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...

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.

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PPB patients continue to represent a diagnostic challenge. Asymptomatic and predominantly cystic PPB remain indistinguishable from CCAM preoperatively. A high index of suspicion for PPB must be considered in any child presenting with cystic lung lesions beyond early infancy, particularly in a child with poor weight gain.

Maintenance of cytoplasmic pH (pH i ) within a narrow physiological range is crucial to normal cellular function. This is of particular relevance to phagocytic cells within the acidic inflammatory microenvironment where the pH i tends to be acid loaded. We have previously reported that a vacuolar-type H ؉ -ATPase (VATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pH i regulatory mechanism within the inflammatory milieu. Since this microenvironment is frequently characterized by the influx of cells known to release inflammatory cytokines, we performed studies to examine the effect of one such mediator molecule, interleukin-1 (IL-1), on pH i regulation in peritoneal macrophages.IL-1 caused a time-and dose-dependent increase in macrophage pH i recovery from an acute acid load. This effect was specific to IL-1 and was due to enhanced plasmalemmal V-ATPase activity. The increased V-ATPase activity by IL-1 occurred following a lag period of several hours and required de novo protein and mRNA synthesis. However, Northern blot analysis revealed that IL-1 did not exert its effect via alterations in the levels of mRNA transcripts for the A or B subunits of the V-ATPase complex. Finally, stimulation of both cAMP-dependent protein kinase and protein kinase C was required for the stimulatory effect of IL-1 on VATPase activity.Thus, cytokines present within the inflammatory milieu are able to modulate pH i regulatory mechanisms. These data may represent a novel mechanism whereby cytokines may improve cellular function at inflammatory sites.

Na+/H + exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na+-driven) Na+/H § exchange caused a cytosolic acidification and activated an outward current, whereas forward (extraceUular Na+-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na § gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H + exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H § conductance." This was supported by measurements of the reversal potential (Erev) of tail cur-" rents, which identified H + (equivalents) as the charge carrier. During Na+/H + exchange, Er,v changed along with the measured changes in pHi (by 60-69 mV/ pH). Moreover, the current and Na § + exchange could be dissociated. Zn 2 § which inhibits the H § conductance, reversibly blocked the currents without altering Na+/H + exchange. In Chinese hamster ovary (CHO) cells, which lack the H + conductance, Na+/H § exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H + conductance, such as macrophages, activation of Na+-H §

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