic conditions were chosen to optimize the detection of H-selective currents, while minimizing the contribution of other ion channels. The voltage protocol used for analysis of the current is illustrated in the inset of Fig. 4A. The holding potential was clamped at -60 mV, and pulses lasting 2.9 s were applied between -90 and +75 mV in 15-mV increments at 30-s intervals. As shown in Fig. 4A, little current was detectable at potentials more negative than -15 mV, while a clear outward current was recorded at this and more positive potentials. Activation was slow and frequently occurred with sigmoidal kinetics, particularly at the lower voltages. The current-voltage relationship, averaged from four experiments, is presented in Fig. 4B. Importantly, addition of Zn (0.5 mM)^2 rapidly and reversibly shifted the current-voltage relationship to more positive potentials. The blockage of the outward currents in adherent patch-clamped cells by Zn is consistent with the fluorescence studies of pH(i) recovery, which suggested the existence of a proton conductive pathway.


Figure 4: Outward-rectifying, slowly activating, Zn-sensitive current. Osteoclasts were patched in the whole-cell configuration with a pipette containing NMG-Asp 6.0. The holding potential was clamped at -60 mV, and pulses lasting 2.9 s were applied between -90 and +75 mV in 15-mV increments at 30-s intervals. A, family of currents from a 39-pF osteoclast patched with a 7-M pipette. The bath was perfused with NMG-Asp pH 7.5. B, patched osteoclasts were perfused with NMG-Asp pH 7.5 solution (solid circles) and a family of current traces like those in A were generated. The bath was next perfused with solution NMG-Asp/Zn pH 7.5 (opentriangles), and another family of currents was recorded. Finally, the bath was perfused with the original Zn-free solution (solidtriangles), and one last current family generated. The magnitude of the current was averaged over the last 150 ms of each voltage pulse. To facilitate comparison between experiments, the average current determined during the +75-mV pulse from the first family of traces was set to 1 and all other currents from each cell are expressed relative to this value. The relative currents of four cells from different animals are plotted versus the applied potential. Steady-state currents prior to application of test pulses were negligible. Values are means ± S.E. (n = 4). Where absent, bars were smaller than the symbols.



While the conditions used above facilitate detection of the conductance, they are unphysiological (low pH(i) and cytosolic media devoid of K). However, the outward current can also be readily elicited at near physiological pH(i) (6.9) and intracellular K concentrations (solution K-Asp 6.9; Table 1). In fact, these conditions were used below to assess the effects of K channel antagonists.

Voltage-driven Changes in Osteoclast pH(i)

To explore the relationship between the observed pH(i) recovery in depolarizing medium and the voltage-gated current seen in the patch-clamping experiments, simultaneous electrophysiological and fluorimetric analyses of single, adherent osteoclasts were performed. The purpose of these experiments was to determine whether the current recorded was, at least in part, carried by H (equivalent) ions. We therefore determined whether the current induced by applied voltages was associated with changes in pH(i). Cells were patch-clamped in the whole-cell configuration with a pipette containing the free acid of BCECF (300 µM) dissolved in solution NMG-Asp low beta 6.5 (see Table 1for details of composition of this and other media). The pH of this solution was 6.5, to approximate the conditions attained in the acid-loaded cells described above. The bathing medium was solution NMG-Asp 7.5. Equilibration of cytosolic pH with the pH of the pipette occurred within minutes once the whole-cell patch configuration was achieved. The holding potential of -60 mV elicited no pH(i) changes, nor did it generate detectable current. Voltage pulses of varying intensity were then applied for periods of 10 s, while pH(i) was simultaneously monitored by fluorescence ratio (Fig. 5). No significant pH(i) change was seen when the voltage was stepped to -30 mV, nor was any current recorded. However, both a modest current followed by a small (approx0.1 pH) yet reproducible intracellular alkalinization were recorded at 0 mV. The currents and associated pH(i) increases became more pronounced at progressively more depolarizing voltages. At +60 mV an alkalinization of nearly 0.3 pH (^4)developed during the 10-s pulse. It must be borne in mind that such pH changes occurred despite the continuity of the cytosol with the comparatively unlimited reservoir of buffer in the patch pipette. These experiments indicate a correlation between the elicited outward current and changes in pH(i), suggesting that at least part of the current is due to proton (equivalent) flux.


Figure 5: Depolarization increases pH and activates an outward current. BCECF-loaded osteoclasts on glass coverslips were patched in the whole-cell configuration at a holding potential of -60 mV. The patch pipette contained NMG-Asp low beta 6.5, and the bath was continually perfused with solution NMG-Asp 7.5. Cells were then subjected to voltage steps lasting 10 s, at 120-s intervals. The voltage protocol is illustrated by the bottom traces. The corresponding whole-cell currents are illustrated in the middle traces. pH was estimated simultaneously in the patched cell by measuring the fluorescence ratio of BCECF and is shown by the top traces. Data are representative of 3 similar experiments.



Characterization of the Outward Current

While the above experiments suggest that the movement of H equivalents represents a significant component of the current, the contribution of other species and the ionic selectivity of the pathway was not delineated. In order to examine the contribution of other ions to the outward current, the reversal potential of tail currents was studied under various conditions. The conductance was initially activated by the application of a depolarizing pulse, the magnitude and duration of which was limited to minimize changes in intracellular pH, which could conceivably affect the properties of the tail currents. Test pulses of varying voltage were then applied and the deactivating currents were recorded. Tail currents from a representative experiment are shown in Fig. 6A. By curve fitting, the instantaneous current at the start of each test pulse was determined. These current values were then plotted against the potential of the test pulse. The reversal potential (E) was then defined by interpolating the zero-current potential. Tail current analysis was performed at varying pH(o) with constant pH(i) (6.0), and representative traces are shown in Fig. 6A. Clearly, E varied markedly as a function of the transmembrane DeltapH. In Fig. 6B, E calculated from four similar experiments was plotted as a function of pH(o). The similarity of the experimental values (circles) with the H equilibrium potential (E(H)+; solidline) (^5)strongly suggests a primary role for H equivalents in carrying the outward current. Further studies, summarized in Table 2, showed that while E was exquisitely sensitive to the concentration of H, it was virtually unaffected by imposition of large inward K or Cl gradients. Moreover substitution of Cs for NMG also had little effect on E. Since Cs is an effective K-channel blocker, these data further rule out the possibility that contaminating K ions contribute significantly to the observed current.


Figure 6: Relationship between the reversal potential of tail currents (E) and the transmembrane pH gradient. Osteoclasts were patched in the whole-cell configuration using patch pipettes filled with NMG-Asp 6.0. The bath was then perfused with NMG-Asp 7.5, NMG-Asp 6.8, or NMG-Asp 6.0. E was determined using the following protocol: the holding potential was -60 mV, outward current was first activated by a 900-ms depolarizing pulse, chosen to activate the current yet minimize pH changes. Test pulses lasting 350 ms were next applied between -90 mV and +30 mV in +10-mV increments. A, representative tail currents from an osteoclast with a capacitance of 60 pF patched with a 7-M pipette. For clarity only currents at E, one inward and one outward current, have been illustrated. B, curve fitting of tail currents like those illustrated in A was used to determine the instantaneous tail current. Linear regression of 3-4 of these instantaneous currents on either side of the zero current level was used to calculate E, which is plotted in panelBversus pH. Each point represents the mean ± S.E. of 3 experiments using cells from different animals. Where absent, error bars were smaller than the symbol. The solid line is the predicted H equilibrium potential (E) (see also Footnote 5).





Table 2also demonstrates the effect of varying pH(i) on the E. Changes to the internal pH of osteoclasts patched in the whole-cell mode were achieved at constant pH(o) by including a fixed concentration of NH(4)/NH(3) in the pipette solution and then altering the external NH(4)/NH(3) concentration to predetermined values. This protocol results in rapid and reversible clamping of pH(i), at levels which can be predicted from the transmembrane NH(4) gradient (Table 2; see Grinstein et al.(1994) for details). As was found for pH(o), changes in pH(i) produced sizable shifts in the E which closely followed the predicted E (see last column of Table 2). Considered in aggregate, these findings provide strong evidence that H (equivalents) are the species primarily responsible for the voltage-dependent outward currents in acid-loaded cells.

Effects of pH(o) and pH(i) on the Outward Current

Fig. 7examines the effect of the extracellular pH (pH(o)) on the current-voltage relationship. Cells were patched with a pipette containing NMG-Asp solution at pH 6.0 and perfused sequentially with NMG-Asp solutions at pH 6.0, 6.8, or 7.5. To facilitate comparison between experiments, the current-voltage relationship was normalized relative to the maximum current recorded in each experiment (generated by depolarization to +75 mV using pH(i) = 6.0 and pH(o) = 7.5). Results derived from three independent experiments indicated that an extracellular acidification markedly reduced the outward current at all voltages where the conductance was active and shifted the threshold for current activation to more positive voltages (Fig. 7).


Figure 7: Effect of pH on H conductance. Osteoclasts patched in the whole-cell configuration were clamped at a holding potential of -60 mV. Pulses lasting 2.9 s were applied between -90 and +75 mV in 15-mV increments at 30-s intervals. The pipette contained solution NMG-Asp 6.0. The bath was perfused with NMG-Asp 7.5 (opencircles), NMG-Asp 6.8 (solidcircles), or NMG-Asp 6.0 (open triangles). Points were calculated by averaging the current during the last 150 ms of each voltage pulse. To facilitate comparison between experiments, the current generated at pH 7.5 during the +75-mV pulse was set to 1 and all other currents are normalized to this value. The relative currents of three cells from different animals are plotted versus the applied potential. Values are means ± S.E. (n = 3). Where absent, bars were smaller than the symbol.



The effect of decreased pH(i) was also examined (Fig. 8). Using the NH/NH gradient protocol, changes to the internal pH were made in single cells at a constant pH = 7.5. Contrary to the effects of altering pH, internal acidification potentiated the current, while shifting the activation threshold to more negative potentials (Fig. 8). These findings can be partially explained by the changes in driving force that accompany alteration in the transmembrane pH gradient, lending further credence to the idea that H equivalents are the primary charge carriers. In addition, however, pH also exerts direct effects on the permeability to H. This property of the conductance has been explored in more detail in macrophages (Kapus et al., 1993).


Figure 8: Effect of pH on H conductance. Osteoclasts patched in the whole-cell configuration were clamped at a holding potential of -60 mV. Pulses lasting 2.9 s were applied between -90 and +75 mV in 15-mV increments at 30-s intervals. The pipette contained solution Cs-Asp/NH(4)-50. The bath was perfused with Cs-Asp/NH(4)-1 (solid circles), Cs-Asp/NH(4)-3 (open triangles), Cs-Asp/NH(4)-9 (solid triangles), or Cs-Asp/NH(4)-15 (open squares). The calculated intracellular pH (see Grinstein et al.(1994) for details) is indicated for each condition. Points were calculated by averaging the current during the last 150 ms of each voltage pulse. Currents were normalized as in Fig. 7. The relative currents of three cells from different animals are plotted versus the applied potential. Values are means ± S.E. (n = 3). Where absent, bars were smaller than the symbol. The intracellular pH was estimated as described under ``Experimental Procedures.''



Possible Role of K Channels

Both inward- and outward-rectifying K channels have been described in osteoclasts (Arkett et al., 1994). The latter bear some interesting similarities to the conductance described above, including susceptibility to inhibition by extracelluar Zn and H. It was therefore conceivable that in our experiments H were traversing K channels, particularly when the latter cation was omitted. To assess this possibility, we measured the effects of several other agents known to affect the osteoclast K outward rectifier. Partial (50 ± 21%) inhibition was obtained with 1 mM 4-aminopyridine. This agent had been shown earlier to inhibit other H-selective channels, possibly by acting as a weak base and altering local pH, since similar results were obtained with other bases (see DeCoursey and Cherny(1994) for review). In contrast, the conductance was only marginally reduced (4.7 ± 0.8%; n = 3) by 5 mM tetraethylammonium and was unaffected by intracellular Cs (geq100 mM), which was in fact used in several of the experiments above (e.g.Fig. 8). These findings, together with the markedly different kinetics of activation and inactivation of the K and H currents, strongly suggest that these ions traverse different channels.


DISCUSSION

The ability to regulate pH(i) within a tight physiological range is crucial for normal cellular function. This is particularly important for active cells such as osteoclasts, since the products of their metabolism tend to deviate pH(i) away from the normal range. The present studies analyzed some of the mechanisms capable of acid extrusion from osteoclasts, which are likely involved in pH(i) homeostasis during acidification of the osteoclast microenvironment or activation of these cells by other means. As previously reported (Teti et al., 1989; Hall and Chambers, 1990), osteoclasts cultured on glass coverslips had a functional, amiloride-sensitive, Na/H antiporter operating in the plasma membrane. However, unlike many other cells, osteoclasts were also able to recover from an acute acid load in Na-free solutions, implicating the presence in these cells of pH regulatory mechanisms other than the antiporter. Of note, the Na-independent recovery was observed mainly in depolarized cells, suggesting that a voltage-sensitive, possibly rheogenic process was involved.

Osteoclasts are known to be endowed with plasmalemmal V-ATPases. These pumps operate physiologically to translocate protons into the extracellular space and are necessary for bone resorption. Our previous studies demonstrated that in peritoneal macrophages, plasmalemmal V-ATPases play a major role in pH(i) homeostasis (Swallow et al., 1990). Based on the common lineage of these two cell types, it was anticipated that V-ATPases would similarly contribute to pH(i) regulation in osteoclasts. However, the recovery noted in Na-free medium was virtually resistant to inhibition by the specific V-ATPase inhibitor, bafilomycin A(1), under conditions and at concentrations where the macrolide induced rapid dissipation of the pH gradient within the intracellular organelles. Thus, the bafilomycin used was active and achieved sufficiently high cellular levels to exert its inhibitory effect. These data do not rule out the possibility that pump isoforms may exist in the plasmalemma which are selectively resistant to bafilomycin. However, bone resorption, which is ostensibly mediated by V-ATPases at the ruffled membrane, has been shown to be susceptible to bafilomycin A(1) (Sundquist et al., 1990).

Several explanations may underlie our failure to detect V-ATPase-mediated H extrusion from these cells. First, the cells were cultured on glass coverslips rather than on bone slices (to permit spectroscopic analysis) and this may preclude their exposure to the normal physiological stimuli required for either physical or functional expression of pumps on the plasmalemma. In other cells, environmental stimuli are known to promote translocation of pumps present in intracellular vesicles to the plasma membrane (Al-Awqati, 1986). Alternatively, bone-dependent stimuli may induce association of activator proteins (Xie et al., 1993) with the pump or dissociate pump inhibitor molecules (Zhang et al., 1992). Second, if the pumps are circumscribed to the portion of the membrane in apposition to the substratum, they may be unable to contribute to pH(i) regulation. Rapid acidification of the underlying secretory space may kinetically or thermodynamically limit the activity of such pumps, rendering them incapable of further H extrusion upon cytosolic acidification. Cytochemical studies defining the localization of V-ATPases under the conditions studied should clarify the explanation. Further, osteoclasts cultured on glass as in the present studies offer an excellent experimental model to study H-pump induction or up-regulation in seemingly ``quiescent'' osteoclasts.

The ability of the cells to undergo Na-independent pH(i) recovery in K medium but not in NMG suggested a role for a voltage-dependent process, possibly a H conductive pathway. Several lines of evidence support the notion that H conductance contributes to pH(i) recovery under these conditions. First, Zn, an inhibitor of conductive proton permeability in other systems, reversibly prevents pH(i) recovery following an acid load. Second, patch clamp studies using the whole-cell configuration showed that an outwardly directed current could be induced when depolarizing pulses were applied to osteoclasts. The current was reversibly blocked by Zn and was associated with cytosolic alkalinization, the magnitude of which correlated with the size of the current. Third, ion substitution experiments demonstrated that the magnitude and direction of the current were virtually unaffected by replacement of the major ionic species with large, impermeant organic or inorganic ions. Importantly, Cs was not inhibitory, making it unlikely that a K conductance was responsible for the current (Ravesloot et al., 1989; Arkett et al., 1992). Further, the slow kinetics of the response were distinctly different from those of the outward-rectifying K current reported earlier in mammalian osteoclasts (Arkett et al., 1992).

The magnitude of the current was found to depend on the transmembrane pH gradient. Indeed, the E derived from analysis of the tail currents at varying pH(o) and/or pH(i) levels correlated well with the calculated E, providing strong indication that H equivalents are the primary charge carriers. Thus, when considered together, the results from both fluorescence measurements and from electrophysiological studies provide convincing evidence that pH(i) recovery in Na-free medium is mediated by a conductance that is extremely selective for H.

Is the magnitude of the current sufficient to account for the observed changes in pH(i)? Under conditions that mimic those in the acid-loaded cells of Fig. 1Fig. 2Fig. 3, the ``steady state'' current at 75 mV averaged 373 ± 53 pA (n = 11), equivalent to 6.7 pA/pF. At 0 mV, the approximate potential of cells in K media, the current density was about 5-fold lower (Fig. 4, Fig. 7, and Fig. 8). Assuming that the current is carried entirely by H equivalents and considering the cellular volume, it is possible to predict the pH change associated with the passage of current, if the buffering capacity of the cells is known. The latter was estimated by pulsing with ammonium and averaged 22.8 ± 3.5 mM/pH in acid-loaded cells (pH(i) 5.9-6.1) and 12.65 ± 2.8 mM/pH (n = 4) at physiological pH(i). Cellular volume was calculated from the measured average diameter of the cells (approx28 µm), assuming that adherent cells are approximately hemispherical. When these parameters are taken into consideration, the H current recorded at approx0 mV is predicted to alkalinize the cells at a rate of 0.3 pH/min, which compares favorably with the actual rates recorded fluorimetrically (Fig. 2B).

The exact physiological role for the observed H conductance in osteoclasts has not been established. A Zn-sensitive H conductance has recently been reported in peritoneal macrophages (Kapus et al., 1993), in neutrophils (DeCoursey and Cherny, 1993), and in granulocytic HL60 cells (Demaurex et al., 1993). The currents in these cell types share many similarities, including outward rectification, sensitivity to internal and external pH, and inhibition by heavy metals (DeCoursey and Cherny, 1993; Demaurex et al., 1993; Kapus et al., 1993). One feature that is common to all these cell types is their ability to undergo a respiratory burst in response to stimulation. It has been suggested that the conductance is required to extrude the protons that are released by the oxidative pathway (Lukacs et al., 1993). Of note, osteoclasts are similarly endowed with an NADPH oxidase and are capable of a respiratory burst (Key et al, 1990; Garrett et al., 1990), so that the H conductance may serve a comparable function. While the conditions required for stimulation of the conductance appear too extreme for it to activate in the resting state, local acidification and/or a depolarization generated by the purportedly electrogenic oxidase may suffice to gate the current. Two stable levels of membrane potential have been reported in osteoclasts, -70 and -15 mV (Mears, 1971; Ferrier et al., 1986; Ravesloot et al., 1989; Sims and Dixon, 1989). The depolarized state, believed to correspond to the activated cell, would promote activation of the conductance at moderately acidic pH(i) (i.e. 6.8-6.5, see Fig. 8), which may result from metabolic acid generation. It is conceivable, therefore that the voltage-sensitive H conductance contributes to translocation of acid equivalents during the early stages of acidification of the lacunae. Subsequently, further extracellular acidification would inactivate the conductance (Fig. 7), preventing detrimental backflux of protons extruded by the pump.

In summary, the present studies show that V-ATPases contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data are consistent with the presence of a Zn- and membrane potential-sensitive Hconductance in the plasma membrane of osteoclasts. In this regard, it is noteworthy that there are reports in the literature showing that Zn can affect bone formation (Yamaguchi et al., 1987) and influences the course of osteoporosis (Fushimi et al., 1993). The mechanism underlying this effect of Zn is unknown but could be related to inhibition of the bone-resorbing activity of osteoclasts. In fact, a recent preliminary report suggests that concentrations of Zn expected to antagonize the conductance potently inhibit osteoclastic bone resorption in vitro (Moonga and Dempster, 1994). Whether this is a direct effect on the H conductance described herein is not known.


FOOTNOTES

*
This work was supported in part by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Scholar of the Medical Research Council of Canada.

Cross-appointed to the Dept. of Biochemistry of the University of Toronto.

**
International Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Div. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028.

(^1)
The abbreviations used are: V-ATPase, vacuolar-type adenosine triphosphatase; pH, cytoplasmic pH; pH, extracellular pH; BCECF, 2`,7`-bis-carboxyethyl-5(6)-carboxyfluorescein; NMG, N-methyl-D-glucammonium; TRAP, tartrate-resistant acid phosphatase; HBSS, Hanks' balanced salt solution; , ohm(s); pF, picofarad(s); MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
The free concentration of Zn, which can form stable complexes with certain anions, was not determined. For this reason, concentrations in the text are only nominal. The total concentration of Zn used in electrophysiological experiments (0.5 mM) was greater than that used in fluorescence determinations, to compensate for the ability of aspartate (the main anion in the patch-clamp experiments) to bind the divalent cation.

(^3)
We cannot rule out the possibility that plasmalemmal and endomembrane V-ATPases differ in their sensitivity to bafilomycin A(1).

(^4)
This value is an average of the whole cell pH. Because of the inordinately large size of these cells, it is most likely that the DeltapH near the membrane was considerably greater.

(^5)
Data were calculated from the bulk pH of the pipette and bath using the Nernst equation.


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