Limits...
Extracellular fluid flow and chloride content modulate H(+) transport by osteoclasts.

Morethson P - BMC Cell Biol. (2015)

Bottom Line: Bone 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.The 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. primoret@icb.usp.br.

ABSTRACT

Background: Bone 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.

Results: The 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.

Conclusions: The 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.

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Related in: MedlinePlus

Mature primary osteoclasts were selected based on their morphology, using phase contrast optic system (Olympus IX70; 200 X) (a). In some experiments, it was performed further confirmation of the cell phenotype by immunofluorescence using antibody against calcitonin receptor (CTR) (confocal microscope Zeis LSM510; 200 X), a specific marker of osteoclasts (b), or by cytochemistry for tartrate resistant acid phosphatase (TRAP) (TRAP kit, Sigma, USA; 200 X) (c)
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Fig7: Mature primary osteoclasts were selected based on their morphology, using phase contrast optic system (Olympus IX70; 200 X) (a). In some experiments, it was performed further confirmation of the cell phenotype by immunofluorescence using antibody against calcitonin receptor (CTR) (confocal microscope Zeis LSM510; 200 X), a specific marker of osteoclasts (b), or by cytochemistry for tartrate resistant acid phosphatase (TRAP) (TRAP kit, Sigma, USA; 200 X) (c)

Mentions: Mature osteoclasts were collected under aseptic conditions from the long bones of newborn Wistar rats. All experimental procedures were performed in accordance with the guidelines of the Standing Committee on Animal Research of the University of São Paulo (Protocol No. 090-35/02). The removed bones were washed with cold α-MEM (Gibco, Grand Island, NE), minced, and cells were detached by repeated pipetting. The debris was allowed to sediment for 30 s, and then the cell suspension was collected. Cells were placed on plastic coverslips at a density of 5 × 106 cells/mL in 300 mOsm/L α-MEM containing 20 IU/L penicillin G, 20 μg/L streptomycin and 0.05 μg/L amphotericin B, 10 % of fetal calf serum and 20 mM N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Cells were kept in a CO2 incubator (Lab-Line Instruments, Melrose Park, IL) at 5 % CO2, pH 7.4 at 37 °C, for at least two hours before experiments. Mature osteoclasts collected from long bones were selected based on their morphology, using phase contrast microscopy (Olympus IX70, Tokyo, Japan). After selected experiments, further confirmation of the cell phenotype was performed using tartrate resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich, St. Louis, MO) or by immunocytochemistry using an antibody against the calcitonin receptor (Abcam, Cambridge, UK), a specific marker for osteoclasts (Fig. 7).Fig. 7


Extracellular fluid flow and chloride content modulate H(+) transport by osteoclasts.

Morethson P - BMC Cell Biol. (2015)

Mature primary osteoclasts were selected based on their morphology, using phase contrast optic system (Olympus IX70; 200 X) (a). In some experiments, it was performed further confirmation of the cell phenotype by immunofluorescence using antibody against calcitonin receptor (CTR) (confocal microscope Zeis LSM510; 200 X), a specific marker of osteoclasts (b), or by cytochemistry for tartrate resistant acid phosphatase (TRAP) (TRAP kit, Sigma, USA; 200 X) (c)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536797&req=5

Fig7: Mature primary osteoclasts were selected based on their morphology, using phase contrast optic system (Olympus IX70; 200 X) (a). In some experiments, it was performed further confirmation of the cell phenotype by immunofluorescence using antibody against calcitonin receptor (CTR) (confocal microscope Zeis LSM510; 200 X), a specific marker of osteoclasts (b), or by cytochemistry for tartrate resistant acid phosphatase (TRAP) (TRAP kit, Sigma, USA; 200 X) (c)
Mentions: Mature osteoclasts were collected under aseptic conditions from the long bones of newborn Wistar rats. All experimental procedures were performed in accordance with the guidelines of the Standing Committee on Animal Research of the University of São Paulo (Protocol No. 090-35/02). The removed bones were washed with cold α-MEM (Gibco, Grand Island, NE), minced, and cells were detached by repeated pipetting. The debris was allowed to sediment for 30 s, and then the cell suspension was collected. Cells were placed on plastic coverslips at a density of 5 × 106 cells/mL in 300 mOsm/L α-MEM containing 20 IU/L penicillin G, 20 μg/L streptomycin and 0.05 μg/L amphotericin B, 10 % of fetal calf serum and 20 mM N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Cells were kept in a CO2 incubator (Lab-Line Instruments, Melrose Park, IL) at 5 % CO2, pH 7.4 at 37 °C, for at least two hours before experiments. Mature osteoclasts collected from long bones were selected based on their morphology, using phase contrast microscopy (Olympus IX70, Tokyo, Japan). After selected experiments, further confirmation of the cell phenotype was performed using tartrate resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich, St. Louis, MO) or by immunocytochemistry using an antibody against the calcitonin receptor (Abcam, Cambridge, UK), a specific marker for osteoclasts (Fig. 7).Fig. 7

Bottom Line: Bone 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.The 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. primoret@icb.usp.br.

ABSTRACT

Background: Bone 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.

Results: The 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus