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.

The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca 2؉ signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. antagonism ͉ nucleotide ͉ second messenger ͉ synthesis

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the G ␣ subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for G s␣ In current pharmacotherapy the input into G proteinregulated signaling is manipulated by targeting the receptor with appropriate agonists and antagonists. Several arguments, however, suggest that elements of the receptor-activated, downstream signaling cascade, in particular G proteins, may be considered as drug targets per se. (i) The molecular diversity of G protein ␣, ␤, and ␥ subunits is large, and the number of distinct ␣␤␥ oligomers that can be produced by combinatorial association of subunit is excessive (1). (ii) The interaction of a given receptor with the cellular complement of G proteins may be governed by both exquisite specificity and promiscuity; in the first case, only one defined oligomer supports the ability of a receptor to regulate an effector in an intact cell (for review see refs. 2 and 3). (iii) On the other hand, many receptors couple to multiple G proteins (2, 3); this is exemplified by the thyrotropin receptor (4), which can activate essentially all G protein ␣ subunits expressed in the thyroid (i.e., members of all subfamilies of G ␣ other than the transducins). Thus, cellular stimulation by a receptor often results in the concerted activation of several distinct G proteins such that multiple effector pathways are recruited to produce the biological response. In theory, it may be desirable to block signaling of a receptor via one type of G protein but not via the other G proteins (resulting in ''biased inhibition of receptor/G protein tandem formation''), a goal that cannot be achieved by receptor antagonists but that may be achieved by compounds that bind selectively to individual G proteins. In addition, several human diseases arise from activation of G protein ␣ subunits by point mutations (for a brief overview, see ref. 5); appropriate G protein antagonists are desirable under these circumstances.Suramin has been shown previously to act directly on G protein ␣ subunits (6) and to block their activation by receptors (7-9). In the present work, we have searched for compounds that inhibit G s␣ directly. Two compounds of remarkable selectivity were identified that suppress the coupling of ␤-ad-

The 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are widely used and well tolerated cholesterol-lowering drugs. In rare cases, side effects occur in skeletal muscle, including myositis or even rhabdomyolysis. However, the molecular mechanisms are not well understood that lead to these muscle-specific side effects. Here, we show that statins cause apoptosis in differentiated human skeletal muscle cells. The prototypical representative of statins, simvastatin, triggered sustained intracellular Ca 2ϩ transients, leading to calpain activation. Intracellular chelation of Ca 2ϩ completely abrogated cell death. Moreover, ryanodine also completely prevented the simvastatin-induced calpain activation. Nevertheless, an activation of the ryanodine receptor by simvastatin could not be observed. Downstream of the calpain activation simvastatin led to a translocation of Bax to mitochondria in a caspase 8-independent manner. Consecutive activation of caspase 9 and 3 execute apoptotic cell death that was in part reversed by the coadministration of mevalonic acid. Conversely, the simvastatin-induced activation of calpain was not prevented by mevalonic acid. These data delineate the signaling cascade that leads to muscle injury caused by statins. Our observations also have implications for improving the safety of this important medication and explain to some extent why physical exercise aggravates skeletal muscle side effects.