cyclic-guanosine-diphosphate-ribose and nicotinamide-guanine-dinucleotide

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50 μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.

Topics: Adenosine Triphosphate; ADP-ribosyl Cyclase 1; Animals; Calcium-Transporting ATPases; Chromatography, High Pressure Liquid; Cyclic ADP-Ribose; Electric Stimulation; Enzyme Inhibitors; Guanine Nucleotides; Guanosine Diphosphate Sugars; In Vitro Techniques; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Contraction; Muscle, Smooth; NAD; Receptors, Cell Surface; Ryanodine; Spectrometry, Fluorescence; Urinary Bladder

Two types of ADP-ribosyl cyclase activity were distinguished in dog and rat cardiac muscles by measuring the enzymatic conversion of NGD (as an NAD analog) into the fluorescent product cyclic GDP-ribose in cardiac muscle subcellular fractions. Both types of activity were confined to membrane fractions isolated from microsomes by sucrose gradient centrifugation. One of the activities co-purified with fractions that were enriched in sarcolemma (SLM), as evidenced by immunodetection of the dihydropyridine receptor, while the other activity was found to co-precipitate with the sarcoplasmic reticulum (SR), that was identified on the basis of its immuno-staining with a ryanodine receptor monoclonal antibody. In certain aspects, the plasma membrane-bound ADP-ribosyl cyclase activity resembled the characteristics of CD38 or CD38-like proteins: it was sensitive to thiols and lectins and was recognized by a monoclonal anti CD38 antibody. The SR enzyme had apparently distinct properties, as it was insensitive to both thiols and lectins and was not recognized by the CD38 antibody. In addition, the SR-associated ADP-ribosyl cyclase was inhibited by endogenous protein kinase C (PKC)-dependent phosphorylation in both dog and rat cardiac SR. The PKC-modulated SR ADP-ribosyl cyclase we describe here might be a principal component of the signal transduction machinery that is responsible for regulation of the intracellular levels of cADPR.

Topics: Adenosine Diphosphate Ribose; ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; Cyclic ADP-Ribose; Dithiothreitol; Dogs; Electrophoresis, Polyacrylamide Gel; Guanine Nucleotides; Guanosine Diphosphate Sugars; Isoenzymes; Membrane Glycoproteins; Myocardium; N-Glycosyl Hydrolases; NAD; NAD+ Nucleosidase; Phosphorylation; Protein Kinase C; Rats; Sarcoplasmic Reticulum