Circular Smooth Muscle Strips Research Articles

Contractility of isolated colonic smooth muscle strips from rats treated with cancer chemotherapy: differential effects of cisplatin and vincristine.

Certain antineoplastic drugs cause gastrointestinal disorders even after the end of treatment. Enteric neuropathy has been associated with some of these alterations. Our goal was to assess the impact of repeated treatment with cisplatin and vincristine on the contractility of circular and longitudinal muscle strips isolated from the rat colon. Two cohorts of male rats were used: in cohort 1, rats received one intraperitoneal (ip) injection of saline or cisplatin (2 mg kg-1 week-1) on the first day of weeks 1-5; in cohort 2, rats received two cycles of five daily ip injections (Monday to Friday, weeks 1-2) of saline or vincristine (0.1 mg kg-1 day-1). Body weight and food and water intake were monitored throughout the study. One week after treatment, responses of colonic smooth muscle strips to acetylcholine (10-9-10-5 M) and electrical field stimulation (EFS, 0.1-20 Hz), before and after atropine (10-6 M), were evaluated in an organ bath. Both drugs decreased body weight gain. Compared to saline, cisplatin significantly decreased responses of both longitudinal and circular smooth muscle strips to EFS, whereas vincristine tended to increase them, although in a non-significant manner. No differences were observed in the muscle response to acetylcholine. Atropine abolished the contractile responses induced by acetylcholine, although those induced by EFS were only partially reduced in the presence of atropine. The findings suggest that although both drugs cause the development of enteric neuropathy, this seems to have a functional impact only in cisplatin-treated animals. Understanding the effects of chemotherapy on gastrointestinal motor function is vital for enhancing the quality of life of cancer patients.

6-OHDA-Induced Changes in Colonic Segment Contractility in the Rat Model of Parkinson's Disease.

Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model. Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K+ Krebs-induced contractility and spontaneous contractions. This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation.

Downregulation of ICCs and PDGFRα+ cells on colonic dysmotility in hirschsprung disease.

To investigate the effect of the distribution and expression of interstitial cells of Cajal (ICCs) and platelet-derived growth factor receptor-α positive (PDGFRα+) cells in different colon segments on colonic motility in children with Hirschsprung disease (HSCR). Smooth muscles of the narrow and dilated segments of the colon were obtained from 16 pediatric patients with HSCR. The proximal margin was set as the control section. The mRNA and protein expressions of c-Kit, PDGFRα, ANO1, and SK3 channels were examined. Circular smooth muscle strips of the colon were prepared for performing electrophysiology experiments using electric field stimulation (EFS) and intervention from different drugs (TTX, NPPB, Apamin, L-NAME, and CyPPA). The mRNA and protein expressions of c-Kit, ANO1, PDGFRα, and SK3 were much lower in the narrow segment than those in the dilated and proximal segments of the colon. The narrow segment showed a considerably spontaneous contraction of the muscle strip. After the EFS, the relaxation response decreased from the proximal to the narrow segment, whereas the contraction response increased. TTX blocking did not cause any significant changes in the narrow segment. In contrast, when NPPB, Apamin, L-NAME, and CyPPA were used to intervene in the muscle strips, the proximal segment showed a more sensitive inhibitory or excitatory response than the narrow segment. Downregulation of the ICCs and PDGFRα+ cells from the proximal to narrow segment may be responsible for the dysmotility of the colon in pediatric HSCR.

Characterization of Cecal Smooth Muscle Contraction in Laying Hens.

The ceca play an important role in the physiology of the gastrointestinal tract in chickens. Nevertheless, there is a gap of knowledge regarding the functionality of the ceca in poultry, especially with respect to physiological cecal smooth muscle contraction. The aim of the current study is the ex vivo characterization of cecal smooth muscle contraction in laying hens. Muscle strips of circular cecal smooth muscle from eleven hens are prepared to investigate their contraction ex vivo. Contraction is detected using an isometric force transducer, determining its frequency, height and intensity. Spontaneous contraction of the chicken cecal smooth muscle and the influence of buffers (calcium-free buffer and potassium-enriched buffer) and drugs (carbachol, nitroprusside, isoprenaline and Verapamil) affecting smooth muscle contraction at different levels are characterized. A decrease in smooth muscle contraction is observed when a calcium-free buffer is used. Carbachol causes an increase in smooth muscle contraction, whereas atropine inhibits contraction. Nitroprusside, isoprenaline and Verapamil result in a depression of smooth muscle contraction. In conclusion, the present results confirm a similar contraction behavior of cecal smooth muscles in laying hens as shown previously in other species.

Role of PGE2 in colonic motility: PGE2 attenuates spontaneous contractions of circular smooth muscle via EP4 receptors in the rat colon

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called ‘giant contractions’ (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.

Peppermint and caraway oils have muscle inhibitory and pro-secretory activity in the human intestine in vitro.

Herbal medicinal products with a broad activity spectrum may be promising alternatives to treat functional gastrointestinal disorders (FGD). Menthacarin® is a drug with a fixed combination of peppermint and caraway oils, which is clinically used to treat FGD-associated symptoms. We studied the effects of peppermint and caraway oils on contractile and secretory activity in 255 human small and large intestinal preparations derived from surgical resections (73 patients). Motility was recorded in circular smooth muscle strips and secretion with the Ussing chamber-voltage clamp technique. Electrical field stimulation evoked nerve induced contractile responses. KEYRESULTS: Peppermint and caraway oil concentrations dependently inhibited muscle contractility as indicated by sustained muscle relaxation and decrease in phasic contractility. These effects occurred in small and large intestinal preparations with IC50 values ranging between 17 and 90µg/mL for peppermint oil and between 7 and 127µg/mL for caraway oil. Neither peppermint nor caraway oil influenced the nerve evoked contractile response. The inhibition of contractile activity, but not the muscle relaxation, was prevented by the L-type calcium channel activator Bay K8644 but not by the neurotoxin tetrodotoxin. Both peppermint oil and caraway oil increased epithelial secretion, which remained in tetrodotoxin. The findings revealed a strong muscle inhibitory and pro-secretory action of peppermint and caraway oils at clinically relevant concentrations. Both actions were nerve-independent. The inhibition of contractility was mediated by inhibition of L-type calcium channels. The effects on muscle and epithelial activity may contribute to the beneficial effects observed in patients with FGD.

Diallyl trisulfide regulates rat colonic smooth muscle contractions by inhibiting L-type calcium channel currents

Diallyl trisulfide (DATS) is an active organosulfide component of allicin and has several beneficial effects, including antimicrobial, antioxidant, cardioprotective and anticancer effects. Few studies have shown the modulatory effect of DATS on L-type calcium channels in rat colonic smooth muscle cells and colonic motility. To investigate the modulatory effect of DATS on L-type calcium channels in rat colonic smooth muscle and colonic contraction, L-type calcium channel currents were recorded, and colonic contractility in longitudinal and circular smooth muscle strips was measured. DATS attenuated L-type calcium channel currents without affecting steady-state activation or inactivation kinetics and inhibited the spontaneous contractions of both longitudinal and circular smooth muscle strips dose-dependently. In conclusion, DATS has an inhibitory effect on the contractions of colonic muscle strips that is related to its regulation of L-type calcium channels.

CAMP Catalyzing Phosphodiesterases Control Cholinergic Muscular Activity But Their Inhibition Does Not Enhance 5-HT4 Receptor-Mediated Facilitation of Cholinergic Contractions in the Murine Gastrointestinal Tract.

Background: As the signal transduction of 5-HT4 receptors on cholinergic neurons innervating smooth muscle is controlled by phosphodiesterase (PDE) 4 in porcine stomach and colon, and human large intestine, the in vivo gastroprokinetic effects of a 5-HT4 receptor agonist might be enhanced by combination with a selective PDE4 inhibitor. The presence of 5-HT4 receptors on cholinergic neurons towards murine gastrointestinal circular muscle was recently shown. If the control of this receptor pathway by PDE4 is also present in mice, this might be a good model for in vivo testing of the combination therapy. Therefore this study investigates the role of cAMP catalyzing PDEs in smooth muscle cell activity and in the intraneuronal signal transduction of the 5-HT4 receptors in the gastrointestinal tract of C57Bl/6J mice.Methods: In circular smooth muscle strips from murine fundus, jejunum, and colon, submaximal cholinergic contractions were induced by either electrical field stimulation (EFS) or by carbachol (muscarinic receptor agonist). The influence of the PDE inhibitors IBMX (non-selective), vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), and rolipram (PDE4) was tested on these contractions and on the facilitating effect of a submaximal concentration of prucalopride (5-HT4 receptor agonist) on EFS-induced contractions.Results: In the three gastrointestinal regions, IBMX and cilostamide concentration-dependently decreased carbachol- as well as EFS-induced contractions. Some inhibitory effect was also observed with rolipram. In the fundus a non-significant trend for an enhancement of the facilitating effect of prucalopride on EFS-induced contractions was observed with IBMX, but none of the selective PDE inhibitors enhanced the facilitating effect of prucalopride in fundus, jejunum or colon.Conclusion: In analogy with the porcine gastrointestinal tract, in murine fundus, jejunum, and colon circular smooth muscle PDE3 is the main regulator of the cAMP turnover, with some contribution of PDE4. In contrast to the porcine gastrointestinal tract, the in vitro facilitation of electrically induced cholinergic contractions by 5-HT4 receptor stimulation could not be enhanced by specific PDE inhibition. The C57Bl/6J murine model is thus not suitable for in vivo testing of a 5-HT4 receptor agonist combined with a selective PDE4 inhibitor.

Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger.

Background and AimsHydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question.MethodsIsometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function.ResultsPorcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice.ConclusionsEndogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.

Mechanism of sodium hydrosulfide modulation of L-type calcium channels in rat colonic smooth muscle cells

Hydrogen sulfide (H2S) can exert different effects on the gastrointestinal tract by modulating ion channels. Previously, we found that H2S donor sodium hydrosulfide (NaHS) regulates colonic motility through L-type calcium channels, but the molecular mechanism remains unknown. The present study was designed to investigate possible mechanisms underlying the modulation of L-type calcium channels by NaHS in rat colonic smooth muscle cells. L-type calcium currents in colonic smooth muscle cells were recorded using the whole-cell patch-clamp technique. Spontaneous contractions of mid-colonic smooth muscle strips were measured in an organ bath system and a biological signal acquisition system. NaHS evoked a significant rightward shift in the steady-state activation curve of L-type calcium channels, changed the shape of the current-voltage (I-V) curve, and decreased the peak current density at 0mV, although it significantly increased with higher stimulatory voltage. The sulfhydryl-modifying reagent DL-dithiothreitol (DTT) enhanced the effects of NaHS on L-type calcium channels, while diamide (DM) and reduced L-glutathione (GSH) alleviated the effects of NaHS. Additionally, NaHS inhibited the spontaneous high-amplitude contractions of both longitudinal and circular smooth muscle strips in a dose-dependent manner. The inhibitory effects were reversible. DTT and GSH enhanced the effects of NaHS, while DM attenuated the effects of NaHS. In conclusion, NaHS modulates L-type calcium channels in rat colonic smooth muscle cells and regulates the contractile activity of colonic smooth muscle, potentially by modifying the free sulfhydryl groups of L-type calcium channels.

Synergy between 5-HT4 receptor stimulation and phosphodiesterase 4 inhibition in facilitating acetylcholine release in human large intestinal circular muscle.

Gastroprokinetic properties of 5-HT4 receptor agonists, such as prucalopride, are attributed to activation of 5-HT4 receptors on cholinergic nerves innervating smooth muscle in the gastrointestinal smooth muscle layer, increasing acetylcholine release and muscle contraction. In porcine stomach and colon, phosphodiesterase (PDE) 4 has been shown to control the signaling pathway of these 5-HT4 receptors. The aim of this study was to investigate the PDE-mediated control of these 5-HT4 receptors in human large intestine. Circular smooth muscle strips were prepared from human large intestine; after incubation with [³H]-choline, electrically induced tritium outflow was determined as a measure for acetylcholine release. The influence of PDE inhibition on the facilitating effect of prucalopride on electrically induced acetylcholine release was studied. The non-selective PDE inhibitor IBMX enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence, while rolipram and roflumilast (PDE4) enhanced the prucalopride-induced facilitation to the same extent as IBMX. In human large intestinal circular muscle, the intracellular pathway of 5-HT4 receptors facilitating cholinergic neurotransmission to large intestinal circular smooth muscle is controlled by PDE4. If the synergy between 5-HT4 receptor agonism and PDE4 inhibition is confirmed in a functional assay with electrically induced cholinergic contractions of human large intestinal circular smooth muscle strips, combination of a selective 5-HT4 receptor agonist with a selective PDE4 inhibitor might enhance the in vivo prokinetic effect of the 5-HT4 receptor agonist in the large intestine.

5-HT4 receptors facilitate cholinergic neurotransmission throughout the murine gastrointestinal tract.

In the gastrointestinal tract of several species, facilitating 5-HT4 receptors were proposed on myenteric cholinergic neurons innervating smooth muscle by invitro study of the effect of the selective 5-HT4 receptor agonist prucalopride on submaximal cholinergic contractions. This was not yet established in the murine gastrointestinal tract. In circular smooth muscle strips from murine fundus, jejunum and colon, contractions were induced by electrical field stimulation in the presence of guanethidine, L-NAME and for colon also MRS 2500. Submaximal contractions were induced to study the influence of prucalopride. Electrical field stimulation at reduced voltage induced reproducible submaximal neurogenic and cholinergic contractions as the contractions were abolished by tetrodotoxin and atropine. Hexamethonium had no systematic inhibitory effect but mecamylamine reduced the responses, suggesting that part of the cholinergic response is due to activation of preganglionic neurons. Prucalopride concentration-dependently increased the submaximal cholinergic contractions in the three tissue types, reaching maximum from 0.03μmol/L onwards. The facilitation in the different series with 0.03μmol/L prucalopride ranged from 41% to 104%, 30% to 76% and 24% to 74% in fundus, jejunum, and colon, respectively. The effect of 0.03μmol/L prucalopride was concentration-dependently inhibited by GR 113808. In the murine gastrointestinal tract, activation of 5-HT4 receptors with prucalopride enhances cholinergic contractions, illustrating facilitation of myenteric cholinergic neurotransmission. The degree of enhancement with prucalopride is of similar magnitude as previously reported in other species, but the effective concentrations are lower than those needed in the gastrointestinal tract of other species.

Supernatants of irritable bowel syndrome mucosal biopsies impair human colonic smooth muscle contractility.

Changes in intestinal motility are likely to contribute to irritable bowel syndrome (IBS) pathophysiology. The aim of the study was to investigate the effects of IBS mucosal supernatants on human colonic muscle contractility. Supernatants were obtained from biopsies of 18 IBS patients-nine with constipation (IBS-C) and nine with diarrhea-predominant IBS (IBS-D)-and nine asymptomatic subjects, used as controls. Colonic circular smooth muscle strips or isolated cells (SMC) were exposed to control or IBS supernatants. Spontaneous phasic contractions on strips and morphofunctional parameters on cells were evaluated in basal conditions and in response to acetylcholine (Ach). Incubation with IBS supernatants was also conducted in the presence of antagonists and inhibitors (namely histamine, protease and prostaglandin antagonists, nuclear factor-kappa B inhibitor, catalase, NADPH oxidase inhibitor, and the cAMP- and/or cGMP-cyclase inhibitors). Exposure to IBS-C and IBS-D supernatants induced a significant reduction in basal tone and Ach-elicited contraction of muscle strips and a significant shortening and impairment of Ach contraction of SMCs. The NADPH oxidase inhibitor prevented the effect of supernatants, while the protease antagonist only IBS-C effect. No effect was observed with the other antagonists and inhibitors. Dilution of IBS-D supernatants partially restored the effects only on SMCs, whereas dilution of IBS-C supernatants significantly reverted the effects on muscle strips and Ach-elicited response on SMC. Supernatants from mucosal biopsies of IBS patients reduce colonic contractility. The observed impairment was concentration dependent, likely occurring through intracellular oxidative stress damage, involving different neuromotor mechanisms depending on the IBS subtype.

Changes in the response to excitatory antagonists, agonists, and spasmolytic agents in circular colonic smooth muscle strips from patients with diverticulosis.

Colonic samples from asymptomatic diverticulosis (DS) patients presented enhanced electrical field stimulation (EFS)-contractions, in an earlier study of ours, suggesting increased endogenous responses. The aim of this study was to explore changes in excitatory neuromuscular transmission and to assess the pharmacodynamics of spasmolytic agents in DS. Circular muscle strips fromsigmoid colon of DS patients (n=30; 69.5±14.8years) and controls (n=32; 64.7±16.2years) were studied using organ baths to evaluate the direct effect of excitatory agonists (carbachol, neurokinin A [NKA] and substance P [SP]), and the effect of antagonists (atropine and NK2 antagonist GR94800) and spasmolytic drugs (otilonium bromide [OB] and N-butyl-hyoscine) on the contractions induced by EFS-stimulation of excitatory motorneurons. qRT-PCR was also performed to compare mRNA expression of M2 , M3 , NK2 receptors and L-type calcium channels. Contractions to carbachol (Emax : 663.7±305.6% control vs 2698.0±439.5% DS; p<0.0005) and NKA (Emax : 387.8±35.6% vs 1102.0±190.1%; p<0.0005) were higher in DS group, without differences for SP. Higher potency for DS patients was observed in the concentration-response curves for atropine (pIC50 =8.56±0.15 control vs pIC50 =9.95±0.18 DS group; p<0.005) and slightly higher for GR94800 (pIC50 =7.21±0.18 control vs pIC50 =7.97±0.32 group; p<0.0001). Lower efficacy (Emax ) and potency (pIC50 ) was observed for spasmolytic drugs in DS, whereas no differences were found regarding the relative expression of the receptors evaluated between groups. The greater response to cholinergic and tachykinergic agonists and greater potency for muscarinic and NK2 antagonists observed in DS might play a role in the spasticity found in diverticular disease.

Modulatory role and mechanism of BDNF and its receptor TrkB in colonic motility disorder in chronic stress rat

To investigate the role and mechanism of BDNF and its receptor TrkB in the disorder of colonic motility in chronic stress rats. A total of 20 male Wistar rats were randomly divided into two groups using to completely random method after weighed, with 10 rats in each group. Chronic water avoidance stress model was established. Then the fecal pellets of water avoidance stress (WAS) group and sham water avoidance stress (SWAS) group were recorded. Enzyme linked immunosorbent assay (ELISA), real time PCR, Western blot and immunohistochemistry were used to detect the expression of BDNF and TrkB in serum and colon muscle. The amplitudes of contractions of circular smooth muscle strips of each group were recorded after the treatment of TTX, BDNF and K252a. The number of fecal pellets had obviously increased in WAS group comparing with SWAS group (P < 0.05). The level of serum BDNF in WAS group was higher than that of SWAS group ((158.30 ± 9.82) vs (84.68 ± 7.80) pg/ml). And the expression of TrkB in the colon muscle in WAS group was higher than that in SWAS group (0.44 ± 0.03 vs 0.30 ± 0.02, P < 0.05). There was no significant difference between the two groups in expression of BDNF mRNA in colon muscle (P > 0.05). TrkB was mainly expressed in the cell nucleus of muscular layer neurons, and the expression of TrkB had obviously increased in WAS rats. The amplitudes of contractions of circular smooth muscle in WAS rats had significantly increased compared with SWAS rats ((0.35 ± 0.02) vs (0.22 ± 0.03) g, P < 0.05). After adding TTX to block the function of enteric nervous, the difference was remaining ((0.89 ± 0.07) vs (0.53 ± 0.06) g, P < 0.05). BDNF was added to the bath and the R value at different time was recorded. The difference had statistically significant at 6 min and 12 min (both P < 0.05). BDNF could induced the contraction peak of the circular smooth muscle. The contraction peak induced by BDNF was delayed and reduced when K252a was added to the bath 30 min before adding BDNF. BDNF plays a modulatory role in the disorder of colonic motility in chronic stress rat by acting on its receptor TrkB.

Trypsin-induced biphasic regulation of tone in the porcine lower esophageal sphincter

The lower esophageal sphincter (LES) plays an important role in coordinated esophageal motility. The present study aimed to elucidate how trypsin affects LES contractility. Porcine LES circular smooth muscle strips were prepared. Contractile responses to trypsin were assessed. Trypsin (300nM) induced a transient contraction. At concentrations of 1μM or higher, trypsin induced biphasic responses, consisting of a transient contraction followed by a transient relaxation. Pretreatment with either 1μM tetrodotoxin or carbenoxolone had no effect on these responses. In contrast, trypsin-induced responses were completely blocked by pretreatment with the serine protease inhibitor. Pretreatment with 10μM FSLLRY-NH2, a PAR2 antagonist, significantly inhibited trypsin-induced biphasic responses. Trypsin (1μM)-induced contractions were partially inhibited by pretreatment with 10μM Y-27632. In addition, trypsin (10μM)-induced relaxation was partially inhibited by pretreatment with 10μM Y-27632, 10μM PD98059 or 10μM SB203580. Trypsin-induced relaxation was abolished by increasing the extracellular K+ concentration to 40mM, but not by pretreatment with l-arginine methyl ester. Furthermore, trypsin-induced relaxation was partially inhibited by pretreatment with 10μM glibenclamide or 1μM 4-aminopyridine. Trypsin causes biphasic regulation of LES tone by directly acting on smooth muscle. Rho-associated protein kinase (ROK) is involved in trypsin-induced contraction, whereas ROK, ERK1/2, p38MAPK, and membrane hyperpolarization are involved in relaxation. The regulation of LES tone by trypsin may play a role in esophageal motility.

Effect of N-butylscopolammonium bromide on equine ileal smooth muscle activity in an ex vivo model.

N-butylscopolammonium bromide (NBB) is an anticholinergic agent used to treat spasmodic colic in horses. Intestinal smooth muscle spasm also occurs in horses with intraluminal intestinal obstructions, such as ileal impactions. The antispasmodic effects of NBB may be useful in managing ileal impactions, but the effects of NBB on equine ileal smooth muscle are unknown. To investigate the effects of NBB on spontaneous and induced contraction of the equine ileum in an ex vivo model. Ex vivo biomechanical study assessing contractile properties in isolate equine ileal smooth muscle with and without exposure to NBB. Ileal tissue was collected from 6 healthy horses after euthanasia, and isolated circular and longitudinal smooth muscle strips were connected to isometric force transducers in organ baths. After equilibration, the effect of NBB (1 nmol/l to 100 μmol/l) on spontaneous and carbachol-induced contraction was determined and compared with responses in control tissue. At ≥30 μmol/l, NBB inhibited spontaneous contractions in all muscle strips that exhibited spontaneous activity. N-butylscopolammonium bromide pre-treatment inhibited carbachol-induced contraction in circular (NBB-treated half maximal effective concentration [EC₅₀] 0.530 × 10(-8) mol/l vs. control EC₅₀ 41.57 × 10(-8) mol/l) and longitudinal muscle strips (NBB-treated EC50 0.243 × 10(-8) mol/l vs. control EC₅₀ 90.84 × 10(-8) mol/l). Abolition of carbachol-induced contraction with NBB was observed at lower concentrations in circular than longitudinal muscle strips. Pretreatment with NBB significantly inhibited carbachol-induced contractions; NBB-treated tissue required greater carbachol concentrations to produce sustained contractions than control muscle strips. Histamine-evoked contraction was not affected by NBB. N-butylscopolammonium bromide inhibits spontaneous and cholinergically mediated contraction in equine ileal smooth muscle strips ex vivo. Thus, NBB might reduce intestinal spasm in equine ileal impactions and could be useful for medical management of these cases, although further study is needed to confirm these effects in vivo.

The effects of eupatilin (stillen®) on motility of human lower gastrointestinal tracts.

Gastrointestinal motility consists of phasic slow-wave contractions and the migrating motor complex (MMC). Eupatilin (Stillen®) has been widely used to treat gastritis and peptic ulcers, and various cytokines and neuropeptides are thought to be involved, which can affect gastrointestinal motility. We performed a study to identify the effects of eupatilin on lower gastrointestinal motility with electromechanical recordings of smooth muscles in the human ileum and colon. Ileum and colon samples were obtained from patients undergoing bowel resection. The tissues were immediately stored in oxygenated Krebs-Ringer's bicarbonate solution, and conventional microelectrode recordings from muscle cells and tension recordings from muscle strips and ileal or colonic segments were performed. Eupatilin was perfused into the tissue chamber, and changes in membrane potentials and contractions were measured. Hyperpolarization of resting membrane potential (RMP) was observed after administration of eupatilin. The amplitude, AUC, and frequency of tension recordings from circular and longitudinal smooth muscle strips and bowel segments of the ileum and colon were significantly decreased after admission of eupatilin. Eupatilin elicited dose-dependent decreases during segmental tension recordings. In conclusion, eupatilin (Stillen®) showed inhibitory effects on the human ileum and colon. We propose that this drug may be useful for treating diseases that increase bowel motility, but further studies are necessary.

Neurotensin and cholecystokinin contract gallbladder circular muscle in chickens

The contractile effects of neurotensin (NT) and cholecystokinin octapeptide (CCK-8) on isolated circular smooth muscle strips of chicken gallbladder were investigated. The NT (0.25-300 nM) produced concentration-dependent contractions on smooth muscle with an EC50 of 8.5 nM (95% confidence limits = 5.3-13.6 nM). In comparison, CCK-8 produced concentration-dependent contractions with an EC50 of 13 nM (95% confidence limits of 9-20 nM). There were no statistical differences in contractile responses when comparing NT and CCK-8 at equimolar concentrations. The NT appears to act directly on smooth muscle tissue in the chicken; the contractile responses were not blocked by 10 µM atropine or tetrodotoxin. A portion of the activity is mediated by extracellular calcium as 100 nM nifedipine inhibited 30% of peptide-induced muscle tension. The NT receptor (NTR) type 1 antagonist SR 48692 (0.1 µM) did not significantly reduce NT potency. The contractile effects of CCK-8 remained unaltered in tissues pretreated with atropine, TTX, or nifedipine. The CCK-A antagonist lorglumide, at a concentration of 1 µM, reduced the contractile potency of CCK-8 by one-half. Avian receptors for NT and CCK may differ pharmacologically from their mammalian counterparts, but their contractile actions on the gallbladder resulting in increased biliary output by flow are further evidence of their role in the postprandial regulation of lipid digestion in chickens.

Developmental origins of colon smooth muscle dysfunction in IBS-like rats

Epidemiological studies show that subsets of adult and pediatric patients with irritable bowel syndrome (IBS) have prior exposures to psychological or inflammatory stress. We investigated the cellular mechanisms of colonic smooth muscle dysfunction in adult rats subjected to neonatal inflammation. Ten-day-old male rat pups received 2,4,6-trinitrobenzene sulfonic acid to induce colonic inflammation. Colonic circular smooth muscle strips were obtained 6 to 8 wk later. We found that about half of the neonate pups subjected to inflammatory insult showed a significant increase in expression of the pore-forming α1C-subunit of Cav1.2b channels in adult life. These were the same rats in whom Vip mRNA increased in the colon muscularis externae. Additional experiments showed reduced interaction of histone deacetylase (HDAC) 3 with α1C1b promoter that increased the acetylation of histone H3 lysine 9 (H3K9) in the core promoter region. Vasoactive intestinal peptide (VIP) treatment of naïve muscularis externae swiftly recruited CREB-binding protein (CBP) to the α1C1b promoter and dissociated HDAC3 from this region to initiate transcription. The CBP interaction with the α1C1b promoter was transient, but the dissociation of HDAC3 persisted to sustain H3K9 hyperacetylation and increase in transcription. Intraperitoneal treatment of adult naïve rats with butyrate mimicked the effects of neonatal colon inflammation. We concluded that neonatal inflammation upregulates VIP in the colon muscularis externae, which modulates epigenetic events at the α1C1b promoter to activate α1C1b gene transcription. Inflammatory insult in early life may be one of the etiologies of smooth muscle dysfunction in adult life, which contributes to the altered motility function in patients with diarrhea-predominant IBS.