Modulation of proton transport in osteoclasts. Effects of acidosis and extracellular fluid flow.

A acidose metabólica causa perda de mineral ósseo e a estimulação mecânica causa remodelamento ósseo adaptativo. A reabsorção óssea que caracteriza essas mudanças ósseas depende da acidificação extracelular pela secreção vetorial de H+ pelos osteoclastos. A H+-ATPase vacuolar em paralelo com o trocador Cl-/H+ (CLC7) são os mecanismos conhecidos envolvidos na reabsorção óssea, entretanto, os osteoclastos também expressam canais para H+ dependentes a voltagem. Este trabalho foi realizado para avaliar a contribuição dos canais para H+ na função celular visando à compreensão de seu relacionamento com a H+-ATPase vacuolar e o CLC7 (1); analisar se o fluxo de fluido extracelular modifica a secreção de H+ (2) e avaliar a diferenciação dos osteoclastos in vitro sob acidose metabólica devido à redução do HCO3- (3). Osteoclastos de ratos Wistar foram obtidos diretamente dos animais ou foram diferenciados in vitro (com M-CSF e RANKL) e semeados sobre vidro, plástico ou substratos mineralizados em α-MEM + 10% SFB, em pH 7,4 ou 6,9, e então mantidos em incubadora com 5% CO2, a 37°C. A diferenciação celular foi avaliada pela contagem de células TRAP-positivas ou de núcleos marcados por DAPI. A secreção de H+ foi avaliada por epifluorescência, utilizando-se BCECF-AM, sensível a pH. Os registros do pH intracelular foram feitos na vigência de soluções tamponadas por HEPES, na ausência de CO2/HCO3- (pH 7,4, 300 mOsm/L H2O, a 37°C), na presença ou ausência de perfusão contínua de fluido extracelular a uma velocidade de 5 ml/min. Na ausência de perfusão, os osteoclastos exibiram variações cíclicas do pHi (acidificação e alcalinização espontâneas), com período de 12 a 45 minutos (n = 35) e amplitude de 0,12 a 1,43 unidades de pHi. As oscilações não foram abolidas por concanamicina (100 mM) (n = 3), por NPPB (100 mM) (n = 3), na ausência de Na+ extracelular (n = 5) ou na ausência de Cl- extracelular (n = 3). O fluxo de fluido aboliu as oscilações e a ausência de Cl- extracelular modificou significativamente seu padrão. Na ausência de perfusão, a secreção de H+ após acidificação intracelular induzida foi abolida por Zn2+ (100 mM) (n = 5). Além disso, na presença de perfusão, a secreção de H+ após acidificação intracelular induzida foi abolida por NPPB (n = 4) e não foi abolida por bafilomicina (200 nm) (n = 3). A acidose metabólica não modifica o número de osteoclastos diferenciados in vitro, entretanto, o tratamento das culturas com Zn2+ causou redução do numero de células mononucleares e aumento relativo do número de osteoclastos multinucleados em relação ao controle tanto em pH 7,4 quanto em pH 6,9.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 Clsecretion 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 RANK- L) 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 amplitude difference between maximal and minimal pHi of 0.12 to 1.43 units pHi. These oscillations were not abolished in the presence of oncanamycin (100 mM) (n = 3), NPPB (100 mM) (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 Zn2+ (100 mM) (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 Zn2+, 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

Additional file 1: Figure S1. of Extracellular fluid flow and chloride content modulate H+ transport by osteoclasts

BCECF-loaded primary osteoclast on confocal microscope Zeiss LSM 510 (200 X). The mature osteoclast was extracted from 2 days-old Wistar rat and incubated with the pH-sensitive dye BCECF-AM (12 μM) for 10 min at 37°C. A. Transmitted light image. B. Fluorescence image of BCECF trapped in the cytosol. Note that nuclei are not fluorescent. C. Merged image of A. and B. (PNG 260 kb

Double disruption of alpha(2A)- and alpha(2C)-adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via beta(2)-adrenoceptor (beta(2)-AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, alpha(2A)-AR and alpha(2C)-AR(alpha(2A)/alpha(2C)-ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In alpha(2A)/alpha(2C)-ARKO versus wild-type (WT) mice, micro-computed tomographic (mu CT) analysis showed increased, better connected, and more plate-shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate-resistant acid phosphatase (TRACP) and receptor activator of NF-kappa B (RANK), which are osteoclast-related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine-regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial beta(2)-AR mRNA expression also was similar in KO and WT littermates, whereas alpha(2A)-, alpha(2B)- and alpha(2C)-AR mRNAs were detected in the tibia of WT mice and in osteoblast-like MC3T3-E1 cells. By immunohistochemistry, we detected alpha(2A)-, alpha(2B)-, alpha(2C)- and beta(2)-ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5-day-old mouse fetuses and 35-day-old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective alpha(2)-AR agonist clonidine and to the nonspecific alpha-AR antagonist phentolamine. These findings suggest that beta(2)-AR is not the single adrenoceptor involved in bone turnover regulation and show that alpha(2)-AR signaling also may mediate the SNS actions in the skeleton. (c) 2011 American Society for Bone and Mineral Research.FAPESP, BrazilFAPESP[05/59557-8]FAPESP[06/52982-8]FAPESP[08/50059-3]FAPESP[03/07327-3]CAPES, Brazi