Inhibitory Junction Potentials Research Articles

β-Nicotinamide Adenine Dinucleotide Is an Enteric Inhibitory Neurotransmitter in Human and Nonhuman Primate Colons

An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5'-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K(+) channels. In murine colon β-nicotinamide adenine dinucleotide (β-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here β-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. A small-volume superfusion assay and high-pressure liquid chromatography with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of β-NAD, ATP, and metabolites. Postjunctional responses to nerve stimulation, β-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of β-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts were assayed by reverse transcription polymerase chain reaction. In contrast to ATP, overflow of β-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by the neurotoxins, tetrodotoxin, and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous β-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179), and (1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS 2500). β-NAD activated nonselective cation currents in SMCs, but failed to activate outward currents. β-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current.

Loss of Kitlow progenitors, reduced stem cell factor and high oxidative stress underlie gastric dysfunction in progeric mice.

Gastrointestinal functions decline with ageing leading to impaired quality of life, and increased morbidity and mortality. Neurodegeneration is believed to underlie ageing-associated dysmotilities but the mechanisms have not been fully elucidated. We used progeric mice deficient in the anti-ageing peptide Klotho to investigate the contribution of key cell types of the gastric musculature to ageing-associated changes in stomach function and the underlying mechanisms. Klotho expression, enteric neurons, interstitial cells of Cajal (ICC), smooth muscle cells and electrical activity were assessed by immunofluorescence, confocal microscopy, 3-dimensional reconstruction, flow cytometry, quantitative RT-PCR, Western immunoblotting and intracellular recordings. Gastric emptying of solids was analysed by the [13C]octanoic acid breath test. Circulating and tissue trophic factors were measured by enzyme immunoassays and quantitative RT-PCR. The role of oxidative stress was investigated in organotypic cultures. Klotho expression was detected in gastric glands, myenteric neurons and smooth muscle cells. Progeric Klotho-deficient mice had profound loss of ICC and ICC stem cells without a significant decrease in neuron counts, expression of neuronal nitric oxide synthase or smooth muscle myosin. Slow wave amplitude and nitrergic inhibitory junction potentials were reduced while solid emptying was unchanged. Klotho-deficient mice were marantic and had low insulin, insulin-like growth factor-I and membrane-bound stem cell factor. Klotho deficiency accentuated oxidative stress and ICC loss. We conclude that Klotho-deficient, progeric mice display a gastric phenotype resembling human ageing and involving profound ICC loss. Klotho protects ICC by preserving their precursors, limiting oxidative stress, and maintaining nutritional status and normal levels of trophic factors important for ICC differentiation.

T2056 Purinergic Inhibitory Junction Potentials are Selectively Attenuated in Regions of Disrupted Motility in the Inflamed Guinea Pig Distal Colon

T2056 Purinergic Inhibitory Junction Potentials are Selectively Attenuated in Regions of Disrupted Motility in the Inflamed Guinea Pig Distal Colon

Critical role of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7) receptors on AH neurons these interneurons also contribute to the generation of the CMMC.

Purinergic and nitrergic neuromuscular transmission mediates spontaneous neuronal activity in the rat colon

Nitric oxide (NO) and ATP mediate smooth muscle relaxation in the gastrointestinal tract. However, the involvement of these neurotransmitters in spontaneous neuronal activity is unknown. The aim of the present work was to study spontaneous neuromuscular transmission in the rat midcolon. Microelectrode experiments were performed under constant stretch both in circular and longitudinal directions. Spontaneous inhibitory junction potentials (sIJP) were recorded. Tetrodotoxin (1 microM) and apamin (1 microM) depolarized smooth muscle cells and inhibited sIJP. N(omega)-nitro-l-arginine (l-NNA, 1 mM) depolarized smooth muscle cells but did not modify sIJP. In contrast, the P2Y(1) antagonist MRS-2500 (1 microM) did not modify the resting membrane potential (RMP) but reduced sIJP (IC(50) = 3.1 nM). Hexamethonium (200 microM), NF-023 (10 microM), and ondansetron (1 microM) did not modify RMP and sIJP. These results correlate with in vitro (muscle bath) and in vivo (strain gauges) data where l-NNA but not MRS-2500 induced a sustained increase of spontaneous motility. We concluded that, in the rat colon, inhibitory neurons regulate smooth muscle RMP and cause sIJP. In vitro, the release of inhibitory neurotransmitters is independent of nicotinic, P2X, and 5-hydroxytryptamine type 3 receptors. Neuronal NO causes a sustained smooth muscle hyperpolarization that is responsible for a constant inhibition of spontaneous motility. In contrast, ATP acting on P2Y(1) receptors is responsible for sIJP but does not mediate inhibitory neural tone. ATP and NO have complementary physiological functions in the regulation of gastrointestinal motility.

All ahead stop! How intestinal motility adapts to cope with inflammation induced ulceration

All ahead stop! How intestinal motility adapts to cope with inflammation induced ulceration

Purinergic neuromuscular transmission is selectively attenuated in ulcerated regions of inflamed guinea pig distal colon

This study was undertaken to investigate neuromuscular transmission in regions of the inflamed colon in which motility is disrupted. Propulsive motility was evaluated in segments of control guinea pigs and those treated 6 days previously with trinitrobenzene sulfonic acid. Intracellular recordings were then obtained from circular muscle cells to examine excitatory and inhibitory junction potentials (EJPs and IJPs). In inflamed preparations, propulsion of fecal pellets was temporarily halted or obstructed at sites of mucosal damage, whereas the propulsive motility was linear in control colons. The amplitudes of evoked and spontaneous IJPs were significantly reduced in ulcerated regions of inflamed preparations, but EJPs were comparable to controls. Pharmacological dissection of the IJP revealed that the purinergic component was reduced, while the nitrergic IJP was slightly increased. Furthermore, the reduction in the purinergic IJP in inflamed preparations persisted in the presence of hexamethonium, suggesting that the deficit involved the inhibitory motor neuron and/or smooth muscle. Nerve fibre density was not altered in the circular muscle, and pre-contracted rings of inflamed colon relaxed normally to ATP, suggesting that the deficit involves altered ATP release and/or degradation. The P2Y(1) receptor antagonist MRS2179 slowed propulsive motility indicating that decreased purinergic neuromuscular transmission could contribute to the inflammation-induced motor deficit. We conclude that purinergic inhibitory neuronal input to the circular muscle is selectively reduced in regions of the colon in experimental colitis where the mucosa is damaged, and this is likely to contribute to altered motility in colitis by diminishing downstream relaxation during the peristaltic reflex.

P2Y1 Receptors Mediate Apamin-Sensitive and -Insensitive Inhibitory Junction Potentials in Murine Colonic Circular Smooth Muscle

Purinergic inhibitory neuromuscular transmission plays an important role in the control of intestinal motility. In most tissues this neurotransmission is apamin-sensitive, but recent studies in human colonic circular smooth muscle (CSM) suggest the presence of apamin-insensitive purinergic inhibitory junction potentials (IJPs). The current studies used conventional intracellular recordings on colonic CSM strips to characterize the purinergic IJPs in murine colonic CSM. P2Y1 receptor expression was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The IJP induced by nerve stimulation (NS) of one and four pulses in neuronal nitric-oxide synthase knockout mice consists of an apamin-sensitive and a dominant apamin-resistant component. These are identical to the IJPs in wild-type and CD1 mice in the presence of N(omega)-nitro-l-arginine methyl ester (200 microM) and were significantly inhibited by alpha,beta-methylene ATP (50 microM), an analog of ATP. IJPs were not affected by the P2X receptor antagonist 2',3'-o-(2,4,6-trinitrophenyl)-ATP (10 microM). Furthermore, apamin-resistant IJPs induced by single-pulse NS were abolished by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 microM), a P2 receptor antagonist; 2'-deoxy-N6-methyl adenosine 3,5-diphosphate (MRS-2179; 10 microM), a selective P2Y1 receptor antagonist; and tetrodotoxin (1 microM). Aboral NS induced apamin-sensitive purinergic IJPs, whereas oral and circumferential NS produced apamin-sensitive and -resistant IJPs, with the latter predominating. RT-PCR and immunohistochemistry confirmed the presence of P2Y1 receptors on smooth muscle and in the myenteric plexus. These data suggest that, depending on stimulus location, activation of P2Y1 receptors produces both apamin-sensitive and apamin-resistant IJPs in murine colonic CSM.

TREK‐1 channels do not mediate nitrergic neurotransmission in circular smooth muscle from the lower oesophageal sphincter

The ionic mechanisms underlying nitrergic inhibitory junction potentials (IJPs) in gut smooth muscle remain a matter of debate. Recently, it has been reported that opening of TWIK-related K(+) channel 1 (TREK-1) K(+) channels contributes to the nitrergic IJP in colonic smooth muscle. We investigated the effects of TREK-1 channel blockers on nitrergic neurotransmission in mouse and opossum lower oesophageal sphincter (LOS) circular smooth muscle (CSM). The effects of TREK-1 channel blockers were characterized pharmacologically in murine and opossum gut smooth muscle using conventional intracellular and tension recordings. In LOS, L-methionine depolarized the resting membrane potential (RMP) but did not inhibit the nitrergic IJP. Cumulative application of theophylline hyperpolarized the RMP and inhibited the nitrergic IJP concentration dependently. The induced membrane hyperpolarization was prevented by pre-application of caffeine, but not by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. 8-Br-cAMP significantly hyperpolarized membrane potential and increased the amplitude of the nitrergic IJP. In opossum LOS muscle strips, L-methionine increased resting tone but had no effect on nerve-mediated LOS relaxation. On the other hand, theophylline markedly inhibited tone. In CSM from mouse proximal colon, L-methionine caused modest inhibition of nitrergic IJPs. TREK-1 channels were not involved in the nitrergic IJP in LOS CSM. Not only does L-methionine have no effect on the nitrergic IJP or LOS relaxation, but the effect of theophylline appears to be due to interruption of Ca(2+)-releasing pathways (i.e. caffeine-like effect) rather than via blockade of TREK-1 channels.

P2Y1 receptors mediate inhibitory neuromuscular transmission in the rat colon

Inhibitory junction potentials (IJP) are responsible for smooth muscle relaxation in the gastrointestinal tract. The aim of this study was to pharmacologically characterize the neurotransmitters [nitric oxide (NO) and adenosine triphosphate (ATP)] and receptors involved at the inhibitory neuromuscular junctions in the rat colon using newly available P2Y(1) antagonists. Organ bath and microelectrode recordings were used to evaluate the effect of drugs on spontaneous mechanical activity and resting membrane potential. IJP and mechanical relaxation were studied using electrical field stimulation (EFS). N(omega)-nitro-L-arginine (L-NNA) inhibited the slow component of the IJP and partially inhibited the mechanical relaxation induced by EFS. MRS2179, MRS2500 and MRS2279, all selective P2Y(1) receptor antagonists, inhibited the fast component of the IJP without having a major effect on the relaxation induced by EFS. The combination of both L-NNA and P2Y(1) antagonists inhibited the fast and the slow components of the IJP and completely blocked the mechanical relaxation induced by EFS. Sodium nitroprusside caused smooth muscle hyperpolarization and cessation of spontaneous motility that was prevented by oxadiazolo[4,3-alpha]quinoxalin-1-one. Adenosine 5'-O-2-thiodiphosphate, a preferential P2Y agonist, hyperpolarized smooth muscle cells and decreased spontaneous motility. This effect was inhibited by P2Y(1) antagonists. The co-transmission process in the rat colon involves ATP and NO. P2Y(1) receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y(1) receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y(1) receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.

Neurotransmission in lower esophageal sphincter ofW/Wvmutant mice

To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/W(v) wild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired IJPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/W(v) mutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/W(v) mutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal Ca2+-dependent signaling initiated by spontaneous Ca2+ release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle.

T1791 Effect of NaHS On Circular Muscle Tone and Spontaneous Inhibitory Junction Potentials (IJPs) in the Mouse Colon

T1791 Effect of NaHS On Circular Muscle Tone and Spontaneous Inhibitory Junction Potentials (IJPs) in the Mouse Colon

Imaging of Nitric Oxide in Nitrergic Neuromuscular Neurotransmission in the Gut

BackgroundNumerous functional studies have shown that nitrergic neurotransmission plays a central role in peristalsis and sphincter relaxation throughout the gut and impaired nitrergic neurotransmission has been implicated in clinical disorders of all parts of the gut. However, the role of nitric oxide (NO) as a neurotransmitter continues to be controversial because: 1) the cellular site of production during neurotransmission is not well established; 2) NO may interacts with other inhibitory neurotransmitter candidates, making it difficult to understand its precise role.Methodology/Principal FindingsImaging NO can help resolve many of the controversies regarding the role of NO in nitrergic neurotransmission. Imaging of NO and its cellular site of production is now possible. NO forms quantifiable fluorescent compound with diaminofluorescein (DAF) and allows imaging of NO with good specificity and sensitivity in living cells. In this report we describe visualization and regulation of NO and calcium (Ca2+) in the myenteric nerve varicosities during neurotransmission using multiphoton microscopy. Our results in mice gastric muscle strips provide visual proof that NO is produced de novo in the nitrergic nerve varicosities upon nonadrenergic noncholinergic (NANC) nerve stimulation. These studies show that NO is a neurotransmitter rather than a mediator. Changes in NO production in response to various pharmacological treatments correlated well with changes in slow inhibitory junction potential of smooth muscles.Conclusions/SignificanceDual imaging and electrophysiologic studies provide visual proof that during nitrergic neurotransmission NO is produced in the nerve terminals. Such studies may help define whether NO production or its signaling pathway is responsible for impaired nitrergic neurotransmission in pathological states.

Modulation of slow waves by transmural nerve stimulation of smooth muscle tissue isolated from the corpus of the guinea-pig stomach

Modulation of slow waves in response to transmural nerve stimulation (TNS) was investigated in smooth muscle preparations isolated from the corpus of the guinea-pig stomach. Single TNS evoked an inhibitory junction potential (i.j.p.) and enhanced the amplitude of the following slow wave. Effects of atropine, N(omega)-nitro-L-arginine (L-NA) and apamin revealed that corpus smooth muscle was innervated by cholinergic excitatory, nitrergic inhibitory and apamin-sensitive inhibitory nerves. In preparations isolated from the upper corpus which generated slow waves of 5-15 mV amplitude, a 1 min train of TNS (0.5 or 1 Hz frequency) increased the amplitude, with further enhancement by L-NA, but inhibition by atropine. In the lower corpus, larger amplitude (20-30 mV) slow waves were generated but these were not altered by a TNS train. However, application of L-NA and neostigmine, or often L-NA alone, resulted in increased frequency and decreased amplitude of slow waves during TNS, with an associated depolarization of the membrane. These changes were inhibited by atropine. In the presence of atropine, TNS reduced slow wave amplitude in an L-NA-sensitive manner. Acetylcholine (ACh) at 1 nM increased the amplitude of slow waves in the upper corpus. In the lower corpus, while low concentrations of ACh (<10 nM) did not increase the frequency and decrease the amplitude of slow waves with an associated depolarization of the membrane, this occurred at high concentrations of ACh (>10 nM). Application of the NO donor, sodium nitroprusside (SNP, 10 nM-1 microM), reduced the amplitude of slow waves. The changes in amplitude of slow waves elicited by ACh or SNP were not associated with a significant change in frequency. These results indicate that in the corpus circular smooth muscle, neural modulation of slow waves appeared to be exerted mainly on the amplitude, but not on the frequency.

Cannabinoid-1 (CB1) receptors regulate colonic propulsion by acting at motor neurons within the ascending motor pathways in mouse colon

Cannabinoid-1 (CB(1)) receptors on myenteric neurons are involved in the regulation of intestinal motility. Our aim was to investigate CB(1) receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Presence of the CB(1) receptor was investigated by RT-PCR, and immunohistochemistry was used for colabeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances, and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in an unpartitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and CB(1)(-/-) mice. Immunostaining with the cytoskeletal marker peripherin showed CB(1) immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed CB(1) on choline acetyltransferase-, calretinin-, and 5-HT-immunopositive myenteric neurons, but nitrergic neurons appeared immunonegative for CB(1) immunostaining. Solitary spindle-shaped CB(1)-immunoreactive cells in between smooth muscle cells lacked specific markers for interstitial cells of Cajal or glial cells. ES elicited neuronally mediated excitatory JP (EJP) and inhibitory JP. Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode 6 and a maximal EJP projection distance of approximately 18 mm. The CB(1) receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber, WIN 55,212-2 reduced EJP at the separated oral sites, proving that CB(1) activation inhibits interneuron-mediated neurotransmission. These effects were absent in the presence of the CB(1) antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in SR141716A-pretreated wild-type or CB(1)(-/-) mice. Activation of the CB(1) receptor modulates excitatory cholinergic neurotransmission in mouse colon by reducing amplitude and spatial spreading of the ascending electrophysiological impulses. This effect on electrophysiological spreading involves CB(1)-mediated effects on motor neurons and ascending interneurons and is likely to underlie the here reported in vivo reduction in colonic propulsion.

Polarized intrinsic neural reflexes in response to colonic elongation

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca(2+) imaging techniques. Full colons were 158.1 +/- 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s(-1)). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca(2+) waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechanosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Purinergic and nitrergic junction potential in the human colon

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).

Neural and anatomical abnormalities of the gastrointestinal system resulting from contusion spinal cord injury

Gastrointestinal (GI) abnormalities resulting from spinal cord injury (SCI) are challenging disorders that have not been examined experimentally using clinically relevant models. In this study, female Sprague–Dawley rats (n=5/group×4: T10–T11 contusion, laminectomy, or naïve) were fasted for 24 h before being submitted to dye recovery assays (Phenol Red solution, 1.5 ml/rat; per oral) on GI emptying/transiting at 48 h or 4 weeks postinjury (p.i.). Compared with controls, SCI significantly increased dye recovery rate (DRR, determined by spectrophotometry) in the duodenum (+84.6%) and stomach (+32.6%), but decreased it in the jejunum (−64.1% and −49.5%) and ileum (−73.6% and −70.1%) at 48 h and 4 weeks p.i., respectively (P≤0.005, ANOVA with post hoc t-test). Electrophysiological analysis revealed that purinergic fast inhibitory junction potential (IJP) was reduced ∼30% in the antrum and duodenum of rats 48 h p.i. (numbers of animals/numbers of tissue samples=3/7; P<0.001), and slow IJP was essentially abolished. Immunocytochemistry consequently uncovered significant reductions in the GI vasoactive intestinal polypeptide and neuronal nitric oxide synthase (i.e. slow IJP mediators) reactivity at 48 h and 4 weeks p.i., suggesting that SCI disrupted interstitial neurotransmission. Importantly, SCI caused discernible atrophy of the GI mucosa and muscle coat (e.g. the two layers of gastric wall were correspondingly 28% and 27% thinner 4 weeks p.i.). We conclude that contusive SCI triggers GI abnormalities with unique pathophysiology and pathology in different segments. Such GI disorders evolve continuously during the entire post-SCI period examined, and may require therapeutic development to target specific underlying mechanisms.

A NEW ELECTROPHYSIOLOGICAL TOOL TO INVESTIGATE THE SPATIAL NEURONAL PROJECTIONS WITHIN THE MYENTERIC ASCENDING REFLEX OF THE MOUSE COLON

1. The intestinal peristaltic reflex is regulated by local microcircuits that, upon activation, result in an oral contraction and anal relaxation of the circular muscle. This contractile response is associated with typical electrophysiological changes in membrane potential resulting from excitatory and inhibitory myenteric pathways. 2. The aim of the present study was to investigate the influence of local electrical stimulation (ES; single pulses, 15 V, 0.3 msec duration) on the ascending gastrointestinal electrophysiological potentials of the mouse colon using a novel 12-channel stimulation electrode in a newly designed model of the ascending myenteric pathways with simultaneous intracellular recording. 3. Local myenteric reflex responses in the proximal colon were initiated by ES (12 bipolar stimulation electrodes (SE) 0.7 mm apart from each other) and excitatory and inhibitory junction potentials (EJP and IJP, respectively) were recorded from circular smooth muscle cells with intracellular recording techniques. In vivo colonic propulsion was determined by measuring the time to expulsion of a 3 mm glass bead inserted 2.5 cm into the distal colon of mouse. 4. Under basal conditions, circular smooth muscle cells displayed a stable membrane potential (-56.7 +/- 6.9 mV; n = 13). Electrical stimulation elicited a tetrodotoxin (3 micromol/L)-sensitive, neuronal-induced EJP (cholinergic; atropine (1 micromol/L) sensitive) and a biphasic IJP. Both the EJP and IJP showed characteristic responses dependent on the distance between stimulation and recording sites. The EJP could be recorded over long distances, resulting in a maximal EJP amplitude at a distance of 10 mm distance (represented by stimulation electrodes (SE) number 6/7) and a maximal projection distance of 18-20 mm. Both components of IJP were maximal during direct stimulation (at SE1; stimulation at the recording site) and gradually decreased to SE6/7 (10 mm). At distances greater than 10 mm apart, ES did not produce IJP. The ganglionic blocker hexamethonium (10-100 micromol/L) concentration dependently abolished all inhibitory junction potentials at distances greater than 10 mm and significantly reduced the amplitude of EJP for the first 10 mm. Colonic propulsion was decreased by hexamethonium (40 mg/kg) and atropine (0.7 mg/kg). 5. Neuronal circuits of the ascending myenteric reflex functionally project distances ranging up to 18-20 mm. Our newly designed setup allows simultaneous electrophysiological investigations of neuronal microcircuitry within the myenteric plexus over short and long distances and enables conclusions to be drawn regarding neuroneuronal and neuromuscular transmission.

Functional evidence for purinergic inhibitory neuromuscular transmission in the mouse internal anal sphincter

The neurotransmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10-60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. l-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 microM), by desensitization of P2Y receptors with adenosine 5'-[beta-thio]diphosphate (ADP-betaS) (10 microM), and by the selective P2Y1 receptor blocker 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179) (10 microM). Relaxation and IJPs were also significantly reduced by the K(+) channel blocker apamin (1 microM). Removal of extracellular potassium (K(o)) increased IJP amplitude to 205% of control, whereas return of K(o) 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 microM) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apamin-sensitive K(+) channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.