Natural occurrence of 2'-phospho-cyclic ADP ribose in mammalian tissues.

2'-Phospho-cyclic ADP-ribose (P-cADPR) is a newly identified Ca2+-mobilizing agent derived from NADP that stimulates intracellular Ca2+ release by a mechanism distinct from inositol 1, 4, 5-trisphosphate. In this report, we show that P-cADPR is an endogenous metabolite in bovine tissues with basal levels ranging from 17.6 to 89.5 fmol/mg protein. The natural occurrence of this Ca2+-mobilizing nucleotide provides a potential link between NADP(H) metabolism and regulation of Ca2+ homeostasis.

Intramolecular ADP-ribose transfer reactions and calcium signalling. Potential role of 2'-phospho-cyclic ADP-ribose in oxidative stress.

Intramolecular ADP-ribose transfer reactions result in the formation of cyclic ADP-ribose (cADPR) and 2'-phospho-cyclic ADP-ribose (P-cADPR) from NAD and NADP, respectively. The potent Ca2+ releasing activity of these cyclic nucleotides has led to the postulation that they function as second messengers of Ca2+ signalling. The synthesis and hydrolysis of cADPR and P-cADPR are catalyzed by NAD(P) glycohydrolases, but the metabolic signals that regulate their metabolism are poorly understood. To investigate the physiological roles of cADPR and P-cADPR, it is essential to have methods that allow the routine measurement of these nucleotides in cellular systems. As described here, a sensitive and selective radioimmunoassay (RIA) for cADPR has been adapted to search for the natural occurrence of P-cADPR in mammalian tissues. Perchloric acid extracts prepared from bovine tissues and purified by anion exchange chromatography were found to contain immunoreactive material which was identified as P-cADPR. P-cADPR may play an important role in oxidative stress as a link between NADP(H) metabolism and alteration of intracellular Ca2+ homeostasis.

trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

NAD glycohydrolases and the metabolism of cyclic ADP-ribose.

Cyclic ADP-ribose is a recently discovered metabolite of NAD that functions in cellular calcium signalling. The discovery that NAD glycohydrolases can catalyze the synthesis and hydrolysis of cyclic ADP-ribose has renewed interest in this class of ADP-ribose transferring enzymes that were discovered over 50 years ago.

Cyclic ADP-ribose. A new component of calcium signaling.

Cyclic ADP-ribose is a recently discovered metabolite of NAD that appears to function in cellular calcium signaling. The discovery that NAD glycohydrolases are bifunctional enzymes that catalyze both the synthesis and hydrolysis of cyclic ADP-ribose raises many questions concerning the mechanisms by which these enzymes function in calcium signaling. Likewise, the identification of human lymphocyte antigen CD 38 as a bifunctional NAD glycohydrolase raises interesting questions concerning the involvement of cyclic ADP-ribose mediated calcium signaling in immune function. The dementia associated with niacin deficiency has been a long-standing curiosity. This signaling mechanism may resolve questions connecting this vitamin deficiency to central nervous system (CNS) function.

Modification of cardiac membrane adenylate cyclase activity and Gs alpha by NAD and endogenous ADP-ribosyltransferase.

The mechanism by which NAD stimulates cardiac adenylate cyclase was investigated. In highly purified canine cardiac sarcolemma, NAD stimulated adenylate cyclase activity in the presence of agents which activate Gs (i.e. 5 mM AlF4-, 10 microM GTP gamma S, 10 microM GppNHp or isoproterenol plus 2 nM GTP gamma S). Furthermore, the EC50 of isoproterenol to stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 microM GTP gamma S or isoproterenol plus 2 nM GTP gamma S produced a selective increase in the radiolabeling of a single 45-kDa protein which was identified as Gs alpha by immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from Gs alpha prelabeled in the presence of AlF4- and [32P]-NAD indicating that an arginine residue on Gs alpha was modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1 or 3-aminobenzamide, inhibited AlF4- stimulated ADP-ribosylation of Gs alpha and NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of Gs alpha was not removed under hypotonic or hypertonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess an ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated Gs alpha.