nitroarginine and vasoactive-intestinal-peptide-(10-28)

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

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.

Topics: Animals; Apamin; Carbachol; Cholinergic Agonists; Chymotrypsin; Electric Stimulation; Enzyme Inhibitors; Gastric Fundus; Guanylate Cyclase; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle Relaxation; Neuropeptides; Neurotransmitter Agents; Nitroarginine; Oxadiazoles; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Quinoxalines; Vasoactive Intestinal Peptide

Participation of the nitric oxide-cyclic GMP pathway in nonadrenergic, noncholinergic (NANC) relaxation induced by electrical field stimulation of longitudinal muscle of the rectum of Wistar-ST rats was studied by using a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). ODQ concentration dependently inhibited the relaxation and at 10 microM, maximally inhibited it by 83%. However, results obtained with N(G)-nitro-L-arginine, L-arginine and exogenously added nitric oxide excluded the participation of nitric oxide in the relaxation. An inhibitor of cyclic GMP-dependent protein kinase (PKG) partially (39%) inhibited the relaxation. ODQ also significantly inhibited the relaxation, which persisted after the PKG inhibitor-treatment, by 85%. The results strongly suggest that ODQ inhibits the NANC relaxation in a cyclic GMP-PKG pathway-independent manner.

Topics: Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Male; Muscle Relaxation; Neuropeptides; Nitroarginine; Oxadiazoles; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Quinoxalines; Rats; Rats, Wistar; Rectum; Vasoactive Intestinal Peptide

Participation of nitric oxide, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) in nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle of various intestinal regions in Sprague Dawley rats (8-week-old) was studied in vitro. Nitric oxide was suggested to participate in NANC relaxation of every intestinal region studied. But the participation was partial and its extent varied among the regions: significant in the proximal colon and rectum, and moderate in the jejunum, ileum and distal colon. Participation of PACAP in NANC relaxation was suggested only in the distal colon, while that of VIP was not detected in any of regions. Results obtained in the present study indicate that extent of participation of nitric oxide in NANC relaxation in Sprague Dawley rat intestine is more significant than those of other strains, Wistar and Wistar-ST.

Topics: Animals; Atropine; Colon; Enzyme Inhibitors; Gastrointestinal Motility; Guanethidine; Ileum; Jejunum; Muscle Contraction; Muscle Relaxation; Neuropeptides; Nitric Oxide; Nitroarginine; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Rats, Sprague-Dawley; Rats, Wistar; Species Specificity; Vasoactive Intestinal Peptide

The mediators of nonadrenergic, noncholinergic (NANC) relaxation of the circular muscle of rat rectum were examined in vitro. In the circular muscle of rat rectum, NG-nitro-L-arginine (L-NOARG) at 10 microM did not affect electrical field stimulation-induced relaxation but at 100 microM it inhibited electrical field stimulation-induced relaxation by about 75% and 1-mM L-arginine reversed the inhibition. Exogenous nitric oxide (NO) (1-10 microM) concentration dependently relaxed the circular muscle. Electrical field stimulation increased the cyclic GMP content of the circular muscle to about twice its resting level. L-NOARG, even at 10 microM, completely inhibited the electrical field stimulation-induced elevation of cyclic GMP content. However, L-arginine at 1 mM did not reverse the inhibition in cyclic GMP content. Inhibitory junction potentials (i.j.ps) induced by electrical field stimulation in the circular muscle cells were not affected by L-NOARG, 100 microM. Apamin ( < or = microM) did not affect the electrical field stimulation-induced relaxation, but almost completely inhibited electrical field stimulation-induced i.j.ps. NO (0.3-10 microM) induced relaxation of the circular muscle with a concomitant decrease in intracellular Ca2+ level ([Ca2+]i). Abundant immunoreactivity of NO synthase was found in the circular muscle layer, in addition to myenteric and submucosal plexus. The results suggest that NO induces NANC relaxation with a concomitant change in [Ca2+]i in the circular muscle of rat rectum. However, the involvement of changes in cyclic GMP level and in membrane potentials in the mechanism was not shown in the present experimental conditions.

Topics: Animals; Arginine; Calcium; Cyclic GMP; Electric Stimulation; Immunohistochemistry; In Vitro Techniques; Male; Membrane Potentials; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Myenteric Plexus; Neuropeptides; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Potassium Channel Blockers; Rats; Rats, Wistar; Rectum; Vasoactive Intestinal Peptide

The effect of vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide-38 (PACAP-38), and PACAP-27 on the release of serotonin (5-HT) into the intestinal lumen and the portal circulation was studied by using in vivo isolated vascularly and luminally perfused rat duodenum. 5-HT levels were determined by HPLC. VIP, PACAP-38, and PACAP-27 reduced the luminal release of 5-HT but did not affect the vascular release of 5-HT. The inhibitory effect caused by VIP, PACAP-38, and PACAP-27 was not affected by either atropine, hexamethonium, TTX, or TTX plus ACh, but it was completely antagonized by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA). The VIP receptor antagonist VIP-(10-28) blocked the effects of VIP, PACAP-38, and PACAP-27. These results suggest that VIP and PACAP exert a direct inhibitory effect on the luminal release of 5-HT from the enterochromaffin (EC) cells via a common receptor site on the EC cells and that this effect is mediated by NO but not by cholinergic pathways. A single injection of TTX, atropine, or hexamethonium reduced the luminal release of 5-HT, whereas a single injection of VIP-(10-28) stimulated the luminal release of 5-HT and this effect was antagonized by atropine, hexamethonium, or TTX. These results suggest that EC cells may receive the direct innervation of cholinergic neurons as well as VIP and/or PACAP neurons, with the former exerting a tonic stimulatory influence and the latter exerting a tonic inhibitory influence on 5-HT release into the intestinal lumen.

Topics: Acetylcholine; Animals; Arginine; Atropine; Chromatography, High Pressure Liquid; Duodenum; Hexamethonium; Humans; In Vitro Techniques; Male; Mesenteric Arteries; Neuropeptides; Nitroarginine; Peptide Fragments; Perfusion; Pituitary Adenylate Cyclase-Activating Polypeptide; Portal Vein; Rats; Rats, Wistar; Serotonin; Swine; Tetrodotoxin; Vasoactive Intestinal Peptide

Despite its widespread distribution and significance in the gut, the role of pituitary adenylate cyclase-activating peptide (PACAP) in internal anal sphincter (IAS) relaxation has not been examined. This study examined the role of PACAP in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of IAS smooth muscle. Circular smooth muscle strips from the opossum IAS were prepared for measurement of isometric tension. The influence of PACAP and vasoactive intestinal peptide (VIP) antagonists and tachyphylaxis on the neurally mediated IAS relaxation was examined either separately or in combination. The release of these neuropeptides in response to NANC nerve stimulation before and after the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine and NO was also investigated. Both PACAP and VIP antagonists caused significant attenuation of IAS relaxation by NANC nerve stimulation. The combination of the antagonists, however, did not have an additive effect on IAS relaxation. VIP tachyphylaxis caused significant suppression of IAS relaxation by NANC nerve stimulation. PACAP and VIP were found to be released by NANC nerve stimulation and exogenous NO. The data suggest the involvement of PACAP in IAS relaxation primarily by the activation of PACAP1/VIP receptor and lack of its independent role in the relaxation. Furthermore, NO may regulate the presynaptic release of PACAP and VIP.

Topics: Anal Canal; Animals; Female; In Vitro Techniques; Isometric Contraction; Male; Muscle Relaxation; Muscle, Smooth; Neuropeptides; Neurotransmitter Agents; Nitroarginine; Opossums; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Tachyphylaxis; Vasoactive Intestinal Peptide

The circuit of myenteric interneurons that regulate excitatory input to longitudinal colonic muscle was identified using dispersed ganglia and longitudinal muscle strips with adherent myenteric plexus from rat distal colon. The preparations enabled measurement of neurotransmitter release from interneurons and/or excitatory motoneurons innervating longitudinal muscle. 1, 1-Dimethyl-4-phenylpiperizinium (DMPP) and somatostatin were used to activate myenteric neurons in dispersed ganglia and muscle strips, respectively. DMPP-stimulated vasoactive intestinal peptide (VIP) release in dispersed ganglia was inhibited by [Met]enkephalin and bicuculline and augmented by naloxone and GABA, implying that inhibitory opioid and stimulatory GABA neurons regulate the activity of VIP interneurons. In muscle strips, VIP stimulated basal and augmented somatostatin-induced substance P (SP) release; the somatostatin-induced increase in SP release was inhibited by VIP-(10-28) and NG-nitro-L-arginine, implying that excitatory VIP neurons regulate tachykinin motoneurons innervating longitudinal muscle. Somatostatin inhibited [Met]enkephalin and stimulated VIP release; basal and somatostatin-stimulated VIP release were inhibited by [Met]enkephalin and bicuculline and augmented by naloxone and GABA, implying that inhibitory pathways linking somatostatin, opioid, and GABA neurons regulate VIP interneurons, which in turn regulate tachykinin and probably cholinergic motoneurons.

Topics: Animals; Bicuculline; Colon; Dimethylphenylpiperazinium Iodide; Enkephalin, Methionine; gamma-Aminobutyric Acid; Ganglia, Autonomic; In Vitro Techniques; Interneurons; Models, Neurological; Muscle, Smooth; Myenteric Plexus; Naloxone; Neurotransmitter Agents; Nitric Oxide; Nitroarginine; Peptide Fragments; Rats; Somatostatin; Substance P; Vasoactive Intestinal Peptide

We investigated the role of capsaicin-sensitive sensory nerves (CPSNs), nitric oxide (NO), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) in gastric adaptive and receptive relaxation in isolated guineapig stomachs. Changes in intragastric volume and pressure were recorded simultaneously in isolated stomachs in baths containing atropine and guanethidine. Adaptive relaxation was induced by luminal distention, and receptive relaxation was induced by electrical vagal stimulation. We found that desensitization to capsaicin inhibited adaptive relaxation, but not vagally induced relaxation. Extraluminal capsaicin induced gastric relaxation. Adaptive relaxation and capsaicin-induced relaxation were reduced by both tetrodotoxin and NG-nitro-L-arginine (LNNA), but not by hexamethonium. The effect of LNNA was partially reversed by co-incubation with L-arginine. Neither CGRP(8-37) nor VIP(10-28) inhibited all responses of adaptive relaxation, vagally induced and capsaicin-induced relaxation. These findings suggest that activation of CPSNs may be involved in adaptive relaxation, and that NO, but not CGRP or VIP, may be involved in the mechanisms of adaptive relaxation and receptive relaxation.

Topics: Adaptation, Physiological; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Electric Stimulation; Enzyme Inhibitors; Ganglionic Blockers; Gastric Mucosa; Guinea Pigs; Hexamethonium; In Vitro Techniques; Male; Miotics; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitroarginine; Peptide Fragments; Stomach; Tetrodotoxin; Vagus Nerve; Vasoactive Intestinal Peptide

1. The mediators of non-adrenergic non-cholinergic (NANC) relaxation of the longitudinal muscle of rat proximal, middle and distal colon were examined in vitro. 2. Electrical transmural stimulation (TMS) of proximal, middle and distal segments of rat colon induced NANC relaxations which were inhibited by tetrodotoxin (1 microM), but not by atropine (1 microM) or guanethidine (4 microM). 3. In the proximal colon, L-nitro-arginine (N5-nitroamidino-L-2,5-diaminopentanoic acid) inhibited the TMS-induced NANC relaxation and L-arginine (1 mM) reversed this inhibition. Nitric oxide (0.3-10 microM) induced relaxation of the proximal segment. 4. NANC relaxation of the proximal segments was still evident after desensitization to vasoactive intestinal peptide (VIP). A VIP antagonist (VIP 10-28, 10 microM) had no effect on the TMS-induced NANC relaxation, which was also resistant to alpha-chymotrypsin (2 units ml-1) and a substance P antagonist ([D-Pro2, D-Trp7,9]substance P, 1 microM). 5. In the middle colon, L-nitro-arginine did not inhibit the TMS-induced NANC relaxation in 6 of 9 preparations tested and partially inhibited the relaxation in the other 3 preparations. L-Arginine did not reverse the partial inhibition. 6. Complete desensitization to VIP was not achieved in the middle colon. The VIP antagonist had no effect on the TMS-induced NANC relaxation. After alpha-chymotrypsin treatment of the segment, desensitization of the segments to substance P, or in the presence of the substance P antagonist, the TMS-induced NANC relaxation was augmented. 7. In the distal colon, L-nitro-arginine did not have any significant effect on the TMS-induced relaxation and nitric oxide did not induce relaxation. The VIP antagonist significantly inhibited TMS-induced NANC relaxation. Alpa-Chymotrypsin-treatment of the distal segments resulted in significant inhibition of NANC relaxation. No desensitization to substance P was achieved. Treatment with the substance P antagonist had no effect. 8. These results suggest that nitric oxide is the mediator of the NANC inhibitory response in the proximal region of rat colon; in the middle colon, substance P acts as an excitatory neurotransmitter, antagonizing the NANC relaxation caused by the mediator of the response, which is still uncertain. Our results suggest that that VIP is the most likely candidate as a NANC transmitter in the distal colon.

Topics: Animals; Arginine; Chymotrypsin; Colon; Electric Stimulation; Gastrointestinal Motility; In Vitro Techniques; Male; Muscle, Smooth; Neurons; Nitric Oxide; Nitroarginine; Peptide Fragments; Rats; Rats, Wistar; Substance P; Vasoactive Intestinal Peptide