Colonic Longitudinal Smooth Muscle Research Articles

Therapeutic Effects of Berberine Hydrochloride on Stress-Induced Diarrhea-Predominant Irritable Bowel Syndrome Rats by Inhibiting Neurotransmission in Colonic Smooth Muscle.

The etiology of diarrhea-predominant irritable bowel syndrome (IBS-D) is complicated and closely related to neurotransmission in the gastrointestinal (GI) tract. Developing new strategies for treating this disease is a major challenge for IBS-D research. Berberine hydrochloride (BBH), the derivative of berberine, is a herbal constituent used to treat IBS. Previous studies have shown that BBH has potential anti-inflammatory, antibacterial, analgesic, and antidiarrheal effects and a wide range of biological activities, especially in regulating the release of some neurotransmitters. A modified IBS-D rat model induced by chronic restraint stress was used in all experiments to study the effects of BBH on the GI tract. This study measured the abdominal withdrawal reflex (AWR) response to graded colorectal distention (CRD; 20, 40, 60, and 80 mmHg) and observed the fecal areas of stress-induced IBS-D model. Experiments were conducted using organ bath techniques, which were performed in vitro using strips of colonic longitudinal smooth muscle. Inhibitory and excitatory neurotransmitter agents were added to each organ bath to observe contractile responses on the strips and the treatment effect exerted by BBH. The IBS-D rat model was successfully induced by chronic restraint stress, which resulted in an increased defecation frequency and visceral hypersensitivity similar to that of humans. BBH could reduce 4-h fecal areas and AWR response to CRD in IBS-D. The stress-induced IBS-D model showed upregulated colonic mRNA expression levels of 5-hydroxytryptamine-3A receptor and downregulated expression levels of neuronal nitric oxide synthase. Meanwhile, BBH could reverse this outcome. The responses of substances that regulate the contraction induced by related neurotransmission in the longitudinal smooth muscle of IBS-D colon (including the agonist of acetylcholine, carbachol; NOS inhibitor, L-NAME; and P2Y1 receptor antagonist, MRS2500) can be inhibited by BBH. In summary, BBH promotes defecation frequency and visceral hypersensitivity in IBS-D and exerts inhibitory effects on contractile responses in colonic longitudinal smooth muscle. Thus, BBH may represent a new therapeutic approach for treating IBS-D.

Effects of experimental colitis in rats on incretin levels, inflammatory markers, and enteric neuronal function.

IntroductionThe aim of the study was to assess the effects of chronic inflammation on incretin levels, inflammatory markers, and enteric neuronal function measured in isolated preparations of smooth muscle of rat.Material and methodsWe induced experimental colitis using 2,4-dinitrobenzenesulfonic acid (DNBS) in 17 Albino male Sprague-Dawley rats, while 16 rats were used as a control. They were housed in temperature-controlled rooms in a 12-h light/dark cycle at 22–24°C and 50 to 60% humidity. We evaluated in both inflamed and healthy rats: fasting plasma glucose concentration, fasting plasma insulin, myeloperoxidase, active glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and GLP-2 levels, adiponectin, and C-reactive protein (CRP). We also evaluated colonic longitudinal smooth muscle contractile activity.ResultsIntrarectal administration of DNBS reduced body weight gain in inflamed rats. We recorded higher levels of fasting plasma glucose, and insulin in inflamed rats. We observed higher levels of myeloperoxidase and CRP, and lower levels of ADN in inflamed rats. We recorded higher levels of GIP, GLP-1, and GLP-2 in inflamed rats compared to the healthy ones. Regarding functional response of colon intestinal smooth muscle after electrical stimulation, we recorded a lower functional response of colon intestinal smooth muscle after electrical stimulation in inflamed rats.ConclusionsWe can conclude that chronic inflammation leads to an increase of incretin levels and to a decrease of functional response of colon intestinal smooth muscle after electrical stimulation.

Shugan Decoction Alleviates Colonic Dysmotility in Female SERT-Knockout Rats by Decreasing M3 Receptor Expression.

BackgroundIrritable bowel syndrome (IBS) is a functional gut disease characterized by visceral hypersensitivity and gut motor dysfunction. Serotonin (5-hydroxytryptamine, 5-HT) is an important enteric neurotransmitter. High levels of 5-HT aggravate IBS symptoms. The serotonin reuptake transporter (SERT) is a membrane-embedded transporter involved in IBS pathogenesis that plays an important role in regulating 5-HT signaling.AimWe investigated whether gut motor function was altered in SERT-knockout (SERT-KO) rats. Additionally, we sought to determine whether Shugan decoction (SGD), a clinically experienced prescription for the treatment of IBS, exerts regulatory effects on intestinal motility in SERT-KO rats, and attempted to identify the mechanisms involved.MethodSERT-KO rats were produced by transcription activator-like effector nuclease (TALEN) technology. Fecal pellet output was measured for ten consecutive days to estimate distal colonic motility. Small intestinal motility was measured by charcoal-meal experiments. The colonic and small intestinal muscle contractile activities were measured by organ bath study. Western blot was used to analyze the muscarinic receptor expression in colon tissue.ResultCompared with that in wild-type (WT) rats, the defecation amount, amplitude of spontaneous contraction, and the tension of ACh-induced contraction of colonic longitudinal smooth muscle in SERT-KO rats were significantly increased. The expression of muscarinic receptor subtype-3 (M3R) in the colons of SERT-KO rats was also elevated. SGD can decrease defecation of SERT-KO rats. Moreover, SGD reduced the amplitude of spontaneous contraction, the frequency and tension of ACh-induced contraction of colonic longitudinal smooth muscle, and the expression of M3R in the colon in SERT-KO rats.ConclusionsSERT-KO rats showed increased defecation accompanied by enhanced colonic motility and M3R expression. The findings suggest that SGD modifies colonic dysmotility and reduces defecation in SERT-KO rats by down-regulating M3R expression in the colon.

Effects of Fengliao-Changweikang in Diarrhea-predominant Irritable Bowel Syndrome Rats and Its Mechanism Involving Colonic Motility.

Background/AimsThis study was designed to investigate the effect of Fengliao-Changweikang (FLCWK) in diarrhea-predominant irritable bowel syndrome (IBS-D) rats and explore its underlying mechanisms.MethodsIBS-D model rats were induced by neonatal maternal separation (NMS) combined with restraint stress (RS). In in vivo experiments, the model rats were randomly divided into 5 groups: NMS + RS, FLCWK (low dose, middle dose, and high dose), and pinaverium bromide. The normal control (no handling) rats were classified as the NH group. The therapeutic effect of FLCWK was evaluated by fecal characteristics, electromyographic response and abdominal withdrawal reflex scores. In in vitro experiments, the model rats were randomly divided into 2 groups: NMS + RS, FLCWK (middle dose), and no handling rats were used as the NH group. The differences in basic tension and ACh-induced tension of isolated colonic longitudinal smooth muscle strips (CLSMs) among the 3 groups were observed. In addition, different inhibitors (nifedipine, TMB-8, L-NAME, methylene blue, and 4-AP) were pretreated to explore the underlying mechanisms.ResultsIn in vivo experiments, fecal characteristics, electromyographic response, and abdominal withdrawal reflex scores significantly improved in the FLCWK group, compared with the NMS + RS group. In in vitro experiments, the basic tension and ACh-induced tension of CLSMs in IBS-D rats were significantly inhibited by FLCWK. After pre-treatment with different inhibitors, the ACh-induced tension of CLSMs in each group showed no significant difference.ConclusionsFLCWK manifested curative effect in IBS-D rats by inhibiting colonic contraction. The underlying mechanisms may be related to regulatory pathway of nitric oxide/cGMP/Ca2+ and specific potassium channels.

Inhibitory effects of patchouli alcohol on stress-induced diarrhea-predominant irritable bowel syndrome.

AIMTo elucidate the mechanism of patchouli alcohol (PA) in treatment of rat models of diarrhea-predominant irritable bowel syndrome (IBS-D).METHODSWe studied the effects of PA on colonic spontaneous motility using its cumulative log concentration (3 × 10−7 mol/L to 1 × 10−4 mol/L). We then determined the responses of the proximal and distal colon segments of rats to the following stimuli: (1) carbachol (1 × 10−9 mol/L to 1 × 10−5 mol/L); (2) neurotransmitter antagonists including Nω-nitro-L-arginine methyl ester hydrochloride (10 μmol/L) and (1R*, 2S*)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (1 μmol/L); (3) agonist α,β-methyleneadenosine 5′-triphosphate trisodium salt (100 μmol/L); and (4) single KCl doses (120 mmol/L). The effects of blockers against antagonist responses were also assessed by pretreatment with PA (100 μmol/L) for 1 min. Electrical-field stimulation (40 V, 2-30 Hz, 0.5 ms pulse duration, and 10 s) was performed to observe nonadrenergic, noncholinergic neurotransmitter release in IBS-D rat colon. The ATP level of Kreb’s solution was also determined.RESULTSPA exerted a concentration-dependent inhibitory effect on the spontaneous contraction of the colonic longitudinal smooth muscle, and the half maximal effective concentration (EC50) was 41.9 μmol/L. In comparison with the KCl-treated IBS-D group, the contractile response (mg contractions) in the PA + KCl-treated IBS-D group (11.87 ± 3.34) was significantly decreased in the peak tension (P < 0.01). Compared with CCh-treated IBS-D rat colon, the cholinergic contractile response of IBS-D rat colonic smooth muscle (EC50 = 0.94 μmol/L) was significantly decreased by PA (EC50 = 37.43 μmol/L) (P < 0.05). Lack of nitrergic neurotransmitter release in stress-induced IBS-D rats showed contraction effects on colonic smooth muscle. Pretreatment with PA resulted in inhibitory effect on L-NAME-induced (10 μmol/L) contraction (P < 0.05). ATP might not be the main neurotransmitter involved in inhibitory effects of PA in the colonic relaxation of stress-induced IBS-D rats.CONCLUSIONPA application may serve as a new therapeutic approach for IBS-D.

Nitrergic signaling via interstitial cells of Cajal and smooth muscle cells influences circular smooth muscle contractility in murine colon.

Regulation of gastrointestinal motility involves excitatory and inhibitory neurotransmission. Nitric oxide (NO), the major inhibitory neurotransmitter, acts via its receptor NO-sensitive guanylyl cyclase (NO-GC). In the GI tract, NO-GC is expressed in several cell types such as smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Using cell-specific knockout mice, we have previously shown that NO-GC modulates spontaneous contractions in colonic longitudinal smooth muscle. However, its detailed role in the colonic circular smooth muscle is still unclear. Myography was performed to evaluate spontaneous contractions in rings of proximal colon (2.5mm) from global (GCKO) and cell-specific knockout mice for NO-GC. Immunohistochemistry and in situ hybridization were used to specify NO-GC expression. Colonic circular smooth muscle showed three different contraction patterns: high-frequency ripples, slow phasic contractions, and large contractions. Ripples formed independently of NO-GC. Slow phasic contractions occurred intermittently in WT, SMC-GCKO, and ICC-GCKO tissue, whereas they were more prominent and prolonged in GCKO and SMC/ICC-GCKO tissue. Tetrodotoxin and the NO-GC inhibitor ODQ transformed slow phasic contractions of WT and single cell-specific knockout into GCKO-like contractions. ODQ increased the frequency of large contractions in WT and ICC-GCKO colon but not in GCKO, SMC-GCKO, and SMC/ICC-GCKO preparations. Tetrodotoxin and hexamethonium abolished large contractions. We conclude that short rings of murine colon can be effectively used to record spontaneous contractions. Although NO-GC in SMC determines smooth muscle tone, concerted action of NO-GC in both SMC and ICC modulates slow phasic contractions and large contractions.

Pharmacological identification of β-adrenoceptor subtypes mediating isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle.

Object We aimed to identify the β-adrenoceptor (β-AR) subtypes involved in isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle using pharmacological and biochemical approaches. Methods Longitudinal smooth muscle was prepared from the male guinea pig ascending colon and contracted with histamine prior to comparing the relaxant responses to three catecholamines (isoprenaline, adrenaline, and noradrenaline). The inhibitory effects of subtype-selective β-AR antagonists on isoprenaline-induced relaxation were then investigated. Results The relaxant potencies of the catecholamines were ranked as: isoprenaline > noradrenaline ≈ adrenaline, whereas the rank order was isoprenaline > noradrenaline > adrenaline in the presence of propranolol (a non-selective β-AR antagonist; 3 × 10−7 M). Atenolol (a selective β1-AR antagonist; 3 × 10−7–10−6 M) acted as a competitive antagonist of isoprenaline-induced relaxation, and the pA2 value was calculated to be 6.49 (95% confidence interval: 6.34–6.83). The relaxation to isoprenaline was not affected by ICI-118,551 (a selective β2-AR antagonist) at 10−9–10−8 M, but was competitively antagonized by 10−7–3 × 10−7 M, with a pA2 value of 7.41 (95% confidence interval: 7.18–8.02). In the presence of propranolol (3 × 10−7 M), the relaxant effect of isoprenaline was competitively antagonized by bupranolol (a non-selective β-AR antagonist), with a pA2 value of 5.90 (95% confidence interval: 5.73–6.35). Conclusion These findings indicated that the β-AR subtypes involved in isoprenaline-induced relaxation of colonic longitudinal guinea pig muscles are β1-AR and β3-AR.

Antispasmodic effect of Bupi Yichang pill on colonic contraction of rats in vitro

ObjectiveTo investigate the pharmacological effect of Bupi Yichang (BPYX) pill on colonic contraction of rats and explore its underlying mechanism. MethodsThe experiments were performed on colonic longitudinal smooth muscle strips (CLSMs) under isometric conditions. CLSMs suspended in tissue chambers were stimulated by KCl (80 mM) or acetylcholine (ACh, 0.1 mM), or exhausting intracellular Ca2+ and internal flow of extracellular Ca2+ to induce muscle contraction and their responses to different doses of BPYX pill were observed. After that, incubation with different inhibitors was conducted to verify its underlying mechanism. ResultsBupi Yichang pill dose-dependently and reversibly inhibited colonic contraction. The antispasmodic effect of BPYC pill was partially blocked by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride (TMB-8, an intracellular Ca2+ antagonist, 500 μM), thapsigargin (a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase, 1 μM), and nifedipine (a voltage-dependent Ca2+ channel blocker, 10 μM) (P < .05). However, there were no significant difference after incubation with ethylene glycol tetraacetic acid (EGTA, a calcium chelating agent, 1 mM), 4-aminopyridine (4-AP, a potassium channel blocker, 50 μM), NG-nitro-l-arginine methyl ester (l-NAME, an inhibitor of nitric oxide synthase, 10 mM), methylene blue (an inhibitor of cyclic guanosine monophosphate, 10 μM) and apamin (a selective blocker of Ca2+-activated K+ channel, 0.1 μM) (P > .05). ConclusionBupi Yichang pill could inhibit colonic contraction of rats in vitro and its antispasmodic effect might be partially mediated by calcium channels.

Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10−5M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10−4M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10μg/ml) increased permeability over 24–48h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.

Immunolocalization of AMPA receptor subunits within the enteric nervous system of the mouse colon and the effect of their activation on spontaneous colonic contractions.

The appropriate expression of specific neurotransmitter receptors within the cellular networks that compose the enteric nervous system (ENS) is central to the regulation of gastrointestinal (GI) functions. While the ENS expression patterns of the neurotransmitter glutamate have been well documented, the localization of its receptors on ENS neurons remains to be fully characterized. We investigated the expression patterns of glutamate receptor AMPA subunits within ENS neurons of the mouse colon and the consequences of their pharmacological activation on spontaneous colonic contractility. RT-PCR was used to detect individual AMPA receptor (GluR 1-4) subunit expression at the mRNA level in mouse colon tissue. Immunohistochemistry and confocal microscopy was used to localize the expression of the GluR1 and 4 subunits in colon tissue. Brain tissue was used as a positive control. Organ bath preparations were used to determine the effect of AMPA receptors activation on the force and frequency of colonic longitudinal smooth muscle spontaneous contractions. GluR1, 3, 4 mRNA was detected in the mouse colon. Immunoreactivity for GluR1 and 4 subunits was detected on the somatic and dendritic surfaces of subpopulations of neurochemically defined ENS neurons. The pharmacological activation of AMPA receptors increased the force but not frequency of spontaneous colonic contractions. Molecularly distinct AMPA receptor subtypes are differentially expressed within the neural networks of the mouse colon and have a direct role in motility. These data provide the rationale for the development of AMPA-selective ligands for the therapeutic delivery to the GIT in motility disorders.

Effect of gingerol on colonic motilityviainhibition of calcium channel currents in rats

To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process. The distal colon was cut along the mesenteric border and cleaned with Ca(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique. Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L, respectively (P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 μmol/L concentration of gingerol, the percentage of gingerol-induced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6% (P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 mV, respectively, at concentrations of 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L (P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was -14.23 ± 1.12 mV in the control group and -10.56 ± 1.04 mV in the 75 μmol/L group (P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93 (P < 0.05) in the control and 75 μmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of -27.43 ± 1.26 mV in the control group and -26.56 ± 1.53 mV in the 75 μmol/L gingerol group (P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68 (P > 0.05) in the 75 μmol/L gingerol group. Gingerol inhibits colonic motility by preventing Ca(2+) influx through L-type calcium channels.

Inhibitory effect of TongXie-YaoFang formula on colonic contraction in rats.

To investigate the pharmacological effect of TongXie-YaoFang (TXYF) formula and its underlying mechanisms. A neonatal maternal separation plus restraint stress (NMS + RS) model of diarrhea-predominant irritable bowel syndrome was developed by subjecting male Sprague-Dawley rats to daily maternal separation from postnatal days 2 to 21 plus restraint stress from days 50 to 59. Rats were randomly divided into two groups (NMS + RS and TXYF formula), and rats with no handling or separation were used as normal controls. Starting from postnatal day 60, rats were administered TXYF formula (9.84 g/100 g body weight) orally twice daily for 14 consecutive days, while the normal and NMS + RS groups were given distilled water. The distinctions of movement index (MI, area under the curve of contraction intensity/min, mg/min) and contraction frequency (CF, number of contractions/min, times/min) of isolated colonic longitudinal smooth muscle strips (CLSMs) in the three groups before and after treatment were observed with a Power Lab system. Different inhibitors were applied, and then 10(-4) mol/L acetylcholine chloride (Ach) was added to CLSMs to induce muscle contraction. Before treatment, the MI of CLSMs in the NMS + RS and TXYF formula groups was similar and both higher than that in the normal group (545.49 ± 73.66 mg/min vs 245.76 ± 34.44 mg/min and 551.09 ± 54.29 mg/min vs 245.76 ± 34.44 mg/min, P < 0.01, respectively). After treatment, the MI in the TXYF formula group was lower than that in the NMS + RS group (261.39 ± 38.59 mg/min vs 533.9 ± 61.63 mg/min, P < 0.01). In the same way, the CF of CLSMs in the NMS + RS and TXYF formula groups was similar and both higher than that in the normal group (3.42 ± 0.25 times/min and 3.31 ± 0.21 vs 1.1 ± 0.17 times/min, P < 0.01) before treatment. After treatment, the CF in the TXYF formula group was lower than that in the NMS + RS group (1.42 ± 0.87 times/min vs 3.11 ± 0.82 times/min, P < 0.01) and similar to that in the normal group (1.42 ± 0.87 times/min vs 1.09 ± 0.13 times/min). When 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride and 4-aminopyridine were added to the bath and equilibrated for 30 min, respectively, and 10(-4) mol/L Ach was added to CLSMs to induce muscle contraction, MI of the CLSMs in the TXYF formula group was lower than that in the normal group (666 ± 36.32 mg/min vs 747.77 ± 49.47 mg/min, and 686.53 ± 39.17 mg/min vs 750.45 ± 29.39 mg/min; P < 0.01, respectively). The MI of CLSMs in the TXYF formula group was lower than that in the normal group after treatment with nifedipine (689.48 ± 30.84 mg/min vs 741.65 ± 32.41 mg/min; P < 0.05). TXYF formula inhibits colon contraction in rats. This may be related to activation of specific potassium channels and inhibition of extracellular calcium internal flow.

Molecular and functional diversity of GABA-A receptors in the enteric nervous system of the mouse colon.

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

Cytokine-induced iNOS and ERK1/2 inhibit adenylyl cyclase type 5/6 activity and stimulate phosphodiesterase 4D5 activity in intestinal longitudinal smooth muscle.

This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1β and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1β and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1β or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pretreated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1β or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1β or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.

Bioengineered Three-Dimensional Physiological Model of Colonic Longitudinal Smooth Muscle In Vitro

The objective of this study was to develop a physiological model of longitudinal smooth muscle tissue from isolated longitudinal smooth muscle cells arranged in the longitudinal axis. Longitudinal smooth muscle cells from rabbit sigmoid colon were isolated and expanded in culture. Cells were seeded at high densities onto laminin-coated Sylgard surfaces with defined wavy microtopographies. A highly aligned cell sheet was formed, to which addition of fibrin resulted in delamination. (1) Acetylcholine (ACh) induced a dose-dependent, rapid, and sustained force generation. (2) Absence of extracellular calcium attenuated the magnitude and sustainability of ACh-induced force by 50% and 60%, respectively. (3) Vasoactive intestinal peptide also attenuated the magnitude and sustainability of ACh-induced force by 40% and 60%, respectively. These data were similar to force generated by longitudinal tissue. (4) Bioengineered constructs also maintained smooth muscle phenotype and calcium-dependence characteristics. This is a novel physiologically relevant in vitro three-dimensional model of colonic longitudinal smooth muscle tissue. Bioengineered three-dimensional longitudinal smooth muscle presents the ability to generate force, and respond to contractile agonists and relaxant peptides similar to native longitudinal tissue. This model has potential applications to investigate the underlying pathophysiology of dysfunctional colonic motility. It also presents as a readily implantable band-aid colonic longitudinal muscle tissue.

576 Contraction of Colonic Longitudinal Smooth Muscle is Dependent on PKC-Mediated Phosphorylation of Tropomyosin

576 Contraction of Colonic Longitudinal Smooth Muscle is Dependent on PKC-Mediated Phosphorylation of Tropomyosin

Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

We show that androgens, testosterone and 5alpha-dihydrotestosterone (DHT), acutely (approximately 40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC(20)) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC(20) phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC(20) by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.

S1241 Development of a Three-Dimensional Physiological Model of Colonic Longitudinal Smooth Muscle In Vitro Lined Up in Longitudinal Axis

S1241 Development of a Three-Dimensional Physiological Model of Colonic Longitudinal Smooth Muscle In Vitro Lined Up in Longitudinal Axis

Somatostatin receptor subtypes mediate contractility on human colonic smooth muscle cells

This study demonstrates the expression of functional somatostatin receptor (sstr) subtypes in human circular and longitudinal colonic smooth muscle cells (SMC). Native somatostatin (SS) and sstr subtype-specific analogues were used to characterize the sstr subtypes present in both cell types by contraction/relaxation studies. Qualitative and quantitative mRNA analysis and immunohistochemistry of sstr subtypes were also carried out. sstr subtype 2 mRNA was expressed in circular SMC, and various levels of subtypes 1, 2 and 3 mRNA were expressed in longitudinal colonic SMC. Native SS and each subtype-specific analogue exerted a modest, but significant, contraction, although inhibition of carbachol-induced contraction (relaxation) was the main effect on SMC from both layers. CH-288, a sstr subtype 1-specific analogue, and octreotide, a sstr subtype 2-specific analogue, were the most effective relaxant analogues on longitudinal and circular SMC, respectively. sstr subtypes display a distinct expression pattern on human colonic SMC; on circular SMC, subtype 2 is the only sstr, whereas sstr subtypes 1, 2 and 3 are expressed on human SMC isolated from the longitudinal layer. The contractile effects of SS are mediated through sstr subtype 2 and sstr subtype 1 on circular and longitudinal human colonic SMC, respectively.