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 o bjective of the present study was to further characterize this putativ e RyR by use of the two well-known cell-impermeant RyR modulators, rut henium red and adenosine 3',5'-cyclic diphosphate ribose (cADPr). We f ound 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 [C a2+]. 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 concentr ation-dependent increase in the magnitude of the cytosolic Ca2+ respon se to extracellular ruthenium red (5 x 10(-9) and 5 x 10(-5) M), a phe nomenon 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 in tact nonleaky cell membrane, these results would favor a plasma membra ne locus of action for the two modulators. Furthermore, pretreatment o f 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-s ensitive 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+ re sponse to caffeine (5 x 10(-4) M), another RyR modulator, was also str ongly attenuated by pretreatment with 5 x 10(-9) M ruthenium red. We c onclude that ruthenium red and cADPr act on plasma membrane-resident s ites and that both these sites interact with the process of divalent c ation sensing.