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      Cyclic ADP-Ribose Contributes to Contraction and Ca 2+ Release by M 1 Muscarinic Receptor Activation in Coronary Arterial Smooth Muscle


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          The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca<sup>2+</sup> release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 µmol/L) and oxotremorine (OXO; 2.5–80 µmol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>i</sub>) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca<sup>2+</sup>]<sub>i</sub> in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca<sup>2+</sup>-free Hanks’ solution. This OXO-induced rise in [Ca<sup>2+</sup>]<sub>i</sub> was significantly reduced by pirenzepine (PIR; an M<sub>1</sub> receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from β-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca<sup>2+</sup> release induced by activation of M<sub>1</sub> muscarinic receptors.

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          Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose.

          Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.
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            Cyclic ADP ribose activation of the ryanodine receptor is mediated by calmodulin.

            Cyclic ADP-ribose (cADPR) is a newly identified nucleotide which can release calcium from a variety of cells, suggesting it is a messenger for mobilizing internal Ca2+ stores. Its cyclic structure has now been confirmed by X-ray crystallography. Available results are consistent with it being a modulator of Ca(2+)-induced Ca2+ release. Here we report that sea urchin egg microsomes purified by Percoll gradients lose sensitivity to cADPR, but the response can be restored by a soluble protein in the supernatant. Purification and characterization of the protein indicate that it is calmodulin. It appears to be sensitizing the Ca2+ release mechanism because caffeine and strontium, agonists of Ca(2+)-induced Ca2+ release, can also mimic calmodulin in conferring cADPR-sensitivity. Although evidence indicates that cADPR may be an activator of the ryanodine receptor, present results point to the importance of accessory proteins such as calmodulin in modulating its activity.
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              Muscarinic receptor-mediated dual regulation of ADP-ribosyl cyclase in NG108-15 neuronal cell membranes.

              Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma x glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30-52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                February 2003
                26 March 2003
                : 40
                : 1
                : 28-36
                Departments of Pharmacology and Toxicology and Physiology, Medical College of Wisconsin, Milwaukee, Wisc., USA
                68936 J Vasc Res 2003;40:28–36
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 6, Tables: 1, References: 41, Pages: 9
                Research Paper


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