Amplitude Of Inhibitory Junction Potentials Research Articles

Intestinal nerve cell injury occurs prior to insulin resistance in female mice ingesting a high-fat diet.

Diabetic patients suffer from gastrointestinal disorders associated with dysmotility, enteric neuropathy and dysbiosis of gut microbiota; however, gender differences are not fully known. Previous studies have shown that a high-fat diet (HFD) causes type two diabetes (T2D) in male mice after 4-8weeks but only does so in female mice after 16weeks. This study seeks to determine whether sex influences the development of intestinal dysmotility, enteric neuropathy and dysbiosis in mice fed HFD. We fed 8-week-old C57BL6 male and female mice a standard chow diet (SCD) or a 72% kcal HFD for 8weeks. We analyzed the associations between sex and intestinal dysmotility, neuropathy and dysbiosis using motility assays, immunohistochemistry and next-generation sequencing. HFD ingestion caused obesity, glucose intolerance and insulin resistance in male but not female mice. However, HFD ingestion slowed intestinal propulsive motility in both male and female mice. This was associated with decreased inhibitory neuromuscular transmission, loss of myenteric inhibitory motor neurons and axonal swelling and loss of cytoskeletal filaments. HFD induced dysbiosis and changed the abundance of specific bacteria, especially Allobaculum, Bifidobacterium and Lactobacillus, which correlated with dysmotility and neuropathy. Female mice had higher immunoreactivity and numbers of myenteric inhibitory motor neurons, matching larger amplitudes of inhibitory junction potentials. This study suggests that sex influences the development of HFD-induced metabolic syndrome but dysmotility, neuropathy and dysbiosis occur independent of sex and prior to T2D conditions. Gastrointestinal dysmotility, neuropathy and dysbiosis might play a crucial role in the pathophysiology of T2D in humans irrespective of sex.

Stress increases descending inhibition in mouse and human colon.

A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues. Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 μM). Intracellular recordings were obtained from colonic circular smooth muscle cells in isolated smooth muscle/myenteric plexus preparations and the inhibitory junction potential (IJP) was elicited by nerve stimulation or balloon distension oral to the site of recording. Water avoidance stress increased the number of fecal pellets compared to control (p < 0.05). WAS also caused a significant increase in IJP amplitude following balloon distension. Stress hormones also increased the IJP amplitude following nerve stimulation and balloon distension (p < 0.05) in control mice but had no effect in colons from stressed mice. No differences were observed with application of ATP between stress and control tissues, suggesting the actions of stress hormones were presynaptic. Stress hormones had a large effect in the nerve stimulated IJP in human colon (increased >50%). Immunohistochemical studies identified alpha and beta adrenergic receptor immunoreactivity on myenteric neurons in human colon. These studies suggest that WAS and stress hormones can signal via myenteric neurons to increase inhibitory neuromuscular transmission. This could lead to greater descending relaxation, decreased transit time, and subsequent diarrhea.

R‐Type Calcium Channels Contribute to Colonic Inhibitory Neuromuscular Transmission

The inhibitory neurotransmitters, ATP and nitric oxide (NO), relax the colonic smooth muscle which is important for propulsion of colonic content. Voltage‐gated Ca2+ channels (VGCC) control neurotransmitter release and regulate colonic motility. The α1E subunit of the R‐type VGCC is expressed by enteric neurons yet its role in regulating colonic motility is unclear. We investigated the role of R‐type VGCCs in colonic inhibitory neuromuscular transmission using alpha1E knockout (KO) mice. Intracellular recordings of inhibitory junction potentials (IJP) were obtained from circular muscle cells in distal colon. IJPs were evoked by trains of electrical stimulation (1s, 10 Hz, 40‐90 volts). IJPs have a fast ATP‐mediated component and a slow NO‐mediated component. Peak amplitude (ATP component) and area under the curve (AUC; both components) of the IJP were compared between wildtype (WT) and KO mice. Resting membrane potential (RMP) was measured as an index of ongoing inhibitory transmission. There was a voltage dependent increase in IJP amplitude and AUC in WT and KO mice. IJP amplitude was similar in WT and KO mice. The IJP AUC was decreased in KO compared to WT. Nifedipine (1 µM; L‐type VGCC blocker) had no effect on these responses. RMP in KO muscle cells was decreased compared with WT (‐44 ± 2 mV in WT and ‐38 ± 1 mV in KO; P &lt; 0.05). Thus, there was a 5.4 mV RMP depolarization in KO mice. Nifedipine did not change RMP of WT or KO cells (‐39 ± 3 mV, WT and ‐40 ± 1 mV in KO). In conclusion, R‐type VGCCs contribute to colonic inhibitory neuromuscular transmission by controlling NO release. In addition, R‐type VGCCs contribute to ongoing release of NO and ATP which maintain colonic muscle tone.

High‐fat Diet Causes Loss of Nitric Oxide Motor Neurons and Impairs Inhibitory Neuromuscular Communication in the Mouse Distal Colon

High‐fat (HF) diet increases the risks of gastrointestinal (GI) motility complications, however, the mechanisms that underlie these complications are still not entirely understood. We tested the hypothesis that HF diet causes oxidative stress, loss of nitric oxide (NO) motor neurons in the myenteric plexus, disrupted inhibitory neuromuscular communication and impaired GI motility.MethodsDistal colon segments from mice fed a control (10 Kcal%) or HF diet (60 Kcal%) were used in vitro. Alexa fluor 546 and 680 conjugated maleimides were used to assess the oxidized/reduced glutathione ratio in myenteric NO neurons. NO neuronal density was assessed using nNOS immunohistochemistry. Basal NO availability was measured using NO induced fluorescence of DAF‐FM diacetate. Electrical field stimulation was applied across varying voltages (60‐120V) and train (200‐1000ms) durations to evoke inhibitory junction potentials (IJPs) in colonic circular smooth muscle cells. Amplitude and area of IJP were examined to assess fast and slow IJPs respectively.ResultsWe observed an increase (17%, p&lt;0.01) in oxidized/reduced glutathione fluorescence ratio in nNOS neurons of HF mice. HF mice had lower (28%, p&lt;0.01) nNOS neurons per ganglionic area than controls. Fluorescence intensity of DAF‐FM diacetate was also lower (20%, p&lt;0.01) in HF mice. HF mice showed a significant loss (52%, p&lt;0.05) in IJP area, when stimulated at higher train duration (800‐1000ms).ConclusionWe conclude that HF diet causes oxidative stress, leading to loss of inhibitory NO neurons, lower NO availability, and disrupted neuromuscular transmission in the mouse distal colon. Supported by NIH (DK094932).

Dynamics of inhibitory co-transmission, membrane potential and pacemaker activity determine neuromyogenic function in the rat colon

Interaction of different neuromyogenic mechanisms determines colonic motility. In rats, cyclic depolarizations and slow waves generate myogenic contractions of low frequency (LF) and high frequency (HF), respectively. Interstitial cells of Cajal (ICC) located near the submuscular plexus (SMP) generate slow waves. Inhibitory junction potential (IJP) consists on a purinergic fast (IJPf) followed by a nitrergic slow (IJPs) component leading to relaxation. In the present study, we characterized (1) the dynamics of purinergic-nitrergic inhibitory co-transmission and (2) its contribution on prolonged inhibition of myogenic activity. Different protocols of electrical field stimulation (EFS) under different pharmacological conditions were performed to characterize electrophysiological and mechanical responses. Smooth muscle cells (SMCs) in tissue devoid of ICC-SMP had a resting membrane potential (RMP) of -40.7 ± 0.7 mV. Single pulse protocols increased purinergic and nitrergic IJP amplitude in a voltage-dependent manner (IJPfMAX = -26.4 ± 0.6 mV, IJPsMAX = -6.7 ± 0.3 mV). Trains at increasing frequencies enhanced nitrergic (k = 0.8 ± 0.2 s, IJPs∞ = -15 ± 0.5 mV) whereas they attenuated purinergic responses (k = 3.4 ± 0.6 s,IJPf∞ = -8.9 ± 0.6 mV). In tissues with intact ICC-SMP, the RMP was -50.0 ± 0.9 mV and nifedipine insensitive slow waves (10.1 ± 2.0 mV, 10.3 ± 0.5 cpm) were recorded. In these cells, (1) nitrergic and purinergic responses were reduced and (2) slow waves maintained their intrinsic frequency and increased their amplitude under nerve-mediated hyperpolarization. Based on the co-transmission process and consistent with the expected results on RMP, prolonged EFS caused a progressive reduction of LF contractions whereas HF contractions were partially insensitive. In conclusion, inhibitory neurons modulate colonic spontaneous motility and the principles determining post-junctional responses are (1) the frequency of firing that determines the neurotransmitter/receptor involved, (2) the transwall gradient and (3) the origin and nature of each myogenic activity

Oxidative stress disrupts purinergic neuromuscular transmission in the inflamed colon

Colitis, induced by trinitrobenzene sulfonic acid (TNBS) in guinea pig, leads to decreased purinergic neuromuscular transmission resulting in a reduction in inhibitory junction potentials (IJPs) in colonic circular muscle. We explored possible mechanisms responsible for this inflammation-induced neurotransmitter plasticity. Previous studies have suggested that the deficit in inflamed tissue involves decreased ATP release. We therefore hypothesized that decreased purinergic transmission results from inflammation-induced free radical damage to mitochondria, leading to decreased purine synthesis and release. Stimulus-induced release of purines was measured using high-performance liquid chromatography, and quantities of all purines measured were significantly reduced in the inflamed colons as compared to controls. To test whether decreased mitochondrial function affects the IJP, colonic muscularis preparations were treated with the mitochondrial ATP synthase inhibitors oligomycin or dicyclohexylcarbodiimide, which resulted in a significant reduction of IJP amplitude. Induction of oxidative stress in vitro, by addition of H2O2 to the preparation, also significantly reduced IJP amplitude. Purinergic neuromuscular transmission was significantly restored in TNBS-inflamed guinea pigs, and in dextran sodium sulfate-inflamed mice, treated with a free radical scavenger. Furthermore, propulsive motility in the distal colons of guinea pigs with TNBS colitis was improved by in vivo treatment with the free radical scavenger. We conclude that oxidative stress contributes to the reduction in purinergic neuromuscular transmission measured in animal models of colitis, and that these changes can be prevented by treatment with a free radical scavenger, resulting in improved motility.

Inhibitory neuromuscular transmission in the mouse distal colon is mediated by SK and calcium activated chloride channels

Neurogenic colonic smooth muscle relaxation is caused by inhibitory junction potentials (IJPs). We attempted to identify neurotransmitter, receptor and ion channel mechanisms that mediate IJPs in the mouse distal colon.MethodsIntracellular recordings were obtained from circular smooth muscle cells in vitro. Electrical field stimulation was used to evoke IJPs. Drugs were applied in the organ bath solution.ResultsThe P2Y1 receptor antagonist MRS‐2179 (10 μM) reduced IJP amplitude (66 ± 4%) and area (24 ± 11%). The nitric oxide synthase (NOS) inhibitor L‐NNA (100 μM) reduced IJP area (33 ± 8%) but not amplitude. Co‐application of MRS‐2179 and L‐NNA blocked IJPs. The SK channel blocker, apamin (0.1 μM) reduced IJP amplitude (36 ± 5%) and area 41 ± 12%. The Ca2+‐activated Cl‐channel blocker, niflumic acid (50 μM) reduced IJP amplitude (52 ± 6%) and area (44 ± 6%). Co‐application of niflumic acid and apamin blocked IJPs. ATP (5 mM) produced a larger hyperpolarization (20 ± 2 mV) compared to β‐NAD (10 ± 3 mV). MRS‐2179 and apamin reduced the ATP response amplitude by 34 ± 8% and 31 ± 1% respectively. MRS‐2179 and apamin reduced the β‐NAD (5 mM) response by 71 ± 14% and 61 ± 5% respectively.ConclusionIJPs are mediated via P2Y1 and NOS mediated pathways, which involve SK and Ca2+‐activated Cl‐channels. Responses caused by β‐NAD more closely mimic the IJP compared to ATP. Supported by NIH (DK094932).

Loss of responsiveness of circular smooth muscle cells from the guinea pig ileum is associated with changes in gap junction coupling

Gap junction coupling and neuromuscular transmission to smooth muscle were studied in the first 4 h after preparations were set up in vitro. Intracellular recordings were made from smooth muscle cells of guinea pig ileum. Fast inhibitory junction potentials (IJPs) were small (1.3 ± 1.0 mV) in the first 30 min but increased significantly over the first 120 min to 15.8 ± 0.9 mV (n = 12, P < 0.001). Comparable increases in slow IJPs and excitatory junction potentials were also observed. During the same period, resting membrane potential depolarized from -58.8 ± 1.4 to -47.2 ± 0.4 mV (n = 12, P < 0.001). Input resistance, estimated by intracellular current injection, decreased in parallel (P < 0.05), and dye coupling, measured by intracellular injection of carboxyfluorescein, increased (P < 0.001). Input resistance was higher and dye coupling was less in longitudinal than circular smooth muscle cells. Gap junction blockers [carbenoxolone (100 μM), 18β-glycyrrhetinic acid (10 μM), and 2-aminoethoxydiphenyl borate (50 μM)] hyperpolarized coupled circular smooth muscle cells, reduced the amplitude of fast and slow IJPs and excitatory junction potentials, increased input resistance, and reduced dye coupling. Local application of ATP (10 mM) mimicked IJPs and showed comparable increases in amplitude over the first 120 min; carbenoxolone and 2-aminoethoxydiphenyl borate significantly reduced ATP-evoked hyperpolarizations in coupled cells. In contrast, synaptic transmission between myenteric neurons was not suppressed during the first 30 min. Gap junction coupling between circular smooth muscle cells in isolated preparations was initially disrupted but recovered over the next 120 min to a steady level. This was associated with potent effects on neuromuscular transmission and responses to exogenous ATP.

Expression Level of Hand2 Affects Specification of Enteric Neurons and Gastrointestinal Function in Mice

Hand2 is a basic helix-loop-helix transcription factor required for terminal differentiation of enteric neurons. We studied Hand2 haploinsufficient mice, to determine whether reduced expression of Hand2 allows sufficient enteric neurogenesis for survival, but not for development of a normal enteric nervous system (ENS). Enteric transcripts that encode Hand2 and the neuron-specific embryonic lethal abnormal vision proteins HuB, HuC, and HuD were quantified. Immunocytochemistry was used to identify and quantify neurons. Apoptosis was analyzed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling procedure. Intracellular microelectrodes were used to record inhibitory junction potentials. Gastrointestinal transit and colonic motility were measured in vivo. Levels of of enteric Hand2 transcripts were associated with genotypes of mice, in the following order: Hand2(+/+) > Hand2(LoxP/+) > Hand2(+/-) > Hand2(LoxP/-). Parallel reductions were found in expression of HuD and in regional and phenotypic manners. Numbers of neurons, numbers of neuronal nitric oxide synthase(+) and calretinin(+), but not substance P(+) or vasoactive intestinal peptide(+) neurons, decreased. No effects were observed in stomach or cecum. Apoptosis was not detected, consistent with the concept that Hand2 inhibits neuronal differentiation, rather than regulates survival. The amplitude of inhibitory junction potentials in colonic circular muscle was similar in Hand2 wild-type and haploinsufficient mice, although in haploinsufficient mice, the purinergic component was reduced and a nitrergic component appeared. The abnormal ENS of haploinsufficient mice slowed gastrointestinal motility but protected mice against colitis. Reduced expression of factors required for development of the ENS can cause defects in the ENS that are subtle enough to escape detection yet cause significant abnormalities in bowel function.

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

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.

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.

Inhibitory purinergic transmission in mouse caecum: Role for P2Y1 receptors as prejunctional modulators of ATP release

Using conventional microelectrode recording techniques, we investigated, in the circular muscle of the mouse caecum, the neurotransmitter(s) involved in the neurally-evoked inhibitory junction potentials (IJPs) and the existence of possible prejunctional mechanisms controlling neurotransmitter release. Electrical field stimulation with single pulses elicited IJPs, consisting only of a "fast" hyperpolarization, while using train stimuli (30-50 Hz) the initial fast hyperpolarization was followed by a slower hyperpolarization. The fast and the slow component were selectively antagonized by apamin, a blocker of calcium-activated potassium channels, and N(omega)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, respectively. Fast IJPs were antagonized also by P2 purinoceptor antagonists, suramin or 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS), P2Y purinoceptor desensitization by adenosine 5'-O-2-thiodiphosphate (ADPbetaS). 2'-Deoxy-N(6)-methyl ADP diammonium salt (MRS 2179), P2Y1 purinoceptor antagonist, at the concentration of 1 microM increased the amplitude of the fast IJP, while at the concentration of 10 microM induced a reduction. 8,8'-[Carbonylbis[imino-3,1-phenylenecarbonylimino (4-fluoro-3,1-phenylene) carbonylimino]] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF 157) and 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyl-oxymethyl)-propyl ester (MRS 2395), P2Y11 and P2Y12 purinoceptor antagonist, were without any effect. ATP-induced hyperpolarization was affected by apamin and by P2Y purinoceptor desensitization, but not by MRS 2179. 2-(Methylthio)ATP tetrasodium salt hydrate (2-MeSATP), P2Y1 purinoceptor agonist, at a concentration which did not cause changes in the membrane potential, reduced the amplitude of the fast IJPs. This effect was prevented by MRS 2179. Paired nerve stimulation, either using single pulses or train stimuli, did not cause any alteration of the second-evoked IJP. In conclusion, in the circular muscle of the mouse caecum, ATP is responsible for the fast IJP while nitric oxide is responsible for the slow IJP. ATP-mediated response is dependent on ADPbetaS-sensitive P2Y receptors, which are in part P2Y1, but not P2Y11 or P2Y12 receptor subtypes. In addition, the most substantial finding of this study is the functional demonstration that ATP released by nerve stimulation activates P2Y1 receptors, located prejunctionally, limiting its release by motoneurons.

Inhibitory neuromuscular transmission mediated by the P2Y1 purinergic receptor in guinea pig small intestine

ATP is a putative inhibitory neurotransmitter responsible for inhibitory junction potentials (IJPs) at neuromuscular junctions (IJPs) in the intestine. This study tested the hypothesis that the purinergic P2Y(1) receptor subtype mediates the IJPs. IJPs were evoked by focal electrical stimulation in the myenteric plexus and recorded with "sharp" intracellular microelectrodes in the circular muscle coat. Stimulation evoked three categories of IJPs: 1) purely purinergic IJPs, 2) partially purinergic IJPs, and 3) nonpurinergic IJPs. Purely purinergic IJPs were suppressed by the selective P2Y(1) purinergic receptor antagonist MRS2179. Purely purinergic IJPs comprised 26% of the IJPs. Partially purinergic IJPs (72% of the IJPs) consisted of a component that was abolished by MRS2179 and a second unaffected component. The MRS2179-insensitive component was suppressed or abolished by inhibition of formation of nitric oxide by N(omega)-nitro-l-arginine methyl ester (l-NAME) in some, but not all, IJPs. An unidentified neurotransmitter, different from nitric oxide, mediated the second component in these cases. Nonpurinergic IJPs were a small third category (4%) of IJPs that were abolished by l-NAME and unaffected by MRS2179. Exogenous application of ATP evoked IJP-like hyperpolarizing responses, which were blocked by MRS2179. Application of apamin, which suppresses opening of small-conductance Ca(2+)-operated K(+) channels in the muscle, decreased the amplitude of the purinergic IJPs and the amplitude of IJP-like responses to ATP. The results support ATP as a neurotransmitter for IJPs in the intestine and are consistent with the hypothesis that the P2Y(1) purinergic receptor subtype mediates the action of ATP.

Synaptic inhibition of smooth muscles of the human colon: Effects of vitamin B6 and its derivatives

Spontaneous activity, which is manifested as slow depolarization waves and action potentials, is observed in most (81%) smooth muscles (SMs) of the circular layer of the human colon. Independently of the type of pathology, inhibitory junction potentials (IJPs) in SMs of various parts of the human colon are evoked by intramural stimulation; ranges of parameters of these potentials were comparable with those observed in muscle intestinal fragments isolated at a distance of several tens of centimeters from the zone of injury. In muscle strips (MSs) of such fragments, pyridoxal-5′-phosphate (PPh) applied in different concentrations caused suppression of IJPs: in the concentration of 1·10−8 to 1·10−4 M it decreased the amplitude, and in the concentrations of 1·10−5 to 1·10−4 M and 1·10−4 M, respectively, it decreased rates of the half-amplitude rise and decay of these potentials. Pyridoxal (1·10−4 M) and 4-pyridoxolic acid (1·10−4 M) also caused a drop in the amplitude of IJPs; however, these agents influenced this parameter to a lesser extent, as compared with the effect of 1·10−4 M PPh. Pyridoxine (1·10−4 M) and pyridoxamine (1·10−4 M) evoked no significant changes in the parameters of IJPs in MSs of the human colon. Our data allow us to hypothesize that the suppressing effect of PPh on IJPs is determined by the presence of a purinergic component present in non-adrenergic inhibition of SMs of the human colon.

Synchronization of enteric neuronal firing during the murine colonic MMC

DiI (1,1'didodecyl-3,3,3',3'-tetramethylindocarbecyanine perchlorate) retrograde labelling and intracellular electrophysiological techniques were used to investigate the mechanisms underlying the generation of spontaneously occurring colonic migrating myoelectric complexes (colonic MMCs) in mice. In isolated, intact, whole colonic preparations, simultaneous intracellular electrical recordings were made from pairs of circular muscle (CM) cells during colonic MMC activity in the presence of nifedipine (1-2 microm). During the intervals between colonic MMCs, spontaneous inhibitory junction potentials (IJPs) were always present. The amplitudes of spontaneous IJPs were highly variable (range 1-20 mV) and occurred asynchronously in the two CM cells, when separated by 1 mm in the longitudinal axis. Colonic MMCs occurred every 151 +/- 7 s in the CM and consisted of a repetitive discharge of cholinergic rapid oscillations in membrane potential (range: 1-20 mV) that were superimposed on a slow membrane depolarization (mean amplitude: 9.6 +/- 0.5 mV; half-duration: 25.9 +/- 0.7 s). During the rising (depolarizing) phase of each colonic MMC, cholinergic rapid oscillations occurred simultaneously in both CM cells, even when the two electrodes were separated by up to 15 mm along the longitudinal axis of the colon. Smaller amplitude oscillations (< 5 mV) showed poor temporal correlation between two CM cells, even at short electrode separation distances (i.e. < 1 mm in the longitudinal axis). When the two electrodes were separated by 20 mm, all cholinergic rapid oscillations and IJPs in the CM (regardless of amplitude) were rarely, if ever, coordinated in time during the colonic MMC. Cholinergic rapid oscillations were blocked by atropine (1 microm) or tetrodotoxin (1 microm). Slow waves were never recorded from any CM cells. DiI labelling showed that the maximum projection length of CM motor neurones and interneurones along the bowel was 2.8 mm and 13 mm, respectively. When recordings were made adjacent to either oral or anal cut ends of the colon, the inhibitory or excitatory phases of the colonic MMC were absent, respectively. In summary, during the colonic MMC, cholinergic rapid oscillations of similar amplitudes occur simultaneously in two CM cells separated by large distances (up to 15 mm). As this distance was found to be far greater than the projection length of any single CM motor neurone, we suggest that the generation of each discrete cholinergic rapid oscillation represents a discreet cholinergic excitatory junction potential (EJP) that involves the synaptic activation of many cholinergic motor neurones simultaneously, by synchronous firing in many myenteric interneurones. Our data also suggest that ascending excitatory and descending inhibitory nerve pathways interact and reinforce each other.

Structural differences in the enteric neural network in murine colon: impact on electrophysiology.

The enteric neural network in the proximal murine colon shows a regularly occurring hypoganglionic region, which is here characterized by using anatomical and electrophysiological techniques. Staining with NADPH diaphorase, methylene blue, and cuprolinic blue in standard whole mounts and three-dimensional gut preparations of the murine proximal colon consistently revealed two hypoganglionic areas surrounded by a dense clustering of enteric neurons. This irregularity in the ganglionic plexus was found to be present in mice of three different genetic backgrounds, as well as in rats. The lack of myenteric ganglia in these regions was associated with an absence of the longitudinal muscle layer, as shown in cross sections. Histochemical identification of interstitial cells of Cajal in Kit(W-lacZ/+) transgenic mice showed Kit-positive cells oriented parallel to both muscle layers of the colon. Kit-positive cells oriented parallel to the longitudinal muscle layers were absent in the hypoganglionic area described. Electrical field stimulation elicited TTX-sensitive inhibitory junction potentials (IJPs), which showed region-specific characteristics. The initial partly apamin-sensitive hyperpolarization was present in all parts of the murine colon, whereas a second sustained NG-nitro-L-arginine-sensitive hyperpolarization was absent in the cecum and decreased from the proximal to the distal colon. Dissecting the hypoganglionic area from the surrounding tissue abolished the otherwise normal inhibitory neurotransmission to the circular muscle (1.6 +/- 1.4 and 2.6 +/- 1.7 mV for the fast and slow component of IJP amplitude in the hypoganglionic area vs. 16.5 +/- 1.9 and 23.7 +/- 2.7 mV for the fast and slow component of IJP amplitude in the neuron-rich area, respectively, P < 0.01, n = 6), whereas dissection of an area of identical size with an intact myenteric network showed normal inhibitory neurotransmission, indicating that the hypoganglionic area receives essential functional neural input from the neuron-rich surrounding tissue. In summary, in the murine and rat proximal colon, a constant and distinct hypoganglionic region is described with important concomitant changes in local electrophysiology.

Neuroeffector transmission to different layers of smooth muscle in the rat penile bulb

1. Using intracellular recording techniques, two distinct layers of smooth muscle were identified in the rat penile bulb. The inner muscle layer (parenchyma) exhibited spontaneous action potentials, while the outer sheet (sac) was electrically quiescent. 2. In the parenchyma, transmural stimulation initiated non-adrenergic, non-cholinergic (NANC) inhibitory junction potentials (IJPs) which were abolished by Nomeganitro-L-arginine (LNA) or 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The amplitude of IJPs was reduced by ouabain, dinitrophenol or decreasing the extracellular potassium concentration ([K+]o) but not by several K+ channel blockers. 3. The parenchyma also received an excitatory innervation mediated by alpha-adrenoceptors which caused a contraction that was not associated with a membrane potential change. 4. In the sac, transmural stimulation initiated two component excitatory junction potentials (EJPs) mediated by alpha-adrenoceptors and associated action potentials. The initial component was more dramatically suppressed than the secondary component by caffeine, ryanodine or cyclopiazonic acid (CPA). Lowering of the extracellular chloride concentration ([Cl-]o) selectively inhibited the rapid component of EJPs, while niflumic acid was less potent. 5. These results suggest that IJPs in the parenchyma result from the release of NO which stimulates sodium pump activity following the activation of guanylate cyclase. In the sac, the activation of alpha-adrenoceptors initiates EJPs by releasing Ca2+ from intracellular stores which activates Ca2+-activated channels.

Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO.

1. The rat colonic circular muscle displays cyclic episodes of myenteric potential oscillations (MPOs), each of them associated with a spontaneous contraction. Nifedipine 1 microM abolished both MPOs and their associated contractions. TTX (1 microM) increased the amplitude and frequency of spontaneous contractions. 2. Electrical field stimulation (EFS) induced a non-adrenergic non-cholinergic (NANC) inhibitory junction potential (IJP), with two phases: an initial fast hyperpolarization (characterized by IJP amplitude) and a sustained hyperpolarization (characterized by IJP duration). 3. Sodium nitroprusside (10 microM) hyperpolarized and abolished spontaneous contractions even in presence of TTX or 1 microM apamin. ATP (100 microM) also hyperpolarized and abolished spontaneous contractions but its effects were decreased by TTX and abolished by apamin. 4. Suramin (100 microM) or apamin reduced the amplitude of the IJPs, but did not affect their duration. Incubation with L-NOARG (1 mM) reduced the duration but not the amplitude of the IJPs. In presence of L-NOARG plus suramin or L-NOARG plus apamin, both duration and amplitude of the IJPs were reduced but a residual IJP could still be recorded. 5. We conclude that the mechanical and electrical cyclic activity of the rat colonic circular muscle is modulated but not originated by the enteric nervous system and involves L-type calcium channel activity. EFS induces release of NANC inhibitory neurotransmitters which hyperpolarize and relax smooth muscle cells. Both ATP and NO are involved in IJP generation: ATP is responsible for the first phase of the IJPs involving activation of apamin-sensitive potassium channels, whereas NO initiates the second phase which is independent of the activation of such channels.

Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum.

1. The role of NK1 and NK3 receptors in synaptic transmission between myenteric neurons during motility reflexes in the guinea-pig ileum was investigated by recording intracellularly the reflex responses of the circular muscle to distension or compression of the mucosal villi. Experiments were performed in a three-chambered organ bath that enabled drugs to be selectively applied to different sites along the reflex pathways. 2. When applied in the recording chamber, an NK1 receptor antagonist, SR140333 (100 nM), reduced by 40-50% the amplitudes of inhibitory junction potentials (i.j.ps) evoked in the circular muscle by activation of descending reflex pathways. This effect was abolished when synaptic transmission in the stimulus region was blocked with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+, leaving only the component of the descending reflex pathway conducted via long anally directed collaterals of intrinsic sensory neurons. 3. SR140333 (100 nM) had no effect on descending reflex i.j.ps when applied to the stimulus region. Ascending reflexes were also unaffected by SR140333 in the stimulus region or between the stimulus and recording sites. 4. Septide (10 nM), an NK1 receptor agonist, enhanced descending reflexes by 30-60% when in the recording chamber. [Sar9,Met(O2)11]substance P had no effect at 10 nM, but potentiated distension-evoked reflexes at 100 nM. 5. A selective NK3 receptor antagonist, SR142801 (100 nM), when applied to the stimulus region, reduced the amplitude of descending reflex responses to compression by 40%, but had no effect on responses to distension. SR142801 (100 nM) had no effect when applied to other regions of the descending reflex pathways. 6. SR142801 (100 nM) only inhibited ascending reflexes when applied at the recording site. However, after nicotinic transmission in the stimulus region was blocked, SR142801 (100 nM) at this site reduced responses to compression. 7. Contractions of the circular muscle of isolated rings of ileum evoked by low concentrations of septide, but not [Sar9,Met(O2)11]substance P, were potentiated by tetrodotoxin (300 nM). 8. Contractile responses evoked by an NK3 receptor agonist, senktide, were non-competitively inhibited by SR142801. After excitatory neuromuscular transmission was blocked, senktide produced inhibitory responses that were also antagonised by SR142801, but to a lesser extent and in an apparently competitive manner. 9. These results indicate that tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons. NK3 receptors play a role in transmission from intrinsic sensory neurons and from ascending interneurons to excitatory motor neurons during motility reflexes.