cADP-ribose potentiates cytosolic Ca2+ elevation and Ca2+ entry via L-type voltage-activated Ca2+ channels in NG108-15 neuronal cells

The effects of cADP-ribose (cADPR), a metabolite of beta-NAD(+), on the elevation of cytoplasmic free Ca2+ concentration ([Ca2+](i)) and Ca2+ flux through voltage-activated Ca2+ channels (VACCs) were studied in NG108-15 neuroblastoma x glioma hybrid cells. NG108-15 cells were pre-loaded with fura-2 and whole-cell patch-clamped. Application of cADPR through patch pipettes did not by itself trigger any [Ca2+](i) rise at the resting membrane potential. A rise in [Ca2+](i) was evoked upon sustained membrane depolarization, and was significantly larger in cADPR-infused cells than in non-infused cells. This potentiation in the [Ca2+](i) elevation was reproduced by infusion of beta-NAD(+), and was blocked by s-bromo-cADPR and antagonized by external application of ryanodine or by pretreatment of cells with FK506. Nicotinamide inhibited beta-NAD(+)-induced, but not cADPR-elicited, potentiation. [Ca2+](i) increases or Ca2+ influx, measured by Mn2+ quenching, elicited by the same protocol of depolarization was blocked completely by nifedipine but not by omega-conotoxin. Ca2+ influx in cADPR- or beta-NAD(+)-infused cells was steeper and greater than that in control cells, and was inhibited partly by ryanodine. In contrast, ryanodine accelerated Ca2+ influx in non-infused cells. These results show that cADPR amplifies both depolarization-induced [Ca2+](i) increase and Ca2+ influx through L-type VACCs. These results suggest that cADPR functions on ryanodine receptors as a direct agonist and also interacts with L-type VACCs as an indirect agonist, i.e. via a retrograde signal.