Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via b 2 -adrenoceptor (b2-AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, a 2A -AR and a 2C -AR (a 2A /a 2C -ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In a 2A /a 2C -ARKO versus wild-type (WT) mice, micro-computed tomographic (mCT) 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-kB (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 b 2 -AR mRNA expression also was similar in KO and WT littermates, whereas a 2A -, a 2B -and a 2C -AR mRNAs were detected in the tibia of WT mice and in osteoblast-like MC3T3-E1 cells. By immunohistochemistry, we detected a 2A -, a 2B -, a 2C -and b 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 a 2 -AR agonist clonidine and to the nonspecific a-AR antagonist phentolamine. These findings suggest that b 2 -AR is not the single adrenoceptor involved in bone turnover regulation and show that a 2 -AR signaling also may mediate the SNS actions in the skeleton. ß

BackgroundBone resorption takes place within the basic multicellular units (BMU), and the surface to be resorbed is isolated from adjacent bone surfaces by a sealing zone between osteoclast membrane and bone matrix, which defines the limits of the resorption lacuna. Considering that the extracellular fluid (ECF) in both BMU and the resorption lacuna can be isolated from its surroundings, I hypothesize that flow and ion composition of the bone ECF in these sites might contribute to the regulation of osteoclast H+ secretion. To investigate this hypothesis, I evaluated the H+ secretion properties of individual osteoclasts and osteoclast-like cells (OCL-cells) and investigated whether changes in flow or chloride content of the extracellular solution modify the H+ secretion properties in vitro.ResultsThe results show that 1) osteoclasts are unable to secrete H+ and regulate intracellular pH (pHi) under continuous flow conditions and exhibit progressive intracellular acidification; 2) the cessation of flow coincides with the onset of H+ secretion and subsequent progressive intracellular alkalinization of osteoclasts and OCL-cells; 3) osteoclasts exhibit spontaneous rhythmic oscillations of pHi in non-flowing ECF, 4) pHi oscillations are not abolished by concanamycin, NPPB, or removal of extracellular Na+ or Cl−; 5) extracellular Cl− removal modifies the pattern of oscillations, by diminishing H+ secretion; 6) pHi oscillations are abolished by continuous flowing of ECF over osteoclasts and OCL-cells.ConclusionsThe data suggest, for the first time, that ECF flow and Cl− content have direct effects on osteoclast H+ secretion and could be part of a mechanism determining the onset of osteoclast H+ secretion required for bone resorption.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0066-4) contains supplementary material, which is available to authorized users.

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ABSTRACT

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 HCO 3 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% CO 2 incubator at 37 o 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 CO 2 /HCO 3-(pH 7.4, 300 mOsm/L H 2 O, at 37 o 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 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 Clmodifies significantly these patterns. In the absence of perfusion, the H + secretion after induced intracellular acidification was abolished by Zn 2+ (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 2+ , 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 H + ATPase activity (1), the proton secretion is stimulated with the interruption of extracellular fluid flow (2), met...