Slow Inhibitory Junction Potentials Research Articles

Diminished enteric neuromuscular transmission in the distal colon following experimental spinal cord injury

Neurogenic bowel following spinal cord injury (SCI) leads to decreased colonic motility, remodeling of the neuromuscular compartment and results in chronic evacuation difficulties. The distal colon of the rat serves a dual role for fluid absorption and storage that is homologous to the descending colon of humans. Dysmotility of the descending colon is one component of neurogenic bowel. We investigated the integrity of the enteric neuromuscular transmission responsible for the generation of excitatory and inhibitory junction potentials (EJPs and IJPs, respectively) in the distal colon of rats. We previously demonstrated a chronic reduction in colonic enteric neurons from rats with acute and chronic high-thoracic (T3) SCI and hypothesized that neurogenic bowel following T3-SCI results from diminished enteric neuromuscular transmission.Immunohistochemical labeling for myenteric neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) neurons demonstrated a significant loss of presumptive nitric oxide (NO) and acetylcholine (ACh) immunoreactive neurons in both 3-day and 3-week injured animals. Colonic neuromuscular transmission in response to transmural electrical stimulation of the colon was significantly reduced 3-days and 3-weeks following SCI in male rats. Specifically, cholinergic-mediated excitatory junction potentials (EJPs) and nitrergic-mediated slow inhibitory junction potentials (IJPs) were significantly reduced while ATP-mediated fast IJPs remained unaffected.We conclude that a reduction in excitatory and inhibitory enteric neuromuscular transmission contributes to neurogenic bowel observed following SCI, and that these loss-of-function changes involve enteric-mediated cholinergic and nitrergic pathways.

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<0.01) in oxidized/reduced glutathione fluorescence ratio in nNOS neurons of HF mice. HF mice had lower (28%, p<0.01) nNOS neurons per ganglionic area than controls. Fluorescence intensity of DAF‐FM diacetate was also lower (20%, p<0.01) in HF mice. HF mice showed a significant loss (52%, p<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).

Myosin Va but Not nNOSα is Significantly Reduced in Jejunal Musculomotor Nerve Terminals in Diabetes Mellitus.

Nitric oxide (NO) mediated slow inhibitory junction potential and mechanical relaxation after electrical field stimulation (EFS) is impaired in diabetes mellitus. Externally added NO donor restore nitrergic function, indicating that this reduction result from diminution of NO synthesis within the pre-junctional nerve terminals. The present study aimed to investigate two specific aims that may potentially provide pathophysiological insights into diabetic nitrergic neuropathy. Specifically, alteration in nNOSα contents within jejunal nerve terminals and a local subcortical transporter myosin Va was tested 16 weeks after induction of diabetes by low dose streptozotocin (STZ) in male Wistar rats. The results show that diabetic rats, in contrast to vehicle treated animals, have: (a) nearly absent myosin Va expression in nerve terminals of axons innervating smooth muscles and (b) significant decrease of myosin Va in neuronal soma of myenteric plexus. In contrast, nNOSα staining in diabetic jejunum neuromuscular strips showed near intact expression in neuronal cell bodies. The space occupancy of nitrergic nerve fibers was comparable between groups. Normal concentration of nNOSα was visualized within a majority of nitrergic terminals in diabetes, suggesting intact axonal transport of nNOSα to distant nerve terminals. These results reveal the dissociation between presences of nNOSα in the nerve terminals but deficiency of its transporter myosin Va in the jejunum of diabetic rats. This significant observation of reduced motor protein myosin Va within jejunal nerve terminals may potentially explain impairment of pre-junctional NO synthesis during EFS of diabetic gut neuromuscular strips despite presence of the nitrergic synthetic enzyme nNOSα.

Sa2046 Effect of RM-131 on Circular Smooth Muscle Cells in Human and Mouse Colon and on Colonic Intraluminal Pressure in Conscious Mice

Background and Aims: RM-131 is a synthetic ghrelin agonist that has been found to significantly accelerate gastric emptying, intestinal transit and colonic filling. The mechanism(s) through which smooth muscle cells of gastrointestinal tract respond to RM-131 is not known. We studied the effect of RM-131 on circular smooth muscle cells of human and mouse colon in vitro and on colonic intraluminal pressure in conscious mice. Methods: SJL/J mice and human colonic tissue were used in this study. Normal human colon was obtained from surgical excess tissue. The use of mice was approved by Mayo Institutional Animal Care and Use Committee and the use of human biopsy specimens was approved by Institutional Review Board. Muscle strips without the mucosa from mouse colon and human colon strips were placed in a recording chamber for intracellular recording. The recording chamber was superfused with oxygenized normal Krebs solution at 37°C. A bipolar stimulating electrode was placed on both sides of the muscle strip for electric field stimulation to elicit inhibitory junction potentials (IJPs). To test the effect of RM-131 on colonic intraluminal pressure in conscious mice, a miniaturized pressure transducer catheter was introduced into the colon such that the middle of the pressure sensor was 2.5 cm proximal to the anus. Results: RM-131 (0.25 μM) significantly hyperpolarized the resting membrane potential for human colonic circular smooth muscle cells by 2.8±0.7 mV (P<0.01, n=9) and in mouse preparations by 3.4±0.4 mV (P<0.01, n=6). RM-131 had no significant effect on either the fast IJP or the slow IJP. Ghrelin (1 μM) also hyperpolarized the resting membrane potential of circular smooth muscle cells in all cells recorded from human preparations by 3.5±1.4 mV (P=0.06, n=3) and in mouse preparations by 3.8±0.8 mV (P<0.01, n=7). Ghrelin also had no significant effect on either the fast IJP or slow IJP. For intraluminal pressure recordings, RM-131 or control saline was applied via intraperitoneal injection. Intraluminal pressure changes with RM-131 or saline were normalized with their pre-control. In the RM-131 treated group, intraluminal pressure was 0.57±0.05 (n=8) which was significantly less compared to the control group (0.82±0.08, n=8), suggesting that RM131 significantly reduced colonic tone. Conclusions: Our results suggest that RM-131 decreases colonic tone by hyperpolarizing the resting membrane potential of circular smooth muscle cells and decreases the excitability of colonic circular smooth muscle cells. These effects of RM-131 are likely the basis for the decrease in colonic intraluminal pressure. The previously reported increase in upper GI contractility together with a decrease in colonic intraluminal pressure position RM-131 for the treatment of functional gastrointestinal disorders.

Sa2047 Estrogen Causes Relaxation of Human Gallbladder via G Protein-Coupled Estrogen Receptors

Background and Aims: RM-131 is a synthetic ghrelin agonist that has been found to significantly accelerate gastric emptying, intestinal transit and colonic filling. The mechanism(s) through which smooth muscle cells of gastrointestinal tract respond to RM-131 is not known. We studied the effect of RM-131 on circular smooth muscle cells of human and mouse colon in vitro and on colonic intraluminal pressure in conscious mice. Methods: SJL/J mice and human colonic tissue were used in this study. Normal human colon was obtained from surgical excess tissue. The use of mice was approved by Mayo Institutional Animal Care and Use Committee and the use of human biopsy specimens was approved by Institutional Review Board. Muscle strips without the mucosa from mouse colon and human colon strips were placed in a recording chamber for intracellular recording. The recording chamber was superfused with oxygenized normal Krebs solution at 37°C. A bipolar stimulating electrode was placed on both sides of the muscle strip for electric field stimulation to elicit inhibitory junction potentials (IJPs). To test the effect of RM-131 on colonic intraluminal pressure in conscious mice, a miniaturized pressure transducer catheter was introduced into the colon such that the middle of the pressure sensor was 2.5 cm proximal to the anus. Results: RM-131 (0.25 μM) significantly hyperpolarized the resting membrane potential for human colonic circular smooth muscle cells by 2.8±0.7 mV (P<0.01, n=9) and in mouse preparations by 3.4±0.4 mV (P<0.01, n=6). RM-131 had no significant effect on either the fast IJP or the slow IJP. Ghrelin (1 μM) also hyperpolarized the resting membrane potential of circular smooth muscle cells in all cells recorded from human preparations by 3.5±1.4 mV (P=0.06, n=3) and in mouse preparations by 3.8±0.8 mV (P<0.01, n=7). Ghrelin also had no significant effect on either the fast IJP or slow IJP. For intraluminal pressure recordings, RM-131 or control saline was applied via intraperitoneal injection. Intraluminal pressure changes with RM-131 or saline were normalized with their pre-control. In the RM-131 treated group, intraluminal pressure was 0.57±0.05 (n=8) which was significantly less compared to the control group (0.82±0.08, n=8), suggesting that RM131 significantly reduced colonic tone. Conclusions: Our results suggest that RM-131 decreases colonic tone by hyperpolarizing the resting membrane potential of circular smooth muscle cells and decreases the excitability of colonic circular smooth muscle cells. These effects of RM-131 are likely the basis for the decrease in colonic intraluminal pressure. The previously reported increase in upper GI contractility together with a decrease in colonic intraluminal pressure position RM-131 for the treatment of functional gastrointestinal disorders.

Effect of endogenous hydrogen sulfide on the transwall gradient of the mouse colon circular smooth muscle

A transwall gradient in resting membrane potential (RMP) exists across the circular muscle layer in the mouse colon. This gradient is dependent on endogenous generation of CO. H2S is also generated in muscle layers of the mouse colon. The effect of endogenously generated H2S on the transwall gradient is not known. The aim was to investigate the role of endogenous H2S. Our results showed that the CSE inhibitor dl-propargylglycine (PAG, 500μm) had no effect on the transwall gradient. However, in preparations pretreated with the nitric oxide synthase inhibitor N-nitro-l-arginine (l-NNA, 200μm) and in nNOS-knockout (KO) mouse preparations, PAG shifted the transwall gradient in the depolarizing direction. In CSE-KO-nNOS-KO mice, the gradient was shifted in the depolarizing direction. Endogenous generation of NO was significantly higher in muscle preparations of CSE-KO mice compared to wild-type (WT) mice. The amplitude of NO-mediated slow inhibitory junction potentials (S-IJPs) evoked by electric field stimulation was significantly higher in CSE-KO mouse preparations compared to the amplitude of S-IJPs in wild-type mouse preparations. CSE was present in all submucosal ganglion neurons and in almost all myenteric ganglion neurons. Eleven per cent of CSE positive neurons in the submucosal plexus and 50% of CSE positive neurons in the myenteric plexus also contained nNOS. Our results suggest that endogenously generated H2S acts as a stealth hyperpolarizing factor on smooth muscle cells to maintain the CO-dependent transwall gradient and inhibits NO production from nNOS.

Resolution and concordance in dissecting the compound inhibitory junction potential

Resolution and concordance in dissecting the compound inhibitory junction potential

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.

Characterization of Ascending Limb of Peristaltic Reflex in Distal Colon of W/W V Wild Type and Mutant Mice

Background: Ascending neuromuscular reflexes induced by distension play an important role in gastrointestinal motility. However, the mechanisms underlying this reflex in colon is incompletely understood. AIM: To investigate nerve stimulation and balloon distention (BD)-mediated ascending responses in circular and longitudinal smooth muscle (CSM; LSM) of murine distal colon. METHODS: Conventional intracellular recordings were performed in colonic LSM and CSM fromW/Wvwild-type and CSM fromW/Wvmutant mice. Responses to nerve stimulation (NS; 4 square wave pulses, each 0.3 ms duration, 20 Hz) and BD (0.1 ml, 10 s) were measured in proximal end of a ~ 1.5 cm segment of distal colon. A balloon was placed in the lumen of distal end and a pair of transmural NS electrodes were positioned between the balloon and recording sites. Tissue preparations were perfused with Krebs' solution at 36oC. RESULTS: In the CSM, NS evoked mono-phasic inhibitory junction potentials (IJP) with amplitude of 35.4 ± 1.8 mV and duration of 795 ± 39 ms, and BD induced depolarization of 5.7 ± 1.8 mV (n = 5) that reached a peak in 4 7 s and was maintained until the termination of the BD. Spontaneous action potentials (APs) were superimposed on the depolarization. Application of atropine (3 μM) to the bath significantly increased the NS-induced IJP from 35.4 ± 1.8 mV to 38.5 ± 1.8 mV, but abolished the BDinduced depolarization. In the LSM, NS produced a biphasic IJP; a fast IJP with amplitude of 22.7 ± 1.6 mV and duration of 1071 ± 42 ms, following by a slow IJP (sIJP) with amplitude of 4.6 ± 1.3 mV and duration of 3236 ± 370 ms (n = 3). BD evoked an initial IJP with amplitude of 4.8 ± 3.8 mV and duration of 489 ± 128 ms, following by a train of APs. MRS-2500 (1 μM), a P2Y1 antagonist, abolished the initial IJP, whereas atropine abolished the APs. In CSM from W/Wv mutant mice, responses to NS were no different from controls. However, the depolarization in response to BD was attenuated and delayed in onset until after the balloon was deflated. CONCLUSIONS: These data indicate that in the murine distal colon: 1) Ascending response induced by BD differs from that induced by the NS; 2) Ascending response evoked by the BD in CSM differs from LSM; 3) Only cholinergic nerves are involved in ascending excitation to both CSM and LSM; 4. Ascending BD, but not NS, responses are impaired in W/Wv mutant mice, suggesting that ICCs may be involved in transducing the BD response.

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.

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.

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.

Regional differences in nitrergic innervation of the smooth muscle of murine lower oesophageal sphincter

Anatomical and pharmacological studies have demonstrated that the lower oesophageal sphincter (LES) is not a simple homogenous circular muscle with uniform innervation. Regional differences have been demonstrated in several species including humans. We investigated, for the first time in mice LES, regionally distinct physiological and pharmacological characteristics of the neuromusculature. Conventional intracellular recordings and pharmacological techniques were employed to evaluate electrical properties and functional innervation of smooth muscle cells. Results from CD1 (control), nNOS((-/-)) and eNOS((-/-)) genetic knockout mice were compared. Smooth muscle of sling and clasp LES displayed unitary membrane potentials of 1- 4 mV. Transmural nerve stimulation produced a monophasic inhibitory junction potential (IJP) in the sling, whereas in the clasp a biphasic IJP, consisting of a brief IJP followed by a long-lasting slow IJP (lsIJP), was induced. Pharmacological interventions and genetically modified mice were used to demonstrate a monophasic apamin-sensitive (purinergic) component in both LES regions. However, the nitrergic IJP was monophasic in the sling and biphasic in the clasp. Unitary membrane potentials and IJPs were not different in CD1 and eNOS((-/-)) mice, suggesting no involvement of myogenic NOS. These data in mouse LES indicate that there are previously unreported regional differences in the IJP and that both the apamin-resistant monophasic and biphasic IJPs are mediated primarily by nitrergic innervation.

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.

ARE GAP JUNCTIONS TRULY INVOLVED IN INHIBITORY NEUROMUSCULAR INTERACTION IN MOUSE PROXIMAL COLON?

1. Gap junctions exist between circular muscle cells of the colon and between interstitial cells of Cajal (ICC) in the myenteric plexus of the gastrointestinal tract. They also probably couple intramuscular ICC with smooth muscle cells. Recent functional evidence for this was found in dye-coupling and myoelectrical experiments. 2. In the present study, we tested the hypothesis of gap junctions putatively being involved in neuromuscular interaction in mouse colon by using different classes of gap junction blockers. 3. Electrical field stimulation of the myenteric plexus elicited tetrodotoxin-sensitive and hexamethonium-independent fast and slow inhibitory junction potentials (fIJP and sIJP, respectively) in circular smooth muscle cells, as evaluated by intracellular recording techniques in impaled smooth muscle cells. Heptanol produced a time-dependent hyperpolarization of the membrane potential (MP) and abolished fIJP and sIJP. Octanol had no effect on the MP and abolished fIJP and sIJP. Carbenoxolone produced a time-dependent depolarization of the MP without any effect on fIJP or sIJP. The connexin 43 mimetic gap junction blocker GAP-27 had no effect on MP, fIJP or sIJP. 4. Based on the presently available gap junction blockers we found no evidence that gap junctions are involved in neuromuscular transmission in mouse colon, as suggested by morphological studies.

Excitatory purinergic neurotransmission in smooth muscle of guinea-pig [corrected] taenia caeci.

Non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmission has been an area of intense interest in gut motor physiology, whereas excitatory NANC neurotransmission has received less attention. In order to further explore excitatory NANC neurotransmission, we performed conventional intracellular recordings from guinea-pig taenia caeci smooth muscle. Tissue was perfused with oxygenated Krebs solution at 35 degrees C and nerve responses evoked by either oral or aboral nerve stimulation (NS) (4 square wave pulses, 0.3 ms duration, 20 Hz). Electrical activity was characterized by slow waves upon which one to three action potentials were superimposed. Oral NS evoked an inhibitory junction potential (IJP) at either the valley or peak of the slow wave. Application of nifedipine (1 microM) abolished slow waves and action potentials, but membrane potential flunctuations (1-3 mV) and IJPs remained unaffected. Concomitant application of apamin (300 nM), a small-conductance Ca(2+)-activated K(+) channel blocker, converted the IJP to an EJP that was followed by slow IJP. Further administration of N(G)-nitro-l-arginine methyl ester (l-NAME, 200 microM), a nitric oxide synthase inhibitor, abolished the slow IJP without affecting the EJP, implying that the slow IJP is due to nitrergic innervation. The EJP was abolished by tetrodotoxin (1 microM), but was not significantly affected by atropine (3 microM) and guanethidine (3 microM) or hexamethonium (500 microM). Substance P (SP, 1 microM) desensitization caused slight attenuation of the EJP, but the EJP was abolished by desensitization with alpha,beta-methylene ATP (50 microM), a P2 purinoceptor agonist that is more potent than ATP at the P2X receptor subtype, suramin (100 microM), a non-selective P2 purinoceptor antagonist, and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM) , a selective P2X purinoceptor antagonist. In contrast, the EJP was unaffected by MRS-2179 (2 microM), a selective P2Y(1) receptor antagonist. Aboral NS evoked an apamin- and l-NAME-sensitive IJP, but virtually no NANC EJP. These data suggest the presence of polarized excitatory purinergic neurotransmission in guinea-pig taenia caeci, which appears to be mediated by P2X purinoceptors, most likely the P2X(1) subtype.

Role of sarcoplasmic reticulum in control of membrane potential and nitrergic response in opossum lower esophageal sphincter.

1. We previously demonstrated that a balance of Ca2+-activated Cl- current (ICl(Ca)) and K+ current activity sets the resting membrane potential of opossum lower esophageal sphincter (LES) circular smooth muscle at approximately -41 mV, which leads to continuous spike-like action potentials and the generation of basal tone. Ionic mechanisms underlying this basal ICl(Ca) activity and its nitrergic regulation remain unclear. Recent studies suggest that spontaneous Ca2+ release from sarcoplasmic reticulum (SR) and myosin light chain kinase (MLCK) play important roles. The current study investigated this possibility. Conventional intracellular recordings were performed on circular smooth muscle of opossum LES. Nerve responses were evoked by electrical square wave pulses of 0.5 ms duration at 20 Hz. 2. In the presence of nifedipine (1 microm), substance P (1 microm), atropine (3 microm) and guanethidine (3 microm), intracellular recordings demonstrated a resting membrane potential (MP) of -38.1+/-0.7 mV (n=25) with spontaneous membrane potential fluctuations (MPfs) of 1-3 mV. Four pulses of nerve stimulation induced slow inhibitory junction potentials (sIJPs) with an amplitude of 6.1+/-0.3 mV and a half-amplitude duration of 1926+/-147 ms (n=25). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific guanylyl cyclase inhibitor, abolished sIJPs, but had no effects on MPfs. Caffeine, a ryanodine receptor agonist, hyperpolarized MP and abolished sIJPs and MPfs. Ryanodine (20 microm) inhibited the sIJP and induced biphasic effects on MP, an initial small hyperpolarization followed by a large depolarization. sIJPs and MPfs were also inhibited by cyclopiazonic acid, an SR Ca2+ ATPase inhibitor. Specific ICl(Ca) and MLCK inhibitors hyperpolarized the MP and inhibited MPfs and sIJPs. 4. These data suggest that (1). spontaneous release of Ca2+ from the SR activates ICl(Ca), which in turn contributes to resting membrane potential; (2). MLCK is involved in activation of ICl(Ca); (3). inhibition of ICl(Ca) is likely to underlie sIJPs induced by nitrergic innervation.

Nitrergic prejunctional inhibition of purinergic neuromuscular transmission in the hamster proximal colon.

Neurogenic ATP and nitric oxide (NO) may play important roles in the physiological control of gastrointestinal motility. However, the interplay between purinergic and nitrergic neurons in mediating the inhibitory neurotransmission remains uncertain. This study investigated whether neurogenic NO modulates the purinergic transmission to circular smooth muscles of the hamster proximal colon. Electrical activity was recorded from circular muscle cells of the hamster proximal colon by using the microelectrode technique. Intramural nerve stimulation with a single pulse evoked a fast purinergic inhibitory junction potential (IJP) followed by a slow nitrergic IJP. The purinergic component of the second IJP evoked by paired stimulus pulses at pulse intervals between 1 and 3 s became smaller than that of the first IJP. This purinergic IJP depression could be observed at pulse intervals <3 s, but not at longer ones, and failed to occur in the presence of NO synthase inhibitor. Exogenous NO (0.3-1 microM), at which no hyperpolarization is produced, inhibited purinergic IJPs, without altering the nitrergic IJP and exogenously applied ATP-induced hyperpolarization. In the presence of both purinoceptor antagonist and nitric oxide synthase (NOS) inhibitor, intramural nerve stimulation with 5 pulses at 20 Hz evoked vasoactive intestinal peptide (VIP)-associated IJPs, suggesting that VIP component may be masked in the IJPs of the hamster proximal colon. Our results suggest that neurogenic NO may modulate the purinergic transmission to circular smooth muscles of the hamster proximal colon via a prejunctional mechanism. In addition, VIP may be involved in the neurotransmitter in the hamster proximal colon.

Host and microbial determinants influence Helicobacter pylori-induced gastric carcinogenesis in a murine model of hypergastrinemia

CB1 receptor agonist anandamide, the synthetic CB1 receptor agonist WIN 55,212-2 and the CBI specific antagonist SR141716 on smooth muscle membrane potentials following electrical neuronal stimulation. Experiments were performed in wild-type as well as in CB1deficient mice. Results: Focal electrical stimulation of the myenteric plexus induced a fast excitatory junction potential (EJP; abolished by atropine) followed by a fast inhibitory junction potential (fliP; reduced by apamin) and a slow inhibitory junction potential (slJP; abolished by L-NNA). None of the used drugs had an influence on the resting membrane potential. Addition of anandamide (10-6-10-5M) in the wildtype abolished the EJP while leaving the flJP and the slJP unchanged. The more potent CB1 agonist WIN 55,212-2 reduced the EJP and the flJP but did not change the sUP. In the CB1 deficient mice anandamide and WIN 55,212-2 had no effect. SR141716 when given alone increased the EJP in wildtype mice while leaving the IJPs in the wildtype and the EJPs and IJPs in the knockout mice unchanged. 5R141716 reversed all anandamide and WIN 55,212-2 effects in the wildtype mice. In the CB 1 defficient mice, the EJFs are significantly increased compared to wildtype, the UPs remein unaltered. Conclusions: We conclude that the CB1 eannabinoidreceptor modulates excitatory and inhibitory neurotranamission in the mouse colon. Since in the CB1 deficient mice EJPs are sigmficantly increased these findings strongly suggesting a physiologic involvement of CB1 receptors in cholinergic neurotransmission. Finally, our study supports that no other receptors than CB1 are involved in the anandamide action on mouse distal colon since the endogenous agonist anandamide had no effect in CB1deficient mice.