AB1529 Sigma-AldrichAnti-Nitric Oxide Synthase I Antibody

The enteric nervous system (ENS) provides the intrinsic neural control of the gastrointestinal tract (GIT) and regulates virtually all GI functions. Altered neuronal activity within the ENS underlies various GI disorders with stress being a key contributing factor. Thus, elucidating the expression and function of the neurotransmitter systems, which determine neuronal excitability within the ENS, such as the GABA-GABAA receptor (GABAAR) system, could reveal novel therapeutic targets for such GI disorders. Molecular and functionally diverse GABAARs modulate rapid GABAergic-mediated regulation of neuronal excitability throughout the nervous system. However, the cellular and subcellular GABAAR subunit expression patterns within neurochemically defined cellular circuits of the mouse ENS, together with the functional contribution of GABAAR subtypes to GI contractility remains to be determined. Immunohistochemical analyses revealed that immunoreactivity for the GABAAR gamma (γ) 2 and alphas (α) 1, 2, 3 subunits was located on somatodendritic surfaces of neurochemically distinct myenteric plexus neurons, while being on axonal compartments of submucosal plexus neurons. In contrast, immunoreactivity for the α4-5 subunits was only detected in myenteric plexus neurons. Furthermore, α-γ2 subunit immunoreactivity was located on non-neuronal interstitial cells of Cajal. In organ bath studies, GABAAR subtype-specific ligands had contrasting effects on the force and frequency of spontaneous colonic longitudinal smooth muscle contractions. Finally, enhancement of γ2-GABAAR function with alprazolam reversed the stress-induced increase in the force of spontaneous colonic contractions. The study demonstrates the molecular and functional diversity of the GABAAR system within the mouse colon providing a framework for developing GABAAR-based therapeutics in GI disorders.

Acute administration of the antitumoral drug cisplatin can induce nausea/emesis and diarrhea. The long-term effects of cisplatin on gastrointestinal motility, particularly after repeated administration, are not well known. Because cisplatin is highly neurotoxic, myenteric neurons can be affected. Our aim was to study the prolonged effects of repeated cisplatin administration in a rat model, focusing on gastrointestinal motor function and myenteric neurons.

BACKGROUND AND PURPOSE: Urocortins (Ucns) 1, 2 and 3 are corticotropin-releasing factor (CRF)-related neuropeptides and may be involved in neural regulation of colonic motor functions. Nevertheless, details of the neural mechanism of action for Ucns have been unclear. We have, here, tested the hypothesis that Ucns act in the enteric nervous system (ENS) to influence colonic motor behaviour.

Muscarinic acetylcholine receptors (MR) are involved in multiple intestinal reflexes. The cellular localization of subtypes of MRs within enteric circuits mediating muscle and mucosal reflexes remains to be demonstrated. This study aimed to localize the three functionally significant subtypes of MRs in human colon.

BACKGROUND: Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum.

Gastrointestinal motility is mainly controlled by the myenteric plexus. The longitudinal muscle-myenteric plexus (LMMP) preparation from the guinea-pig ileum is the best characterised adult gastrointestinal preparation; it has also been studied in old and neonatal animals, but not at weaning, when milk is substituted with the food typical of adult animals. We used LMMP preparations from weanling and adult guinea-pigs to study different functional parameters and immunohistochemically identified subpopulations of myenteric neurones, including the excitatory motor neurones to the longitudinal muscle (LM-EMN). Excitatory stimuli (low-frequency electrical stimulation, acetylcholine, substance P, and naloxone in morphine-tolerant preparations) produced similar responses in weanling and adult guinea-pigs. The endogenous cannabinoid anandamide, but not the synthetic cannabinoid agonist WIN 55,212-2 or the opioid morphine, inhibited the electrically stimulated twitches less efficaciously, and in vitro tolerance to morphine was also lower in weanling compared to adult animals. The packing densities of the calbindin-immunoreactive neurones (sensory neurones) and of neurones immunoreactive to both calretinin (CR) and neurofilament triplet protein (NFT; ascending interneurones) were slightly but significantly lower in weanling animals, whereas those of the neurones immunoreactive to CR but not NFT (LM-EMN) or immunoreactive to nitric oxide synthase (mainly inhibitory motor neurones) were comparable to the adult. Although guinea-pigs are relatively mature and can even ingest solid food at birth, their myenteric plexus is still not fully mature at the standard time of weaning. The nutritional, behavioural and environmental changes associated with weaning may be essential to attain full maturation of the myenteric plexus and gastrointestinal motility.

Neuromuscular transmission is crucial for normal gut motility but little is known about its postnatal maturation. This study investigated excitatory/inhibitory neuromuscular transmission in vitro using ileal nerve-muscle preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 months postnatal) guinea pigs. In tissues from neonates and adults, nicotine (0.3-30 micromol L(-1)) contracted longitudinal muscle preparations in a tetrodotoxin (TTX) (0.3 micromol L(-1))-sensitive manner. The muscarinic receptor antagonist, scopolamine (1 micromol L(-1)), reduced substantially nicotine-induced contractions in neonatal tissues but not adult tissues. In the presence of N(omega)-nitro-l-arginine (NLA, 100 micromol L(-1)) to block nitric oxide (NO) mediated inhibitory neuromuscular transmission, scopolamine-resistant nicotine-induced contractions were revealed in neonatal tissues. NLA enhanced the nicotine-induced contractions in neonatal but not in adult tissues. Electrical field stimulation (20 V; 0.3 ms; 5-25 Hz, scopolamine 1 micromol L(-1) present) caused NLA and TTX-sensitive longitudinal muscle relaxations. Frequency-response curves in neonatal tissues were left-shifted compared with those obtained in adult tissues. Immunohistochemical studies revealed that NO synthase (NOS)-immunoreactivity (ir) was present in nerve fibres supplying the longitudinal muscle in neonatal and adult tissues. However, quantitative studies demonstrated that fluorescence intensity of NOS-ir nerve fibres was higher in neonatal than adult tissues. Nerve fibres containing substance P were abundant in longitudinal muscle in adult but not in neonatal tissues. Inhibitory neuromuscular transmission is relatively more effective in the neonatal guinea pig small intestine. Delayed maturation of excitatory motor pathways might contribute to paediatric motility disturbances.

The canonical transient receptor potential (TRPC) family of ion channels is implicated in many neuronal processes including calcium homeostasis, membrane excitability, synaptic transmission, and axon guidance. TRPC channels are postulated to be important in the functional neurobiology of the enteric nervous system (ENS); nevertheless, details for expression in the ENS are lacking. Reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry were used to study the expression and localization of TRPC channels. We found mRNA transcripts, protein on Western blots, and immunoreactivity (IR) for TRPC1/3/4/6 expressed in the small intestinal ENS of adult guinea pigs. TRPC1/3/4/6-IR was localized to distinct subpopulations of enteric neurons and was differentially distributed between the myenteric and submucosal divisions of the ENS. TRPC1-IR was widely distributed and localized to neurons with cholinergic, calretinin, and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to both cholinergic and noncholinergic secretomotor neurons in the submucosal plexus. TRPC3-IR was found only in the submucosal plexus and was expressed exclusively by neuropeptide Y-IR neurons. TRPC4/6-IR was expressed in only a small population of myenteric neurons, but was abundantly expressed in the submucosal plexus. TRPC4/6-IR was coexpressed with both cholinergic and nitrergic neurochemical codes in the myenteric plexus. In the submucosal plexus, TRPC4/6-IR was expressed exclusively in noncholinergic secretomotor neurons. No TRPC1/3/4/6-IR was found in calbindin-IR neurons. TRPC3/4/6-IR was widely expressed along varicose nerve fibers and colocalized with synaptophysin-IR at putative neurotransmitter release sites. Our results suggest important roles for TRPC channels in ENS physiology and neuronal regulation of gut function.

Recently, an antibody against the choline transporter (CHT), an essential molecule involved in ACh uptake, was used to label cholinergic nerves in the central nervous system; however, the enteric nervous system (ENS) was not examined. The present study localised CHT immunoreactivity (CHT-IR) within the rat ileum ENS and determined whether it colocalised with immunoreactivity for markers of cholinergic, tachykinergic and nitrergic circuitry. Segments of rat ileum were fixed, prepared for sectioning or whole-mounts and incubated with anti-CHT antisera followed by a fluorescent secondary antibody. Samples were double-labelled with antibodies to nitric oxide synthase, substance P (SP), common choline acetyltransferase (cChAT) and vesicular acetylcholine transporter (VAChT). CHT-IR was present in varicosities of nerve fibres in the myenteric plexus and muscle layers of rat ileum. In the myenteric ganglia, CHT-IR was found in nerve fibres and the cytoplasm of some nerve cell bodies. In the myenteric ganglia, no CHT/cChAT-immunoreactive neurons were present. A small number of CHT/SP-immunoreactive neurons and CHT/SP-immunoreactive nerve fibres clustered around unlabelled neurons. CHT-IR colocalised with VAChT-IR in the myenteric plexus but only half of the CHT-immunoreactive myenteric nerve fibres were VAChT-immunoreactive and half of VAChT-immunoreactive fibres were CHT-immunoreactive. In the circular muscle, 75% of CHT-immunoreactive fibres were VAChT-immunoreactive. Thus, the anti-CHT antiserum labels neurons and nerve fibres in the rat ENS. It does not label cholinergic cChAT-immunoreactive neurons, although it does immunostain cholinergic VAChT-immunoreactive nerve fibres and a population of nerves that are not VAChT-immunoreactive.

The peptide CART is widely expressed in central and peripheral neurons, as well as in endocrine cells. Known peripheral sites of expression include the gastrointestinal (GI) tract, the pancreas, and the adrenal glands. In rodent pancreas CART is expressed both in islet endocrine cells and in nerve fibers, some of which innervate the islets. Recent data show that CART is a regulator of islet hormone secretion, and that CART null mutant mice have islet dysfunction. CART also effects GI motility, mainly via central routes. In addition, CART participates in the regulation of the hypothalamus-pituitary-adrenal-axis. We investigated CART expression in porcine pancreas, GI-tract, adrenal glands, and thyroid gland using immunocytochemistry.CART immunoreactive (IR) nerve cell bodies and fibers were numerous in pancreatic and enteric ganglia. The majority of these were also VIP IR. The finding of intrinsic CART containing neurons indicates that pancreatic and GI CART IR nerve fibers have an intrinsic origin. No CART IR endocrine cells were detected in the pancreas or in the GI tract. The adrenal medulla harboured numerous CART IR endocrine cells, most of which were adrenaline producing. In addition CART IR fibers were frequently seen in the adrenal cortex and capsule. The capsule also contained CART IR nerve cell bodies. The majority of the adrenal CART IR neuronal elements were also VIP IR. CART IR was also seen in a substantial proportion of the C-cells in the thyroid gland. The majority of these cells were also somatostatin IR, and/or 5-HT IR, and/or VIP IR.CART is a major neuropeptide in intrinsic neurons of the porcine GI-tract and pancreas, a major constituent of adrenaline producing adrenomedullary cells, and a novel peptide of the thyroid C-cells. CART is suggested to be a regulatory peptide in the porcine pancreas, GI-tract, adrenal gland and thyroid.