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.

Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring and potent Ca2+-mobilizing agent. Structural analogues are currently required as pharmacological tools for the investigation of this topical molecule, but modifications to date have concentrated primarily upon the purine ring. Two novel dehydroxylated analogues of cADPR have now been prepared from chemically synthesized nicotinamide adenine dinucleotide (NAD+) precursors modified in the ribose moiety linked to adenine. ADP-ribosyl cyclase of Aplysia californica catalyzed the conversion of 2'A-deoxy-NAD+ and 3'A-deoxy-NAD+ into the corresponding 2'A-deoxy-cADPR and 3'A-deoxy-cADPR analogues, respectively. These analogues were used to assess the effect of 2'- and 3'-hydroxyl group deletion in the adenosine ribose moiety of cADPR on the Ca2+-releasing potential of cADPR. These compounds were found to have comparatively markedly different activities as agonists for Ca2+ mobilization in sea urchin egg homogenate. 2'A-Deoxy-cADPR is similar to cADPR, whereas 3'A-deoxy-cADPR is at least 100-fold less potent, indicating that the 3'A-hydroxyl group, but not the 2'A-hydroxyl group, is essential for calcium releasing activity. EC50 values recorded were 32 nM, 58 nM, and 5 microM for cADPR, 2'A-deoxy-cADPR, and 3'A-deoxy-cADPR, respectively. Moreover, 200 nM 2'A-deoxy-cADPR was required to desensitize the cADPR-sensitive Ca2+ channel to a subsequent addition of 100 nM cADPR, but 20 microM 3'A-deoxy-cADPR was required to produce the same desensitizing effect. This is in accordance with the 100-fold lower potency exhibited by the latter analogue. To further investigate the importance of the 3'-hydroxyl group, we have also synthesized 3'A-O-methyl-cADPR, in which the 3'-hydroxyl group of adenosine has been methylated and its ability potentially to donate a hydrogen atom in a hydrogen bond has been removed. Although inactive in releasing Ca2+, 3'A-O-methyl-cADPR inhibited cADPR-induced Ca2+ release in a dose-dependent manner with an approximate IC50 value of 5 microM, whereas 3'-O-methyladenosine had no effect. This further supports the requirement of a 3'-OH group for Ca2+ releasing activity. In addition, however, it suggests that this group may not be crucial for ligand-receptor recognition. Thus, replacement of the hydrogen atom of the hydroxyl with a methyl group effects a change of activity from an agonist to an antagonist of cADPR-induced Ca2+ release. Two other analogues with modifications in the 2' and/or 3' positions, 3'-cADPR phosphate and 2',3'-cyclic-cADPR phosphate, were synthesized and tested for their Ca2+-mobilizing activity in sea urchin egg homogenates. Both analogues were inactive with respect to both agonistic and antagonistic activities on the cADPR-sensitive Ca2+ release mechanism. These are the first steps toward a wider structure-activity relationship for cADPR, and this is the first study to implicate a crucial role for the adenosine ribose hydroxyl groups of cADPR in the biological activity of this cyclic nucleotide. Additionally, this...

These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.

Oscillations of Ca2؉ in heart cells are a major underlying cause of important cardiac arrhythmias, and it is known that Ca 2؉ -induced release of Ca 2؉ from intracellular stores (the sarcoplasmic reticulum) is fundamental to the generation of such oscillations. There is now evidence that cADP-ribose may be an endogenous regulator of the Ca 2؉ release channel of the sarcoplasmic reticulum (the ryanodine receptor), raising the possibility that cADP-ribose may influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose, an antagonist of cADP-ribose, suppressed oscillatory activity associated with overloading of intracellular Ca 2؉ stores in cardiac myocytes exposed to high doses of the ␤-adrenoreceptor agonist isoproterenol or the Na