Pain is a common and debilitating symptom of inflammatory bowel disease (IBD). Based on evidence that peripheral GABAA receptor (GAR) inhibition plays an important role in establishing colonic afferent excitability and nociceptive threshold, we hypothesized that the increase in pain associated with IBD is due to, at least in part, a decrease in peripheral GAR-mediated inhibition. Acute colitis was induced with 5 days of dextran sodium sulfate (DSS, 3%) in the drinking water. Visceral sensitivity was assessed with the visceromotor response (VMR) evoked with balloon distention of the colon in control and DSS-treated mice before and after intracolonic administration of GAR agonist muscimol, the high-affinity GAR preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP), the GAR positive allosteric modulator diazepam, or the GAR antagonists gabazine and bicuculline. Low concentrations of muscimol or THIP increased the VMR in DSS-treated mice but not in control mice. However, high concentrations of muscimol decreased the VMR in both control and DSS-treated mice. Diazepam decreased the VMR in both DSS-treated and control mice. By contrast, at a concentration of gabazine that blocks only low-affinity GAR, there was no effect on the VMR in either DSS-treated or control mice, but at concentrations of the antagonist that block low-affinity and high-affinity GAR, the VMR was increased in control mice and decreased in DSS-treated mice. Furthermore, bicuculline increased the VMR in control mice but decreased it in DSS-treated mice. These data suggest that activating of low-affinity GAR or blocking high-affinity GAR may be effective therapeutic strategies for the management of pain in IBD.

While the presence of GABA receptors on primary afferents has been well described, most functional analyses have focused on the regulation of transmitter release from central terminals and/or signalling in the sensory neuron cell body. Evidence that GABA receptors are transported to peripheral terminals and that there are several sources of GABA in the colon raise the possibility that GABA signalling in the periphery may influence colonic afferent excitability. GABA_A and GABA_B are present and functional in the colon, where exogenous agonists decrease the excitability of colonic afferents and suppress visceral nociception. Endogenous GABA release within the colon is sufficient to establish the resting excitability of colonic afferents as well as the behavioural response to noxious stimulation of the colon, primarily via GABA_A receptors. Peripheral GABA receptors may serve as a viable target for the treatment of visceral pain.

It is well established that GABA receptors at the central terminals of primary afferent fibres regulate afferent input to the superficial dorsal horn. However, the extent to which peripheral GABA signalling may also regulate afferent input remains to be determined. The colon was used to explore this issue because of the numerous endogenous sources of GABA that have been described in this tissue. The influence of GABA signalling on colonic afferent excitability was assessed in an ex vivo mouse colorectum pelvic nerve preparation where test compounds were applied to the receptive field. The visceromotor response (VMR) evoked by noxious colorectal distension was used to assess the impact of GABA signalling on visceral nociception, where test compounds were applied directly to the colon. Application of either GABA_A or GABA_B receptor agonists attenuated the colonic afferent response to colon stretch. Conversely, GABA_A and GABA_B receptor antagonists increased the stretch response. However, while the noxious distension-induced VMR was attenuated in the presence of GABA_A and GABA_B receptor agonists, the VMR was only consistently increased by GABA_A receptor antagonists. These results suggest that GABA receptors are present and functional in the peripheral terminals of colonic afferents and activation of these receptors via endogenous GABA release contributes to the establishment of colonic afferent excitability and visceral nociception. These results suggest that increasing peripheral GABA receptor signalling could be used to treat visceral pain.

γ-Aminobutyric Acid (GABA) and its receptors, GABAA,B,C, are expressed in several locations along the gastrointestinal tract. Nevertheless, a role for GABA in enteric synaptic transmission remains elusive. In this study, we characterized the expression and function of GABA in the myenteric plexus of the mouse ileum. About 8% of all myenteric neurons were found to be GABA-immunoreactive (GABA+) including some Calretinin+ and some neuronal nitric oxide synthase (nNOS+) neurons. We used Wnt1-Cre;R26R-GCaMP3 mice, which express a genetically encoded fluorescent calcium indicator in all enteric neurons and glia. Exogenous GABA increased the intracellular calcium concentration, [Ca2+]i of some myenteric neurons including many that did not express GABA or nNOS (the majority), some GABA+, Calretinin+ or Neurofilament-M (NFM)+ but rarely nNOS+ neurons. GABA+ terminals contacted a significantly larger proportion of the cell body surface area of Calretinin+ neurons than of nNOS+ neurons. Numbers of neurons with GABA-induced [Ca2+]i transients were reduced by GABAA,B,C and nicotinic receptor blockade. Electrical stimulation of interganglionic fiber tracts was used to examine possible effects of endogenous GABA release. [Ca2+]i transients evoked by single pulses were unaffected by specific antagonists for each of the 3 GABA receptor subtypes. [Ca2+]i transients evoked by 20 pulse trains were significantly amplified by GABAC receptor blockade. These data suggest that GABAA and GABAB receptors are not involved in synaptic transmission, but suggest a novel role for GABAC receptors in modulating slow synaptic transmission, as indicated by changes in [Ca2+]i transients, within the ENS.

We have shown that the kinetics of conversion of intestinal crypt cell populations to a partially or wholly mutant phenotype are consistent with a model in which each crypt contains an infrequently dividing 'deep' stem cell that is the progenitor of several more frequently dividing 'proximate' stem cells. An assumption of our model is that each deep stem cell exists in a growth inhibitory niche. We have used information from the literature to develop a model for a quiescent intestinal stem cell niche. This niche is postulated to be primarily defined by an enteroendocrine cell type that maintains stem cell quiescence by secretion of growth inhibitory peptides such as somatostatin and guanylin/uroguanylin. Consistent with this model, there is evidence that the proteins postulated as defining a growth-inhibitory stem cell niche can act as intestinal tumour suppressors. Confirmation that a growth-inhibitory niche does exist would have important implications for our understanding of intestinal homeostasis and tumorigenesis.

The incretin hormone, glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells following food ingestion. Its secretion is triggered by a range of nutrients, including fats, carbohydrates and proteins. We reported previously that Na(+)-dependent glutamine uptake triggered electrical activity and GLP-1 release from the L-cell model line GLUTag. However, whereas alanine also triggered membrane depolarization and GLP-1 secretion, the response was Na+ independent. A range of alanine analogues, including d-alanine, beta-alanine, glycine and l-serine, but not d-serine, triggered similar depolarizing currents and elevation of intracellular [Ca2+], a sensitivity profile suggesting the involvement of glycine receptors. In support of this idea, glycine-induced currents and GLP-1 release were blocked by strychnine, and currents showed a 58.5 mV shift in reversal potential per 10-fold change in [Cl-], consistent with the activation of a Cl(-)-selective current. GABA, an agonist of related Cl- channels, also triggered Cl- currents and secretion, which were sensitive to picrotoxin. GABA-triggered [Ca2+]i increments were abolished by bicuculline and partially impaired by (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), suggesting the involvement of both GABA(A) and GABA(C) receptors. Expression of GABA(A), GABA(C) and glycine receptor subunits was confirmed by RT-PCR. Glycine-triggered GLP-1 secretion was impaired by bumetanide but not bendrofluazide, suggesting that a high intracellular [Cl-] maintained by Na(+)-K(+)-2Cl- cotransporters is necessary for the depolarizing response to glycine receptor ligands. Our results suggest that GABA and glycine stimulate electrical activity and GLP-1 release from GLUTag cells by ligand-gated ion channel activation, a mechanism that might be important in responses to endogenous ligands from the enteric nervous system or dietary sources.

To investigate the expression between gamma-aminobutyric acid (GABA) and glutamate decarboxylase and its relation with differentiation and maturation of jejunal epithelial cells in rat jejunum.

Immunohistochemical expression of GABA and glutamate decarboxylase (GAD, including two isoforms, GAD65 and GAD67) was investigated in rat jejunum. Meanwhile, double staining was performed with GAD65 immunohistochemistry, followed by lectin histochemistry of fluorescent wheat germ agglutinin. Furthermore, evaluation of cell kinetics in jejunum was conducted by (3)H-thymidine autoradiography and immunohistochemistry using a monoclonal antibody to proliferating cell nuclear antigen (PCNA).

The cells showing positive immunoreactivity GABA and GAD65 were mainly distributed in the villi in rat jejunum, while jejunal epithelial cells were negative for GAD67. Positive GABA or GAD65 staining was mainly located in the cytoplasm and along the brush border of epithelial cells in the middle and upper portions. In addition, a few GABA and GAD65 strongly positive cells were scattered in the upper two thirds of jejunal villi. Double staining showed that GAD65 immunoreactivity was not found in goblet cells. (3)H-thymidine-labeled nuclei were found in the lower and middle portions of jejunal crypts, which was consistent with PCNA staining. Therefore, GABA and GAD65 were expressed in a maturation or functional zone.

The characteristic expression of GABA and GAD suggests that GABA might be involved in regulation of differentiation and maturation of epithelial cells in rat jejunum.

1 We characterized the effects of intravenous GABA and preferential GABAA (muscimol), GABAB (R-baclofen and SKF-97541) and GABAC agonists (imidazole-4-acetic acid) on gastric acid secretion in urethane-anesthetized mice implanted with a gastric cannula, and determined the role of vagal cholinergic mechanisms, and gastrin and somatostatin by using peptide immunoneutralization, the SSTR2 antagonist, PRL-2903, and SSTR2 knockout mice. 2 The selective GABA(B) agonists R-baclofen (0.1-3 mg kg(-1), i.v.) and SKF-97541 (0.01-0.3 mg kg(-1), i.v.) induced a dose-related stimulation of gastric acid secretion. SKF-97541 was about 10 times more potent than R-baclofen stimulating gastric acid secretion. Neither GABA (0.1-100 mg kg(-1), i.v.) nor muscimol (0.1-3 mg kg(-1)) nor imidazole-4-acetic acid (0.1-10 mg kg(-1)) affected basal gastric acid secretion. 3 Stimulatory effects of SKF-97541 (0.1 mg kg(-1), i.v.) were blocked by the selective GABAB antagonist, 2-hydroxysaclofen, cholinergic blockade with atropine, subdiaphragmatic vagotomy or gastrin immunoneutralization. 4 Somatostatin immunoneutralization or SSTR2 blockade with PRL-2903 enhanced the secretory response to SKF-97541 (0.1 mg kg(-1), i.v.) by 78 and 105%, respectively. 5 In SSTR2 knockout mice, SKF-97541 (0.1 mg kg(-1), i.v.) increased basal gastric acid secretion by 48%. Neither GABA nor muscimol nor imidazole-4-acetic acid modified basal gastric acid secretion in SSTR2 knockout mice. 6 These results indicate that, in mice, stimulation of GABAB receptors increases gastric acid secretion through vagal- and gastrin-dependent mechanisms. Somatostatin implication might be secondary to the release of gastrin and the increase in gastric luminal acidity.

The modifying effect of dietary administration of defatted rice-germ and gamma-aminobutyric acid (GABA)-enriched defatted rice-germ on azoxymethane (AOM)-induced colon carcinogenesis was investigated in two experiments with male F344 rats. In the first experiment (the pilot study), the effects of the defatted rice-germ, the GABA-enriched defatted rice-germ and rice-germ on AOM-induced (15 mg/kg body wt once a week for 3 weeks) formation of aberrant crypt foci (ACF) were examined. The latter two preparations (2.5% in the diet) significantly inhibited ACF formation (P < 0.005). In the second experiment, a long-term study of the effects of rice-germ was done. One group was treated with AOM alone, four groups received the carcinogen and were fed the diets containing 2.5% rice-germ or 2.5% GABA-enriched defatted rice-germ for 5 (initiation phase) or 30 weeks (post-initiation phase), two groups were treated with rice-germ or GABA-enriched defatted rice-germ alone and one group was kept on the basal diet. At the termination of the study, dietary exposure to rice-germ during the initiation phase significantly reduced the incidence of colonic adenocarcinoma (71 versus 29%, P < 0.01). GABA-enriched defatted rice-germ or rice-germ during the post-initiation phase also decreased the frequency of colonic adenocarcinoma (71 versus 20%, GABA-enriched defatted rice-germ feeding, P < 0.01; 27%, rice-germ feeding, P < 0.01). These data suggest that constituents of rice-germ are possible dietary preventatives for human colon cancers.

GABAergic neurons occur in the myenteric plexus and submucosa and their innervations of the gut, where GABA stimulates motor neurons, and non-neural cells via "central type" GABA(A) receptors. These receptors occur on half of the neurons in the rat intestine. The GABA(A) receptor is a ligand-gated chloride channel constructed from different subunit families (alpha, beta, gamma, delta, epsilon). In rat these exist as subtypes, alpha1-6, beta1-3, gamma1-3 and delta, defining the clinically relevant pharmacological features of GABA(A) receptors. However, the identity, distribution, and abundance of enteric GABA(A) receptor subunits are unknown. To identify and map the regional expression of GABA(A) receptor subunit messenger RNAs in the enteric nervous system, we assayed enteric RNA from the ileum of Sprague-Dawley rats by reverse transcription-polymerase chain reaction for alpha1-6, beta 1-3, gamma1-3, and delta subunit messenger RNAs. Subunit messenger RNA localization, was probed by in situ hybridization. Reverse transcription-polymerase chain reaction analysis of RNA from myenteric and submucosal nerve layers revealed the expression alpha1, alpha3, beta2, beta3, gamma1 and gamma3 subunit messenger RNAs. Little alpha4 and alpha6 and no alpha2, beta1, gamma2 or delta subunit messenger RNA were detected. In situ hybridization revealed that transcripts for alpha1, alpha3, alpha5 and beta2 subunits occur in both myenteric and submucous ganglia. However, beta3 messenger RNA was found only in myenteric plexus. The gamma1 subunit messenger RNA was also restricted to the cells in the myenteric plexus while gamma3 was found in cells of both nerve layers. In this study of the subunit messenger RNA expression profile of GABA(A) receptors within the enteric nerve layers we show an abundant, diverse and widespread distribution that is unique in comparison to the CNS. The distinctive and heterogeneous distribution of enteric GABA(A) subunits may be important in the integration of neural control of gut function.

1. Gastrointestinal neuroendocrine (NE) cells synthesize, store and secrete gamma-aminobutyric acid (GABA). Recently, an autocrine-paracrine function of GABA has been proposed for secretion from NE cells. 2. To search for functional GABAA receptors in NE gut cells, we performed whole-cell and perforated-patch-clamp studies in the intestinal cholecystokinin (CCK)-secreting NE cell line STC-1. 3. Application of GABA evoked currents in STC-1 cells. These effects were mimicked by muscimol, an agonist of GABAA receptors, and blocked by picrotoxin or bicuculline, antagonists of GABAA receptors. The GABA- or muscimol-activated currents reversed near 0 mV, which under the recording conditions used was consistent with the activation of the GABAA receptor-Cl- channel complex. 4. In contrast to the effect on most neurons, GABA as well as muscimol led to a (reversible) depolarization of the membrane potential of STC-1 cells. Membrane depolarization in turn activated voltage-gated Ca2+ channels and increased intracellular Ca2+ concentrations in STC-1 cells. 5. In accordance with the observed membrane depolarization and activation of voltage-gated Ca2+ channels, both GABA and muscimol stimulated Ca2+-dependent CCK release. In contrast, bicuculline inhibited the GABA-induced secretion of CCK. 6. Using the reverse transcription-polymerase chain reaction (RT-PCR), mRNA of the GABAA receptor subunits alpha2, alpha3, alpha5, beta1, beta3 and delta could be detected in STC-1 cells. 7. In summary, we have shown that the CCK-secreting gut NE cell line STC-1 expresses functional GABAA receptors and that GABA stimulates CCK release. Thus, GABA is involved in the fine tuning of CCK secretion from the gut NE cell line STC-1.

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, synthesised from glutamate by glutamate decarboxylase (GAD), in the central nervous system. Two forms of GAD, designated GAD 65 and GAD 67, are encoded by distinct genes and have been demonstrated in the mammalian brain. GABA has been postulated to be synthesised in neurons of the enteric nervous system (ENS), but evidence for its role as an enteric neurotransmitter is equivocal. We therefore aimed to determine whether GAD 65 and GAD 67 messenger RNAs (mRNAs) and proteins were expressed in the ileum of mice, rats and guinea pigs. Using an RNase protection assay, both GAD 65 and GAD 67 mRNAs were detected in the rodent small intestine. Antisera specific for GAD 65 or GAD 67, used in immunoblot analyses, revealed GAD 65-like and GAD 67-like immunoreactivity in rat and guinea pig ileum. Anti-GAD 65 antisera detected a major band of 65 kDa. Anti-GAD 67 antisera detected a major band of 55 kDa, which probably represented a breakdown product, and a minor band of 67 kDa. Analysis of immunoblot extracts of rat and guinea pig ileum revealed more GAD 67-like than GAD 65-like immunoreactivity. GAD enzymatic activity was high in the rat and guinea-pig brain, and low in the whole and dissected ileum. These results demonstrate that both GAD 65 and GAD 67 genes are transcribed and translated in the ileum of three rodent species and lend indirect support to the postulate that GABA is synthesised by neurons of the ENS and intestinal endocrine cells.

GABAB receptors are a distinct subclass of receptors for the major inhibitory transmitter 4-aminobutanoic acid (GABA) that mediate depression of synaptic transmission and contribute to the inhibition controlling neuronal excitability. The development of specific agonists and antagonists for these receptors has led to a better understanding of their physiology and pharmacology, highlighting their diverse coupling to different intracellular effectors through Gi/G(o) proteins. This review emphasises our current knowledge of the neurophysiology and neurochemistry of GABAB receptors, including their heterogeneity, as well as the therapeutic potential of drugs acting at these sites.