Regulation of gastrointestinal motility involves excitatory and inhibitory neurotransmission. Nitric oxide (NO), the major inhibitory neurotransmitter, acts via its receptor NO-sensitive guanylyl cyclase (NO-GC). In the GI tract, NO-GC is expressed in several cell types such as smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Using cell-specific knockout mice, we have previously shown that NO-GC modulates spontaneous contractions in colonic longitudinal smooth muscle. However, its detailed role in the colonic circular smooth muscle is still unclear.

Myography was performed to evaluate spontaneous contractions in rings of proximal colon (2.5 mm) from global (GCKO) and cell-specific knockout mice for NO-GC. Immunohistochemistry and in situ hybridization were used to specify NO-GC expression.

Colonic circular smooth muscle showed three different contraction patterns: high-frequency ripples, slow phasic contractions, and large contractions. Ripples formed independently of NO-GC. Slow phasic contractions occurred intermittently in WT, SMC-GCKO, and ICC-GCKO tissue, whereas they were more prominent and prolonged in GCKO and SMC/ICC-GCKO tissue. Tetrodotoxin and the NO-GC inhibitor ODQ transformed slow phasic contractions of WT and single cell-specific knockout into GCKO-like contractions. ODQ increased the frequency of large contractions in WT and ICC-GCKO colon but not in GCKO, SMC-GCKO, and SMC/ICC-GCKO preparations. Tetrodotoxin and hexamethonium abolished large contractions.

We conclude that short rings of murine colon can be effectively used to record spontaneous contractions. Although NO-GC in SMC determines smooth muscle tone, concerted action of NO-GC in both SMC and ICC modulates slow phasic contractions and large contractions.

There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 μm) and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA; 100 μm) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP(-/-) mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 μm), absent in the VIP(-/-) mouse and mimicked by exogenous VIP (1-100 nm). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP(-/-) mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged period of anal relaxation.

Inflammation is the response of vascularized tissues to injury, irritation and infection. Nearly always, the inflammatory response is successfully resolved and, when necessary, a process of wound healing is initiated. Nowhere in the body is this homeostatic process more challenging than in the gastrointestinal (GI) tract, where the microbial flora sits in very close proximity to the mucosal immune system, separated only by an epithelial cell barrier. Delicate regulatory systems of the mucosal immune system determine mucosal permeability and response to bacterial flora, and aberrations in this system result in acute or chronic inflammatory conditions. Examples of such are two commonly occurring inflammatory GI disorders: inflammatory bowel disease and postoperative ileus. Inflammatory bowel disease is the result of a chronic and excessive mucosal immune response, whereas postoperative ileus represents a transient condition of GI tract paralysis that is the result of an inflammatory response to abdominal surgery. The clinical management of both conditions is very challenging and depends heavily on the possibility of modulating the host immune response. In this brief report, we highlight the role of neuropeptides in GI physiology and immune regulation, discuss a recently discovered endogenous anti-inflammatory pathway mediated by the ChemR23 receptor and speculate on the therapeutic potential of peptides that bind G-protein-coupled receptors in the management of inflammation in the GI tract.

In numerous mammals, nitric oxide (NO) influences the activity of the exocrine and endocrine pancreas. In this study, immunocytochemistry was utilized to investigate the expression of neuronal nitric oxide synthase (nNOS) in the pancreas of sheep. In double immunocytochemical staining, the co-localization of nNOS with vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or substance P (SP) was studied. The presence of nNOS was confined to the intrapancreatic neurones (9.6 +/- 1.3%) as well as to nerve fibres of the endocrine pancreas and intrapancreatic ganglia. nNOS-immunoreactive (IR) neurones were round and oval in shape and predominantly (83.3 +/- 2.6%) belonged to the middle-size group (25-50 mum). Numerous, fine islets supplying nNOS-IR nerve terminals were devoid of VIP, SP or NPY. Moderately numerous, non-varicose nNOS-IR nerve fibres of intrapancreatic ganglia frequently expressed VIP or NPY, but not SP; 2.2 +/- 0.6% of nNOS-IR intrapancreatic neurones displayed lack of VIP, whereas 7.5 +/- 0.8% were VIP-IR. All nNOS-IR neurones were devoid of SP. The frequencies of nNOS-IR/NPY-IR and nNOS-IR/NPY-negative intrapancreatic neurones were 2.2 +/- 0.4% and 6.1 +/- 1.1%, respectively. Comparison with other mammals indicated that nitrergic innervation of the ovine pancreas is species-determined and may be a reflection of the ruminants' digestion specificity. The possible origin of nNOS-IR nerve fibres and functional significance of NO in the pancreas of sheep were discussed.

Microscopic observation of intramural nerves in the frog esophagus, fixed and stained with OsO(4) and ZnI(2), revealed that nerve cell bodies and bundles connecting the nerve cell bodies formed loose and irregular networks. The nerve cell bodies were mostly lying singly in the nerve bundles, with occasional observations of two closely linked nerve cell bodies. Isolated circular and longitudinal segments of esophageal muscle were spontaneously rhythmically contractile, with a frequency of 2.2-3.0 per min. This was not altered by tetrodotoxin (TTX). In longitudinal muscle segments, transmurally applied electrical stimulation produced contractile responses which were not inhibited by atropine or guanethidine, but were reduced in amplitude by TTX, suggesting a nonadrenergic-noncholinergic (NANC) excitatory innervation in the esophagus muscle. In circular muscle segments, transmural application of brief electrical stimulation evoked two types of mechanical response: a biphasic response consisting of an initial relaxation and a following contraction (type I) and a contraction alone (type II). These mechanical responses were not modulated by either atropine or guanethidine. In the type I response, TTX abolished the relaxation component, suggesting that this was produced by non-adrenergic non-cholinergic (NANC) inhibitory nerve excitation. In about half of the type II responses, the amplitude of the contraction was significantly reduced by TTX, suggesting that a part of the contraction was produced by activation of NANC excitatory nerves. Thus, the esophageal smooth muscle of the frog demonstrates myogenic activity, and is innervated by both excitatory and inhibitory NANC nerves.

Vasoactive intestinal peptide (VIP) relaxes smooth muscle by interacting with receptors coupled to cAMP- or cGMP-signalling pathways. Their relative contribution to human gastric relaxation is unknown. This study aimed at investigating, in terms of biological activity, receptor expression and related signalling pathways, the action of VIP separately on the human fundus and the antrum. VIP caused greater relaxation of smooth muscle cells (SMC) and strips of the antrum presenting on the former a higher efficacy and potency (ED(50): 0.53 +/- 0.17 nmol L(-1)) than on the fundus (ED(50): 3.4 +/- 1.4 nmol L(-1)). On both fundus and antrum strips, its effect was tetrodotoxin insentitive. Reverse transcriptase-polymerase chain reaction analysis showed the sole expression of VPAC2 and natriuretic peptide clearance receptors, with VPAC2 being more abundant in the antrum. Functional regional differences in receptor-related signalling pathways were found. Activation of the cAMP-pathway by forskolin or its inhibition by adenylate cyclase (2'5'-dideoxyadenosine) or kinase (Rp-cAMPs) inhibitors had more pronounced effects on antrum SMC. Activation of the cGMP-pathway by sodium nitroprusside or its inhibition by guanylate cyclase (LY83583) or kinase (KT5823) inhibitors had more effects on fundus SMC, on which a higher expression of endothelial nitric oxide synthase was found. In conclusion, regional differences in VIP action on human stomach are related to distinct myogenic properties of SMC of the antrum and the fundus.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal motility. These peptides cause a variety of postjunctional responses including membrane hyperpolarization and inhibition of contraction. Neuropeptides released from enteric motor neurons can elicit responses by direct stimulation of smooth muscle cells as opposed to other transmitters that rely on synapses between motor nerve terminals and interstitial cells of Cajal. Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP(1-38) with confocal microscopy and patch-clamp technique. Localized Ca2+ transients (Ca2+ puffs) were observed in colonic myocytes, and these events coupled to spontaneous transient outward currents (STOCs). VIP and PACAP increased Ca2+ transients and STOC frequency and amplitude. Application of dibutyryl cAMP had similar effects. The adenylyl cyclase blocker MDL-12,330A alone did not affect spontaneous Ca2+ puffs and STOCs but prevented responses to VIP. Disruption of A-kinase-anchoring protein (AKAP) associations by application of AKAP St-Ht31 inhibitory peptide had effects similar to those of MDL-12,330A. Inhibition of ryanodine receptor channels did not block spontaneous Ca2+ puffs and STOCs but prevented the effects of dibutyryl cAMP. These findings suggest that regulation of Ca2+ transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of Ca2+ transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels.

Mediators of neurogenic responses of the gastric antrum were studied in wild-type and pituitary adenylate cyclase-activating polypeptide (PACAP) -knockout (KO) mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild-type mouse antrum induced a triphasic response; rapid transient relaxation and contraction, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by L-nitroarginine and atropine, respectively. The sustained relaxation was significantly inhibited by a PACAP receptor antagonist, PACAP(6-38). The antral strips prepared from PACAP-KO mice unexpectedly exhibited a tri-phasic response. However, the sustained relaxation was decreased to about one-half of that observed in wild-type mice. PACAP(6-38) inhibited EFS-induced sustained relaxation (33.5% of control) in PACAP-KO mice. Anti-peptide histidine isoleucine (PHI) serum partially (the 30% inhibition) or significantly (the 60% inhibition) inhibited the sustained relaxations in the wild-type and PACAP-KO mice, respectively. The immunoreactivities to the anti-PACAP and anti-PHI serums were found in myenteric ganglia of the mouse antrum. These results suggest that nitric oxide and acetylcholine mediate the transient relaxation and contraction, respectively, and that PACAP and PHI separately mediate the sustained relaxation in the antrum of the mouse stomach.

Gastrointestinal (GI) smooth muscle responses to stimulation of the nonadrenergic noncholinergic inhibitory nerves have been suggested to be mediated by polypeptides, ATP, or another unidentified neurotransmitter. The discovery of nitric-oxide (NO) synthase inhibitors greatly contributed to our understanding of mechanisms involved in these responses, leading to the novel hypothesis that NO, an inorganic, gaseous molecule, acts as an inhibitory neurotransmitter. The nerves whose transmitter function depends on the NO release are called "nitrergic", and such nerves are recognized to play major roles in the control of smooth muscle tone and motility and of fluid secretion in the GI tract. Endothelium-derived relaxing factor, discovered by Furchgott and Zawadzki, has been identified to be NO that is biosynthesized from l-arginine by the constitutive NO synthase in endothelial cells and neurons. NO as a mediator or transmitter activates soluble guanylyl cyclase and produces cyclic GMP in smooth muscle cells, resulting in relaxation of the vasculature. On the other hand, NO-induced GI smooth muscle relaxation is mediated, not only by cyclic GMP directly or indirectly via hyperpolarization, but also by cyclic GMP-independent mechanisms. Numerous cotransmitters and cross talk of autonomic efferent nerves make the neural control of GI functions complicated. However, the findingsrelated to the nitrergic innervation may provide us a new way of understanding GI tract physiology and pathophysiology and might result in the development of new therapies of GI diseases. This review article covers the discovery of nitrergic nerves, their functional roles, and pathological implications in the GI tract.

We have examined the mechanisms of cAMP-induced gallbladder relaxation by recording isometric tension and membrane potential in the intact tissue, and global intracellular calcium concentrations ([Ca(2+)](i)) and F-actin content in isolated myocytes. Both the phosphodiesterase (PDE) inhibitor, IBMX (100 microM) and the adenylate cyclase activator, forskolin (2 microM) caused decreases in basal tone that exhibited similar kinetics. IBMX and forskolin both caused concentration dependent, right-downward shifts in the concentration-response curves of KCl and cholecystokinin (CCK). IBMX and forskolin elicited a membrane hyperpolarization that was almost completely inhibited by the ATP-sensitive K(+) channel (K(ATP)) channel blocker, glibenclamide (10 microM). IBMX also induced an increase in large-conductance Ca(2+)-dependent K(+) (BK) channel currents, although the simultaneous blockade of BK and K(ATP) channels did not block IBMX- and forskolin-induced relaxations. Ca(2+) influx activated by L-type Ca(2+) channel activation or store depletion was also impaired by IBMX and forskolin, indicating a general impairment in Ca(2+) entry mechanisms. IBMX also decreases [Ca(2+)](i) transients activated by CCK and 3,6-Di-O-Bt-IP(4)-PM, a membrane permeable analog of inositol triphosphate, indicating an impairment in Ca(2+) release through IP(3) receptors. Ionomycin-induced [Ca(2+)](i) transients were not altered by IBMX, but the contractile effects of the Ca(2+) ionophore were reduced in the presence of IBMX, suggesting that cAMP can decrease Ca(2+) sensitivity of the contractile apparatus. A depolymerization of the thin filament could be reason for this change, as forskolin induced a decrease in F-actin content. In conclusion, these findings suggest that multiple, redundant intracellular processes are affected by cAMP to induce gallbladder relaxation.

1. We examined the role of the NO/cyclic GMP (cyclic GMP) pathway in nitric oxide (NO)- and vasoactive intestinal peptide (VIP)-induced relaxation of feline lower oesophageal sphincter (LES). Furthermore, it was studied whether methylene blue, LY83583 and ODQ, which are soluble guanylate cyclase (sGC) inhibitors, could inhibit NO-induced relaxation. 2. The nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine (L-NNA) had no effect in sodium nitropruside (SNP)-induced relaxation, but 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1)-induced relaxation was decreased by the pretreatment of L-NNA, which showed that SIN-1, not SNP, could activate NOS to cause relaxation. Methylene blue and LY83583 did not inhibit the relaxation by SNP and SIN-1. However, the more specific sGC inhibitor ODQ blocked the relaxation induced by NO donors. 3. To identify the relationship of NOS, sGC and adenylate cyclase in VIP-induced relaxation, tissue were pretreated with L-NNA and ODQ and SQ22536. These inhibitors produced significant inhibition of this response to VIP. The adenylyl cyclase inhibitor SQ 22536 also inhibited relaxation by VIP. 4. In conclusion, our data showed that SNP- and SIN-1-induced relaxation was mediated by sGC. Of sGC inhibitors, methylene blue and LY83583 were not adequate for the examination of NO donor-induced feline LES smooth muscle relaxation. VIP also caused relaxation by the pathway involving NO and cGMP and cAMP.

1. The neurotransmitters involved in NANC relaxation and their possible interactions were investigated in mouse isolated stomach, recording the motor responses as changes of endoluminal pressure from whole organ. 2. Field stimulation produced tetrodotoxin-sensitive, frequency-dependent, biphasic responses: rapid transient relaxation followed by a delayed inhibitory component. 3. The inhibitor of the synthesis of nitric oxide (NO), l-NAME, abolished the rapid relaxation and significantly reduced the slow relaxation. Apamin, blocker of Ca2+-dependent K+ channels, or ADPbetaS, which desensitises P2y purinoceptors, reduced the slow relaxation to 2-8 Hz, without affecting that to 16-32 Hz or the fast relaxation. alpha-Chymotrypsin or vasoactive intestinal polypeptide 6-28 (VIP6-28), antagonist of VIP receptors, failed to affect the fast component or the delayed relaxation to 2-4 Hz, but antagonised the slow component to 8-32 Hz. 4. Relaxation to sodium nitroprusside was not affected by l-NAME, apamin or ADPbetaS, but was reduced by alpha-chymotrypsin or VIP6-28. Relaxation to VIP was abolished by alpha-chymotrypsin, antagonised by VIP6-28, but was not affected by l-NAME, apamin or ADPbetaS. Relaxation to ATP was abolished by apamin, antagonised by ADPbetaS, but was not affected by l-NAME or alpha-chymotrypsin. 5. The present results suggest that NO is responsible for the rapid relaxation and partly for the slow relaxation. ATP is involved in the slow relaxation evoked by low frequencies of stimulation. VIP is responsible for the slow relaxation evoked by high frequencies of stimulation. The different neurotransmitters appear to work in parallel, although NO could serve also as a neuromodulator that facilitates release of VIP.

The inhibitory neurotransmission of the stomach was investigated in isolated guinea-pig gastric fundus. In preparations treated with guanethidine (1 micro mol L-1) and p-fluoro-hexahydro-sila-difenidol (1 micro mol L-1), electrical stimulation evoked neurogenic inhibitory responses not modified by hexamethonium (100 micro mol L-1), suggesting that inhibitory postganglionic non-adrenergic non-cholinergic (NANC) nerve fibres are involved. The nitric oxide (NO)-synthase inhibitor Nomega-nitro-l-argininine-methyl-ester hydrochloride (1-100 micro mol L-1) and the soluble guanylyl cyclase inhibitor ODQ (0.1-3 micro mol L-1) also abolished such relaxant response, suggesting the involvement of NO/Cyclic Guanosine 3',5' monophosphate (cGMP) system as the final mechanism of muscle relaxation. The alpha2-adrenoceptor agonist, UK 14 304 (10 nmol L-1-10 micro mol L-1) did not influence the electrical field stimulation (EFS)-evoked NANC responses. These latter responses were also refractory to a variety of receptor agonists and antagonists, acting at Gamma Aminobutyric Acid (GABA), serotonin 5HT1a, opioid micro , delta and kappa, muscarinic M1 and M2, histamine H2 and H3 and cannabinoid receptors. The NANC response was insensitive to the P/Q-type Ca2+-channel blocker omega-agatoxin TK (1 nmol L-1-0.1 micro mol L-1), but partially inhibited by the N-type Ca2+-channel blocker omega-conotoxin GVIA (0.1 nmol L-1-0.1 micro mol L-1), and by the L-type Ca2+-channel blockers nifedipine and calcicludine (0.1 nmol L-1-0.1 micro mol L-1). These data suggest that the NANC relaxation of the isolated guinea-pig gastric fundus is mediated by NO as the final inhibitory (neuro)transmitter at the longitudinal smooth muscle cells. The mechanism(s) promoting NO production is/are Ca2+-dependent, but apparently insensitive to presynaptic modulation. Both N- and L-type channels seem to occur in nitrergic nerve endings, where they contribute to trigger NO diffusion at the synaptic cleft.

Neurogenic ATP and nitric oxide (NO) may play important roles in the physiological control of gastrointestinal motility. However, the interplay between purinergic and nitrergic neurons in mediating the inhibitory neurotransmission remains uncertain. This study investigated whether neurogenic NO modulates the purinergic transmission to circular smooth muscles of the hamster proximal colon. Electrical activity was recorded from circular muscle cells of the hamster proximal colon by using the microelectrode technique. Intramural nerve stimulation with a single pulse evoked a fast purinergic inhibitory junction potential (IJP) followed by a slow nitrergic IJP. The purinergic component of the second IJP evoked by paired stimulus pulses at pulse intervals between 1 and 3 s became smaller than that of the first IJP. This purinergic IJP depression could be observed at pulse intervals <3 s, but not at longer ones, and failed to occur in the presence of NO synthase inhibitor. Exogenous NO (0.3-1 microM), at which no hyperpolarization is produced, inhibited purinergic IJPs, without altering the nitrergic IJP and exogenously applied ATP-induced hyperpolarization. In the presence of both purinoceptor antagonist and nitric oxide synthase (NOS) inhibitor, intramural nerve stimulation with 5 pulses at 20 Hz evoked vasoactive intestinal peptide (VIP)-associated IJPs, suggesting that VIP component may be masked in the IJPs of the hamster proximal colon. Our results suggest that neurogenic NO may modulate the purinergic transmission to circular smooth muscles of the hamster proximal colon via a prejunctional mechanism. In addition, VIP may be involved in the neurotransmitter in the hamster proximal colon.

Gastric electrical stimulation (GES) improves symptoms in patients with gastroparesis. However, the underlying mechanisms remain unclear. To determine if GES at proximal and distal stomach could affect the biomechanical properties of the stomach, thus contributing to the beneficial effect of GES. Four pairs of electrodes were implanted along the greater curvature of the stomach in seven dogs. Gastric tone and compliance was assessed with a barostat. Measurements were obtained randomly during control and proximal and distal stimulation (4 mA, 375 ms and 6/18 cpm). Data as mean or median (25-75th percentiles). Gastric compliance was not affected by proximal and distal GES. Gastric tone was significantly reduced during proximal GES: 82.0 (66.8, 89.1) mL vs control 49.7 (39.6,75.9) mL at 6 cpm (P = 0.016), and 90.6 (54.5, 117.9) mL vs control 62.8 (39.6, 75.9) mL at 18 cpm (P = 0.031). Tone was not affected by distal GES at 6 cpm: 95.8 (46.3, 106.7) mL vs control 75.2 (49.7, 86.1) mL (P = 0.47) and at 18 cpm: 80.4 (38.1, 170.3) mL vs control 62.8 (44.6, 156.3) mL (P = 0.44). Proximal GES induces gastric relaxation. This effect, if seen also in humans, may explain, in part, the symptomatic improvement associated with GES therapy in patients with gastroparesis.

This study examined whether alterations of the spontaneous and evoked mechanical activity are present in the stomach of the mdx mouse, the animal model for Duchenne muscular dystrophy. The gastric mechanical activity from whole-organ of normal and mdx mice was recorded in vitro as changes of intraluminal pressure. All gastric preparations developed spontaneous tone and phasic contractions, although the tone of the mdx preparations was significantly greater. Atropine reduced the tone of the two preparations by the same degree. Nomega-nitro-l-arginine methyl ester (l-NAME) significantly increased the tone and spontaneous contractions only in the stomach from normal animals, but did not affect on the mdx preparations. Effects ofl-NAME on tone and contractility were preserved in the presence of tetrodotoxin. In both types of tissues electrical field stimulation (EFS) induced a biphasic response: cholinergic contraction followed by slow relaxation. In nonadrenergic noncholinergic conditions, EFS induced a rapid relaxation followed by a slow component in both types of tissues. l-NAME abolished the rapid component, reduced the slow component and unmasked tachychinergic contractions. No significant difference was found in evoked responses. The enteric neurotransmission is preserved in mdx gastric preparations, although alterations in the ongoing production of nitric oxide are present.

Regulation of vasoactive intestinal peptide (VIP) release by nitric oxide (NO) was investigated in the hamster jejunum. Electrical field stimulation and applied NO (3-100 microM) evoked biphasic hyperpolarizations consisting of an initial transient hyperpolarizing component followed by a second more slowly developing component (late component). The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (200 microM) abolished the biphasic inhibitory junction potential evoked by electrical field stimulation. The NO scavenger oxyhemoglobin (50 microM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 microM) abolished both components of the inhibitory junction potentials and the NO-induced hyperpolarizations. VIP(6-28) (1 microM), which abolished VIP (3 microM)-induced hyperpolarizations, also inhibited the late components of the inhibitory junction potentials and the NO-induced hyperpolarizations. ODQ inhibited VIP release and cAMP production by electrical field stimulation and NO application. N(6)-2,0-Dibutyryladenosine 3',5'-cyclic monophosphate (0.1-3 mM) caused a membrane hyperpolarization. These results suggest that NO may stimulate VIP release from enteric nerves in the hamster jejunum. In addition, we propose that NO and NO-stimulated VIP contribute to the early and late components of the inhibitory junction potentials, respectively, in the circular smooth muscle cells of the hamster jejunum.

In many gastrointestinal tissues nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) both play a role as inhibitory non-adrenergic non-cholinergic neurotransmitters. As the mode of interaction between NO and VIP remains controversial, the aim of this study was to investigate the interplay between NO and VIP in the mouse gastric fundus and to evaluate the nitric oxide synthase (NOS) isoform involved in VIP-induced relaxation by using inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) knockout mice. The influence of NOS inhibitors on the relaxant effect of VIP was determined in isolated smooth muscle cells and smooth muscle strips of wild-type and knockout mice. In isolated smooth muscle cells from wild-type, eNOS knockout and nNOS knockout mice, the relaxation induced by VIP (10(-9) M) was inhibited by approximately 70-95 % by both the non-selective NOS inhibitor N(G)-nitro-L-arginine (L-NA; 10(-4) M) and the selective inducible NOS inhibitor N-(3-(aminomethyl)-benzyl)acetamidine (1400W; 10(-6) M). In cells isolated from iNOS knockout mice, VIP still induced full relaxation but it was not influenced by L-NA or 1400W. In smooth muscle strips from wild-type and knockout mice, the concentration-dependent relaxation by VIP (10(-9) to 3 x 10(-7) M) was not influenced by L-NA or 1400W. These results suggest that the experimental method determines the influence of NOS inhibitors on the relaxant effect of VIP. iNOS, probably induced by the isolation procedure, might be involved in the relaxant effect of VIP in isolated smooth muscle cells but not in classic smooth muscle strips.

1. Our previous results showed that the non-selective nitric oxide synthase (NOS) inhibitor L-N(G)-nitroarginine (L-NOARG) and the selective inducible NOS (iNOS) inhibitor N-(3-(acetaminomethyl)-benzyl)acetamidine (1400W) inhibited the relaxant effect of vasoactive intestinal polypeptide (VIP) in isolated smooth muscle cells of the mouse gastric fundus, suggesting the involvement of iNOS. The identity of the NOS isoform involved in the VIP-induced relaxation in isolated smooth muscle cells of the mouse gastric fundus was now further investigated by use of antisense oligodeoxynucleotides (aODNs) to iNOS. 2. Incubation of isolated smooth muscle cells with fluorescein isothiocyanate (FITC)-labelled aODNs showed that nuclear accumulation occurs quickly and reaches saturation after 60 min. The in vivo intravenous administration of aODNs to iNOS, 24 and 12 h before murine tumour necrosis factor alpha (mTNFalpha) challenge, significantly reduced the nitrite levels induced by the mTNFalpha challenge. 3. Intravenous administration of aODNs to iNOS in mice, 24 and 12 h before isolation of the gastric smooth muscle cells, decreased the inhibitory effect of the NOS inhibitors L-NOARG and 1400W on the relaxant effect of VIP, whereas neither saline nor sODNs had any influence. 4. Preincubation of the isolated smooth muscle cells with aODNs almost abolished the inhibitory effect of L-NOARG and 1400W on the VIP-induced relaxation, whereas sODNs failed. 5. These results illustrate that the inhibitory effect of NOS inhibitors in isolated smooth muscle cells of the mouse gastric fundus is due to inactivation of iNOS. iNOS, probably induced by the isolation procedure of the smooth muscle cells, seems involved in the relaxant effect of VIP in isolated gastric smooth muscle cells.

We examined the characteristics of the non-adrenergic non-cholinergic (NANC) nerve induced relaxation and the possible interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) on the basal tone of the circular muscle of the rat gastric fundus. Electrically induced NANC relaxations were partly inhibited by N(omega)-nitro-L-arginine (100 microM), whereas sodium nitroprusside (SNP; 10 microM) and VIP (5 nM) induced relaxations were not affected. 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; 5 microM) also inhibited the responses to electrical stimuli to a similar extent as N(omega)-nitro-L-arginine but not VIP. However, AMT plus N(omega)-nitro-L-arginine did not give an additional inhibition above that of each drug alone on NANC relaxations, and dexamethasone (10 microM) had no effect on NANC nerve induced relaxations. 1H-[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM), a selective inhibitor of guanylate cyclase, abolished the responses to NANC nerve stimulation and SNP, while VIP responses were not influenced. N-ethylmaleimide (100 microM), an adenylate cyclase inhibitor, attenuated relaxations to NANC nerve stimulation, VIP and isoproterenol (1 nM), while having no effect on those to SNP, but in combination with N(omega)-nitro-L-arginine, there was no additional inhibition on the responses to nerve stimulation. Alpha-chymotrypsin (10 u ml(-1)) severely diminished VIP induced relaxations, but did not reduce electrical responses. In conclusion, these results suggest that NO is involved in the relaxations induced by short-term electrical stimulation. However, another possible unidentified transmitter that can trigger the accumulation of cyclic GMP is not entirely ruled out and there is no interaction between NO and VIP in the circular muscle strip of the rat gastric fundus, even in the basal state of the tissue.

1. The subcellular mechanisms involved in the effect of nitric oxide (NO) on the release of vasoactive intestinal polypeptide (VIP) were examined in synaptosomes isolated from rat small intestine. 2. VIP release was stimulated by the NO donor SNAP (10(-7)-10(-4) M) in an oxyhaemoglobin-sensitive manner. The presence of the guanylate cyclase inhibitor ODQ (10(-5) M), or inhibition of protein kinase G (PKG) by KT 5823 (3 x 10(-6) M) or Rp-8Br-PET-cGMPS (5 x 10(-7) M), antagonized the SNAP-induced VIP release, suggesting a regulatory role of PKG, confirming previously published data from enteric ganglia. This finding was further supported by the fact that direct PKG activation by the stable cGMP analogue 8-pCPT-cGMP stimulated VIP secretion to the same extent as SNAP. 3. Basal VIP secretion was enhanced in the presence of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase 5 (PDE 5), suggesting a functional role of PDE 5 in NO-cGMP signalling. Supportive evidence for this finding was obtained by demonstration of the presence of PDE 5 using RT-PCR. 4. Stimulation of endogenous NO production by L-arginine was also effective in releasing VIP. The effect was abolished in the presence of KT 5823, but was insensitive to oxyhaemoglobin (10(-3) M), suggesting that an interaction between NO and VIP is likely to occur within the same nerve terminal rather than between terminals. 5. NO synthesis was not affected by VIP (10(-8)-10(-5) M), suggesting that there is no feedback regulation between the NO and the VIP pathways. 6. These findings support the notion that an anatomical and functional interrelationship exists between NO and VIP in enteric nerve terminals and that complex signalling mechanisms involving PKG and PDE 5 contribute to NO-induced VIP release.

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.

VIP and NO co-localized in many of the same neurons, are co-released by some of the same physiological stimuli. In this study we seek the divergent roles in relation to tissue injury between the neurotransmitters within 24 h after burn injury. Forty-four subjects were examined. Fourteen were mechanical trauma patients with mean injury severity score (ISS) of 27, 15 burns patients with mean per cent total burn surface area (%TBSA) of 18%, and 15 healthy controls. Patients plasma were withdrawn immediately on admission (OA) and 24 h post-injury (PI). Controls fasted (>10 h) the night before morning sampling. Enzyme-linked immunosorbent assay (ELISA) technique suitable for the measurements of NO and VIP was used. For each comparison between the patients and control groups, NO(2)(-)/NO(3)(-) plasma levels were higher in burn (P<0.001) and trauma (P<0.0005) than controls. VIP was higher in trauma (P<0.05) but not in burns (P=NS). Trauma and human burn injuries are associated with increase levels of NO productions. While VIP rose in trauma, it remained unchanged in burns. The relationship between VIP and NO observed under physiological conditions in thermal and trauma injury may be of importance in wound healing.

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.

Peptides involved in the endocrine and enteric nervous systems as well as in the central nervous system exert concerted action on gastrointestinal motility. Mechanical and chemical stimuli which induce peptide release from the epithelial endocrine cells are the earliest step in the initiation of peristaltic activities. Gut peptides exert hormonal effects, but peptide-containing stimulatory (Ach/substance P/tachykinin) and inhibitory (VIP/PACAP/NO) neurons are also involved in the induction of ascending contraction and descending relaxation, respectively. The dorsal vagal complex (DVC), located in the medulla of the brainstem, constitutes the basic neural circuitry of vago-vagal reflex control of gastrointestinal motility. Several gut peptides act on the DVC to modify vagal cholinergic reflexes directly (PYY and PP) or indirectly via afferent fibers in the periphery (CCK and GLP-1). The DVC is also a primary site of action of many neuropeptides (such as TRH and NPY) in mediating gastrointestinal motor activities. The identification over the last few years of a number of neuropeptide systems has greatly changed the field of feeding and body weight regulation. By exploring the brain and gut systems that employ recently identified peptidergic molecules, it will be possible to elaborate on the central and peripheral pathways involved in the regulation of gastrointestinal motility.

The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10(-9) M) was inhibited by 94% by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA, 10(-4) M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10(-6) M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10(-6) M), the glucocorticoid dexamethasone (10(-5) M) and three protein kinase A inhibitors: (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS; 10(-6) M), ¿(8R,9S, 11S)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a, g]cycloocta[cde]-trin-den-1-one¿ (KT5720; 10(-6) M) and N-(2-(p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10(-5) M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored. By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase. Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. N(G)-nitro-L-arginine (L-NOARG: 300 microM) enhanced the basal tone, abolished relaxations to 0.3 - 3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced ( approximately 50%) those elicited by 10 - 50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies > or =1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies. Relaxations to graded low mechanical distension (< or =1 g) were insensitive to tetrodotoxin (TTX: 1 microM) and L-NOARG (300 microM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent ( approximately 25%). In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO. British Journal of Pharmacology (2000) 129, 12 - 20

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.