Hashii M, Minabe Y, Higashida H
Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan.
Biochem J. 2000 Jan 15;345 Pt 2(Pt 2):207-15.
The effects of cADP-ribose (cADPR), a metabolite of beta-NAD(+), on the elevation of cytoplasmic free Ca(2+) concentration (Ca(2+)) and Ca(2+) influx through voltage-activated Ca(2+) channels (VACCs) were studied in NG108-15 neuroblastomaxglioma 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 Ca(2+) rise at the resting membrane potential. A rise in Ca(2+) was evoked upon sustained membrane depolarization, and was significantly larger in cADPR-infused cells than in non-infused cells. This potentiation in the Ca(2+) elevation was reproduced by infusion of beta-NAD(+), and was blocked by 8-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. Ca(2+) increases or Ca(2+) influx, measured by Mn(2+) quenching, elicited by the same protocol of depolarization was blocked completely by nifedipine but not by omega-conotoxin. Ca(2+) 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 Ca(2+) influx in non-infused cells. These results show that cADPR amplifies both depolarization-induced Ca(2+) increase and Ca(2+) 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.
研究了β-NAD(+)的代谢产物环ADP-核糖（cADPR）对NG108-15神经母细胞瘤×胶质瘤杂交细胞胞质游离Ca(2+)浓度（Ca(2+)）升高以及通过电压激活Ca(2+)通道（VACCs）的Ca(2+)内流的影响。用fura-2预负载NG108-15细胞并进行全细胞膜片钳记录。通过膜片吸管施加cADPR本身在静息膜电位时不会引发任何Ca(2+)升高。持续的膜去极化会诱发Ca(2+)升高，且在注入cADPR的细胞中比未注入的细胞明显更大。这种Ca(2+)升高的增强作用可通过注入β-NAD(+)重现，并被8-溴-cADPR阻断，且可被细胞外施加的ryanodine拮抗或被用FK506预处理细胞所拮抗。烟酰胺抑制β-NAD(+)诱导的增强作用，但不抑制cADPR引发的增强作用。通过相同去极化方案引发的、用Mn(2+)淬灭法测量的Ca(2+)增加或Ca(2+)内流被硝苯地平完全阻断，但不被ω-芋螺毒素阻断。注入cADPR或β-NAD(+)的细胞中的Ca(2+)内流比对照细胞更陡峭且更大，并部分被ryanodine抑制。相反，ryanodine加速了未注入细胞中的Ca(2+)内流。这些结果表明，cADPR放大了去极化诱导的Ca(2+)增加以及通过L型VACCs的Ca(2+)内流。这些结果提示，cADPR作为直接激动剂作用于ryanodine受体，并且还作为间接激动剂与L型VACCs相互作用，即通过逆行信号。
