nitroarginine and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

The neurotransmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10-60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. l-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 microM), by desensitization of P2Y receptors with adenosine 5'-[beta-thio]diphosphate (ADP-betaS) (10 microM), and by the selective P2Y1 receptor blocker 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179) (10 microM). Relaxation and IJPs were also significantly reduced by the K(+) channel blocker apamin (1 microM). Removal of extracellular potassium (K(o)) increased IJP amplitude to 205% of control, whereas return of K(o) 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 microM) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apamin-sensitive K(+) channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anal Canal; Animals; Apamin; Electric Stimulation; Enteric Nervous System; Enzyme Inhibitors; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Mice; Mice, Inbred BALB C; Motor Neurons; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Neural Inhibition; Neuromuscular Junction; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Potassium; Potassium Channel Blockers; Potassium Channels; Purinergic P2 Receptor Antagonists; Purines; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Synaptic Transmission; Tetrodotoxin; Thionucleotides

Neurotransmitters released by myenteric neurons regulate movements of intestinal smooth muscles. There has been little pharmacological evidence for a role of purinergic mechanisms in the non-adrenergic, non-cholinergic (NANC) relaxation of the human large intestine. We used P(2) purinoceptor antagonists to assess whether such receptors are involved in the NANC relaxation of the circular muscle of the human sigmoid colon. It was also investigated whether the guanylate cyclase enzyme mediates the NANC response. Human colonic circular strips were tested in organ bath experiments with isotonic recording. NANC, non-nitrergic relaxations induced by electrical field stimulation (1 and 10 Hz, in the presence of atropine, guanethidine, and 100 microM N(G)-nitro-L-arginine [L-NOARG]) were strongly inhibited by a combination of the P(2) purinoceptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-sulfonic acid (PPADS) (50 microM) and suramin (100 microM). PPADS plus suramin was ineffective in the absence of L-NOARG. L-NOARG alone significantly reduced the NANC relaxation to electrical stimulation. PPADS plus suramin strongly inhibited the relaxation in response to exogenous alpha,beta-methylene ATP. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (3 microM) inhibited the NANC relaxation, but did not add to its reduction by L-NOARG. L-NOARG was still slightly effective in the presence of ODQ. Vasoactive intestinal polypeptide tachyphylaxis failed to influence the non-nitrergic NANC relaxation. It is concluded that nitric oxide (NO) and ATP co-mediate, in a non-additive manner, the NANC relaxation. NO probably acts through the guanylate cyclase, though a small fraction of its effect might be mediated by other mechanisms. Activators of the guanylate cyclase other than NO do not seem to participate in the NANC relaxation.

Topics: Adenosine Triphosphate; Colon, Sigmoid; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Guanylate Cyclase; Humans; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth; Myenteric Plexus; Neurotransmitter Agents; Nitrergic Neurons; Nitric Acid; Nitric Oxide; Nitroarginine; Oxadiazoles; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinoxalines; Receptors, Purinergic P2; Statistics, Nonparametric; Suramin

The transmitters involved in the non-nitrergic component of the non-adrenergic, non-cholinergic (NANC) inhibitory response of the rat small intestinal longitudinal muscle to electrical field stimulation of its nerves is a matter of controversy. The present study is the first one to utilise a combination of a nitric oxide synthase inhibitor and a P(2) purinoceptor antagonist for studying this response. We found that the P(2) purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 5x10(-5) M) abolished the non-nitrergic NANC relaxation to electrical field stimulation (10 Hz). PPADS alone provided a significant, moderate inhibitory action. PPADS specifically inhibited relaxations due to exogenous adenosine 5'-triphosphate (ATP) or alpha,beta-methylene ATP. The guanylate cyclase blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10(-6) M) did not add to the inhibitory action of N(G)-nitro-L-arginine on field stimulation-induced relaxation. ODQ abolished the relaxant effect of the nitric oxide donors nitroglycerin or sodium nitroprusside. These data indicate that: (1) nitric oxide and ATP fully account for the field stimulation-induced relaxation in the rat ileal strip under the experimental conditions of this study, and (2) no ODQ-sensitive guanylate cyclase-mediated mechanism is involved in the non-nitrergic component of the NANC relaxation.

Topics: Animals; Electric Stimulation; Enzyme Inhibitors; Female; Guanylate Cyclase; Ileum; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Synthase; Nitroarginine; Nitroglycerin; Nitroprusside; Oxadiazoles; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinoxalines; Rats; Rats, Wistar; Receptors, Purinergic P2; Tetrodotoxin

We have investigated the mechanisms underlying acute changes in gastric motor function triggered by endotoxemia. In fundal strips from rats pre-treated with endotoxin (40 microg/kg, i.p. 30 min), mechanical activity was analyzed and the source of nitric oxide (NO) was visualized by confocal microscopy of tissue loaded with the fluorescent dye DAF-FM. NOS expression was determined by quantitative RT-PCR and Western blot, and enzyme activity by the citrulline assay. Strips from endotoxin-treated rats were hypo-contractile. This was prevented by pre-incubation with the neurotoxin tetrodotoxin, the gangliar blocker hexamethonium, or non-selective and neuronal-specific NOS inhibitors (L-NOARG and TRIM, respectively). The soluble guanylyl cyclase (sGC) inhibitor ODQ and the inhibitor of small conductance Ca2+-activated K+ channels apamin prevented relaxation induced by endotoxin, nicotine, exogenous NO (DETA-NONOate), and the NO-independent sGC activator BAY 41-2272. NO synthesis was observed in neuronal soma, axons, and nerve endings of the myenteric plexus in the fundus of endotoxin-treated rats and was prevented by L-NAME, tetrodotoxin, and hexamethonium. nNOS and iNOS mRNA and protein contents were unchanged. Our findings demonstrate synthesis of NO in post-ganglionic myenteric neurons during early endotoxemia that mediates gastric hypo-contractility. The effect of NO is mediated via sGC and small conductance Ca2+-activated K+channels.

Topics: Animals; Apamin; Autonomic Fibers, Postganglionic; Carbachol; Dexamethasone; Endotoxemia; Gastric Fundus; Gastrointestinal Motility; Guanylate Cyclase; Microscopy, Confocal; Nerve Tissue Proteins; Nicotine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Nitroso Compounds; Peptide Fragments; Potassium Channels, Calcium-Activated; Pyrazoles; Pyridines; Pyridoxal Phosphate; Rats; Suramin; Tetrodotoxin; Vasoactive Intestinal Peptide

1. Conflicting views exist on whether ATP is a neurotransmitter in the enteric nervous system. We investigated the role of ATP in enteric transmission in circular muscle strips of the mouse jejunum. 2. On PGF2alpha-precontracted muscle strips and in the presence of atropine and guanethidine, electrical field stimulation (EFS, 1-8 Hz) of nonadrenergic noncholinergic (NANC) nerves induced transient relaxations that were abolished by the nerve-conductance blocker tetrodotoxin. The NO synthase blocker l-nitroarginine (l-NOARG) partially inhibited the NANC relaxations to EFS, but fast-twitch relaxations to EFS were still observed in the presence of l-NOARG. 3. In the presence of l-NOARG, ATP, the P2X receptor agonist alphabetaMeATP and the P2Y receptor agonist ADPbetaS relaxed jejunal muscle strips. Tetrodotoxin did not affect the relaxation to ATP and ADPbetaS, but inhibited that to alphabetaMeATP. 4. The l-NOARG-resistant NANC relaxations to EFS were almost abolished by apamin, a blocker of small-conductance Ca2+ activated K+ channels, and by suramin and PPADS, blockers of P2 purinoceptors. Relaxations to ATP were almost abolished by apamin and suramin but not affected by PPADS. 5. Desensitisation of alphabetaMeATP-sensitive P2X receptors, the P2X receptor blocker Evans blue and the P2X1,2,3 receptor blocker NF 279 inhibited the l-NOARG-resistant NANC relaxations to EFS and that to alphabetaMeATP without affecting the relaxation to ADPbetaS. Brilliant blue G, a P2X2,5,7 receptor blocker, did not affect the relaxations to EFS. 6. Desensitisation of P2Y receptors and MRS 2179, a P2Y1 receptor blocker, virtually abolished the l-NOARG-resistant NANC relaxations to EFS and the relaxation to ADPbetaS without affecting the relaxation to alphabetaMeATP. 7. Dipyridamole, an adenosine uptake inhibitor, or theophylline and 8-phenyltheophylline, blockers of P1 and A1 purinoceptors, respectively, did not affect the purinergic NANC relaxations to EFS. 8. Our results suggest that ATP or a related purine acts as an inhibitory NANC neurotransmitter in the mouse jejunum, activating P2 but not P1 purinoceptors. Relaxations to the purinergic NANC neurotransmitter mainly involve P2Y receptors of the P2Y1 subtype that are located postjunctionally. Purinergic NANC neurotransmission also involves P2X receptors, most likely of the P2X1 and P2X3 subtype, located pre- and/or postjunctionally.

Topics: Adenosine Triphosphate; Animals; Apamin; Electric Stimulation; Enzyme Inhibitors; In Vitro Techniques; Jejunum; Mice; Muscle Relaxation; Muscle, Smooth; Neurotransmitter Agents; Nitric Oxide Synthase; Nitroarginine; Protein Isoforms; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Suramin; Synaptic Transmission; Thionucleotides

The contractile effect of capsaicin in the guinea-pig small intestine involves an activation of enteric cholinergic neurons. Our present data show that the P(2) purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) significantly reduces the contractile response to capsaicin (2 microM) in the presence, but not in the absence, of the tachykinin receptor antagonists [O-Pro(9), (Spiro-gamma-lactam)Leu(10), Trp(11)]physalaemin (1-11) (GR 82334; 3 microM) and (S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)-N -methylacetamide (SR 142804: 100 nM) (for blocking tachykinin NK1 and NK3 receptors, respectively). PPADS (30 microM) fails to influence submaximal cholinergic contractions evoked by cholecystokinin octapeptide (CCK-8; 2-3 nM) or senktide (1 nM), or the direct smooth muscle-contracting effect of histamine (100-200 nM). A higher concentration (300 microM) of PPADS is also without effect against the stimulatory action of cholecystokinin octapeptide. This means that PPADS can probably be safely used as a purinoceptor antagonist in intestinal preparations. The putative pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist PACAP-(6-38) (3 microM) significantly reduces the contractile effect of PACAP-(1-38) (10 nM) and abolishes that of vasoactive intestinal polypeptide (VIP; 10 nM). PACAP-(6-38) (3 microM) fails to influence the effect of capsaicin (2 microM) both in the absence and in the presence of tachykinin receptor antagonists. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM) also fails to inhibit the capsaicin-induced motor response. We conclude that an endogenous ligand of PPADS-sensitive P(2) purinoceptors (possibly ATP), but not a VIP/PACAP-like peptide or NO, is involved in the nontachykininergic activation of cholinergic neurons in the course of the capsaicin-induced contraction.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Capsaicin; Enzyme Inhibitors; Guinea Pigs; Ileum; In Vitro Techniques; Intestine, Small; Muscle Contraction; Muscle, Smooth; Neurokinin-1 Receptor Antagonists; Neuropeptides; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Physalaemin; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Pyridoxal Phosphate; Receptors, Neurokinin-3; Sincalide; Substance P; Vasoactive Intestinal Peptide

The effect of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM) and the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 50 microM) was investigated on the non-adrenergic, non-cholinergic (NANC) relaxant response of the guinea-pig isolated taenia caeci to electrical field stimulation at 1 or 10 Hz, under isotonic recording conditions. Either drug alone caused an about 50% inhibition, while combining the two drugs nearly abolished the response at both frequencies. The inhibitory effect of L-NOARG (100 microM) was partly reversed by L-arginine (30 mM). PPADS, but not L-NOARG, inhibited the relaxant effect of exogenous ATP, but not that of the nitric oxide donor sodium nitroprusside. It is concluded that both nitric oxide and ATP are involved in the mediation of NANC relaxation in the taenia caeci, in an apparently additive manner.

Topics: Adenosine Triphosphate; Animals; Colon; Electric Stimulation; Female; Guinea Pigs; In Vitro Techniques; Male; Muscle Relaxation; Nitric Oxide; Nitroarginine; Pyridoxal Phosphate