Search

Infusions of lipids into the small intestine potently suppress ongoing feeding. Prior work has identified potential roles for gut extrinsic vagal and non-vagal sensory innervation in mediating the ability of gut lipid infusions to reduce food intake, but the local biochemical processes underlying gut lipid sensing at the level of the small intestine remain unclear. This manuscript will summarize recent progress in the identification and characterization of several candidate gut lipid sensing molecules important in the negative feedback control of ingestion, including the fatty acid translocase CD36, peroxisome proliferator-activated receptor alpha (PPAR-α), and the fatty acid ethanolamide oleoylethanolamide (OEA). In addition, this manuscript addresses a larger role for gut lipid sensing in the overall control of energy availability by modulating not only food intake but also hepatic glucose production., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Restless legs syndrome (RLS) is linked to gastrointestinal disorders. The prevalence of irritable bowel syndrome (IBS) and small intestinal bacterial overgrowth (SIBO) in RLS patients was determined., Methods: RLS subjects were recruited from unbiased ads that did not mention gastrointestinal symptoms. RLS diagnosis was confirmed by a neurologist and utilized the International RLS Study Group criteria. General population controls (GPC) were spouses of gastrointestinal clinic patients and were excluded for RLS. Completely healthy controls (CHC) were excluded for RLS and gastrointestinal symptoms. IBS was diagnosed by Rome II criteria. SIBO was diagnosed by the lactulose breath test (LBT)., Results: There were 32 RLS subjects (23F/9M; 57 yo), 25 GPC (13F/12M; 58 yo) and 30 CHC (19F/11M; 44 yo). Twenty-nine had RLS unassociated with other GI diseases, one had celiac disease, and two had gastric resections. IBS was diagnosed in 28% of RLS subjects compared to 4% GPC (p=0.0317). SIBO was diagnosed in 69% of RLS subjects compared to 28% of GPC (p=0.0033) and 10% of CHC. Using a false positive rate of 10%, 59% of positive LBT results are associated with RLS., Conclusions: IBS and SIBO are common in RLS. Three hypotheses developed are (a) RLS patients are selectively immunocompromised or genetically predisposed and thus more subject to SIBO; (b) SIBO leads to autoimmune changes, and subsequent auto-antibodies attack brain and/or peripheral nerves and (c) SIBO inflammation leads to increased hepcidin and CNS iron deficiency which, in turn, leads to RLS. These hypotheses bear further investigation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Oxidants, produced e.g. during inflammation, alter gastrointestinal functions finally leading to diarrhoea and/or tissue damage. There is only scarce information about the action of oxidants on enteric neurones, which play a central role in the regulation of many gastrointestinal processes. Therefore, the effect of an oxidant, H(2)O(2), on cultured rat myenteric neurones was studied with the whole-cell patch-clamp and imaging (fura-2) techniques. H(2)O(2) (5 mmol/l) induced an increase in the cytosolic Ca(2+) concentration. Both an intracellular release via IP(3) and ryanodine receptors as well as a Gd(3+)-sensitive Ca(2+) influx contributed to this response. Measurement of the membrane potential revealed that the neuronal membrane hyperpolarized by 11.3+/-0.8 mV in the presence of H(2)O(2). Inhibition of Ca(2+)-dependent K(+) channels prevented this hyperpolarization. Voltage-clamp experiments revealed a second action of the oxidant, i.e. a strong inhibition of the fast Na(+) current responsible for the generation of action potentials. This effect seemed to be mediated by the hydroxyl radical (*OH), as Fe(2+) (100 micromol/l), which leads to the generation of this radical from H(2)O(2) via the Fenton reaction, strongly potentiated the action of an ineffective concentration (100 micromol/l) of the oxidant. Protein phosphorylation/dephosphorylation seems to be involved in the mechanism of action of H(2)O(2), as the protein phosphatase inhibitor calyculin A (100 nmol/l) strongly reduced the inhibition of Na(+) current by H(2)O(2). This effect was mimicked by the protein phosphatase 2A specific inhibitor endothall (100 nmol/l), whereas the PP1 blocker tautomycin (3 nmol/l) was less effective. These results suggest that H(2)O(2) reduces the excitability of rat myenteric neurones by a change of basal membrane potential and an inhibition of Na(+) currents.

5-HT released by gastrointestinal mucosa and enteric interneurons has powerful effects on gut behavior. However, the targets of 5-HT-containing neurons within enteric circuits are not well characterized. We used antisera against 5-HT and selected markers of known enteric neuron types to investigate the connections made by 5-HT-containing neurons in the guinea-pig jejunum. Confocal microscopy was used to quantify the number of 5-HT-immunoreactive varicosities apposed to immunohistochemically identified cell bodies. Large numbers of varicosities were identified apposing cholinergic secretomotor neurons, immunoreactive for neuropeptide Y, in both myenteric and submucous plexuses. Subgroups of neurons identified by calretinin (ascending interneurons) and nitric oxide synthase (descending interneurons and inhibitory motor neurons) immunoreactivity were also apposed by many varicosities. Longitudinal muscle motor neurons (calretinin immunoreactive) and AH/Dogiel type II (sensory) neurons (calbindin immunoreactive) were apposed by small numbers of varicosities. Combined retrograde tracing and immunohistochemistry were used to identify excitatory circular muscle motor neurons; these were encircled by 5-HT-immunoreactive varicosities, but the appositions could not be quantified. We suggest that 5-HT-containing interneurons are involved in secretomotor pathways and pathways to subgroups of other interneurons, but not longitudinal muscle motor neurons. There also appear to be connections between 5-HT-containing interneurons and excitatory circular muscle motor neurons. Physiological evidence demonstrates a functional connection between 5-HT-containing interneurons and AH/Dogiel type II neurons, but few 5-HT-immunoreactive varicosities were observed apposing calbindin-immunoreactive cell bodies. Taken together these results suggest that neural 5-HT may have significant roles in excitatory pathways regulating both motility and secretion.

In a previous study, we found that secretagogue-stimulated electrolyte secretion was attenuated by dietary and serosal zinc in piglet small intestinal epithelium in Ussing chambers. Several studies show that the enteric nervous system (ENS) is involved in regulation of electrolyte and/or fluid transport in intestinal epithelium from many species. The aim of the present study is to examine the mechanisms behind the attenuating effect of zinc on electrolyte secretion and to study whether the ENS is involved in this effect of zinc in vitro. Twenty-four piglets (six litters of four piglets) were allocated randomly to one of two dietary treatments consisting of a basic diet supplemented with 100 mg zinc/kg (Zn(100)) or 2500 mg zinc/kg (Zn(2500)), as ZnO. All the piglets were killed at 5-6 days after weaning and in vitro experiments with small intestinal epithelium in Ussing chambers were carried out. Furthermore, zinc, copper, alkaline phosphatase (AP) and metallothionein (MT) in mucosa, liver, and plasma were measured. These measurements showed that zinc status was increased in the Zn(2500) compared to the Zn(100) fed piglets. The in vitro studies did not confirm previous findings of attenuating effects of dietary zinc and zinc in vitro on the 5-HT induced secretion. But it showed that the addition of zinc at the serosal side attenuated the forskolin (FSK) (cAMP-dependent) induced ion secretion in epithelium from piglets fed with Zn(100) diet. Blocking the ENS with lidocaine or hexamethonium apparently slightly reduced this effect of zinc in vitro, but did not remove the effect of zinc. Consequently, it is suggested that zinc attenuates the cAMP dependent ion secretion mainly due to an effect on epithelial cells rather than affecting the mucosal neuronal pathway.

We tested the hypothesis that ATP is an enteric neurotransmitter that acts at P2Y1 excitatory purinergic receptors on intestinal secretomotor neurons to evoke neurogenic mucosal secretion in the guinea pig. Ussing chamber methods for studying neurogenic intestinal secretion were used to test the hypothesis. Application of ATP evoked concentration-dependent increases in short circuit current (Isc) indicative of stimulation of electrolyte secretion. MRS2179, a selective P2Y1 purinergic receptor antagonist, suppressed the ATP-evoked responses in a concentration-dependent manner with an IC50 of 0.9+/-0.1 microM. Tetrodotoxin or a selective vasoactive intestinal peptide (VPAC1) receptor antagonist suppressed or abolished the ATP-evoked responses. A selective VPAC1 receptor antagonist also suppressed Isc responses evoked by electrical field stimulation of the secretomotor neurons. Secretory responses to ATP were not suppressed by scopolamine, piroxicam nor selective adenosine receptor antagonists. Region-specific differences in responses to ATP corresponded to regional differences in the expression of mRNA transcripts for the P2Y1 receptor. Post-receptor signal transduction for the P2Y1-evoked responses involved stimulation of phospholipase C and an IP3/Ca2+-calmodulin/protein kinase C signaling cascade. Our evidence suggests that ATP is released as a neurotransmitter to stimulate neurogenic mucosal secretion by binding to P2Y1 receptors expressed by VIP-ergic secretomotor neurons.

We investigated whether substance P modulates pacemaker currents generated in cultured interstitial cells of Cajal of murine small intestine using whole cell patch-clamp techniques at 30 degrees C. Interstitial cells of Cajal generated spontaneous inward currents (pacemaker currents) at a holding potential of -70 mV. Tetrodotoxin, nifedipine, tetraethylammonium, 4-aminopyridine, or glibenclamide did not change the frequency and amplitude of pacemaker currents. However, divalent cations (Ni2+, Mn2+, Cd2+, and Co2+), nonselective cationic channel blockers (gadolinium and flufenamic acid), and a reduction of external Na+ from normal to 1 mM inhibited pacemaker currents indicating that nonselective cation channels are involved in their generation. Substance P depolarized the membrane potential in current clamp mode and produced tonic inward pacemaker currents with reduced frequency and amplitude in voltage clamp mode. [D-Arg1, D-Trp7,9, Leu11] substance P, a tachykinin NK1 receptor antagonist, blocked these substance P-induced responses. Furthermore, [Sar9, Met(O2)11] substance P, a specific tachykinin NK1 receptor agonist, depolarized the membrane and tonic inward currents mimicked those of substance P. Substance P continued to produce tonic inward currents in external Ca2+-free solution or in the presence of chelerythrine, a protein kinase C inhibitor. However, substance P-induced tonic inward currents were blocked by thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum or by an external 1 mM Na+ solution. Our results demonstrate that substance P may modulate intestinal motility by acting on the interstitial cells of Cajal by activating nonselective cation channels via the release of intracellular Ca2+ induced by tachykinin NK1 receptor stimulation., (Copyright 2004 Elsiever B.V.)

Electrophysiologic recording methods were used to study the actions of galanin on synaptic transmission in the submucous plexus of guinea pig ileum. Exposure to galanin resulted in concentration-dependent suppression of slow noradrenergic inhibitory postsynaptic potentials and fast nicotinic excitatory postsynaptic potentials in the majority of neurons. Failure of galanin to suppress nicotinic depolarizing responses to micropressure pulses of acetylcholine and failure to suppress hyperpolarizing responses to micropressure pulses of norepinephrine suggested that galanin acted at presynaptic inhibitory receptors to suppress release of acetylcholine and norepinephrine. Galanin suppressed slow excitatory postsynaptic potentials in eight of eight neurons with AH (after-hyperpolarization) type electrical behavior and in none of 26 neurons with S (synaptic) type electrical behavior. Suppression of excitatory neurotransmission in AH neurons was always associated with membrane hyperpolarization. Excitatory responses caused by experimentally applied substance P were also inhibited by galanin. Galanin-(1-16) and galanin-like peptide mimicked the inhibitory actions of galanin on neurotransmission. The selective galanin GAL2 receptor agonist [D-Trp(2)]galanin was inactive. The chimeric peptides, galanin-(1-13)-spantide I, galantide, galanin-(1-13)-neuropeptide Y(25-36) amide, galanin-(1-13)-bradykinin-(2-9)amide and galanin-(1-13)-Pro-Pro-Ala-Leu-Ala-Leu-Ala amide all produced varying degrees of suppression of the synaptic potentials. The evidence suggests that the galanin GAL1 receptor, but not the galanin GAL2 receptor, mediated the presynaptic and postsynaptic inhibitory actions of galanin.

Conventional intracellular microelectrodes, neuronal tracer injection techniques and immunohistochemistry were used to study the actions of cysteinyl leukotrienes (CysLTs) on electrical and synaptic behavior of enteric neurons in guinea-pig stomach and small intestine. Bath application of leukotriene C(4), leukotriene D(4) or leukotriene E(4) evoked a slowly activating depolarizing response in most of the myenteric and submucous plexus neurons in the small intestine while no effect was observed in gastric neurons. The depolarization evoked by cysteinyl leukotrienes in intestinal neurons was associated with increased input resistance and enhanced excitability. Suppression of hyperpolarizing after-potentials occurred in AH type neurons. The depolarizing action of cysteinyl leukotrienes was resistant to tetrodotoxin and cyclooxygenase inhibitors. Neither the CysLT(1) receptor antagonists (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY 171883) and alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV 5901), nor the dual CysLT(1)/CysLT(2) receptor antagonist 6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BAY u9773) significantly altered the depolarizing action of the cysteinyl leukotrienes. Neurotransmission was unaffected by the cysteinyl leukotrienes. The results suggested involvement of cysteinyl leukotrienes in enteric immuno-neural communication through excitatory actions on enteric neurons. The receptor mediating these effects was distinct from currently recognized cysteinyl leukotriene receptor subtypes (CysLT(1) and CysLT(2) receptors) and may represent a new receptor subtype.

AH neurons are intrinsic sensory neurons of the intestine that exhibit two types of slow synaptic event: slow excitatory postsynaptic potentials which increase their excitability for about 2-4 min, and sustained slow postsynaptic excitation which can persist for several hours, and may be involved in long-term changes in the sensitivity of the intestine to sensory stimuli. The effects of the neurokinin-3 tachykinin receptor antagonist, SR142801, on these two types of synaptic event in AH neurons of the myenteric ganglia of guinea-pig small intestine were compared. Slow excitatory postsynaptic potentials were evoked by stimulation of synaptic inputs at 10-20 Hz for 1s, and sustained slow postsynaptic excitation was evoked by stimulation of inputs at 1Hz for 4 min. SR142801 (1microM) reduced the amplitude of the slow excitatory postsynaptic potential to 26% of control, and also reduced the increase in input resistance and the extent of anode break excitation associated with the slow excitatory postsynaptic potential. In contrast, SR142801 did not reduce the increase in excitability, the increase in input resistance or the depolarisation that occur during the sustained slow postsynaptic excitation. SR142801 did not change the resting membrane potential or the resting input resistance. We conclude that tachykinins, acting through neurokinin-3 receptors, are involved in the generation of the slow excitatory postsynaptic potential, but not in the sustained slow postsynaptic excitation, and that the release of transmitters from synaptic inputs to AH neurons is frequency coded.

The inhibition of PGE(2) synthesis resulting from sustained NSAIDs therapy has been linked to gastrointestinal irritations and ulceration. The multiple physiological effects of PGE(2) in the gut are mediated through the activation of four receptors termed EP(1-4). The aim of the study was to determine the precise distribution of the four prostaglandin E(2) receptors in the rat stomach, small intestine, and colon. We used non-radioactive in situ hybridization techniques on paraffin-embedded tissue. Mucous cells of the stomach and goblet cells of the small intestine and colon were found to express mRNA for all four EP subtypes. A positive hybridization signal for EP(1), EP(3), and EP(4) was detected in the parietal cells of the stomach whereas the chief cells expressed low levels of EP(1) and EP(3). The EP(1) and EP(3) receptor mRNA could also be detected in the muscularis mucosa, longitudinal muscle and enteric ganglias of the stomach and small intestine. However, close examination of the enteric ganglias indicated that most of the positive labeling was localized to the glial cells, although some neurons did express EP(3). In conclusion, we have detailed the distribution of prostanoid EP receptors in the gut at the cellular level, giving new insights to the role of prostaglandins in gastrointestinal functions.

Biophysical properties of A-type K(+) currents (I(A)) in myenteric neurons from guinea-pig small intestine were studied. I(A) was present in both AH- and S-type myenteric neurons. Reduction of external Ca(2+) did not affect the current. Current density was 13.5+/-10.2 pA/pF in 68 AH-type neurons and 23.4+/-8.2 pA/pF in 31 S-type neurons. S-type neurons appeared to be a homogeneous group based on density of I(A). AH-type neurons were subdivided into two groups with current densities of 9.4+/-4.3 and 25.4+/-4.3 pA/pF. All other biophysical properties of the current were not statistically different for AH- and S-type neurons. Steady-state activation and inactivation curves showed half-activation potentials at -7 mV (k=15. 0 mV) and -86 mV (k=11.5 mV). The curves overlapped at potentials near the resting potential of approximately -55 mV. Time constants for activation ranged from 3.6 to 0.52 ms at test potentials between -20 and 50 mV. Inactivation time constants fell between 41.5 and 11 ms at test potentials between -20 and 50 mV. Time constants for recovery from inactivation fit a double-exponential curve with fast and slow recovery times of 11 and 550 ms. 4-Aminopyridine suppressed I(A) when it was activated at -20 mV following a pre-pulse to -110 mV. Addition of Zn(2+) in the external solution resulted in a concentration-dependent shift of the activation and inactivation curves in the depolarized direction. Zn(2+) slowed the activation and inactivation kinetics of I(A) by factors of 3.3- and 1.2-fold over a wide range of potentials. Elevation of external H(+) suppressed the effect of Zn(2+) with a pK of 7.3-7.4. The effects of Zn(2+) were interpreted as not being due to surface charge screening, because the affinity of Zn(2+) for its binding site on the A-channel was estimated to be between 170 and 312 microM, while the background concentration of Mg(2+) was 10 mM. The enteric nervous system is perceived as an independent integrative nervous system (brain-in-the-gut) that is responsible for local organizational control of motility and secretory patterns of gut behavior. AH- and S-type neurons are synaptically interconnected to form the microcircuits of the enteric nervous system. The results suggest that I(A) is a significant determinant of neuronal excitability for both the firing of nerve impulses and the various synaptic events in the two types of neurons.

Patch-clamp recording was used to study rectifying K+ currents in myenteric neurons in short-term culture. In conditions that suppressed Ca2+ -activated K+ current, three kinds of voltage-activated K+ currents were identified by their voltage range of activation, inactivation, kinetics and pharmacology. These were A-type current, delayed outwardly rectifying current (I(K),dr) and inwardly rectifying current (I(K),ir). I(K),ir consisted of an instantaneous component followed by a time-dependent current that rapidly increased at potentials negative to -80 mV. Time-constant of activation was voltage-dependent with an e-fold decrease for a 31-mV hyperpolarization amounting to a decrease from 800 to 145 ms between -80 and -100 mV. I(K),ir did not inactivate. I(K),ir was abolished in K+ -free solution. Increases in external K+ increased I(K),ir conductance in direct relation to the square root of external K+ concentration. Activation kinetics were accelerated and the activation range shifted to more positive K+ equilibrium potentials. I(K),ir was suppressed by external Cs+ and Ba2+ in a concentration-dependent manner. Ca2+ and Mg+ were less effective than Ba2+. I(K),ir was unaffected by tetraethylammonium ions. I(K),dr was activated at membrane potentials positive to - 30 mV with an e-fold decrease in time-constant of activation from 145 to 16 ms between -20 and 30 mV. It was half-activated at 5 mV and fully activated at 50 mV. Inactivation was indiscernible during 2.5 s test pulses. I(K),dr was suppressed in a concentration-, but not voltage-dependent manner by either tetraethylammonium or 4-aminopyridine and was insensitive to Cs+. The results suggest that I(K),ir may be important in maintaining the high resting membrane potentials found in afterhyperpolarization-type enteric neurons. They also suggest importance of I(K),ir channels in augmentation of the large hyperpolarizing after-potentials in afterhyperpolarization-type neurons and the hyperpolarization associated with inhibitory postsynaptic potentials. I(K),dr in afterhyperpolarization-type enteric neurons has overall kinetics and voltage behaviour like delayed rectifier currents in other excitable cells where the currents can also be distinguished from A-type and Ca2+ -activated K+ current.

Sprague-Dawley rats (weight 130-150 g) were sensitized by an intraperitoneal injection of 1 mg chicken egg albumin with 0.25 ml Freund's adjuvant to stimulate immunoglobulin E antibody production. Leukocyte migration inhibitory factor was used as an indicator of animal sensitization. In acute electrophysiological experiments on sensitized animals, an intra-arterial or intraluminal chicken egg albumin (100 microg) challenge evoked a 10% enhancement of the activity of mesenteric nerves of the small intestine, regardless of the injection site chosen. Afferent nerve activity in control animals was not changed during the chicken egg albumin challenge. Morphometry at the light microscope level showed activation of mast cell degranulation after the antigen challenge to presensitized rats. Intraluminal injections of a stimulator of mast cell degranulation, compound 48/80 (20-30 mg), were found to increase afferent discharges in intact rats. An antagonist of H1 histamine receptors, clemastine, reduced the effect of compound 48/80. The results obtained provide direct evidence for the stimulation of sensory nerve endings by mast cell mediators released during mast cell degranulation.

Acute inflammation of the intestine is associated with motility changes. We investigated the acute effect of inflammatory mediators such as interleukin-1beta, interleukin-2 and tumor necrosis factor-alpha (TNF-alpha) on electrically stimulated ascending and descending reflex responses of the rat small intestine. Exogenous application of interleukin-1beta caused a concentration-dependent inhibition of the oral contraction (0.1 ng/ml: -22.9+/-3.8%, 10 ng/ml: -57.0+/-7.4%, P < 0.05, n=10) but had no effect on anal relaxation. The interleukin-1 receptor antagonist alone had no significant effect on the reflex response, but prevented the inhibitory effect of interleukin-1beta (10 ng/ml: -3.9+/-11.4%, n=8). Interleukin-2 and TNF-alpha had no significant effect on the oral contractile and the anal inhibitory response (n.s., n=10). Using reverse transcriptase polymerase chain reaction (RT-PCR) the presence of mRNA of the interleukin-1 receptor was demonstrated in the rat small intestine. Preincubation of the preparation with indomethacin (10(-6) M), the histamine H1 receptor antagonist, pyrilamine (10(-8) M), and the histamine H3 receptor antagonist, clobenpropit (10(-8) M), decreased the oral contraction by 60.1+/-7.7%, 42.8+/-6.9% and 44.4+/-14.2% as well as the anal relaxation. These data suggest that acute administration of interleukin-1beta inhibits the ascending and descending contractile reflex pathway and this effect seems not to be mediated by prostaglandins or histamine receptors.

The aim of this work was to study the effects of benzalkonium chloride (BAC) treatment on the small intestine and its functioning in rats surgically prepared with Thiry-Vella intestinal loop. The loops were treated with either BAC, which ablated much of the myenteric plexus and extrinsic innervation, or with physiological saline (SAL). In vivo drinking experiments were performed to examine the effect on fluid intake and behavioral indices of distending the loop with a balloon. Spontaneous motility and its changes induced by acetylcholine (ACh) and histamine (His) were studied on isolated stripes in vitro. Finally, samples from the loops were examined histologically. Though reduction of the cell number was less than expected and no differences of the thickness of the muscular layer between the two groups was observed, BAC treatment altered the pattern of spontaneous activity and also the sensitivity to ACh and His in isolated stripes. In vivo distension of the SAL-treated loops reduced fluid intake and produced signs of aversivity; these effects were absent in the BAC-treated group. Our results show that despite the differences in the degree of ablation from those obtained by others, BAC treatment can be used to study the mechanisms underlying the effects of the enteral stimuli on the behavior.

The projections, connections, morphology and electrophysiological features of the myenteric interneurons with somatostatin immunoreactivity in the guinea-pig small intestine have been established using retrograde tracing, immunohistochemistry, confocal microscopy and intracellular recording. After application of the fluorescent dye, 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), to the myenteric plexus, up to 900 nerve cell bodies were labelled in each preparation. Somatostatin-immunoreactive neurons accounted for 13% of all retrogradely labelled cells and were located up to 70 mm orally. When DiI was applied to the submucous ganglia, many myenteric neurons were labelled and 8% of all retrogradely labelled cells were somatostatin immunoreactive and were located up to 60 mm oral to the DiI application sites. These neurons had ovoid cell bodies, a single axon, several long filamentous dendrites and received close contacts from 40-200 somatostatin-immunoreactive varicosities. Intracellular recordings revealed that these cells had features of both S (i.e. with Synaptic inputs) and AH (i.e. neurons with After Hyperpolarization) cells, receiving fast excitatory synaptic inputs, having characteristic "sag" in their response to hyperpolarizing current pulses and sometimes a long afterhyperpolarization following soma action potentials. It is concluded that somatostatin-immunoreactive neurons have distinct electrophysiological features and form very long anally directed interneuronal chains that connect with both myenteric and submucous neurons.

The influence of caloric load and nutrient composition on small intestinal satiety was investigated in six dogs with chronic esophageal fistulas. Dogs received small bowel infusion of a mixed nutrient liquid meal, a fat, carbohydrate, and a protein solution over two hours. Each infusion was given over a range of caloric densities which represented between 0 to 20% of the total 24 h caloric requirement for each animal. Satiety was measured by sham feeding at the end of the infusion. Infusion of 0.25 cal/ml (equivalent to 5% of the 24 h caloric requirement) of a liquid mixed nutrient meal (Isocal) significantly suppressed sham feeding (volume sham fed: control 265 +/- 28 ml/min; Isocal 0.25 cal/ml 218 +/- 52 ml/min, p < 0.05). Oleate and dextrose polymer also significantly reduced sham feeding at a caloric concentration of 0.25 cal/ml (volume sham fed: control 265 +/- 28 ml/min; oleate 112 +/- 28 ml/min; oleate 112 +/- 9 ml/min; dextrose polymer 190 +/- 11 ml/min), whereas peptone did not significantly suppress sham feeding until a solution with 0.5 cal/ml was given. These studies demonstrate that caloric load of intestinal nutrients must reach a threshold to produce satiety in sham feeding dogs. The threshold caloric load is different for the three nutrient groups, with fats requiring the lowest caloric load to produce satiety. These provide insight into the mechanisms by which small intestinal signals might contribute to regulation of the inter-meal interval.

Effects of histamine and related agonists and antagonists on formation of cAMP were determined for enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine. Formation of cAMP was stimulated by histamine in both dose- and time-dependent manners. The stimulatory action of histamine was suppressed by the histamine H2 receptor antagonist, cimetidine. The histamine H1 receptor antagonists, tripelennamine or pyrilamine also suppressed the stimulatory action of histamine, but only at concentrations 3-4 orders higher than required for cimetidine. Formation of cAMP was stimulated dose-dependently by the histamine H2 receptor agonist, dimaprit. The histamine H1 receptor agonist, 2-methyl-histamine, also stimulated cAMP production, but required a threshold concentration 4-5 orders higher than dimaprit. We conclude that histamine acts at the histamine H2 receptor subtype to stimulate adenylate cyclase and the formation of cAMP in myenteric ganglia of the guinea-pig small bowel.

Intracellular recording of electrical and synaptic behavior of neurons in the enteric nervous system of guinea-pig small intestine was used to evaluate actions of DQ-2511 (3-[[[2-(3, 4-dimethoxyphenyl)ethyl]carbamoyl]methyl]amino-N-methylbenzamide). DQ-2511 is a new drug with gastrointestinal prokinetic action. DQ-2511 was most effective in the nanomolar range. The drug depolarized some of the neurons and this was accompanied by increased input resistance and augmented excitability. DQ-2511 in nanomolar concentrations increased the amplitude of fast excitatory postsynaptic potentials at nicotinic synapses. Slow inhibitory postsynaptic potentials, produced by release of norepinephrine from sympathetic postganglionic fibers, were suppressed by DQ-2511. This appeared to reflect presynaptic suppression of release of norepinephrine because postsynaptic responses to exogenously applied norepinephrine were unaffected. The results suggest that the prokinetic action of DQ-2511 on gastrointestinal transit might emerge from actions that augment excitatory synaptic transmission in the microcircuits of the enteric nervous system while suppressing inhibitory sympathetic neurotransmission.

Neurons within the myenteric plexus of the guinea-pig ileum were impaled using conventional intracellular electrodes. Points of stimulation within the surrounding ganglia and connectives which gave rise to fast excitatory synaptic potentials were mapped using a movable monopolar stimulating electrode. Cells were then injected with the intracellular marker, biocytin, and processed for multiple label immunohistochemistry to reveal their morphologies, chemical contents and, hence, their functional classes. Of 65 neurons belonging to the S electrophysiological class, 53 received fast excitatory synaptic inputs from stimulation at sites at least 2 mm away in a directly circumferential direction. These inputs almost certainly arise from stimulation of the circumferentially-directed axons of the Dogiel type II/AH-neurons, which are thought to be intrinsic sensory neurons. The majority of cells which projected anally and were immunoreactive for nitric oxide synthase (19/25), all neurons which ramified in the tertiary plexus and were identified as longitudinal muscle motor neurons (6/6) and all neurons identified as excitatory motor neurons innervating the circular muscle (12/12) received inputs from these circumferentially-directed pathways. However only one of six descending filamentous interneurons impaled received such inputs, suggesting they may be differentially innervated. The conduction velocities of circumferentially-directed axons giving rise to fast excitatory post synaptic potentials were estimated to be 0.41 +/- 0.10 m/s (mean +/- standard deviation, n = 21). The conduction velocities estimated for longitudinally-directed pathways were 0.55 +/- 0.25 m/s (n = 29). Thus, the majority of myenteric neurons receive fast excitatory synaptic input from putative intrinsic sensory neurons which project circumferentially around the intestine.

Localized distension of the intestine evokes an ascending excitatory reflex and a descending inhibitory reflex in the circular muscle layer. The sequential activation of these two reflexes is believed to underlie the motor pattern of peristalsis, which is responsible for the co-ordinated propulsion of intestinal contents. In this study we have shown that the initiation of peristalsis involves mechanisms additional to those mediating the ascending excitatory reflex. A short length of guinea-pig small intestine was mounted in a partitioned organ bath so that the lumen was occluded by the partition, but neuronal continuity was maintained. The anal segment was distended by intraluminal fluid infusion to evoke a peristalsis; in the oral segment, an isotonic transducer was used to record circular muscle contractions due to ascending excitatory reflexes. Stepwise distension of the anal segment with 5 microliters increments at 10 s intervals, or with a large, single-step infusion, elicited both the ascending excitatory reflex and peristalsis, when carried out at 3 min intervals. The threshold volume for the ascending excitatory reflex was smaller than the threshold for peristalsis with either incremental or single-step distensions. The ascending excitatory reflex appeared with a shorter delay than peristalsis. Tetrodotoxin (0.6 microM) or hexamethonium (100 microM) added to the oral compartment abolished the ascending excitatory reflex but not peristalsis. These drugs abolished both the ascending excitatory reflex and peristalsis when added to the anal compartment. When stimuli were delivered at 1 min intervals, peristalsis failed completely after the first trial, but the ascending excitatory reflex persisted, at a slightly reduced amplitude. When the anal segment was distended to just-subthreshold volume, electrical field stimulation (0.25-0.5 ms, 1-5 Hz for 1 s), delivered at 3 min intervals, evoked ascending excitatory responses but not peristalsis. Higher frequency stimulation (10 Hz) consistently evoked both peristalsis and the ascending excitatory responses. When trains of electrical stimulation were repeated at 1 min intervals, peristalsis quickly failed, but the ascending excitatory response persisted, although reduced in amplitude. The initiation of peristalsis can be dissociated from the ascending excitatory reflex by its threshold volume, by the duration of distension or the intensity of electrical stimulation required, and by its susceptibility to fatigue with repeated mechanical or electrical stimuli. This suggests that the ascending excitatory reflex may be part of the mechanism underlying the initiation of peristalsis, but that additional mechanisms must also be involved. Peristalsis should not be regarded as a reflex response but rather as an all-or-nothing motor pattern, triggered by mechanical stimulation, similar to other co-ordinated motor patterns in vertebrates and invertebrates.

The influence of intra-arterial injections of 5-hydroxytryptamine, histamine and prostaglandin E2 on afferent impulse activity of mesenteric nerves of small intestine was studied. In anaesthetized cats 5-hydroxytryptamine (10(-5)-10(-4) M) and histamine (10(-5)-10(-3) M) were shown to increase the impulse activity in a dose-dependent manner. Metergolin, 5-hydroxytryptamine antagonist, suppressed the effect of 5-hydroxytryptamine. Clemastine and cimetidine, antagonists of H1 and H2 histamine receptors respectively, distinctly diminished excitatory histamine effects. Prostaglandin E2 (10-30 micrograms/kg) enhanced the afferent impulse activity. The results suggest that afferents of the small intestine may be involved in reception of inflammatory and immune responses.

The mode and site of action of galanin were examined in the guinea pig small intestine. Galanin (3 x 10(-9)-10(-7) M) inhibited the twitch contractions of longitudinally and circularly oriented muscle strips mediated by the stimulation of cholinergic neurons, but not the contractions mediated by direct stimulation of smooth muscle cells with carbachol. Galanin (3 x 10(-9)-10(-7) M) inhibited both the electrically stimulated and the tetrodotoxin-resistant high K+ (40 mM)-induced increase of [3H]acetylcholine outflow from the ileal strips preloaded with [3H]choline, in a concentration dependent fashion. The inhibitory effect of galanin was antagonized by galantide and produced self-desensitization. The spontaneous and stimulated outflow of [3H]noradrenaline and [3H]gamma-aminobutyric acid were not affected by galanin even at 10(-7) M. Thus, galanin inhibits the motility of guinea pig ileum by inhibition of acetylcholine release from the enteric cholinergic neurons. Galanin may act on the specific receptor located on soma-dendritic regions and nerve terminals of cholinergic neurons.

Endogenous alkaline phosphatase activity has been localized histochemically on the surface of enteric neurons of the guinea-pig small intestine by both light and electron microscopy. The enzyme activity was associated with some myenteric neurons that had Dogiel type I morphology, and the histochemical reaction products typically formed a honeycomb-like structure on labelled cell bodies. No Dogiel type II neurons in the myenteric plexus or submucous neurons showed alkaline phosphatase reactivity. Nerve fibres reactive for alkaline phosphatase were present in the myenteric plexus and ran in bundles in the circular muscle and deep muscular plexus. In addition, reactive varicose axons supplied the submucous plexus and non-ganglionated plexus of the mucosa. The results of interruption of the enteric neuronal pathways demonstrated that alkaline phosphatase-reactive myenteric neurons project anally to other myenteric ganglia, to the circular muscle and to the submucous plexus. Sequential enzyme histochemistry showed that virtually all alkaline phosphatase-reactive neurons also contained nitric oxide synthase, revealed by NADPH-diaphorase reactivity. It was estimated that 14-18% of all myenteric neurons showed alkaline phosphatase reactivity. About one-third of nitric oxide synthase-containing myenteric neurons, however, did not contain alkaline phosphatase activity. At the ultrastructural level, alkaline phosphatase activity was associated specifically with the plasma membranes of nerve cell bodies, axons and dendrites of some myenteric neurons. Reactive nerve fibres made close appositions with non-reactive submucous neurons and, within myenteric ganglia, predominantly with other alkaline phosphatase-reactive neurons. In addition to its presence in neurons, alkaline phosphatase reactivity was also present in some endothelial cells in blood vessels in the submucosa and in capillary pericytes. It is concluded, on the basis of the projections and neurochemistry, that in the guinea-pig small intestine alkaline phosphatase activity is associated with nitric oxide synthase-containing neurons which include inhibitory motor neurons to the circular muscle, and anally-directed interneurons to other myenteric and submucous neurons.

Intracellular recordings were made from 73 guinea-pig submucosal neurons using neurobiotin-filled microelectrodes; subsequently, neuropeptide immunoreactivity, morphology and nerve fibre projections were determined. Five distinct groups of cells could be distinguished: S cells with inhibitory input (61%), S cells without inhibitory input (19%), AH cells (8%), S-AH cells (5%), and glial networks. S cells with inhibitory input were immunoreactive for vasoactive intestinal polypeptide and showed Dogiel Type III morphology with the axon branching and coursing through two to 12 ganglia; varicosities and tufts of varicosities were observed surrounding other cell bodies as well as over blood vessels. S cells without inhibitory input primarily were immunoreactive for neuropeptide Y; they also showed Dogiel Type III morphology and similar, though shorter, axonal projections and varicose features surrounding other neurons. AH cells, which most likely contained substance P, lacked synaptic input and exhibited Dogiel Type II morphology; they branched more extensively than S cells and also formed varicose tufts within other ganglia. S-AH cells combined electrophysiological properties of S cells with inhibitory input and AH cells and did not show consistent morphological or histochemical characteristics. Typical glial networks were observed; in addition, on two occasions unusual networks of dye and electrical coupling between S cells without inhibitory input and a glial complex were observed. These results suggest that vasoactive intestinal polypeptide-containing S cells may act as interneurons which mediate a slow excitatory synaptic potential; that neuropeptide Y-containing S cells, which are known to be cholinergic, may play a role as cholinergic interneurons mediating the nicotinic fast excitatory synaptic potential; and that AH neurons also may provide cholinergic innervation to other submucosal neurons in addition to their previously described dual projections into mucosa and myenteric plexus.

Peptide YY immunoreactivity was detected in neuronal elements in the upper digestive tract of the rat, cat, ferret and pig by immunocytochemistry and radioimmunoassay combined with high-performance liquid chromatography. The two peptide YY antisera used do not recognize the related peptides neuropeptide Y and pancreatic polypeptide. In the rat peptide YY-immunoreactive nerve fibres were virtually restricted to the stomach smooth muscle of the minor curvature where they were numerous. Peptide YY-immunoreactive nerve cell bodies occurred in small ganglia on the serosal surface of the minor curvature. In the cat and ferret peptide YY-immunoreactive nerve fibres occurred in the circular smooth muscle layer of both the minor and major curvatures of the stomach and also in the upper small intestine; such fibres were numerous also in myenteric ganglia in these regions. In the pig, they were few but had roughly the same distribution as in the cat and ferret, except that they were quite numerous in thick muscle bundles close to the oesophagogastric junction. The presence of peptide YY-immunoreactive nerve cell bodies within the myenteric ganglia along the upper digestive tract of cat, ferret and pig, and in serosal ganglia of the rat stomach, indicates that at least some of the peptide YY-immunoreactive fibres demonstrated originate in or close to the gut wall. Double-staining experiments revealed that virtually all peptide YY-containing neurons and nerve fibres were distinct from those storing neuropeptide Y. Peptide YY-immunoreactive endocrine cells were encountered not only in the lower intestines but also in the stomach of the four species studied. In the antrum such cells were numerous and constituted a subpopulation of the gastrin-containing cells. In the oxyntic mucosa they were few and contained somatostatin. Radioimmunoassay revealed peptide YY-like peptides in gastric mucosa and smooth muscle from the upper digestive tract of all four species examined. The results of high-performance liquid chromatography suggest that the peptide YY-like material in the upper digestive tract is distinct from neuropeptide Y and pancreatic polypeptide and identical with authentic peptide YY except in the antral mucosa where only a small proportion of the peptide YY-immunoreactive material eluted like authentic peptide YY.

CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]- 1-azabicyclo[2.2.2.]octan-3-amine], a novel nonpeptide antagonist of the substance P receptor, was evaluated for blocking action at substance P receptors on myenteric neurons of guinea-pig small intestine. Intracellular electrophysiological recording was used to determine actions of the drug on excitatory responses to substance P, on slow and fast excitatory postsynaptic potentials and action potential initiation and propagation in the neurons. CP-96,345 suppressed responses to substance P. It also suppressed spike initiation and propagation in the neuronal processes, as well as in the somal membranes. The effects of the drug on substance P responses could not be attributed to an action at substance P receptors. The mechanism of action appeared to be a nonselective local anesthetic effect on initiation and propagation of action potentials.

The electrophysiological actions of corticotropin-releasing hormone (CRH) on myenteric neurons from the guinea-pig ileum were studied by intracellular microelectrode recording. CRH, when applied by micropressure ejection or in the medium (0.2-20 nM) evoked prolonged depolarization in 21 of 42 S/type 1 neurons and in 28 of 40 AH/type 2 neurons. These responses were associated with increased input resistance and augmented excitability. The post-spike hyperpolarization in AH/type 2 cells was suppressed during the CRH-evoked responses. The reversal potential of the response to CRH was about -90 mV, consistent with the closure of potassium channels by the peptide. The CRH-induced depolarization was prevented by incubation in 10 microM 5'-N-ethylcarboxamidoadenosine (NECA, an adenosine analog) suggesting that the response was mediated by stimulation of adenylate cyclase and elevation of cAMP. CRH reduced the amplitude of fast nicotinic excitatory postsynaptic potentials. This appeared to be a postsynaptic action because the peptide also reduced the responses to exogenously applied acetylcholine. These results suggest that CRH can directly influence intestinal function by acting on myenteric neurons.

This study was designed to test the hypothesis that stimulation of adenylate cyclase and elevation of cAMP is involved in the signal transduction process for substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, cholecystokinin or gastrin releasing peptide in myenteric ganglia. Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to study changes in levels of cAMP in response to application of the brain-gut peptides in the presence and absence of forskolin. Application of substance P and calcitonin gene-related peptide were found to increase intraganglionic cAMP in a dose-dependent fashion when a phosphodiesterase inhibitor was present. The ED50 values for substance P and calcitonin gene-related peptide were 5 microM and 0.75 microM, respectively. The presence of forskolin in the incubation medium resulted in significant upward shifts of the dose-response curves for both peptides. Neither vasoactive intestinal peptide, cholecystokinin nor gastrin releasing peptide stimulated increases in intraganglionic cAMP under the same experimental conditions used for substance P and calcitonin gene-related peptide.

Incubation of isolated rat intestinal segments with hydrogen peroxide (H2O2) led to a decreased beta-adrenoceptor response. The maximal relaxation induced by isoprenaline was lowered while the EC50 remained unaffected. The effect of H2O2 in the small intestine increased slightly from duodenum to ileum. In the ileum, 10(-4) M H2O2 led to a 10% decrease of the maximal relaxation due to isoprenaline and 1 mM decreased the maximal response to about 50%. We further investigated the level at which the isoprenaline response was impaired. The relaxation caused by the stable cAMP analog, dibutyryl-cAMP, or by the adenylate cyclase activator, forskolin, was not affected or affected less than by isoprenaline. When the response to isoprenaline was expressed relative to the maximal response to dibutyryl-cAMP or forskolin, pretreatment with H2O2 led to a decreased isoprenaline response relative to the response to dibutyryl-cAMP or forskolin. This might indicate that exposure to H2O2 leads to a disturbance in receptor-mediated cAMP production. The adenylate cyclase unit is probably not affected since the response to forskolin is relatively resistant to H2O2. Our conclusion is that pretreatment of isolated intestinal segments with H2O2 leads to disturbed beta-adrenoceptor coupling, probably due to altered membrane integrity.

Isolated segments of guinea-pig small intestine were set up in a partitioned bath to study the enteric excitatory reflex evoked by distension. The gut was distended by a rubber balloon inserted at the aboral end and contractions of the circular muscle were recorded at the oral end. The oral and aboral ends of the gut were separated by an intermediate compartment of the bath. Inflation of the intraluminal balloon with 0.075-0.35 ml water elicited reproducible and distension-dependent contraction. This enteric orally directed (ascending) excitatory reflex was abolished by tetrodotoxin irrespective of the compartment in which it was applied. Hyoscine (0.3 microM) almost abolished the enteric excitatory reflex when it was applied to the oral compartment. This indicates that the transmission from the final motor neurons to the circular muscle is mainly cholinergic, acting via muscarinic receptors. Hyoscine had no effect on the enteric excitatory reflex when added to the intermediate compartment. When hyoscine was added to the aboral compartment, it decreased the enteric excitatory reflex elicited by low distension stimuli to 70% of control and decreased the enteric excitatory reflex elicited by higher distension stimuli to 95% of control. This indicates that ganglionic transmission involving muscarinic receptors at the site of distension in the aboral bath contributes to the enteric excitatory reflex. Hexamethonium (100 microm) greatly depressed the enteric excitatory reflex when applied to any compartment indicating that nicotinic transmission is most important in the afferent, intermediate and efferent components of the reflex and that the reflex pathway involves a polysynaptic chain of cholinergic interneurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical double and triple staining techniques were employed on whole mounts of the submucosal plexus from normal Wistar and non-diabetic BB rat jejunum and ileum, to determine the patterns of co-localization of vasoactive intestinal polypeptide-, peptide histidine-isoleucine-, somatostatin-, neuropeptide Y-, calcitonin gene-related peptide-, substance P-, and galanin-immunoreactive nerves. Neuropeptide Y immunoreactivity was found in 38% of submucosal plexus neurons, within the same neuronal elements as vasoactive intestinal polypeptide immunoreactivity (39% of submucosal plexus neurons) and peptide histidine-isoleucine immunoreactivity. A small population (1% of submucosal plexus neurons) containing vasoactive intestinal polypeptide- and peptide histide isoleucine-like immunoreactivity without NPY-like immunoreactivity was also observed. A significant population of fibers containing vasoactive intestinal polypeptide and galanin immunoreactivity were observed in the mucosa and submucosa, although no cell bodies were detected which contained both neuropeptides. Galanin-like immunoreactivity was seen in a small (2% of submucosal plexus neurons) population, not co-localized with any of the other neuropeptides examined. All somatostatin-immunoreactive neuronal elements (18% of submucosal plexus neurons) contained calcitonin gene-related peptide immunoreactivity, just over half of which also contained substance P immunoreactivity. An additional 25% of submucosal plexus neurons contained calcitonin gene-related peptide- without somatostatin-like immunoreactivity and 28% of submucosal plexus neurons contained substance P without somatostatin-like immunoreactivity. Some degree of co-localization was seen between calcitonin gene-related peptide- and substance P-like immunoreactivity, however, this could not be directly quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal secretion was evoked in periarterially denervated jejunal segments of anesthetized rats and cats by exposing the intestines to the heat stable (ST) toxins from a strain of Escherichia coli producing both STa and STb toxins. The secretion was significantly inhibited and to about the same relative extent by the addition of each one of the three following drugs: hexamethonium (i.v., rats), lidocaine (applied on the serosal surface, rats) and tetrodotoxin (intra-arterial, cats). Atropine inhibited fluid secretion in some experiments. It is proposed that a nervous mechanism is mediating part of the secretory response to Escherichia coli heat stable toxins, since three different drugs, which influence nervous activity in different ways, significantly diminished the secretory response. A model for the secretory nervous reflex(es) within the enteric nervous system is proposed; Escherichia coli heat stable toxins activate a "receptor cell" in the epithelium, which then stimulates surrounding dendritic nerve endings via the release of unknown substance(s). A nicotinic receptor is involved but further characteristics of the nervous reflex(es) remain to be elucidated.

The actions of the duodenal ulcerogen cysteamine in different regions of the rat stomach and small intestine were investigated utilizing isolated gut-bath preparations. Applied cysteamine caused concentration-dependent contractions of forestomach strips, primarily by direct actions at cholinergic muscarinic sites on the longitudinal muscle, and to a lesser extent indirectly via stimulation of intrinsic cholinergic motor nerves. In contrast to its gastric actions, applied cysteamine evoked nerve-mediated relaxations of segments of whole duodenum, jejunum and ileum. These actions were concentration-dependent and due to stimulation of the non-adrenergic, non-cholinergic (NANC) inhibitory nerves. In addition, cysteamine selectively inhibited nicotinic and GABA-ergic stimulation of the NANC inhibitory nerves, as well as electrically evoked cholinergic contractions. The interactions of cysteamine with the NANC inhibitory nerves appears to be via sites distinct from nicotinic or GABA receptors, and may have involved toxic effects.

We have analyzed changes in the distributions of terminals with vasoactive intestinal polypeptide (VIP)-like immunoreactivity, and accumulations in severed processes, that occur after lesions of intrinsic and extrinsic nerve pathways of the guinea-pig small intestine. The observations indicate that enteric vasoactive intestinal polypeptide immunoreactive neurons have the following projections. Nerve cell bodies in the myenteric plexus provide varicose processes to the underlying circular muscle; the majority of these pathways, if they extend at all in the anal or oral directions, do so for distances of less than 1 mm. Nerve cell bodies of the myenteric plexus also project anally to provide terminals to other myenteric ganglia. The lengths of the majority of these projections are between 2 and 10 mm, with an average length of about 6 mm. Processes of myenteric neurons also run anally in the myenteric plexus and then penetrate the circular muscle to provide varicose processes in the submucous ganglia at distances of up to 15 mm, the average length being 9-12 mm. In addition, there is an intestinofugal projection of myenteric neurons whose processes end around nerve cell bodies of the coeliac ganglia. A similar projection from the colon supplies the inferior mesenteric ganglia. The nerve cell bodies in submucous ganglia give rise to a subepithelial network of fibres in the mucosa and also supply terminals to submucous arterioles. It is concluded that vasoactive intestinal polypeptide is contained in neurons of a number of intrinsic nerve pathways, influencing motility, blood flow and mucosal transport. The myenteric neurons that project to prevertebral sympathetic ganglia may be involved in intestino-intestinal reflexes.

The hypothesis that gamma-aminobutyric acid (GABA) is a peripheral neurotransmitter was tested by determining whether GABA is present in enteric neurons. GABA was detected in the quail gut using high pressure liquid chromatography. GABA-like immunoreactivity was found in both enteric neural plexuses. Only a few neuronal perikarya, but many fibers were stained in each. GABA-immunoreactive neurons survived for over a week in vitro; therefore, they must be intrinsic to the bowel. These and earlier observations indicate that GABA is a peripheral (enteric) as well as a central neurotransmitter.

Stimulation of the sectioned cervical vagal nerve of anaesthetized cats (ether-chloralose), pretreated with guanethidine and atropine, in the peripheral direction produced gastric relaxation as well as jejunal and ileal contraction. The administration of indomethacin markedly enhanced intestinal tone and the amplitude of spontaneous phasic activity while the basal gastric motility was essentially unchanged. This suggests that endogenous prostaglandins exert an inhibitory influence on intestinal motility. The vagally induced gastric relaxation was significantly inhibited by indomethacin, with could suggest that prostaglandins modulate non-adrenergic, non-cholinergic inhibitory neurotransmission in the stomach.

Somatostatin and dopamine beta-hydroxylase have been localized in the coeliaco-mesenteric ganglia, in mesenteric nerves and in the wall of the guinea-pig small intestine. Nerve lesions were used to determine the sources of the nerves. Nerve cell bodies in the coeliaco-mesenteric ganglia with immunoreactivity for both somatostatin and dopamine beta-hydroxylase project to the intestine via the mesenteric nerves. Most of their terminals are in the submucous ganglia, where they make up the full complement of noradrenergic terminals, and in the mucosa where other noradrenergic terminals, not containing somatostatin immunoreactivity, are also present. The small number of noradrenergic fibres present in the tertiary component of the myenteric plexus and in the circular muscle all show immunoreactivity for somatostatin. The noradrenergic fibres supplying the mesenteric and intestinal blood vessels and those ramifying in the myenteric ganglia do not contain somatostatin. The numerous somatostatin-immunoreactive nerves in the enteric plexuses that do not contain dopamine beta-hydroxylase come from enteric nerve cell bodies. These results, considered in the context of other published work, indicate that post-ganglionic sympathetic noradrenergic neurons are chemically coded according to the target tissue they supply and suggest that neurons that were hitherto thought to be neurochemically equivalent, but which serve different functions, are in fact chemically distinct.

A histochemical technique was used to stain the myenteric neurons in the intact wall of the small intestine of mice, guinea-pigs and sheep. The length and diameter of the small intestine and the total serosal surface area were also obtained. Myenteric neurons were counted on large whole-mount preparations of the muscularis externa. Counts were carried out also on the submucosal plexus, on a more limited scale. In the mouse a spatial density of 10,600 myenteric neurons per cm2 was found. The small intestine was 33 cm long and measured on average 11.5 mm in circumference, the total outer surface (serosal surface) amounting to about 38.0 cm2. The total number of myenteric neurons in the small intestine was calculated as about 403,000. In the guinea-pig the length of the small intestine was 145 cm, the average circumference 22 mm and the total outer surface area about 319 cm2. The neuronal packing density was 8600/cm2, and the total number of myenteric neurons about 2,750,000. In the sheep the small intestine was about 2100 cm long with an average circumference of 60 mm and a total surface area of about 12,600 cm2. The ganglion neuron density was about 2500/cm2, and the total number of myenteric neurons in the small intestine was calculated as about 31,500,000. Thus, in the sheep the small intestine contained about 11 times as many myenteric neurons as the guinea-pig and about 80 times as many as the mouse. The differences are in the same direction as, but not proportional to, the differences in body weight and in the length of the intestine. The neuronal spatial density was highest in the mouse and lowest in the sheep, and in the sheep the neurons were markedly larger, and gathered in ganglia that were larger and further apart from one another, than in the mouse, while they had intermediate values in the guinea-pig. A new way of expressing neuronal packing densities is presented together with the proposal of an arbitrary but reproducible unit of intestinal length (a segment whose length is equal to its diameter). In the submucosal plexus the neuronal density was about 3000/cm2 in the guinea-pig, about 8700/cm2 in the mouse and about 4500/cm2 in the sheep. In the mouse the submucosal neuron density decreased gradually along the length of the small intestine.

Quantitative parameters of the discharge patterns of burst-type units and single-spike units were increased, decreased or unchanged after application of morphine. The results were the same when morphine was added in the presence of naloxone. The results suggest that morphine had no consistent effect on the spike discharge of continuously active neurons in Auerbach's plexus of cat small intestine.

gamma-Aminobutyric acid (GABA) is known as a neurotransmitter in the central nervous system and in the enteric nervous system. The effects and the sites of action of GABA and of its GABAA (muscimol) and GABAB (baclofen) agonists were determined on intestinal motility of unanesthetized rats chronically fitted with intraparietal electrodes in the duodeno-jejunum and fasted for 8 h. GABA (6 mg/kg i.p.) induced a biphasic response i.e. a primary inhibition followed by a period of irregular spiking activity. Muscimol (4 mg/kg i.p.) inhibited the cyclic motor profile while baclofen (4 mg/kg i.p.) had a stimulatory effect chiefly at the duodenal level. Only baclofen intracerebroventricularly administered (1 microgram i.c.v.) was able to reproduce the intestinal motor effects observed after systemic injections. Bicuculline (a specific GABAA antagonist) blocked the inhibition induced by GABA and muscimol; atropine (i.p. and i.c.v.) antagonized the irregular spiking activity induced by GABA and baclofen. It is concluded that the dual effect of GABA can be explained by an action at 2 subtypes of receptors: GABAA and GABAB. Stimulation of GABAA receptors induced peripherally mediated inhibition of the duodeno-jejunal motility. On the contrary stimulation of the GABAB receptors increased and disrupted duodenal cyclic motility by a central action involving central and peripheral muscarinic receptors.

Whole mounts of guinea-pig small intestine were used to examine the distribution of neurons with enkephalin-like immunoreactivity and the effects of microsurgical lesions on these neurons. The enkephalin neurons are intrinsic to the intestine. Cell bodies are found in the myenteric ganglia; processes are in the myenteric plexus, circular muscle (including deep muscular plexus) and submucosa, but not in the mucosa. The cell bodies have one prominent process and several short processes, the latter occasionally are seen to give rise in turn to fine, faint processes. The prominent processes provide fibres to the circular muscle and deep muscular plexus beneath and just anal (up to about 2 mm) to the cell bodies. Fibres in the submucous ganglia come from the overlying myenteric plexus. Orally-directed processes (possibly dendrites) of myenteric cell bodies provide the varicose fibres in the myenteric ganglia. These processes are 3.5-4 mm long. The enkephalin neurons represent a population of enteric neurons, with a distinct distribution and projections, which does not correspond to any of the other populations of enteric neurons that have been studied.

Changes in the distribution of 5-hydroxytryptamine-like immunoreactivity have been examined in enteric neurons at various times after microsurgical lesions of the enteric plexuses. In the myenteric plexus, varicose immunoreactive nerve fibres disappeared or were reduced in number in ganglia anal to an interruption of the myenteric plexus. Up to about 2 mm on the anal side, all varicose immunoreactive fibers disappeared from the ganglia. At about 14-16 mm below an interruption, there were about 50% of the normal number of fibres in the myenteric ganglia and at about 24 mm the innervation was normal. In the submucosa, fibres immunoreactive for 5-hydroxytryptamine were absent from an area on the anal side following interruption of the myenteric plexus. From consideration of the pattern of disappearance, it is deduced that some myenteric nerve cell bodies send immunoreactive axons in an anal direction to supply submucous ganglia. The axons run for about 8 mm in the myenteric plexus, enter the submucosa and then run for a further 4 mm approximately. Thus, varicose fibres immunoreactive for 5-hydroxytryptamine, which occur around the enteric ganglion cells of both plexuses arise from nerve cell bodies in myenteric ganglia than send their axons in an anal direction.

This study examines the role of different types of small intestinal distention of affective behavior. "Isovolemic" and volumetric distention were hypothesized to have different effects in rats. Both stimuli decreased fluid consumption in a free drinking situation equally. Behavioral correlates, however, were clearly different. Changes of the gut volume were accompanied by behavioral elements characteristic to aversivity while such changes were not observed when the distention was isovolemic. These results indicate that the cessation of intake, i.e., satiety, is not the consequence of the interference of concurrent aversive behavior. In contrast with these findings, isovolemic distention proved to be mildly unpleasant when tested by the taste-aversion method, although the effect was clearly distinct from that of volume changes. The results suggest that not avrsivity but discomfort may be a steady, inherent concomitant factor of physiological mechanoceptive gut stimuli.

Release of [3H]acetylcholine was studied in the presence of neurotensin (NT) on the myenteric plexus-longitudinal muscle strips (MPLM) from the guinea pig small intestine. In the dose range from 3 pM to 3 nM, NT induced a dose-related increase in the efflux of [3H]acetylcholine from the MPLM. This release was completely inhibited by 1.5 X 10(-6) M tetrodotoxin. The output of acetylcholine evoked by NT (7.5 X 10(-11) M) was significantly depressed by the addition of somatostatin (6.1 X 10(-7) M) or methionine-enkephalin (8.7 X 10(-8) M). Release of newly synthesized [3H]acetylcholine from the isolated synaptic terminals (synaptosomes) was also examined in the presence of NT (1.5 X 10(-7) M) or 20 mM KCl. Release of [3H]acetylcholine was significantly elevated by either stimulants. It was concluded that neurotensin stimulated ACh release via a neurogenic mechanism, probably by a presynaptic site of action on myenteric cholinergic neurons. Despite a paucity of NT-containing neurons in the myenteric plexus, results from this study support a neuromodulatory role for NT in the regulation of local cholinergic neuronal activity in the gut.

Gastrin-releasing peptide is a neuropeptide with a wide distribution in the rat small intestine. Most of the GRP-containing fibers are intramural in origin. Local severing of myenteric GRP neurons by circumferential removal of the outer longitudinal muscle layer together with the adherent myenteric ganglia (myectomy) in a segment of the rat jejunum resulted in the disappearance of GRP fibers from the myectomized circular muscle and from myenteric ganglia and both muscle layers for approximately 10 mm anally to the lesion. As examined at different time intervals up to 60 weeks postoperatively fine-varicose GRP fibers of a normal appearance were found to return gradually to the portion anal to the lesion beginning at 20 weeks, first in the more distal portions and then (after 40-60 weeks) also in the more proximally located portions. Also the circular muscle in the myectomized segment became reinnervated during this time period. These fibers were notably coarse, more numerous than in control circular muscle, and arranged in thick bundles (hyperinnervation). Such nerve bundles were particularly frequent 40 weeks after the operation. The results indicate a remarkable plasticity of enteric neurons.

Vasoactive intestinal polypeptide nerve processes and cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the inner circular smooth muscle coat and mainly in the mucosa, but were absent in the longitudinal muscle layer. Submucosal blood vessels were often surrounded by immunoreactive vasoactive intestinal polypeptide positive nerves, in close associations (distance less than 40 mn) to blood vessel basement membranes and to smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labeled fibers surrounded unstained neural cell bodies. The synaptic vesicles of vasoactive intestinal polypeptide positive terminals were 35-40 nm in diameter and some dense core vesicles (80-120 nm in diameter) were also observed in the same profiles. These observations suggest that vasoactive intestinal polypeptide nerves may participate in regulating smooth muscle activity and local blood flow in the small intestine.