Noncholinergic Inhibitory Neurons Research Articles

Multidisciplinary approach to nitric oxide signaling: Focus on the gastrointestinal and the central nervous system.

Nitric oxide (NO), with close historical ties to cardiovascular physiology, is an important endogenous mediator, the most potent natural vasorelaxant known, involved in many biological functions . NO acts as an intra/intercellular signalling molecule and is a mediator in almost all organ systems . NO is known to occur in many cells types, including vascular endothelial cells, neurons, and epithelial cells . The transmitter of nonadrenergic, noncholinergic (NANC) inhibitory neurons has been the subject of hundreds of investigations over the past 3 decades. Recent evidence suggests that NO may serve as a NANC inhibitory signaling molecule in the gastrointestinal (GI) tract. NO serves as the primary enteric inhibitory neurotransmitter in GI muscles, and nitrergic neurons regulate gut tone, phasic contractile amplitude and frequency, and inhibitory reflexes . In the central nervous system (CNS) NO is involved in some major processes such as memory through longterm potentiation (LTP) and learning . This gasotransmitter also contributes to a pathogenesis of epilepsy. The role of NO in the generation of epilepsy is contradictory since there is evidence of its proconvulsive and anticonvulsive effects . In this review, we will discuss about the possible role of NO as neurotransmitter in the GI and CNS, with focus on the contribution of NO-mediated signaling pathways in the GI motility and CNS excitability. Physiological functions of nitric oxide

Functional evidence for GABA as modulator of the contractility of the longitudinal muscle in mouse duodenum: Role of GABA A and GABA C receptors

We investigated, in vitro, the effects of γ-aminobutyric acid (GABA) on the spontaneous mechanical activity of the longitudinal smooth muscle in mouse duodenum. GABA induced an excitatory effect, consisting in an increase in the basal tone, which was antagonized by the GABA A-receptor antagonist, bicuculline, potentiated by (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA), a GABA C-receptor antagonist and it was not affected by phaclofen, a GABA B-receptor antagonist. Muscimol, GABA A receptor agonist, induced a contractile effect markedly reduced by bicuculline, tetrodotoxin (TTX), hexamethonium and atropine. Cis-4-aminocrotonic acid (CACA), a specific GABA C receptor agonist, induced an inhibitory effect, consisting in the reduction of the amplitude of the spontaneous contractions and muscular relaxation, which was antagonised by TPMPA, GABA C-receptor antagonist, TTX or N ω-nitro- l-arginine methyl ester (L-NAME), nitric oxide (NO) synthase inhibitor, but not affected by hexamethonium. In conclusion, our study indicates that GABA is a modulator of mechanical activity of longitudinal muscle in mouse duodenum. GABA may act through neuronal presynaptic receptors, namely GABA A receptors, leading to the release of ACh from excitatory cholinergic neurons, and GABA C receptors increasing the release of NO from non-adrenergic, non-cholinergic inhibitory neurons.

Effect of Drugs with Various Mechanisms of Action on Propulsive Activity of the Small Intestine

We studied the effect of drugs with various mechanisms of action on propulsive activity of the small intestine in healthy rats. Blockade of the major inhibitory influences realized via nonadrenergic noncholinergic inhibitory effector neurons was not followed by stimulation of intestinal transit. Propulsive activity of the small intestine increased upon treatment with drugs, whose effects are realized via acetylcholine or acetylcholine and serotonin.

Glucose does not activate nonadrenergic, noncholinergic inhibitory neurons in the rat stomach

We reported previously that intravenously administered d-glucose acts in the central nervous system to inhibit gastric motility induced by hypoglycemia in anesthetized rats. The purpose of this study was to determine whether this effect is due to inhibition of dorsal motor nucleus of the vagus (DMV) cholinergic motoneurons, which synapse with postganglionic cholinergic neurons, or to excitation of DMV cholinergic neurons, which synapse with postganglionic nonadrenergic, noncholinergic (NANC) neurons, particularly nitrergic neurons. Three approaches were employed: 1) assessment of the efficacy of d-glucose-induced inhibition of gastric motility in hypoglycemic rats with and without inhibition of nitric oxide synthase [10 mg/kg iv nitro-l-arginine methyl ester (l-NAME)], 2) assessment of the efficacy of intravenous bethanechol (30 mug.kg(-1).min(-1)) to stimulate gastric motility in hypoglycemic rats during the time of d-glucose-induced inhibition of gastric motility, and 3) determination of c-Fos expression in DMV neurons after intravenous d-glucose was administered to normoglycemic rats. Results obtained demonstrated that l-NAME treatment had no effect on d-glucose-induced inhibition of gastric motility; there was no reduction in the efficacy of intravenous bethanechol to increase gastric motility, and c-Fos expression was not induced by d-glucose in DMV neurons that project to the stomach. These findings indicate that excitation of DMV cholinergic motoneurons that synapse with postganglionic NANC neurons is not a significant contributing component of d-glucose-induced inhibition of gastric motility.

P2 receptors in the murine gastrointestinal tract

The actions of adenosine, adenosine 5′-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), α,β-methylene ATP (α,β-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y 1, P2Y 2, P2Y 4, P2X 1 and P2X 2 receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y 1 mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP≥2-MeSATP>α,β-meATP>ATP=UTP=adenosine. This is suggestive of P2Y 1-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to α,β-meATP. As ATP and UTP are equipotent, the presence of a P2Y 2 receptor is indicated. ATP responses were inhibited by the P2Y 1-selective antagonist MRS 2179, and suramin. P2Y 1 receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y 1 mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N ω-nitro- l-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>α,βmeATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X 2 receptors. Immunoreactivity to P2X 2 receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y 1 receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y 1, P2Y 2 and a novel P2Y receptor subtype responsive to α,β-meATP in smooth muscle, while P2X 2 receptors mediate contraction of colonic smooth muscle.

Mediators and intracellular mechanisms of NANC relaxation of smooth muscle in the gastrointestinal tract.

Tension of the skeletal muscle is solely controlled by excitatory cholinergic motor neurons. By contrast, the peripheral smooth muscle tissues are dual-controlled by nonadrenergic noncholinergic (NANC) inhibitory neurons as well as excitatory neurons. In the gastrointestinal tract, many possible candidates, including peptides, amino acids, nucleotides and gaseous compounds, for the inhibitory mediator and mechanisms of their inhibitory control have been extensively studied. This article introduces the diversity of inhibitory mediators and their probable intracellular mechanisms on the basis of recent findings on this theme, many of which are unexpected interesting findings.

Chronological change of distribution in nitric oxide and peptidergic neurons after rat small intestinal transplantation

Background/Purpose: Nitric oxide (NO) has been considered one of the putative neurotransmitters of nonadrenergic, noncholinergic inhibitory neurons. To examine the effect of transplantation on NO neurons in the intestine, the distribution of NO neurons was examined and compared with that of peptide-containing neurons. Methods: A jejunal graft measuring about 20 cm was harvested from a Lewis rat, syngeneically transplanted as a Thiry-Vella loop, and later was replaced to the recipient small bowel 20 days after transplantation. Tissue specimens of the grafts were taken on days 1, 3, 6, 10, and 20, and 1 year after transplantation (n = 5 each). The distribution of the neurons was examined immunohistochemically, using antisera against protein gene product 9.5 (PGP 9.5, general neuronal marker), brain nitric oxide synthase (bNOS), vasoactive intestinal polypeptide (VIP), and substance P. In addition, NADPH diaphorase staining also was performed to visualize NO neurons. Results: In the PGP 9.5 immunoreactivities, no significant difference in the distribution was observed among the controls and on any day after transplantation. However, the NADPHd activities markedly decreased in muscle layers, especially in the deep muscular layer on day 1 and 3, but quickly recovered by day 6. The distribution of bNOS immunoreactivities was almost same as that of NADPHd staining. The VIP and substance P immunoreactivities also decreased on day 1, and thereafter gradually recovered, and then became normal on day 20. Conclusions: Both the NO and peptidergic neurons markedly decreased just after transplantation, and the NO neurons recovered faster than the peptidergic neurons. These findings suggested that NO neurons might play an important role in the adaptation process of the graft in the early period after transplantation.

Intracisternal or microinjection of TRH into DMN does not activate non-adrenergic, non-cholinergic inhibitory (NANC) neurons of the rat stomach

Intracisternal or microinjection of TRH into DMN does not activate non-adrenergic, non-cholinergic inhibitory (NANC) neurons of the rat stomach

5-HT-induced jejunal motor activity: enteric locus of action and receptor subtypes.

The role of 5-hydroxytryptamine (5-HT), its enteric locus of action, and receptor subtypes involved in the regulation of jejunal contractions were investigated by close intra-arterial infusions in conscious dogs. Close intra-arterial infusions of 5-HT in short segments of the jejunum stimulated phasic contractions that were blocked completely by atropine, partially by tetrodotoxin, and not affected by hexamethonium. This response was also blocked significantly by 5-HT2A and 5-HT2C receptor antagonists but was not affected by 5-HT1A/5-HT1B, 5-HT3, and 5-HT4 receptor antagonists. Spontaneous phase III contractions were inhibited significantly by 5-HT2A and 5-HT2C receptor antagonists, not affected by 5-HT1A/5-HT1B and 5-HT3 receptor antagonists, and enhanced by 5-HT4 receptor antagonists. Repeated close intra-arterial infusions of 5-HT over several days stimulated giant migrating contractions. We conclude that in the conscious state, 5-HT acts on 5-HT2A and 5-HT2C receptors located on postsynaptic cholinergic neurons in the canine jejunum to stimulate phasic contractions and phase III activity. The 5-HT4 receptors in the canine small intestine may be localized on nonadrenergic, noncholinergic inhibitory neurons; these receptors suppress the amplitude and duration of phase III activity.

Neuronal tachykinin NK2 receptors mediate release of non-adrenergic non-cholinergic inhibitory transmitters in the circular muscle of guinea-pig colon.

The aims of this study were: (i) verify the usefulness of the recently described non-peptide antagonist, SR 142801, for blocking tachykinin NK3 receptors in the circular muscle of the guinea-pig colon and (ii) after occlusion of NK3 receptors by SR 142801, test the hypothesis that tachykinins may activate non-adrenergic non-cholinergic inhibitory neurons via non-NK3 receptors. In sucrose gap, we found that SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced atropine (1 microM)-sensitive depolarization, action potentials and contractions of circular muscle of guinea-pig colon without affecting the cholinergic excitatory junction potential and contraction produced by single pulse electrical field stimulation. Likewise, SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced non-adrenergic non-cholinergic hyperpolarization and relaxation of the circular muscle, without affecting the non-adrenergic non-cholinergic inhibitory junction potentials and relaxation produced by single pulse electrical field stimulation. Therefore, SR 142801 is a suitable tool to occlude neuronal NK3 receptors in guinea-pig colon. In the presence of SR 142801 (0.1 microM), atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM) and nifedipine (1 microM) superfusion with neurokinin A (0.3 microM) produced depolarization on which a series of inhibitory junction potentials were superimposed. The incidence, number and amplitude of the inhibitory junction potentials evoked by neurokinin A was partly reduced by pretreatment with either apamin (0.1 microM) or L-nitroarginine (30 microM) and was totally blocked by pretreatment with apamin plus L-nitroarginine or by tetrodotoxin (1 microM). None of these treatments affected the depolarization and contraction produced by neurokinin A. The NK1 receptor selective antagonist, GR 82,334 (3 microM), did not affect the responses to neurokinin A, which were abolished by the NK2 receptor-selective antagonist GR 94,800 (0.1 microM). Substance P (0.3 microM) produced a large depolarization of the membrane but was poorly effective in producing superimposed inhibitory junction potentials. The NK1 receptor-selective agonist [Sar9]substance P sulfone (0.3 microM) produced large depolarization without inducing superimposed inhibitory junction potentials, while the NK2 receptor-selective synthetic agonist [beta-Ala8]neurokinin A(4-10) (0.3 microM) produced depolarization and superimposed inhibitory junction potentials. We conclude that neurokinin A, in addition to direct excitation and contraction of circular muscle activates, via neuronal NK2 receptors, inhibitory non-adrenergic non-cholinergic motorneurons. Thus, neuronal NK2 receptors should be considered as targets for endogenous tachykinins in enteric circuitries leading to descending relaxation in guinea-pig colon.

Nitric oxide is involved in non-adrenergic, non-cholinergic inhibitory neurotransmission in rat duodenum.

1. In rat duodenum, electrical field stimulation (EFS) induced a relaxation due to activation of non-adrenergic, non-cholinergic (NANC) inhibitory intramural neurones. 2. Nitric oxide synthase (NOS) inhibitors, N omega-nitro-L-arginine (L-NNA) and N omega-nitro-L-arginine methyl ester (L-NAME), caused a dose-dependent reduction in amplitude of the NANC relaxation. Responses to low frequencies of stimulation were more sensitive to NOS inhibitors than those to high frequencies. 3. Effects induced by NOS inhibitors were stereospecific since D-NNA and D-NAME did not affect NANC relaxation. L-arginine, but not D-arginine, partially prevented the effects induced by NOS inhibitors on NANC relaxation. 4. The nitrovasodilator drug, sodium nitroprusside, caused muscle relaxation which was not affected by preincubation with either tetrodotoxin (TTX), L-NNA or L-NAME. 5. alpha-Chymotrypsin reduced relaxations elicited by stimulation of NANC nerves, especially when high frequencies of stimulation were used. The residual NANC relaxation was further reduced by NOS inhibitors. In the same way, alpha-chymotrypsin was able to further reduce the relaxation observed after NOS inhibitors. 6. These results suggest that nitric oxide (NO) and a peptide are involved in NANC relaxation of rat duodenal smooth muscle. NO and peptidergic pathways act in parallel to produce muscle relaxation and they are preferentially activated by stimuli at low and high frequencies, respectively.

Evidence for coexistence of ATP and nitric oxide in non-adrenergic, non-cholinergic (NANC) inhibitory neurones in the rat ileum, colon and anococcygeus muscle.

The possible coexistence of the two non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitters, adenosine 5'-triphosphate and nitric oxide in the myenteric plexus was investigated using whole-mount preparations of rat ileum, proximal colon and anococcygeus muscle. The presence of adenosine 5'-triphosphate in neurones was examined using the quinacrine fluorescence technique. After localizing and taking photographs of quinacrine-fluorescent neurones and nerve fibres, the same tissues were then fixed and processed for NADPH-diaphorase activity, a marker for nitric oxide-containing neurones. We have demonstrated for the first time that almost all quinacrine-fluorescent myenteric neurones in the proximal colon are also NADPH-diaphorase reactive, while only a subpopulation of quinacrine-fluorescent neurones in ileum and anococcygeus muscle were also NADPH-diaphorase reactive.

Evidence for coexistence of ATP and nitric oxide in non-adrenergic, non-cholinergic (NANC) inhibitory neurones in the rat ileum, colon and anococcygeus muscle

Evidence for coexistence of ATP and nitric oxide in non-adrenergic, non-cholinergic (NANC) inhibitory neurones in the rat ileum, colon and anococcygeus muscle

Role of M1 muscarinic receptors in the parasympathetic control of colonic motility in cats and rabbits.

1. The effects of pirenzepine (a selective blocking agent of M1 muscarinic receptors) were studied on excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) elicited on colonic smooth muscle by stimulation of efferent parasympathetic nerve fibres in anaesthetized cats and rabbits. 2. Pirenzepine (25 micrograms kg-1 to 0.2 mg kg-1, i.v.) decreased the amplitude of EJPs or abolished them. In pirenzepine, parasympathetic stimulation elicited IJPs in most cases. 3. In both species, pirenzepine initiated spontaneous IJPs, indicating an increase in the activity of the non-adrenergic, non-cholinergic (NANC) inhibitory neurones. 4. The results suggest that M1 muscarinic receptors are involved in synaptic transmission within intramural plexuses, at interneuronal synapses in the parasympathetic excitatory pathway to colonic smooth muscle, but are not involved in the pathway to the NANC inhibitory neurones. The facilitation of IJPs by pirenzepine suggests that, under normal physiological conditions, NANC neurones are tonically inhibited by an intramural nervous circuit involving M1 muscarinic receptors.

Peptide YY induces nerve-mediated responses in the guinea pig intestine

The actions of peptide YY (PYY) were studied in longitudinal organ-bath preparations of the guinea pig intestine. PYY induced concentration-dependent (10(-9)-5 x 10(-8) M) relaxations of tissue from the duodenum, jejunum, ileum, and colon. These responses were unaffected by adrenergic blockade and atropine treatment but could be prevented by tetrodotoxin. The pharmacology of PYY actions in segments of the small and large intestine indicated the involvement of intrinsic nonadrenergic, noncholinergic inhibitory neurones in the relaxation response to this peptide. All tissues could be made tachyphylactic to PYY without affecting their ability to respond to the direct acting muscle relaxants ATP or papaverine. Moreover, nicotinic ganglion stimulated relaxations and cholinergic nerve-mediated contractions were also unaffected. These results show applied PYY to have potent neurogenic actions in the guinea pig intestine with some similarities to PYY actions in the rat intestine.

Effects of gabexate mesilate (FOY) on the gallbladder, sphincter of Oddi and duodenum of the normal and gastorectomized dogs.

FOY induced a dose-dependent inhibitory response on the gallbladder, sphincter of Oddi and duodenum of normal and gastrectomized dogs, although it induced an excitatory response in some dogs. The inhibitory response was not reduced or terminated by pretreatment with atropine, guanethidine, hexamethonium or/and proglumide. The FOY-induced inhibitory response reversed to the excitatory response in the sphincter of Oddi and duodenum by pretreatment with tetrodotoxin, but not in the gallbladder. These results suggested that the FOY-induced inhibitory response of the sphincter of Oddi and duodenum was caused by stimulation of nonadrenergic noncholinergic inhibitory neurons, not by FOY-induced cholecystokinin secretion. The excitatory response was induced by direct stimulation of their smooth muscles. The inhibitory response of the gallbladder to FOY was induced by direct stimulation of the smooth muscles.

Adaptation of the mechanisms controlling gastric motility following chronic vagotomy in the ferret

Changes in gastric motility were studied in the urethane-anaesthetized ferret following acute or chronic (3 weeks) vagotomy. The stomach was divided into the corpus and antrum and the effects of vagotomy on tone, frequency and contraction amplitude were investigated separately in the two gastric regions. In the corpus tone is kept at low levels by vagal activation of nonadrenergic, non-cholinergic (NANC) inhibitory neurones and also tonic sympathetic inhibition of intramural cholinergic activity. Frequency of contractions is also low due to tonic inhibition of cholinergic neurones by the vagus but not the sympathetic nervous system. There appears to be little vagal involvement in contraction amplitude but there is sympathetic inhibition of this parameter again via inhibition of cholinergic neurones. In the antrum there is no vagally driven inhibition of tone but a sympathetic inhibition of cholinergic neurones tends to reduce tone in the intact animal. Frequency of contractions does not appear to be extrinsically modulated. The vagus is tonically excitatory with regard to contraction amplitude in the antrum whereas the sympathetic nervous system is inhibitory, again via inhibition of cholinergic neurones. After chronic vagotomy some adaptation appears to take place within the surviving control systems in both the corpus and the antrum. Changes in cholinergic function have been suggested previously and are corroborated in this study. In addition novel alterations in intrinsic NANC systems and the remaining sympathetic innervation have been demonstrated in both regions of the stomach which tended to reduce the effects of vagotomy and return values for the parameters measured toward those observed in intact animals. The contribution of the cholinergic, adrenergic and NANC neurotransmitter systems to the post-vagotomy motility patterns differed in the corpus and antrum.

Cisapride inhibits motility of the sphincter of Oddi in the Australian possum

Cisapride has been shown in a number of recent studies to have prokinetic effects on the gastrointestinal tract. Studies of its action on the biliary tract have been limited. In this study we determined the effect of cisapride on sphincter of Oddi motility of the Australian brush tailed possum in vivo, and evaluated possible mechanisms for its effect. Cisapride produced a dose-dependent inhibition of sphincter of Oddi phasic contractions. The inhibitory effect was not blocked by atropine, phentolamine, propranolol, methysergide, or hexamethonium administration, or after cervical truncal vagotomy. Tetrodotoxin, however, abolished the sphincter response to cisapride. We conclude that cisapride has an inhibitory effect on the possum sphincter of Oddi. This inhibitory effect is mediated via nonadrenergic, noncholinergic inhibitory neurons and is similar to the inhibitory effect of cholecystokinin on the sphincter of Oddi.

Participation of the parasympathetic and sympathetic nerves in regulation of gallbladder motility in the dog.

The participation of the parasympathetic and sympathetic nerves in the canine gallbladder motility was examined. Efferent stimulation of the parasympathetic (vagus) and sympathetic (celiac) nerves caused contraction or inhibition of the neck, body and fundus of the gallbladder. The contractile response induced by vagus nerve stimulation was reduced by subthreshold efferent stimulation of the celiac nerve, while the inhibitory response was neither reduced nor enhanced by subthreshold efferent stimulation of the celiac nerve. The contractile and inhibitory response induced by celiac nerve stimulation was not reduced in the neck, body and fundus by subthreshold efferent stimulation of the vagus nerve. The contractile response to vagus nerve stimulation was reversed to a relaxant response by atropine administration, which was reduced or abolished by hexamethonium. It is suggested that the vagus nerve-induced contractile response in the canine gallbladder is modulated by sympathetic nerves presynaptically at the vagus nerve endings in the enteric ganglion, but the vagus nerve-induced relaxant response, which probably was induced by non-adrenergic non-cholinergic inhibitory neurons, is not modulated by the sympathetic nerves.

Non-adrenergic, non-cholinergic inhibitory innervation to the gastrointestinal tract

The smooth muscles of the gastrointestinal tract and blood vessels are innervated by non-adrenergic, non-cholinergic (NANC) inhibitory neurons. The transmitter(s) in relation to NANC inhibitory neurons remains unknown, but there are two main working hypotheses, the purinergic and VIPergic nerve hypotheses, at present. Although there is a large amount of data supporting the purinergic hypothesis, definitive evidence is still lacking. VIP seems to be regarded as a likely candidate for the neurotransmitters of some NANC inhibitory neurons. However, the data presented are still incomplete. For the purinergic hypothesis, the discrepancies seem to be greater in the stomach and oesophagus and those for the VIPergic hypothesis, in the guinea pig taenia coli. Moreover, there are many examples of the co-existence of peptides or of peptides and synthesizing enzymes of amines or acetylcholine in the gastrointestinal tract. Therefore, it is possible that NANC inhibitory neurons liberate more than one active inhibitory substance and/or there are different types of NANC inhibitory neurons in the gastrointestinal tract. Much more evidence seem to be needed before the neurotransmitter(s) of NANC inhibitory neurons in the gastrointestinal tract and blood vessels can be identified.