Soluble alpha v beta 3-integrin ligands raise [Ca2+]i in rat osteoclasts and mouse-derived osteoclast-like cells

Abstract

We evaluated the possible involvement of intracellular Ca2+ concentration ([Ca2+]i) changes in the action of alpha v beta 3-ligands, known to regulate osteoclast function. Rat osteoclasts or mouse osteoclast-like cells, as examined by microfluorimetry and fura 2, showed a transient [Ca2+]i increase when perfused with (all 0.1 microM) vitronectin, osteopontin, polypeptide echistatin, fibronectin, and Arg-Gly-Asp-Asp and Arg-Gly-Asp-Ser peptides (10(-4) M) but not with laminin, collagen I, collagen IV, or [Ala24]echistatin, in which Ala was substituted for Arg in the Arg-Gly-Asp complex. The threshold for echistatin was 10 pM, the 50% effective concentration was 1 nM, and the median [Ca2+]i increase was 420 nM above the resting level (217 +/- 22 nM) at saturating concentration of 0.1 microM. Echistatin did not cause Mn2+ influx, and 10 microM nifedipine, 10 nM omega-conotoxin, 5 mM Ni2+, or Cd2+ did not prevent [Ca2+]i change. However, extracellular Ca2+ was needed for the [Ca2+]i increase, probably enabling ligand-integrin interaction. Polyclonal and monoclonal (LM609) antibody as well as depletion of [Ca2+]i stores with 5 microM thapsigargin and Ca(2+)-free medium abolished the [Ca2+]i increase, after restoring extracellular Ca2+. Furthermore, the LM609 antibody induced a Ca2+ signal in the presence or absence of extracellular Ca2+, suggesting that the alpha v beta 3-ligand interaction is mediated at least partially by Ca2+ mobilized from intracellular stores.