Detection and characterization of endothelin in transformed human osteoblast cell culture medium

Abstract

Endothelin-1 (ET-1), a 21 amino acid peptide originally purified from conditioned medium of cultures of porcine aortic endothelial cells, is recognized also as a product of many other cells such as epithelial cells, glial cells, and neurons. It is now recognized that at least ET-1 plays an important role in bone metabolism. It has been shown that ET-1 inhibits osteoclast bone resorption by a direct effect on cell motility and it can also activate phospholipase C in the osteoblast. Furthermore, several studies have shown that ET-1 stimulates the formation of inositol phosphates, the synthesis of DNA, the mobilization of calcium from extra- and intracellular pools, the activation of phospholipase D, and the stimulation of tyrosine phosphorylation in osteoblast-like (MC3T3-E1 and UMR-106) cells. The aim of the present study was to detect and characterize the presence of endothelin in transformed human osteoblast cell culture medium (HTb96) by radio-immunoassay and chromatography methods. Immunoreactive endothelin (IR-ET) was undetectable in the medium incubated at 0.5 and 1 h and was 3.2±0.2 fmol/10⁵ cells (mean ± SEM, n=6) at 2 h, 9.5±0.5 fmol/10⁵ cells at 6 h, 19.8 ± 2.1 fmol/10⁵ cells at 24 h, and 23.7 ± 2.0 fmol/10⁵ cells at 48 h, respectively. Sephadex G-25 superfine chromatography and fast protein liquid chromatography studies showed that >90% of IR-ET in the culture medium coeluted with synthetic ET-1. These results show that ET-1 could be formed by transformed human osteoblasts. Further studies should be conducted to elucidate the physiological role of endothelins as possible autocrine, paracrine, or endocrine factors in calcium and bone metabolism.