Extracellularly applied ruthenium red and cADP ribose elevate cytosolic Ca2+ in isolated rat osteoclasts

We demonstrated recently that the divalent cation-sensing receptor on the osteoclast, the Ca2+ receptor (CaR), is a functional component of a cell surface-expressed ryanodine receptor-like molecule (RyR). The objective of the present study was to further characterize this putative RyR by use of the two well-known cell-impermeant RyR modulators, ruthenium red and adenosine 3',5'-cyclic diphosphate ribose (cADPr). We found that, when applied extracellularly, ruthenium red (5 x 10(-8)-10(-4) M) and cADPr (5 x 10(-6) M) triggered an elevation of cytosolic [Ca2+]. Depolarization of the cell membrane by the application of 0.1 M K+ in the presence of 5 x 10-(-6) M valinomycin resulted in a concentration-dependent increase in the magnitude of the cytosolic Ca2+ response to extracellular ruthenium red (5 x 10(-9) and 5 x 10(-5) M), a phenomenon that was not seen when osteoclasts were hyperpolarized using 5 X 10(-3) M K+ with 5 X 10(-6) M valinomycin. In the presence of an intact nonleaky cell membrane, these results would favor a plasma membrane locus of action for the two modulators. Furthermore, pretreatment of osteoclasts with either modulator resulted in a markedly attenuated cytosolic Ca2+ transient elicited in response to the CaR agonist Ni2+, thus confirming an interaction between the cADPr- and ruthenium red-sensitive sites and the osteoclast CaR. The inhibition of the cytosolic Ca2+ response to Ni2+ induced by ruthenium red remained unchanged in the face of membrane potential changes. Finally, the cytosolic Ca2+ response to caffeine (5 x 10(-4) M), another RyR modulator, was also strongly attenuated by pretreatment with 5 x 10(-9) M ruthenium red. We conclude that ruthenium red and cADPr act on plasma membrane-resident sites and that both these sites interact with the process of divalent cation sensing.