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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

Changes in intracellular pH (pHi) of osteoclasts. The pHi of primary osteoclasts labeled with the pH-sensitive dye BCECF-AM (12 μM) was monitored along time in order to evaluate the H+ secretion under flow and no-flow conditions after the NH4Cl prepulse, which causes an acid load to the cells. Experiments were performed in the absence HCO3− to inhibit base transporters. a. Flowing standard HEPES-buffered solution (5 mL/min) after acid loading is related to a discrete H+ secretion and a small ΔpHi. b. Non-flowing HEPES-buffered solution (0 mL/min) after acid loading is related to a transient H+ secretion and a higher ΔpHi than in flow condition
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Fig2: Changes in intracellular pH (pHi) of osteoclasts. The pHi of primary osteoclasts labeled with the pH-sensitive dye BCECF-AM (12 μM) was monitored along time in order to evaluate the H+ secretion under flow and no-flow conditions after the NH4Cl prepulse, which causes an acid load to the cells. Experiments were performed in the absence HCO3− to inhibit base transporters. a. Flowing standard HEPES-buffered solution (5 mL/min) after acid loading is related to a discrete H+ secretion and a small ΔpHi. b. Non-flowing HEPES-buffered solution (0 mL/min) after acid loading is related to a transient H+ secretion and a higher ΔpHi than in flow condition

Mentions: As previously described by Boron and De Weer [26], applying a solution containing NH4Cl initially causes an increase in pHi. Initially NH3 diffuses across the cell membrane and combines with a H+ inside the cell; NH4+ can also move into the cell through K+ transporters, bringing extra H+, however, this process is slower. The removal of NH4Cl solution causes a decrease in pHi. The intracellular NH3 rushes out the cell, leaving the H+ inside. The cell responds to this fall in pHi by activating acid extruding mechanisms. In a set of experiments, I used bis-carboxyethyl-carboxyfluorescein (BCECF-AM) fluorescence to monitor pHi changes using NH4Cl prepulse technique. Indeed I was able to monitor pHi changes following acid load (Figs. 2 and 3), in both osteoclasts and OCL-cells. A BCECF-loaded osteoclast is shown in (Additional file 1: Figure S1).Fig. 2


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

Morethson P - BMC Cell Biol. (2015)

Changes in intracellular pH (pHi) of osteoclasts. The pHi of primary osteoclasts labeled with the pH-sensitive dye BCECF-AM (12 μM) was monitored along time in order to evaluate the H+ secretion under flow and no-flow conditions after the NH4Cl prepulse, which causes an acid load to the cells. Experiments were performed in the absence HCO3− to inhibit base transporters. a. Flowing standard HEPES-buffered solution (5 mL/min) after acid loading is related to a discrete H+ secretion and a small ΔpHi. b. Non-flowing HEPES-buffered solution (0 mL/min) after acid loading is related to a transient H+ secretion and a higher ΔpHi than in flow condition
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Changes in intracellular pH (pHi) of osteoclasts. The pHi of primary osteoclasts labeled with the pH-sensitive dye BCECF-AM (12 μM) was monitored along time in order to evaluate the H+ secretion under flow and no-flow conditions after the NH4Cl prepulse, which causes an acid load to the cells. Experiments were performed in the absence HCO3− to inhibit base transporters. a. Flowing standard HEPES-buffered solution (5 mL/min) after acid loading is related to a discrete H+ secretion and a small ΔpHi. b. Non-flowing HEPES-buffered solution (0 mL/min) after acid loading is related to a transient H+ secretion and a higher ΔpHi than in flow condition
Mentions: As previously described by Boron and De Weer [26], applying a solution containing NH4Cl initially causes an increase in pHi. Initially NH3 diffuses across the cell membrane and combines with a H+ inside the cell; NH4+ can also move into the cell through K+ transporters, bringing extra H+, however, this process is slower. The removal of NH4Cl solution causes a decrease in pHi. The intracellular NH3 rushes out the cell, leaving the H+ inside. The cell responds to this fall in pHi by activating acid extruding mechanisms. In a set of experiments, I used bis-carboxyethyl-carboxyfluorescein (BCECF-AM) fluorescence to monitor pHi changes using NH4Cl prepulse technique. Indeed I was able to monitor pHi changes following acid load (Figs. 2 and 3), in both osteoclasts and OCL-cells. A BCECF-loaded osteoclast is shown in (Additional file 1: Figure S1).Fig. 2

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