Phagocytes generate large amounts of metabolic acid during activation. Therefore, the presence of a conductive pathway capable of H + extrusion has been suggested (Henderson, L. M., J. B. Chappell, and O. T. G. Jones. 1987. Biochemical Journal. 246:325-329.). In this report, electrophysiological and fluorimetric methods were used to probe the existence of a H + conductance in murine peritoneal macrophages. In suspended cells, recovery of the cytosolic pH (pHi) from an acid-load in Na + and HCO~-free medium was detectable in depolarizing but not in hyperpolarizing media. The rate of alkalinization was potentiated by the rheogenic ionophore valinomycin. These findings are consistent with the existence of a conductive H + (equivalent) pathway. This notion was confirmed by patchclamping and fluorescence ratio measurements of single adherent cells. When voltage was clamped in the whole-cell configuration, depolarizing pulses induced a sizable outward current which was accompanied by cytosolic alkalinization. Several lines of evidence indicate that H + (equivalents) carry this current: (a) the conductance was unaffected by substitution of the major ionic constituents of the intraand/or extracellular media, (b) the reversal potential of the tail currents approached the H + equilibrium potential; and (c) the voltage-induced currents and pHi changes were both Zn z+ sensitive and had similar time course and potential dependence. The peak whole-ceU current displayed marked outward rectification and was exquisitely H + selective. At constant voltage, the H + permeability was increased by lowering pHi but was inhibited by extracellular acidification. Together with the voltage dependence of the conductance, these features ensure that H + extrusion can occur during activation, while potentially deleterious acid uptake is precluded. The properties of the conductance appear ideally suited for pHi regulation during phagocyte activation, because these ceils undergo a sustained depolarization and an incipient acidification when stimulated. Comparison of the magnitude of the current with the amount of metabolic acid generated during macrophage activation indicates that the conductance is sufficiently large to contribute to the H + extrusion required for maintenance of pHi.

H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in the case of phagocytes. When stimulated, these cells undergo a sustained depolarization, while generating large amounts of metabolic acid. In addition, conductive H+ fluxes may also provide counterions to neutralize the activity of electrogenic enzymes, as suggested for the phagocyte NADPH oxidase.

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H ؉ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na ؉ /H ؉ antiport activity. In contrast, chronic acidosis activated vacuolar H ؉ pumps in the osteoclasts. Although only ϳ5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6.5 for 4 -6 h were able to recover from the acute acid load by means of bafilomycin A 1 -sensitive proton extrusion. Conversely, the H ؉ -selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H ؉ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.Bone resorption is a multistep process involving migration of osteoclasts and/or osteoclast precursors to the bone surface, attachment to the bone matrix, and subsequent degradation of the underlying bone mineral by local acidification of the osteoclast-bone interface. When resorbing bone, osteoclasts display a specialized attachment zone, called the clear zone, which delimits a sealed compartment characterized by the presence of an extensive ruffled cell membrane (1). Demineralization of the bone matrix requires acidification of this extracellular compartment. Two lines of evidence suggest that the primary cellular mechanism responsible for this acidification is a vacuolar type H ϩ -ATPase (V-ATPase) 1 localized to the ruffled border of these cells. First, immunohistochemical studies demonstrated a marked accumulation of V-ATPases on the ruffled membrane of osteoclasts adherent to bone (2, 3). Second, the bone-resorbing capacity of osteoclasts is effectively inhibited by the specific V-ATPase inhibitor bafilomycin A 1 (4, 5). Considered together, these observations indicate a central role for the plasmalemmal V-ATPase in osteoclastic bone resorptio...

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

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 (pHi) 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, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-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 pHi, 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.