Mechanisms of cyclic ADP-ribose-induced Ca^<2+> mobilization and its physiological role

在许多组织系统中，钙通道/红嘌呤受体（RyRs）是环adp核糖（cADPR）作用的潜在靶点。然而，其在细胞刺激下的合成调控、cADPR激活RyRs的机制及其功能作用尚不清楚。我们已经证明了cADPR的合成是由camp依赖机制刺激的。越来越多的证据表明FK506结合蛋白（FKBP）12/12.6在RyRs通道门控调控中起重要作用。据信，与ryr的分离激活或延迟了它们的失活。本研究旨在利用cADPR拮抗剂8Br-cADPR、FK506和雷帕霉素，确定RyRs的辅助蛋白FKBP是否在cADPR诱导的RyR通道激活中发挥作用，以及cADPR是否与几种细胞（牛肾上腺染色质细胞、犬腮腺细胞和人中性粒细胞）中的信使作用的生理相关性。cADPR拮抗剂8Br-cADPR、FK506和雷帕霉素可抑制cADPR诱导的Ca^<2+>释放，但咖啡因和瑞诺定诱导的Ca^<2+>释放不受抑制。此外，抗fkbp 12/12.6抗体也能减弱cadpr诱导的Ca^<2+>释放。有证据表明cADPR可能是FKBP-RyR复合物的配体，导致ryr介导的Ca^<2+>释放的动态调节。ACh和fMLP在完整细胞中引起双期细胞内游离Ca^<2+>浓度（[Ca^<2+>]i）升高，最初短暂升高，随后持续升高。8Br-cADPR、FK506和雷帕霉素特异性降低了刺激诱导的[Ca^<2+>]i升高的持续阶段，而环孢素A没有特异性降低，提示cADPR有助于持续的[Ca^<2+>]_i升高。8Br-cADPR、FK506和雷帕霉素降低了ach诱导的肾上腺儿茶酚胺释放和fmlp诱导的中性粒细胞迁移，这些结果证明cADPR的合成受到细胞刺激的调节，cADPR/Ca^<2+>诱导的Ca^<2+>释放通路对刺激诱导的反应形成了正反馈Less

The calcium channels/ryanodine receptors (RyRs) are potential/putative target of cyclic ADP-ribose (cADPR) action in many tissue systems. However, the regulation of its synthesis in response to cell stimulation, and the mechanisms by which cADPR activates RyRs, and its functional roles are still unclear. We have demonstrated that cADPR synthesis is stimulated by cAMP-dependent mechanism. There is increasing evidence indicating that FK506 binding proteins (FKBP)12/12.6 play an important role in the regulation of the RyRs channel gating. It is believed that dissociation from RyRs activates or delays their inactivation. The present study was designed to determine whether FKBP, an accessory protein of the RyRs, plays a role in cADPR-induced activation of RyR channels and whether the physiological relevance of cADPR to the messenger role in several cells (bovine adrenal chromaffin cells, canin parotide cells and human neutrophils) using a cADPR antagonist, 8Br-cADPR, and FK506 and rapamycin … More which bind to FKBPs and dissociate them from the RyRIn permeabilized cells, cADPR-induced Ca^<2+> release but not caffeine-and ryanodine -induced Ca^<2+> release was inhibited by a cADPR antagonist, 8Br-cADPR, and FK506 and rapamycin. Furthermore, anti-FKBP 12/12.6 antibody also attenuated the cADPR-induced Ca^<2+> release. The evidence suggests that cADPR may be the ligand for FKBP-RyR complex, resulting in a dynamic regulation of RyR-mediated Ca^<2+> release. ACh and fMLP causes biphasic intracellular free Ca^<2+> concentration ([Ca^<2+>]i) rise, an initial transient rise followed by sustained rise, in intact cells. 8Br-cADPR, FK506 and rapamycin but not cyclospolin A specifically reduced the sustained phases of stimulation-induced [Ca^<2+>]i rise, suggesting that cADPR contributes to sustained [Ca^<2+>]_i rise. 8Br-cADPR, FK506, and rapamycin reduced ACh-induced adrenal catecholamine release and fMLP-induced neutrophils migrationThese results provide evidence that the synthesis of cADPR is regulated by cell stimulation, and the cADPR/Ca^<2+>-induced Ca^<2+> release pathway forms a positive feedback to stimulation-induced response Less