Role of the Second-Messenger Cyclic-Adenosine 5′-Diphosphate-Ribose on Adrenocorticotropin Secretion from Pituitary Cells

We examined the role of the second-messenger cyclic-ADP-ribose (cADPR) on the regulation of ACTH secretion using AtT20 corticotroph tumor cell line. We found that the cADPR antagonist, 8-Br-cADPR, substantially diminished the secretion of ACTH induced by CRH and potassium in these cells, whereas xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. In addition, the cADPR agonist, 3-deaza-cADPR, augmented ACTH secretion. The presence of the components of the cADPR system, namely ryanodine receptor, CD38, and cADPR itself, was determined in AtT20 cells. Furthermore, we observed that antagonists of the ryanodine channel and cADPR system can decrease the potassium-induced Ca2+ transients in these cells. These results suggest that cADPR is a second messenger in pituitary cells and regulates ACTH secretion by a mechanism dependent on activation of the ryanodine channel by extracellular Ca2+.

Selected Contribution: Effect of volatile anesthetics on cADP-ribose-induced Ca2+ release system

Volatile anesthetics have multiple actions on intracellular Ca2+homeostasis, including activation of the ryanodine channel (RyR) and sensitization of this channel to agonists such as caffeine and ryanodine. Recently it has been described that the nucleotide cADP-ribose (cADPR) is the endogenous regulator of the RyR in many mammalian cells, and cADPR has been proposed to be a second messenger in many signaling pathways. I investigated the effect of volatile anesthetics on the cADPR signaling system, using sea urchin egg homogenates as a model of intracellular Ca2+ stores. Ca2+ uptake and release were monitored in sea urchin egg homogenates by using the fluo-3 fluorescence technique. Activity of the ADP-ribosyl cyclase was monitored by using a fluorometric method using nicotinamide guanine dinucleotide as a substrate. Halothane in concentrations up to 800 μM did not induce Ca2+ release by itself in sea urchin egg homogenates. However, halothane potentiates the Ca2+ release mediated by agonists of the ryanodine channel, such as ryanodine. Furthermore, other volatile anesthetics such as isoflurane and sevoflurane had no effect. Halothane also potentiated the activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca2+ release was decreased about three times by addition of 800 μM halothane. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to halothane. In contrast, all the volatile anesthetics used had no effect on the activity of the enzyme that synthesizes cADPR. I propose that the complex effect of volatile anesthetics on intracellular Ca2+ homeostasis may involve modulation of the cADPR signaling system.

Palmitoyl-CoA potentiates the Ca2+ release elicited by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs that ultimately activates the ryanodine channel. We now report that certain long-chain acyl-CoA derivative metabolites (14-18 carbons in length), such as palmitoyl-CoA, greatly potentiate the effect of cADPR on Ca2+ release. Furthermore, in higher concentrations, palmitoyl-CoA and other closely related long-chain acyl-CoA derivatives trigger Ca2+ release apparently through the ryanodine channel in sea urchin egg homogenates. Palmitoyl-CoA-induced Ca2+ release was suppressed by ruthenium red, spermine, and the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide, which all prevent activation of the ryanodine channel, but not by heparin or thionicotinamide-NADP. In addition, cADPR was able to desensitize the sea urchin egg homogenates to the subsequent Ca2+ release induced by palmitoyl-CoA and vice versa. In contrast, neither inositol 1,4,5-trisphosphate (IP3) nor the newly identified Ca2+ release agonist nicotinate adenine dinucleotide phosphate was able to desensitize the homogenate to palmitoyl-CoA, indicating that palmitoyl-CoA probably acts selectively by activating the ryanodine channel, but, unlike cADPR, palmitoyl-CoA might act directly on this channel. Finally, we found that palmitoyl-CoA was able to counteract the inhibitory effect of Mg2+ and spermine, which, in physiological concentrations, suppress specifically the cADPR-induced Ca2+ release. We propose that palmitoyl-CoA, present in micromolar concentrations, may trigger Ca2+ release through the ryanodine channel and, in lower concentrations, may increase the sensitivity of the Ca2+ release system to cADPR. Thus palmitoyl-CoA may serve as a regulatory link between the intermediary metabolism and the cADPR-induced Ca2+ release signaling pathway.

Specific modulation of cyclic ADP-ribose-induced Ca2+ release by polyamines

Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs. However, the regulation of the cADPR-induced Ca2+ release system is not yet fully elucidated. We now report that spermine and related polyamines, in physiological concentrations, were able to inhibit the Ca2+ release induced by cADPR in sea urchin egg homogenate bioassays, as measured using the Ca2+ indicator fluo 3, but had no effect on the Ca2+ release induced by D-myo-inositol 1,4,5-trisphosphate (IP3) or by nicotinate adenine dinucleotide phosphate (NAADP). Spermine was a more potent inhibitor of the cADPR-induced Ca2+ release than spermidine and putrescine. Spermine inhibited not only the release induced by cADPR but also the Ca2+ release induced by caffeine and ryanodine. Finally, pretreatment of the sea urchin egg homogenates with caffeine or Sr2+ and Ca2+ prevented the inhibitory effect of spermine on cADPR-induced Ca2+ release. We propose that polyamines, which are present in millimolar concentrations in fertilized eggs, are specific inhibitors of the ryanodine channel and perhaps may serve as endogenous regulators of the cADPR-induced Ca2+ release system.