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Morethson P. Modulation of Proton Transport in Osteoclasts. Effects of Acidosis and Extracellular Fluid Flow [PhD Thesis]. Sao Paulo: Instituto de Ciências Biomédicas da Universidade de São Paulo; 2011. Metabolic acidosis can cause a loss of bone mineral and the mechanic stimulation can cause adaptative bone remodeling. The bone resorption characteristic of these bone changes aforementioned depends on the extracellular acidification by osteoclastmediated proton secretion. The H secretion by vacuolar H-ATPase together with Cl secretion through a Cl/H exchanger (CLC7) are the known mechanisms involved in the bone resorption; however, osteoclasts also express voltage-gated proton channels. The proposed aims of these work were to evaluate the contribution of proton channels in the osteoclast function for better understanding its relation with vacuolar H-ATPase and CLC7 (1); to analyze whether the flow of extracellular fluid modifies the H secretion or not (2); and to analyse the osteoclast differentiation in vitro under metabolic acidosis due to HCO3 reduction (3). Osteoclasts were freshly isolated or generated from bone marrow precursor cells (using M-CSF and RANKL) from of Wistar rats. The cells were placed on glass coverslips, plastic coverslips, or on mineralized substrate in α-MEM + 10% FBS, pH 7.4 or 6.9, and then maintained in a 5% CO2 incubator at 37 C. The differentiation was analyzed by counting of TRAP-stained cells or DAPIstained nuclei. The H secretion was analysed by epifluorescence, using the pHsensitive dye BCECF-AM. The intracellular pH record was done using a standard HEPES-buffered solution free of CO2/HCO3 (pH 7.4, 300 mOsm/L H2O, at 37 C), with or without continuous perfusion of extracellular fluid at a rate of 5 ml/min. In the absence of perfusion, the osteoclasts exhibit cyclic pHi variations (spontaneous acidification and alkalinization), with a period of 12 to 45 minutes (n = 35) and amplitudedifference between maximal and minimal pHiof 0.12 to 1.43 units pHi. These oscillations were not abolished in the presence of concanamycin (100 mM) (n = 3), NPPB (100 μM) (n = 3), in the absence of Na (n = 5) or in the absence of Cl (n = 3) in the extracellular solution. The fluid flow itself abolished the pH oscillations and the absence of extracellular Cl modifies significantly these patterns. In the absence of perfusion, the H secretion after induced intracellular acidification was abolished by Zn (100 μM) (n = 5). In addition, in the presence of perfusion, the H secretion after induced intracellular acidification was abolished by NPPB (n = 4) and was not abolished by bafilomycin (200 nm) (n = 3). Metabolic acidosis does not modify the number of osteoclasts differentiated in vitro, however, when the cell culture was treated with Zn, there was a significant reduction in the number of mononuclear cells and a relative increase in the number of multinucleated osteoclasts compared to control, both in pH 7.4 and pH 6.9 medium. Our data show that the proton secretion in osteoclasts depends on CLC7 and voltage-gated proton channels and does not depend on HATPase activity (1), the proton secretion is stimulated with the interruption of extracellular fluid flow (2), metabolic acidosis does not improve the osteoclast differentiation but Zn has a modulatory effect (3).

Modulação do transporte de prótons em osteoclastos: efeitos da acidose e do fluxo de fluido extracelular.