Cyclic Diphosphate Ribose Research Articles

Similarities and Contrasts in Ryanodine Receptor Localization and Function in Osteoclasts and Striated Muscle Cells

This review compares ryanodine receptor (RyR)-mediated Ca(2+) signaling processes in muscle and osteoclast cells. In muscle, RyR-mediated release of an intracellularly stored, sarcoplasmic reticular (SR), Ca(2+) is triggered by voltage-sensitive dihydropyridine receptor (DHPR)-L-type Ca(2+) channels either through an allosteric coupling with the RyR in skeletal muscle or a Ca(2+)-induced Ca(2+) release initiated by extracellular Ca(2+) entry in cardiac muscle. Both cell subtypes are nevertheless capable of Ca(2+)-induced SR Ca(2+) release with cardiac muscle additionally showing a store overload-induced Ca(2+) release (SOICR) driven by SR luminal Ca(2+) under some pathological conditions. Osteoclasts similarly show cytosolic Ca(2+) elevations driven by release of intracellular Ca(2+) stores that culminate in motile activity in turn modifying bone resorptive activity. However, such triggering is controlled by ambient Ca(2+) rather than membrane potential with features strongly suggestive of control by a surface membrane Ca(2+) receptor. Yet common actions of the RyR-specific agents perchlorate, dantrolene Na, ryanodine, caffeine, adenosine 3',5'-cyclic diphosphate ribose (cADPr) and ruthenium red implicate RyR in signaling in all these cell types. These findings were reconciled by reports confirming and uniquely localizing a cell surface rather than microsomal osteoclastic RyR that might itself detect ambient Ca(2+) possibly through its otherwise intraluminal positioned low-affinity Ca(2+)-binding site in parallel with the SOICR mechanism in cardiac muscle. Such a mechanism could interact with other osteoclast processes transferring Ca(2+) between cytosol, intracellular stores and extracellular space and be integrated with systemic processes regulating Ca(2+) homeostasis.

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.

Ryanodine-induced calcium release from hepatic microsomes and permeabilized hepatocytes

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a second messenger that releases Ca2+ from hepatocyte microsomes. The toxic alkaloid ryanodine modulates Ca2+ release via a receptor (RyR) identified in a variety of cell systems, but its regulation and functional significance in liver are undefined. Similarly, the role in hepatocyte Ca2+ regulation of adenosine 5'-cyclic diphosphate-ribose (cADPR), which is the putative endogenous ligand for RyR in other cell systems, has not been defined. Utilizing microsomes and permeabilized cells, we have investigated Ca2+ regulation in hepatocytes and, in particular, effects of ryanodine, cADPR, and other putative modulators on Ca2+ release and compared these with Ins(1,4,5)P3-induced Ca2+ release. Ryanodine at > or = 50 microM released 20% of microsomal Ca2+, and, in contrast to Ins(1,4,5)P3, no potentiation was observed with guanosine 5'-triphosphate and polyethylene glycol. Ins(1,4,5)P3-induced Ca2+ release was demonstrable after maximal ryanodine-induced Ca2+ release, suggesting that distinct Ca2+ stores are involved. cADPR (5 microM) did not induce Ca2+ release, alone or in combination with calmodulin or hepatic cytosol, nor did it influence ryanodine-induced release, in microsomes or permeabilized hepatocytes (in which ryanodine released 25% of the sequestered Ca2+). Ryanodine-induced Ca2+ release in microsomes was not influenced by 20 mM caffeine, which itself did not mobilize Ca2+, but was prevented by 500 microM tetracaine, which was shown to induce Ca2+ release. We conclude that ryanodine is capable of mobilizing Ca2+ in the hepatocyte from microsomal stores that are distinct from those that can be regulated by Ins(1,4,5)P3 but that cADPR has no such effect. These data suggest that cADPR does not serve as the endogenous ligand for RyR in liver cells or that the site of action of ryanodine in hepatocyte microsomes is distinct from that in other cell types.