ryanodine and 8-aminoadenosine-cyclic-3--5--(hydrogen-phosphate)-5--ribofuranosyl-ester

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca(2+) mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC(50) of 1 nM and peak contraction of 57 +/- 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10(-6) M. The EC(50) of cADPR caused contraction (18 +/- 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 muM) induced contraction of LSM strips that averaged 67 +/- 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.

Topics: Animals; Calcium Channel Blockers; Calcium Signaling; Cyclic ADP-Ribose; Didelphis; Dose-Response Relationship, Drug; Esophagus; In Vitro Techniques; Isometric Contraction; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tetracaine

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.

Topics: Animals; Arteries; Calcium; Cell Membrane; Cells, Cultured; Cyclic ADP-Ribose; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Isoproterenol; Isoquinolines; Peptides; Potassium Channels; Rats; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sulfonamides; Time Factors; Vasodilator Agents

Alongside the well-studied inositol 1,4,5 trisphosphate and ryanodine receptors, evidence is gathering that a new intracellular release mechanism, gated by the pyridine nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP), is present in numerous organisms, ranging from plant to mammalian cells (reviewed in [1]). Most cells have been shown to express at least two Ca(2+)-release mechanisms controlled by different messengers, and this can lead to redundancy, convergence, or divergence of responses. One exception appears to be muscle and heart contractile tissues. Here, it is thought that the dominant intracellular channel is the ryanodine receptor, while IP(3) receptors are poorly expressed and their role appears to be negligible. We now report that NAADP receptors are functional and abundant in cardiac microsomes. NAADP binds specifically and with high affinity (130 pM and 4 nM) to two sites on cardiac microsomes and releases Ca(2+) with an apparent EC(50) of 323 +/- 14 nM. Furthermore, binding experiments show that this receptor displays both positive and negative cooperativity, a peculiarity unique among intracellular Ca(2+) channels. Therefore, we show that the heart possesses multiple mechanisms to increase the complexity of Ca(2+) signaling and that NAADP may be integral in the functioning of this organ.

Topics: Adenosine Diphosphate Ribose; Animals; Calcium; Calcium Channel Blockers; Cyclic ADP-Ribose; Diltiazem; Indicators and Reagents; Kinetics; Microsomes; Myocardium; NADP; Protein Binding; Radioligand Assay; Receptors, Cell Surface; Ruthenium Red; Ryanodine; Verapamil

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.

Topics: Adenosine Diphosphate Ribose; Animals; Calcium; Calcium Channel Blockers; Cyclic ADP-Ribose; Electric Stimulation; Intracellular Membranes; Male; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine

It has been suggested that cyclic adenosine 5'-diphosphoribose (cADPR) directly activates the cardiac isoform of the ryanodine receptor (RyR)/Ca2+ release channel. We have previously shown that selective activation of RyR/Ca2+ release channel by superoxide anion radical (O2.-) is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O2.- -triggered Ca2+ release through the RyR/Ca2+ release channel of cardiac sarcoplasmic reticulum (SR). We now demonstrate that although the effect of O2.- on Ca2+ efflux from RyR/Ca2+ release channel at higher concentrations ( >5 microM) is due to its ability to produce a loss in function of calmodulin thereby decreasing calmodulin inhibition, O2.- radicals at lower concentrations (<5 microM) may be able to stimulate Ca2+ release only in the presence of calmodulin from the SR via increased cADPR synthesis; it is also shown that cADPR is a modulator that can activate the Ca2+-release mechanism when it is in a sensitized state by the presence of calmodulin, possibly, at physiological concentration. In addition, the SR vesicles immediately upon addition of cADPR, but not NAD+, did exhibit Ca2+ efflux stimulation. When heart homogenate was incubated with O2.-, conversion of NAD+ into cADPR was stimulated; the reduction of homogenate Ca2+ uptake (by increasing Ca2+ efflux through RyR/Ca2+ release channel) occurred. Thus O2.- radical is responsible for cADPR formation from NAD+ in the cellular environment outside of the SR of heart muscle. The results presented here provide the first evidence of a messenger role for O2.- radical in cADPR-mediated Ca2+ mobilization in myocardium.

Topics: Adenosine Diphosphate Ribose; Animals; Calcium; Calcium Channel Blockers; Calcium Radioisotopes; Calmodulin; Cyclic ADP-Ribose; Dogs; Electron Spin Resonance Spectroscopy; Hypoxanthine; Myocardium; NAD; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Superoxide Dismutase; Superoxides; Xanthine Oxidase

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADPR) from beta-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from beta-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca2+ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, beta-NAD, into unfertilized oocytes. The changes induced by beta-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca2+ chelator, indicating the effects were due to Ca2+ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca2+ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca2+ oscillation while cADPR did not. A similar Ca2+ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP3 and NAADP could likewise inhibit such an InsP3-induced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocyte.

Topics: Adenosine Diphosphate Ribose; ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Animals; Antigens, CD; Antigens, Differentiation; Calcium; Chelating Agents; Cyclic ADP-Ribose; Electrophysiology; Fertilization; Inositol 1,4,5-Trisphosphate; NAD; NAD+ Nucleosidase; NADP; Oocytes; Patch-Clamp Techniques; Ryanodine; Signal Transduction; Urochordata