Amplification of depolarization‐induced and ryanodine‐sensitive cytosolic Ca2+ elevation by synthetic carbocyclic analogs of cyclic ADP‐ribose and their antagonistic effects in NG108‐15 neuronal cells

We synthesized analogs modified in the ribose unit (ribose linked to N1 of adenine) of cyclic ADP‐ribose (cADPR), a Ca2+‐mobilizing second messenger. The biological activities of these analogs were determined in NG108‐15 neuroblastoma × glioma hybrid cells that were pre‐loaded with fura‐2 acetoxymethylester and subjected to whole‐cell patch‐clamp. Application of the hydrolysis‐resistant cyclic ADP‐carbocyclic‐ribose (cADPcR) through patch pipettes potentiated elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) at the depolarized membrane potential. The increase in [Ca2+]i evoked upon sustained membrane depolarization was significantly larger in cADPcR‐infused cells than in non‐infused cells and its degree was equivalent to or significantly greater than that induced by cADPR or β‐NAD+. 8‐chloro‐cADPcR and two inosine congeners (cyclic IDP‐carbocyclic‐ribose and 8‐bromo‐cyclic IDP‐carbocyclic‐ribose) did not induce effects similar to those of cADPcR or cADPR. Instead, 8‐chloro‐cADPcR together with cADPR or cADPcR caused inhibition of the depolarization‐induced [Ca2+]i increase as compared with either cADPR or cADPcR alone. These results demonstrated that our cADPR analogs have agonistic or antagonistic effects on the depolarization‐induced [Ca2+]i increase and suggested the presence of functional reciprocal coupling between ryanodine receptors and voltage‐activated Ca2+ channels via cADPR in mammalian neuronal cells.