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Endothelin inhibits osteoclastic bone resorption by a direct effect on cell motility: implications for the vascular
A S Alam1, A Gallagher, V Shankar
1Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, United Kingdom.
Insights
Endothelin-1 (ET-1) significantly inhibits osteoclast bone resorption and motility. This peptide, produced by bone marrow endothelial cells, may regulate osteoclast activity.
Area of Science:
- Bone Biology
- Cellular Physiology
- Endocrinology
Background:
- Endothelin (ET)-producing endothelial cells are abundant in bone marrow.
- These cells are located near bone-resorbing osteoclasts, suggesting a potential interaction.
Purpose of the Study:
- To investigate the effect of ET-1 on osteoclast function, specifically bone resorption and cell motility.
- To determine the mechanism of ET-1 action on osteoclasts.
Main Methods:
- Osteoclast isolation from neonatal rat long bones.
- Quantification of bone resorption using morphometry on a bone substrate.
- Measurement of tartrate-resistant acid phosphatase activity.
- Assessment of cell motility via time-lapse video analysis.
- Measurement of cytosolic free calcium levels ([Ca2+]i) using indo 1-based microspectrofluorimetry.
Main Results:
- ET-1 demonstrated a concentration-dependent inhibition of osteoclastic bone resorption (EC50 = 2.5 nM).
- ET-1 significantly inhibited osteoclast motility (EC50 = 7.9 nM) without affecting cell spread area.
- These inhibitory effects were reversible, and cell viability was maintained.
- ET-1 did not elevate intracellular calcium levels ([Ca2+]i) at tested concentrations.
- Acid phosphatase secretion was not inhibited by ET-1.
Conclusions:
- ET-1 specifically interacts with an osteoclast receptor to inhibit bone resorption and cell motility.
- The effective concentration of ET-1 suggests a potential physiological role in regulating osteoclast activity.
- Locally produced ET-1 from bone marrow endothelial cells may be a key regulator of osteoclast function.
Abstract:
The abundance of endothelin (ET)-producing endothelial cells in bone marrow and the proximity of these cells to bone-resorbing osteoclasts prompted us to evaluate the action of ET-1 on osteoclast function. Osteoclasts disaggregated from neonatal rat long bones were settled onto devitalized cortical bone substrate, and resorption was quantified by morphometry. The supernatant tartrate-resistant acid phosphatase activity was determined by a spectrophotometric method using paranitrophenol phosphate as substrate. Cell motility was quantified by time lapse video- and computer-assisted image processing using an empirical procedure for morphometric analysis. Cytosolic free calcium levels ([Ca2+]i) were measured in single cells by an indo 1-based microspectrofluorimetric method. Using the area of bone resorbed per slice as response, we found that ET-1 caused a significant (P = 0.011) concentration-dependent inhibition of osteoclastic bone resorption (EC50 = 2.5 nM) without inhibiting acid phosphatase secretion. Exposure of isolated osteoclasts to ET-1 also led to a marked concentration-dependent inhibition of osteoclast motility (EC50 = 7.9 nM; P = 0.013; t1/2 = 18 min) without significant effects on cell spread area. These effects of ET-1 were reversible after removing the peptide, and the cells remained viable during the experiments. In addition, ET-1 did not elevate [Ca2+]i at the concentrations tested. The results suggest that ET-1 specifically interacts with an osteoclast receptor to inhibit osteoclastic bone resorption and cell motility. As the concentration of ET-1 required for osteoclast inhibition was similar to that reported for smooth muscle contraction, it is possible that ET-1, produced locally from the bone marrow endothelial cell, might play a primary role in osteoclast regulation.