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

We demon-strated 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 × 10^-8-10^-4 M) and cADPr 75 × 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 × 10^-6 M valinomycin resulted in a concentration-dependent increase in the magnitude of the cytosolic Ca²⁺ response to extracellular ruthenium red (5 × 10^-9 and 5 × 10^-5 M^-10 a phenomenon that was not seen when osteoclasts were hyperpolarized using 5 × 10^-3 M K⁺ with 5 × 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 Ca²⁺ 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 × 10^-4 M), another RyR modulator, was also strongly attenuated by pretreatment with 5 × 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.