SOLUBLE ALPHA(V)BETA(3)-INTEGRIN LIGANDS RAISE [CA2+](I) IN RAT OSTEOCLASTS AND MOUSE-DERIVED OSTEOCLAST-LIKE CELLS

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 mu M) 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 [Ala(24)]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 mu M. Echistatin did not cause Mn2+ influx, and 10 mu M 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 mu M thapsigargin and Ca2+-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.