REGULATION OF CYTOPLASMIC PH IN OSTEOCLASTS - CONTRIBUTION OF PROTON PUMPS AND A PROTON-SELECTIVE CONDUCTANCE

Osteoclasts resorb bone by secreting protons into an extracellular res orption zone through vacuolar-type proton pumps located in the ruffled border, The present study was undertaken to evaluate whether proton p umps also contribute to intracellular pH (pH(i)) regulation. Fluoresce nce imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit os teoclasts. The fluorescence of single osteoclasts cultured on glass co verslips and loaded with a pH-sensitive indicator was measured in nomi nally HCO3--free solutions. When suspended in Na+-rich medium, the cel ls recovered from an acute acid load primarily by means of an amilorid e sensitive Na+/H+ antiporter. However, rapid recovery was also observ ed 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 contras t, pH(i) recovery in high K+ medium was largely attributed to a Zn2+-s ensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configu ration. Depolarizing pulses induced a slowly developing outward curren t and a concomitant cytosolic alkalinization. Determination of the rev ersal 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(i), consistent w ith a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contri bute minimally to the recovery of cytoplasmic pH from intracellular ac id loads. Instead, the data indicate the presence of a pH- and membran e potential-sensitive H+ conductance in the plasma membrane of osteocl asts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.