Microbiota-derived tryptophan indoles increase after gastric bypass surgery and reduce intestinal permeability in vitro and in vivo.

BACKGROUND: The diet and microbiome contribute to metabolic disease in part due to increased intestinal inflammation and permeability. Dietary tryptophan is metabolized by both mammalian and bacterial enzymes. Using in vitro, in vivo models, and clinical data, we tested whether bacterial tryptophan indole derivatives underlie the positive benefits of microbiota on inflammation that is associated with metabolic disease. METHODS: In high-fat diet (HFD)-fed mice intestinal permeability and plasma endotoxin levels were measured after indole-3-propionic acid (IPA; 20 mg kg-1 p.o. for 4 days). Tryptophan derivatives effect on permeability and gene expression were assessed in T84 intestinal cell monolayers, in the presence or absence of pro-inflammatory cytokines. Plasma tryptophan metabolites were analyzed from lean, or obese T2D subjects undergoing Roux-en-Y gastric bypass surgery (RYGB). KEY RESULTS: IPA reduced the increased intestinal permeability observed in HFD-fed mice. Of 16 metabolites tested in vitro, only IPA, and tryptamine reduced T84 cell monolayer permeability compromised by pro-inflammatory cytokines. In T84 cells, IPA reversed the IFN-γ induced increase of fructose transporter SLC2A5 (GLUT5) mRNA, but not induction of inflammatory or metabolic genes. In obese subjects, IPA levels were reduced relative to lean counterparts, and these levels were increased by 3 months after RYGB. CONCLUSIONS AND INFERENCES: The novel findings are that obese subjects have lower levels of IPA, a solely bacterially derived tryptophan derivative, and IPA improved intestinal barrier function in vitro and DIO mice. Reduced plasma IPA levels and reversal by surgery may be a consequence of intestinal indole-producing microbiota but underlying mechanisms warrant further investigation.

Contribution of FcRn binding to intestinal uptake of IgG in suckling rat pups and human FcRn-transgenic mice.

Immunoglobulin G (IgG) is transcytosed across intestinal epithelial cells of suckling mammals by the neonatal Fc receptor (FcRn); however, the contribution of FcRn vs. FcRn-independent uptake to serum IgG levels had not been determined in either rat pups or human (h)FcRn-expressing mice (Tg276 and Tg32). In isoflurane-anesthetized rodents, serum levels were determined after regional intestinal delivery of human monoclonal antibodies (hIgG) with either wild-type (WT) Fc sequences or variants engineered for different FcRn binding affinities. Detection of full-length hIgG was by immunoassay; intestinal hFcRn and hIgG localization was by immunocytochemistry. High (µg/ml) serum levels of hIgG were detected after proximal intestinal delivery (0.1-10 mg/kg) in 2-wk-old rats. Human FcRn was visualized in epithelial cells of Tg276 mice, but low serum hIgG levels (<10 ng/ml) were obtained. In rat pups, intraintestinal hIgG1 WT administration resulted in dose-related and saturable uptake, whereas uptake of a low FcRn-binding affinity variant was nonsaturable. There were no differences in hIgG levels from systemic and hepatic portal vein serum samples, and intense hIgG immunostaining was noted in villi enterocytes and within lymphatic lacteal-like vessels. This study demonstrated that FcRn-mediated uptake in rat pups accounted for ~80% of serum hIgG levels and that IgG enters the circulation via the lymph and not the hepatic portal vein. The remaining uptake though the immature intestine is nonreceptor mediated. Intestinal epithelial cell hFcRn expression occurred in Tg276 mice, but receptor-mediated transport of IgG was not observed. The suckling rat pup intestine is a mechanistic model of FcRn-IgG-mediated transcytosis.

Modulation of gastrointestinal function by MuDelta, a mixed µ opioid receptor agonist/ µ opioid receptor antagonist.

BACKGROUND & PURPOSE: Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound ('MuDelta'), could normalize GI motility without constipation. EXPERIMENTAL APPROACH: MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified. KEY RESULTS: δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model. CONCLUSIONS AND IMPLICATIONS: MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.

Prokineticin-1 evokes secretory and contractile activity in rat small intestine.

BACKGROUND: Prokineticins 1 and 2 (PROK1 and PROK2) are so named because they contract gastrointestinal smooth muscle, yet little else is known about their role in gastrointestinal function. Therefore, we used a combination of approaches to elucidate the mechanisms by which PROK1 alters ileal contractility and secretion in rats. METHODS: RT-PCR and immunofluorescence were used to determine PROK and receptor (PK-R) mRNA levels and PK-R1 localization, respectively. Upper GI transit and fluid secretion were determined in vivo. Contractility and intestinal epithelial ion transport were assessed in isolated ileal segments. KEY RESULTS: In the gastric fundus, PROK1 mRNA is highly expressed (70-fold >PROK2 mRNA) whereas the ileum has the highest mRNA expression of its receptor. PK-R1 immunoreactivity is visualized in ileal crypt cells, and in submucosal and myenteric neurons. In ileal segments, PROK1 evokes biphasic contractile responses consisting of an early, TTX-sensitive response (EC(50) = 87.8 nmol L(-1)) followed by a late, TTX-insensitive (EC(50) = 72.4 nmol L(-1)) component that is abolished in mucosa-free preparations. Oral administration of PROK1 enhances small bowel transit (111 +/- 3% of control) and fluid secretion (340 +/- 90% of control) and in muscle-stripped ileal preparations increases short-circuit current (EC(50) = 8.2 nmol L(-1)) in a TTX-insensitive manner. The PROK1-evoked Cl- secretion is reduced by piroxicam (non-selective cyclooxygenase inhibitor), and a prostaglandin EP(4) receptor antagonist (AH23848), but not a thromboxane receptor antagonist (GR32191B). CONCLUSIONS & INFERENCES: These results demonstrate that PROK1 has oral prokinetic and secretogogue activity and that it acts on the intestinal mucosa via PK-R1 and prostaglandin receptors to mediate these effects.

Stimulation of neuronal receptors, neuropeptides and cytokines during experimental oil of mustard colitis.

Oil of mustard (OM), administered intracolonically, produces severe colitis in mice that is maximized within 3 days. The purpose of this study was to characterize the cytokine response, and to establish expression patterns of enteric neuronal mediators and neuronal receptors affected during active colitis. We measured the changes in the mRNA levels for neuronal receptors and mediators by real-time PCR, and cytokine and chemokine protein levels in the affected tissue. Significant increases in neuronal receptors, such as transient receptor potential A1 (TRPA1), cannabinoid type 1 receptor, neurokinin 1 receptor (NK1R) and delta-opioid receptor; prokineticin-1 receptor; and soluble mediators, such as prodynorphin, proenkephalin1, NK1, prokineticin-1 and secretory leukocyte protease inhibitor, occurred. Significant increases in cytokines, such as interleukin (IL)-1beta, IL-6 and granulocyte macrophage colony stimulating factor (GM-CSF), and in chemokines, such as macrophage chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1alpha) and Kupffer cell derived chemokine (KC), were detected, with no changes in T-cell-derived cytokines. Furthermore, immunodeficient C57Bl/6 RAG2(-/-) mice exhibited OM colitis of equal severity as seen in wt C57Bl/6 and CD-1 mice. The results demonstrate rapidly increased levels of mRNA for neuronal receptors and soluble mediators associated with pain and inflammation, and increases in cytokines associated with macrophage and neutrophil activation and recruitment. Collectively, the data support a neurogenic component in OM colitis coupled with a myeloid cell-related, T- and B-cell-independent inflammatory component.

Vanilloid receptor 1 antagonists attenuate disease severity in dextran sulphate sodium-induced colitis in mice.

Neurogenic mechanisms have been implicated in the induction of inflammatory bowel disease (IBD). Vanilloid receptor type 1 (TRPV1) has been visualized on nerve terminals of intrinsic and extrinsic afferent neurones innervating the gastrointestinal tract and local administration of a TRPV1 antagonist, capsazepine, reduces the severity of dextran sulphate sodium (DSS)-induced colitis in rats (Gut 2003; 52: 713-9(1)). Our aim was to test whether systemically or orally administered TRPV1 antagonists attenuate experimental colitis induced by 5% DSS in Balb/c mice. Intraperitoneal capsazepine (2.5 mg kg(-1), bid), significantly reduced the overall macroscopic damage severity compared with vehicle-treated animals (80% inhibition, P < 0.05); however, there was no effect on myeloperoxidase (MPO) levels. An experimental TRPV1 antagonist given orally was tested against DSS-induced colitis, and shown to reverse the macroscopic damage score at doses of 0.5 and 5.0 mg kg(-1). Epithelial damage assessed microscopically was significantly reduced. MPO levels were attenuated by approximately 50%, and diarrhoea scores were reduced by as much as 70%. These results suggest that pharmacological modulation of TRPV1 attenuates indices of experimental colitis in mice, and that development of orally active TRPV1 antagonists might have therapeutic potential for the treatment of IBD.

Central vagal stimulation evokes gastric volume changes in mice: a novel technique using a miniaturized barostat.

We have developed a novel technique to measure gastric volume in vivo in mice; this will be invaluable for revealing gastric alterations in genetically modified mice models, thus expanding our understanding of the mechanisms underlying functional disorders. Experimental data on gastric tone currently available has focused on rats using isovolumetric techniques to measure pressure changes, whereas clinical studies use barostatic techniques to measure volume changes. For better translational approaches, we assessed the feasibility of using a miniaturized barostat to measure gastric volume changes in urethane-anaesthetized and unanaesthetized-decerebrate mice. Additionally, we assessed whether central vagal stimulation alters gastric volume in urethane-anaesthetized mice. Nitric oxide donor sodium nitroprusside (1mg kg-1 i.p.) increased gastric volume (+134 +/- 20 microL), whereas the cholinergic agonist carbachol (3 microg kg-1 i.p.) decreased gastric volume (-153 +/- 20 microL). Similar responses were obtained in urethane-anaesthetized and unanaesthetized-decerebrate animals. Microinjection of L-glutamate (25 nmol) into dorsal motor nucleus of the vagus (DMV) altered gastric volume; microinjection into rostral DMV led to gastric contraction (-83 +/- 18 microL) while stimulation of caudal DMV resulted in gastric relaxation (+95 +/- 16 microL). This reveals a functional organization of DMV in mice. This study validates barostatic techniques for application to mice. An understanding of gastric contractility and tone is clinically relevant as impaired gastric accommodation reflex may be an underlying cause of functional dyspepsia.

Cannabinoid1 receptor in the dorsal vagal complex modulates lower oesophageal sphincter relaxation in ferrets.

Delta9-tetrahydrocannabinol (delta9-THC) is an effective anti-emetic; however, other potential gastrointestinal therapeutic effects of delta9-THC are less well-known. Here, we report a role of delta9-THC in a vago-vagal reflex that can result in gastro-oesophageal reflux, that is, gastric distension-evoked lower oesophageal sphincter (LOS) relaxation. Oesophageal, LOS and gastric pressures were measured using a miniaturized, manometric assembly in decerebrate, unanaesthetized ferrets.Gastric distension (30 ml) evoked LOS relaxation (70 +/- 8% decrease from baseline). Delta9-THC administered systemically (0.2 mg kg-1, iv.) or directly to the dorsal hindbrain surface (0.002 mg),significantly attenuated the nadir of the gastric distention-evoked LOS relaxation, and time to reach maximal response. Similar increases to maximal effect were observed after treatment with the cannabinoid receptor agonist WIN 55,212-2 (0.2 mg kg-1 iv.). The effect of systemic delta9-THC on gastric distention-evoked LOS relaxation was reversed by a selective cannabinoid1 (CBI) receptor antagonist, SR141617A (1 mg kg-1 i.v.). Since this reflex is vagally mediated, we used a CB1 receptor antiserum and immunocytochemistry to determine its distribution in ferret vagal circuitry. CBI receptor staining was present in cell bodies within the area postrema, nucleus tractus solitarius (NTS) and nodose ganglion. Intense terminal-like staining was noted within the NTS and dorsal motor vagal nucleus (DMN). Neither nodose ganglionectomy nor vagotomy altered the CB1 receptor terminal-like staining in the dorsal vagal complex. Retrogradely labelled gastric- or LOS-projecting DMN neurones did not express CBI receptors within their soma. Therefore, CBI receptor staining in the NTS and DMN is not due to primary vagal afferents or preganglionic neurones. These novel findings suggest that delta9-THC can modulate reflex LOS function and that the most likely site of action is via the CBI receptor within the NTS. This effect of delta9-THC may have implications in treatment of gastro-oesophageal reflux and other upper gut disorders.

Central neurocircuitry associated with emesis.

Ingestion of toxin, traumatic events, adverse drug reactions, and motion can all result in nausea and emesis. In addition, cyclic vomiting syndrome is quite prevalent in the pediatric population. Coordination of the various autonomic changes associated with emesis occurs at the level of the medulla oblongata of the hindbrain. Chemosensitive receptors detect emetic agents in the blood and relay this information by means of neurons in the area postrema to the adjacent nucleus tractus solitarius (NTS). Abdominal vagal afferents that detect intestinal luminal contents and gastric tone also terminate in the NTS (gelatinosus, commissural, and medial subnuclei). The NTS is viscerotopically organized into subnuclei that subserve diverse functions related to swallowing (subnucleus centralis), gastric sensation (subnucleus gelatinosus), laryngeal and pharyngeal sensation (intermediate and interstitial NTS), baroreceptor function (medial NTS), and respiration (ventrolateral NTS). Neurons from the NTS project to a central pattern generator (CPG), which coordinates the sequence of behaviors during emesis, as well as directly to diverse populations of neurons in the ventral medulla and hypothalamus. Thus, it is critical to realize that there is not an isolated "vomiting center," but rather groups of loosely organized neurons throughout the medulla that may be activated in sequence by a CPG. The newer antiemetic agents appear to block receptors in the peripheral endings of vagal afferents to reduce "perception" of emetic stimuli and/or act in the dorsal vagal complex. A primary site of action of 5-HT(3)-receptor antagonists is by means of the vagal afferents. Neurokinin-1 receptor (NK(1)R) antagonists are antiemetics, because they act at a site in the dorsal vagal complex. Part of their effectiveness may be the result of inhibition of the NK(1)R on vagal motor neurons to prevent fundic relaxation, which is a prodromal event essential for emesis. Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive component of marijuana, can be therapeutically useful as an antiemetic. The site of action of Delta(9)-THC is on cannabinoid CB1 receptors in the dorsal vagal complex. However, it decreases fundic tone and antral motility. It is not easy to predict the potential antiemetic effects of drugs that alter motility. Although antiemetic drugs are available for management of acute chemotherapeutic-induced emesis, few treatments are effective for delayed emesis or cyclic vomiting syndrome.

Receptors and transmission in the brain-gut axis. II. Excitatory amino acid receptors in the brain-gut axis.

In the last decade, there has been a dramatic increase in academic and pharmaceutical interest in central integration of vago-vagal reflexes controlling the gastrointestinal tract. Associated with this, there have been substantial efforts to determine the receptor-mediated events in the dorsal vagal complex that underlie the physiological responses to distension or variations in the composition of the gut contents. Strong evidence supports the idea that glutamate is a transmitter in afferent vagal fibers conveying information from the gut to the brain, and the implications of this are discussed in this themes article. Furthermore, both ionotropic and metabotropic glutamate receptors mediate pre- and postsynaptic control of glutamate transmission related to several reflexes, including swallowing motor pattern generation, gastric accommodation, and emesis. The emphasis of this themes article is on the potential therapeutic benefits afforded by modulation of these receptors at the site of the dorsal vagal complex.

Site of action of GABA(B) receptor for vagal motor control of the lower esophageal sphincter in ferrets and rats.

BACKGROUND & AIMS: Stimulation of gamma-aminobutyric acid B metabotropic receptors (GBRs) by baclofen reduces the incidence of transient lower esophageal sphincter (LES) relaxations. The GBR effect may be a result of a central site of action in the dorsal vagal complex, where upper gastrointestinal vagal reflexes are integrated. Therefore, we first localized GBR immunostaining in the dorsal vagal complex. Next, we tested the hypothesis that baclofen modulates LES motor tone via GBR expressed by vagal efferent neurons. METHODS: An antibody against the human GBR1b isoform was characterized and used for immunocytochemistry in rats and ferrets. Functional studies involved microinjection of L-glutamate into the caudal dorsal motor nucleus of the vagus to evoke an LES relaxation in decerebrate unanesthetized ferrets. RESULTS: In both species, GBR1b was expressed in preganglionic motor neurons and, in ferrets, the receptor was highly expressed in identified LES-projecting preganglionic neurons. GBR1b immunostaining was also pronounced in the subnucleus centralis of the nucleus tractus solitarius. This distribution implicates GBR in control of the esophageal phase of swallowing at the level of the central program generator. In functional studies, centrally evoked LES relaxation (-73% +/- 8% mm Hg) was significantly attenuated after 7 micromol/kg intravenous baclofen (-37% +/- 10%; N = 5). CONCLUSIONS: These data all suggest that GBR agonists inhibit LES relaxation via a site of action associated with vagal motor outflow to the LES.

Organization and neurochemistry of vagal preganglionic neurons innervating the lower esophageal sphincter in ferrets.

The motor control of the lower esophageal sphincter (LES) is critical for normal swallowing and emesis, as well as for the prevention of gastroesophageal reflux. However, there are surprisingly few data on the central organization and neurochemistry of LES-projecting preganglionic neurons. There are no such data in ferrets, which are increasingly being used to study LES relaxation. Therefore, we determined the location of preganglionic neurons innervating the ferret LES, with special attention to their relationship with gastric fundus-projecting neurons. The neurochemistry of LES-projecting neurons was also investigated using two markers of "nontraditional" neurotransmitters in vagal preganglionic neurons, nitric oxide synthase (NOS), and dopamine (tyrosine hydroxylase: TH). Injection of cholera toxin B subunit (CTB)-horseradish peroxidase (HRP) into the muscular wall of the LES-labeled profiles throughout the rostrocaudal extent of the dorsal motor nucleus of the vagus (DMN) The relative numbers of profiles in three regions of the DMN from caudal to rostral are, 43 +/- 5, 67 +/- 11, and 113 +/- 30). A similar rostrocaudal distribution occurred after injection into the gastric fundus. When CTB conjugated with different fluorescent tags was injected into the LES and fundus both labels were noted in 56 +/- 3% of LES-labeled profiles overall. This finding suggests an extensive coinnervation of both regions by vagal motor neurons. There were significantly fewer LES-labeled profiles that innervated the antrum (16 +/- 9%). In the rostral DMN, 15 +/- 4% of LES-projecting neurons also contained NADPH-diaphorase activity; however, TH immunoreactivity was never identified in LES-projecting neurons. This finding suggests that NO, but not catecholamine (probably dopamine), is synthesized by a population of LES-projecting neurons. We conclude that there are striking similarities between LES- and fundic-projecting preganglionic neurons in terms of their organization in the DMN, presence of NOS activity and absence of TH immunoreactivity. Coinnervation of the LES and gastric fundus is logical, because the LES has similar functions to the fundus, which relaxes to accommodate food during ingestion and preceding emesis, but has quite different functions from the antrum, which provides mixing and propulsion of contents for gastric emptying. The presence of NOS in some LES-projecting neurons may contribute to LES relaxation, as it does in the case of fundic relaxation. The neurologic linkage of vagal fundic and LES relaxation may have clinical relevance, because it helps explain why motor disorders of the LES and fundus frequently occur together.

Orphanin FQ/nociceptin and [Phe(1)Psi(CH(2)-NH)Gly(2)] nociceptin(1-13)-NH(2) stimulate gastric motor function in anaesthetized rats.

Orphanin FQ/nociceptin (OFQ/N) is a preferred endogenous ligand for the orphan opioid receptor-like-1 receptor. This peptide has been reported to increase intestinal, but not gastric, motor activity. In the present study, OFQ/N (0.6-60 nmol kg(-1) i.v.) increased intragastric pressure and antral contractility and, as expected, decreased blood pressure in anaesthetized rats. The gastric motor effects of OFQ/N (6 nmol kg(-1)) were not affected by inhibition of nitric oxide synthase or opioid receptor blockade. OFQ/N (6 nmol kg(-1)) evoked gastric motor increases and hypotension were not affected by prior administration of its derivative [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) unless the pseudopepotide was administered shortly (5 min) prior to OFQ/N. This putative antagonist (6-300 nmol kg(-1)) alone increased antral motility with approximately 100 fold lower potency than OFQ/N. Neither bilateral vagotomy nor spinal cord transection altered OFQ/N-evoked increases in intragastric pressure and antral contractility. In conclusion, OFQ/N induces gastric motor excitation in addition to its known effects to increase intestinal motility. The gastric responses to OFQ/N are not dependent on 'classical' opioid receptor activation or nitric oxide, similar to the case for the intestines. The primary site of action of OFQ/N on the stomach is probably via enteric nerves, since central descending vagal or sympathetic pathways are not necessary for OFQ/N to increase gastric motility. The gastric motor effects of the derivative [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) are similar to OFQ/N, although with lower potency. The effects of the derivative as a partial agonist or antagonist in different experimental paradigms may reflect tissue OFQ/N receptor reserve.

Substance P in the dorsal motor nucleus of the vagus evokes gastric motor inhibition via neurokinin 1 receptor in rat.

Many gastrointestinal stimuli result in gastric fundic relaxation. This information is integrated at the interface of vagal afferents and efferents in the dorsal vagal complex. Substance P (SP) is present in this region, and the neurokinin(1) receptor (NK(1)R) is highly expressed in preganglionic neurons of the dorsal motor nucleus of the vagus (DMN). However, its functional effects on vagal motor output to the stomach have not been investigated. Therefore, we determined the gastric motor effects of stereotaxic microinjection of SP and selective tachykinin receptor agents into the DMN of anesthetized rats. Dose-related decreases in intragastric pressure and antral motility were obtained on the microinjection of SP (135 and 405 pmol) into the DMN, without cardiovascular changes. Similar decreases in intragastric pressure were noted after the microinjection of [Sar(9),Met(O(2))(11)]SP (NK(1)R agonist; 135 pmol) but not senktide (NK(3)R agonist; 135 pmol) or vehicle. The gastric motor inhibition evoked by SP (135 pmol) was attenuated by prior microinjection of 2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-yl)-a mine (GR203040; 1 nmol; NK(1)R antagonist). Vagotomy or hexamethonium (15 mg/kg i.v.) completely abolished the gastric relaxation evoked by SP (135 pmol) microinjected into the DMN. We conclude that SP acts on NK(1)R preganglionic cholinergic vagal neurons in the DMN, which control enteric nonadrenergic noncholinergic motor inhibition of the fundus. The potential relevance is that an antiemetic site of action of NK(1)R antagonists may be in the DMN to prevent excitation of neurons controlling fundic relaxation, which is an essential prodromal component of emesis.

Central control of lower esophageal sphincter relaxation.

The lower esophageal sphincter is innervated by both parasympathetic (vagus) and sympathetic (primarily splanchnic) nerves; however, the vagal pathways are the ones that are essential for reflex relaxation of the lower esophageal sphincter (LES), such as that which occurs during transient LES relaxations. Vagal afferent sensory endings from the distal esophagus and LES terminate in the hindbrain nucleus tractus solitarius. The preganglionic motor innervation of the LES arises from the dorsal motor nucleus of the vagus. Together these nuclei comprise the dorsal vagal complex within which there is a neural network coordinating reflex control of the sphincter. Vagal efferent preganglionic neurons to the gastrointestinal tract are organized viscerotopically in the dorsal motor nucleus of the vagus. Stimulation of the dorsal motor nucleus of the vagus caudal to the opening of the fourth ventricle results in relaxations, whereas stimulation in the rostral portion of the nucleus evokes contractions of the LES. Few details are known about the neural circuitry that links sensory information from the stomach and esophagus within the nucleus tractus solitarius to these separate populations of neurons within the dorsal motor nucleus of the vagus. The motor vagal preganglionic output is primarily cholinergic, which ultimately stimulates excitatory or inhibitory motor neurons that control the smooth muscle tone. Excitatory neurons evoke muscarinic receptor-mediated muscle contraction. Inhibitory neurons evoke nitric oxide or vasoactive intestinal polypeptide-mediated relaxation of the lower esophageal sphincter. However, other neurotransmitters are found in vagal preganglionic neurons, including norepinephrine/dopamine and nitric oxide. A subpopulation of nitric oxide synthase-containing vagal preganglionic neurons innervate the upper gastrointestinal tract and mediate relaxation. The neurotransmitters and circuitry controlling lower esophageal sphincter pressure are important to characterize, because part of the dorsal vagal complex is outside of the blood-brain barrier and is a potential target for pharmacologic intervention in the treatment of such disorders as gastroesophageal reflux disease.

Excitation of dorsal motor vagal neurons evokes non-nicotinic receptor-mediated gastric relaxation.

Vagal stimulation results in both gastric motor excitatory and non-adrenergic non-cholinergic (NANC) inhibitory responses. The NANC pathway involves preganglionic cholinergic neurons, which act through nicotinic receptors to ultimately evoke gastric smooth muscle relaxation via release of nitric oxide (NO) and other neurotransmitters. Within the dorsal motor nucleus of the vagus (DMN), some preganglionic neurons also contain NO synthase. The NO synthase-containing neurons innervate the gastric fundus where adaptive relaxation occurs. This study tests the hypothesis that chemical stimulation of vagal motor neurons in animals, in which nicotinic receptors are blocked, evokes an NO-dependent gastric relaxation. A cell body excitant, N-methyl-D-aspartate (NMDA, 0.03-3 nmol), was microinjected into the DMN in anesthetized rats while recording intragastric pressure (IgP). The first group received NMDA before and after administration of a ganglionic blocker, hexamethonium bromide (15 mg/kg, i.v.) and atropine (1.0 mg/kg). Significant dose-dependent increases in IgP and gastric motility occurred before hexamethonium after the 0.3 and 3 nmol doses of NMDA. After hexamethonium, 0.3 and 3 nmol NMDA evoked significant decreases in IgP. A second group of rats was hexamethonium-pretreated and received NMDA microinjection into the DMN before and after an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (10 mg/kg, i.v.). The NMDA-evoked decrease in IgP was completely abolished by the NO synthase inhibitor. These data support the novel idea that NO synthase-containing preganglionic neurons mediate gastric relaxation that is independent of nicotinic receptors.

Delta9-tetrahydrocannabinol inhibits gastric motility in the rat through cannabinoid CB1 receptors.

We investigated involvement of the autonomic nervous system in gastric motor and cardiovascular responses to delta9-tetrahydrocannabinol (delta9-THC) in anesthetized rats. Intravenously administered delta9-THC evoked long-lasting decreases in intragastric pressure and pyloric contractility, bradycardia, and hypotension. The changes in gastric motor function and bradycardia were abolished by vagotomy and ganglionic blockade, whereas spinal cord transection prevented the hypotensive response. Administered intravenously alone, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide, a putative cannabinoid CB1 receptor antagonist, evoked transient decrease in intragastric pressure, and hypertension that was associated with bradycardia. However, this agent completely blocked the gastric motor and cardiovascular responses to intravenous delta9-THC. Application of delta9-THC to the dorsal surface of the medulla resulted in small and short-lasting decreases in gastric motor and cardiovascular function. We conclude that the decrease in gastric motor function and bradycardia are partially due to an action of delta9-THC in the dorsal medulla and that intact vagal nerves are required. The hypotension was mediated through sympathetic pathways. Both gastric motor and cardiovascular effects of peripherally administered delta9-THC seem to be mediated through cannabinoid CB1 receptors.

Vagally-regulated gastric motor activity: evidence for kainate and NMDA receptor mediation.

Blockade of GABA(A) receptors in the dorsal vagal complex produces marked gastric motor excitation. This effect is abolished by a prior microinjection of a non-selective excitatory amino acid receptor antagonist. Here we present functional evidence for kainate and NMDA receptor-mediated gastric excitation in the dorsal vagal complex. Microinjections into the dorsal vagal complex were performed in alpha-chloralose-anesthetized rats using multi-barrelled glass micropipettes while recording intragastric pressure and motility. Kainic acid (30 and 100 pmol in 30 nl) and NMDA (100 and 300 pmol) produced dose-related increases in intragastric pressure and motility. The gastric responses to kainate (30 pmol) and NMDA were selectively abolished by prior microinjection 6,7-dinitroquinoxaline-2,3-dione (600 pmol, 60 nl) and DL-2-amino-5-phosphanopentanoic acid (2 nmol), respectively. Atropine (1 mg/kg, i.v.) pretreatment blocked kainate-, NMDA- and L-glutamate-induced gastric excitation. Thus, both kainate- and NMDA-receptors in the dorsal vagal complex can independently cause vagally-mediated gastric motor excitation.

Gastric motor and cardiovascular effects of insulin in dorsal vagal complex of the rat.

Insulin-binding sites exist in the lower brain stem of the rat, raising the possibility that the circulating hormone may have cardiovascular and gastric effects at this site. Therefore, we investigated the autonomic effects of applying rat insulin to the surface of the dorsal medulla (0.3 and 3 microU/rat) or microinjecting it into the dorsal vagal complex (DVC) (0.1-10 nU/site) in anesthetized rats. Application of rat insulin to the surface (3 microU/rat) and its microinjection into the DVC (1 and 10 nU/site) both evoked marked, albeit transient, increases in intragastric pressure, pyloric and greater curvature contractile activity, and blood pressure. Much higher doses of human (100 mU) and porcine insulin (3 mU) were needed to evoke modest changes in gastric motor and cardiovascular function when applied to the surface of the dorsal medulla. In addition, a 1,000-fold higher dose of porcine insulin (10 microU) in the DVC was not enough to mimic the autonomic effects of rat insulin microinjected into the same site. The excitatory gastric motor effects of rat insulin in the lower brain stem were abolished by vagotomy, whereas spinal cord transection blocked insulin-evoked increases in blood pressure. To test whether the gastric motor effects of rat insulin in the lower brain stem were caused by potential contamination with pancreatic polypeptide, we microinjected rat pancreatic polypeptide into the DVC at a single dose of 2 pmol. Only a modest increase in intragastric pressure in response to the hormone was observed. Thus it is likely that insulin, through its action in the lower brain stem, may be implicated in the pathogenesis of gastrointestinal and cardiovascular complications in hyperinsulinemia. In addition, species variations in the amino acid sequence of insulin may affect its biological activity in the brain of different species.