An Arg-Gly-Asp peptide stimulates Ca²⁺ efflux from osteoclast precursors through a novel mechanism

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on ⁴⁵Ca²⁺ efflux from osteoclast precursors. ⁴⁵Ca²⁺-loaded osteoclast precursors were treated with GRGDSP (170 μM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca²⁺ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 μg/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca²⁺ efflux returned by 1 h. The Ca²⁺ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca²⁺- adenosinetriphosphatase (Ca²⁺-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2- naphthalenesulfonamide, and the absence of an effect of Na⁺/Ca²⁺ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N- [2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca²⁺ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca²⁺ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca²⁺ efflux from osteoclasts through activation of protein- tyrosine kinases and suggest that GRGDSP-stimulated Ca²⁺ efflux is mediated via the plasma membrane Ca²⁺-ATPase.