Contraction Of Colonic Smooth Muscle Research Articles

Nitric oxide and ion channels mediate L-cysteine-induced inhibition of colonic smooth muscle contraction.

Previous studies have suggested that L-cysteine regulates gut motility through hydrogen sulfide. However, the mechanisms involved in the L-cysteine-induced response have not been extensively studied. This study aimed to investigate the underlying mechanisms of action of L-cysteine on spontaneous contraction of rat colon. Longitudinal and circular muscle strips from rat middle colon were prepared to measure the spontaneous contractile activities of colon in an organ bath system. Whole-cell voltage-clamp techniques were applied to record the currents of L-type voltage-dependent Ca2+ channels (VDCCs) and voltage-gated K+ channels (Kv) in isolated smooth muscle cells (SMCs) from colon. L-cysteine inhibited the spontaneous contraction of longitudinal and circular muscle strips from the rat colon in a concentration-dependent manner. The inhibition induced by L-cysteine was significantly decreased by inhibitors of H2S synthesis (p < 0.05). Furthermore, the suppression induced by L-cysteine was partially attenuated by tetrodotoxin, L-NNA and glibenclamide (p < 0.05). Whole-cell voltage-clamp recordings showed that L-cysteine caused a remarkable reduction in the peak currents of VDCCs and significantly increased the membrane currents of Kv channels in isolated SMCs (p < 0.05). We concluded that L-cysteine inhibits the contractile activities of smooth muscle strips from the rat colon. The relaxation in response to L-cysteine may be in part mediated by a nitrergic pathway and by inhibiting the VDCCs in combination with a direct activation of the KV channels and KATP channels.

Estrogen inhibits colonic smooth muscle contractions by regulating BKβ1 signaling.

The estrogen inhibits colonic smooth muscle contractions, which may lead to constipation. However, the mechanisms of inhibition are poorly understood. Therefore, the present study examined the effect of estrogen on rat colonic smooth muscle contractions and its potential association with the large-conductance Ca2+-activated K+ channels β1 (BKβ1) subunit. Twenty-four female Sprague Dawley rats were randomly assigned to 4 groups. After 2 weeks of intervention, the contraction activity of isolated colonic smooth muscle and the expression of BKβ1 in colonic smooth muscle of rats were detected. Additionally, in order to investigate the effects of estrogen on BKβ1 expression and calcium mobilization, in vitro experiments were conducted using rat and human colonic smooth muscle cells (SMCs). BKβ1 shRNA was used to investigate whether calcium mobilization is affected by BKβ1 in colonic SMCs. To explore the relationship between ERβ and BKβ1, serial deletions, site-directed mutagenesis, a dual-luciferase reporter assay, and chromatin immunoprecipitation assays were employed. In response to E2, colonic smooth muscle strips showed a decrease in tension, while IBTX exposure transiently increased tension. Furthermore, in these muscle tissues, BKβ1 and α-SMA were found to be co-expressed. The E2 group showed significantly higher BKβ1 expression. In cultured colonic SMCs, the expression of BKβ1 was found to increase in the presence of E2 or DPN. E2 treatment reduced Ca2+ concentrations, while BKβ1 shRNA treatment increased Ca2+ concentrations relative to the control. ERβ-initiated BKβ1 expression appears to occur via binding to the BKβ1 promoter. These results indicated that E2 may upregulate BKβ1 expression via ERβ and inhibit colonic smooth muscle contraction through ERβ by directly targeting BKβ1.

Estrogen increases the expression of BKCa and impairs the contraction of colon smooth muscle via upregulation of sphingosine kinase 1.

Estrogen (E2) may impair the contraction of colonic smooth muscle (SM) leading to constipation. Large conductance Ca2+ -activated K+ channels (BKCa ) are widely expressed in the smooth muscle cells (SMCs) contributing to hyperpolarization and relaxation of SMCs. Sphingosine kinase 1 (SphK1) is known to influence the expression of BKCa . We aimed to elucidate the potential underlying molecular mechanism of BKCa and SphK1 that may influence E2-induced colonic dysmotility. In ovariectomized rats, SMcontraction and expression of BKCa , SphK1, sphingosine-1-phosphate receptor (S1PR) were analyzed after the treatment with vehicle, BSA-E2, E2, and E2receptor antagonist. The role of BKCa , SphK1, and S1PR in E2-induced SMdysmotility was investigated in rat colonic SMCs. The effect of SphK1 on SMcontraction as well as on the expression of BKCa and S1PR was analyzed in SphK1 knock-out mutant mice and wild-type (WT) mice treated with or without E2. The E2-treated group exhibited a weak contraction of colonic SMand a delayed colonic transit. The treatment with E2 significantly upregulated the expression of BKCa , SphK1, S1PR1, and S1PR2, but not S1PR3, in colon SMand SMCs. Inhibition of BKCa , SphK1, S1PR1, and S1PR2 expression attenuated the effect of E2 on Ca2+ mobilization in rat colon SMCs. WTmice treated with E2 showed impaired gastrointestinal motility and enhanced expression of BKCa , S1PR1, and S1PR2 compared with those without E2 treatment. Conversely, in SphK1 knock-out mice treated with E2, these effects were partially reversed. E2 increased the release of S1P which in turn could have activated S1PR1 and S1PR2. Loss of SphK1 attenuated the effect of E2 on the upregulation of S1PR1 and S1PR2 expression. These findings indicated that E2 impaired the contraction of colon SMthrough activation of BKCa via the upregulation of SphK1 and the release of S1P. In the E2-induced BKCa upregulation, S1PR1 and S1PR2 might also be involved. These results may provide further insights into a therapeutic target and optional treatment approaches for patients with constipation.

A novel model of adenine-induced chronic kidney disease-associated gastrointestinal dysfunction in mice: The gut-kidney axis

Although constipation is a common complication of chronic kidney disease (CKD), there is no animal model that can be used to study the association between renal impairment and gastrointestinal function without interfering with the gastrointestinal tract of the model. Therefore, we determined whether adenine could induce CKD in association with gastrointestinal dysfunction. Six-week-old ICR mice were intraperitoneally injected with saline, 25, 50, or 75 mg adenine/kg body weight for 21 days. Blood urea nitrogen (BUN), plasma creatinine, and renal histopathology were evaluated. Defecation status was evaluated from defecation frequency and fecal water content. Colonic smooth muscle contraction was measured by the organ bath technique, and transepithelial electrical resistance (TEER) was measured using an Ussing chamber. In the 50 mg/kg treatment group, BUN and creatinine were significantly increased compared with control, and inflammatory cell infiltration, glomerular necrosis, tubular dilatation, and interstitial fibrosis were observed in renal tissues. Mice in this group also showed a significant decrease in defecation frequency, fecal water content, colonic motility index, and TEER. Overall, 50 mg/kg of adenine was the best dose to induce CKD with associated constipation and intestinal barrier impairment. Therefore, this adenine administration model can be recommended for CKD-associated gastrointestinal dysfunction research.

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127μM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

Estrogen-regulated expression of SK3 channel in rat colonic smooth muscle contraction

AimsEstrogen can induce inhibition of colonic smooth muscle contraction in male and female mice, which may lead to constipation; however, the mechanisms of inhibition are poorly understood. Hence, this study investigated the effect of estrogen on rat colonic smooth muscle contraction and role of small-conductance Ca2+-activated K+ 3 (SK3) and transcription factors (Sp1 and Sp3) in the underlying mechanisms. Main methodsThe experiment included 24 female Sprague-Dawley (SD) rats divided into 4 groups. The rats were oophorectomized surgically, and a silicone tube containing blank solvent, 0.3 mg/mL estrogen (E2), equal-concentration of estrogen and estrogen receptor antagonist (EI), and bovine serum albumin-E2 (BSA-E2) was implanted. The rats were sacrificed on day 14. The molecular insights were confirmed using real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analyses to determine the effect of estrogenic stimulation on gene and protein expression analyses, respectively. Key findingsThe E2 group showed significantly greater SK3 expression (P < .005) compared with other groups and significantly lowers smooth muscle cell (SMC) contractility (P < .005). Estrogen stimulation and SK3 overexpression resulted in a significant decrease (P < .05) in Ca2+ mobilization in the E2 group versus the control group. Further, the E2 group showed significantly higher Sp1 mRNA (P < .05) but lower Sp3 mRNA expression (P < .05) and protein expression (P < .001) compared with other groups. SignificanceE2 may promote SK3 expression by its genomic effect and inhibit colonic contraction by affecting SK3 expression via an interaction between Sp1 and Sp3.

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.

The role of nitric oxide in the inhibitory effects of exogenous hydrogen sulfide on rat colonic smooth muscle contraction

Objective: To investigate the role of nitric oxide (NO) in hydrogen sulfide (H(2)S)-induced inhibition upon colonic smooth muscle contraction. Methods: Immunohistochemistry was applied to observe the distribution of H(2)S-producing enzymes CBS and CSE in adult male Wistar rats. Organ bath system was used to observe the spontaneous contraction of colonic smooth muscle. Patch clamp technique was applied to record currents of L-type calcium channel (I(Ca,L)) in smooth muscle cells. Results: Specific immunoreactivity for CSE and CBS was observed in mucosa, smooth muscle and enteric plexus of rat proximal colon. NaHS elicited relaxation in a concentration-dependent manner upon muscle contraction in the presence of tetrodotoxin. The NaHS IC(50) of LM was 917.6 μmol/L (95% CI: 776.3-1 085 μmol/L, n=6) and the NaHS IC(50) of CM was 730.4 μmol/L (95% CI: 592.2-900.8 μmol/L, n=6). The SNP-induced relaxation in muscle strips was partially reversed by NaHS (P<0.05). Instead, the relaxation caused by NaHS was decreased by the sGC inhibitor ODQ but affected neither by NO precursor L-arginine, the NO inhibitor L-NNA nor the competitive cGMP antagonist PET-cGMP. NaHS (100 μmol/L) increased I(Ca,L) while NaHS (300 μmol/L) decreased the peak I(Ca,L) with modifying the ion channel characteristics (P<0.05). Conclusions: Exogenous hydrogen sulfide might have a dual effect on colonic motility and its inhibitory effect might be independent of NO signaling system. L-type calcium channel may play an important role during the process of H(2)S modulating colonic contraction.

Optogenetic analysis of neuromuscular transmission in the colon of ChAT-ChR2-YFP BAC transgenic mice.

Propulsion of luminal content along the gut requires coordinated contractions and relaxations of gastrointestinal smooth muscles controlled by the enteric nervous system. Activation of excitatory motor neurons (EMNs) causes muscle contractions, whereas inhibitory motor neuron (IMN) activation causes muscle relaxation. EMNs release acetylcholine (ACh), which acts at muscarinic receptors on smooth muscle cells and adjacent interstitial cells of Cajal, causing excitatory junction potentials (EJPs). IMNs release ATP (or another purine) and nitric oxide to cause inhibitory junction potentials (IJPs) and muscle relaxation. We used commercially available choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-yellow fluorescent protein (YFP) bacterial artificial chromosome (BAC) transgenic mice, which express ChR2 in cholinergic neurons, to study cholinergic neuromuscular transmission in the colon. Intracellular microelectrodes were used to record IJPs and EJPs from circular muscle cells. We used blue light stimulation (BLS, 470 nm, 20 mW/mm2) and electrical field stimulation (EFS) to activate myenteric neurons. EFS evoked IJPs only, whereas BLS evoked EJPs and IJPs. Mecamylamine (10 µM, nicotinic cholinergic receptor antagonist) reduced BLS-evoked IJPs by 50% but had no effect on electrically evoked IJPs. MRS 2179 (10 µM, a P2Y1 receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and Nω-nitro-l-arginine (100 µM, nitric oxide synthase inhibitor) isolated the EJP, which was blocked by scopolamine (1 µM, muscarinic ACh receptor antagonist). Immunohistochemistry revealed ChAT expression in ~88% of enhanced YFP (eYFP)-expressing neurons, whereas 12% of eYFP neurons expressed nitric oxide synthase. These data show that cholinergic interneurons synapse with EMNs and IMNs to cause contraction and relaxation of colonic smooth muscle.NEW & NOTEWORTHY Electrical stimulation of interganglionic connectives has been used widely to study synaptic transmission in the enteric nervous system. However, electrical stimulation will activate many types of neurons and nerve fibers, which complicates data interpretation. Optogenetic activation of enteric neurons using genetically modified mice expressing channelrhodopsin-2 in cholinergic neurons offers a new approach that provides more specificity for nerve stimulation when studying myenteric plexus nerve circuitry.

Activity within specific enteric neurochemical subtypes is correlated with distinct patterns of gastrointestinal motility in the murine colon.

The enteric nervous system in the large intestine generates two important patterns relating to motility: 1) propagating rhythmic peristaltic smooth muscle contractions referred to as colonic migrating motor complexes (CMMCs) and 2) tonic inhibition, during which colonic smooth muscle contractions are suppressed. The precise neurobiological substrates underlying each of these patterns are unclear. Using transgenic animals expressing the genetically encoded calcium indicator GCaMP3 to monitor activity or the optogenetic actuator channelrhodopsin (ChR2) to drive activity in defined enteric neuronal subpopulations, we provide evidence that cholinergic and nitrergic neurons play significant roles in mediating CMMCs and tonic inhibition, respectively. Nitrergic neurons [neuronal nitric oxide synthase (nNOS)-positive neurons] expressing GCaMP3 exhibited higher levels of activity during periods of tonic inhibition than during CMMCs. Consistent with these findings, optogenetic activation of ChR2 in nitrergic neurons depressed ongoing CMMCs. Conversely, cholinergic neurons [choline acetyltransferase (ChAT)-positive neurons] expressing GCaMP3 markedly increased their activity during the CMMC. Treatment with the NO synthesis inhibitor Nω-nitro-l-arginine also augmented the activity of ChAT-GCaMP3 neurons, suggesting that the reciprocal patterns of activity exhibited by nitrergic and cholinergic enteric neurons during distinct phases of colonic motility may be related.NEW & NOTEWORTHY Correlating the activity of neuronal populations in the myenteric plexus to distinct periods of gastrointestinal motility is complicated by the difficulty of measuring the activity of specific neuronal subtypes. Here, using mice expressing genetically encoded calcium indicators or the optical actuator channelrhodopsin-2, we provide compelling evidence that cholinergic and nitrergic neurons play important roles in mediating coordinated propagating peristaltic contractions or tonic inhibition, respectively, in the murine colon.

Su1575 – Estrogen Increases the Expression of BkCA and Impairs the Contraction of Colon Smooth Muscle via Up-Regulation of SPHK1

Su1575 – Estrogen Increases the Expression of BkCA and Impairs the Contraction of Colon Smooth Muscle via Up-Regulation of SPHK1

Altered Gut Motility in Mice Lacking Neuropilin 2 in Smooth Muscle

Introduction/ObjectivesProfound changes in gut motility and colonic smooth muscle contraction are observed in inflammatory bowel disease states such as colitis. However, the mechanisms that contribute to altered motility and contractility under these circumstances are incompletely defined. Recent data from our group have identified the smooth muscle of the gastrointestinal tract as a major site of expression of neuropilin 2 (Nrp2) in vivo. Nrp2 and the related molecule neuropilin 1 are 130–140 kDa transmembrane receptors for the class 3 semaphorin (SEMA3) family of axonal guidance regulators and for members of the vascular endothelial growth factor (VEGF) family that regulate angiogenesis. Based on recent data from our group implicating Nrp2 as a novel regulator of bladder smooth muscle contractility, the objective of this study was to determine the functional significance of Nrp2 in gastrointestinal smooth muscle.MethodsTwo mouse models were used to explore a role for Nrp2 in gastrointestinal motility: (i) SM22alpha‐Cre; Nrp2 fl/fl mice to enable inducible smooth muscle‐specific deletion of Nrp2 in vivo under controlled conditions and (ii) wild type Balb/c mice exposed to dextran sodium sulfate (DSS) to evoke acute colitis. Gut tissues were subjected to isometric tension testing to characterize contractility. Gut transit was evaluated by charcoal meal assay. Nrp2 levels were assessed by immunohistochemical staining and immunoblot analysis of tissues and of primary colonic smooth muscle cells exposed to inflammatory cytokines.ResultsSmooth muscle‐specific deletion of Nrp2 led to an increase in force generation of colonic rings, upon exposure to electrical field stimulation or treatment with the cholinergic agonist, carbachol. Nrp2 deletion also led to elevated gut transit as assessed by charcoal meal assay. In response to colitis, Nrp2 levels were found to decrease in the distal colon, in parallel with elevated contractility of distal colon rings. In addition, treatment of primary human colonic smooth muscle cells with IL‐4, representative of the TH2 inflammatory milieu associated with DSS colitis, decreased Nrp2 levels while treatment with TNF‐a, characteristic of the TH1 response in colitis increased Nrp2 levels.ConclusionsThese observations suggest that dynamic alterations in Nrp2 under conditions of inflammation may underlie altered contractility of intestinal smooth muscle, resulting in dysmotility. Moreover, these findings suggest that Nrp2 may represent a novel therapeutic target for restoration of normal gut motility.Support or Funding InformationHarvard Digestive Diseases Center P&amp;F Grant (RMA); R01 DK104641 (RMA, DRB, MPS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Central 5-hydroxytryptamine (5-HT) mediates colonic motility by hypothalamus oxytocin-colonic oxytocin receptor pathway

Gut-derived 5-hydroxytryptamine (5-HT) is well known for its role in mediating colonic motility function. However, it is not very clear whether brain-derived 5-HT is involved in the regulation of colonic motility. In this study, we used central 5-HT knockout (KO) mice to investigate whether brain-derived 5-HT mediates colonic motility, and if so, whether it involves oxytocin (OT) production in the hypothalamus and OT receptor in the colon. Colon transit time was prolonged in KO mice. The OT levels in the hypothalamus and serum were decreased significantly in the KO mice compared to wild-type (WT) controls. OT increased colonic smooth muscle contraction in both KO and WT mice, and the effects were blocked by OT receptor antagonist and tetrodotoxin but not by hexamethonium or atropine. Importantly, the OT-induced colonic smooth muscle contraction was decreased significantly in the KO mice relative to WT. The OT receptor expression of colon was detected in colonic myenteric plexus of mice. Central 5-HT is involved in the modulation of colonic motility which may modulate through its regulation of OT synthesis in the hypothalamus. Our results reveal a central 5-HT - hypothalamus OT - colonic OT receptor axis, providing a new target for the treatment of brain-gut dysfunction.

Tyrosine kinase Pyk2 is involved in colonic smooth muscle contraction via the RhoA/ROCK pathway.

The contraction of gastrointestinal (GI) smooth muscles is regulated by both Ca(2+)-dependent and Ca(2+) sensitization mechanisms. Proline-rich tyrosine kinase 2 (Pyk2) is involved in the depolarization-induced contraction of vascular smooth muscle via a Ca(2+) sensitization pathway. However, the role of Pyk2 in GI smooth muscle contraction is unclear. The spontaneous contraction of colonic smooth muscle was measured by using isometric force transducers. Protein and phosphorylation levels were determined by using western blotting. Pyk2 protein was expressed in colonic tissue, and spontaneous colonic contractions were inhibited by PF-431396, a Pyk2 inhibitor, in the presence of tetrodotoxin (TTX). In cultured colonic smooth muscle cells (CSMCs), PF-431396 decreased the levels of myosin light chain (MLC20) phosphorylated at Ser19 and ROCK2 protein expression, but myosin light chain kinase (MLCK) expression was not altered. However, Y-27632, a Rho kinase inhibitor, increased phosphorylation of Pyk2 at Tyr402 and concomitantly decreased ROCK2 levels; the expression of MLCK in CSMCs did not change. The expression of P(Tyr402)-Pyk2 and ROCK2 was increased when CSMCs were treated with Ach. Pyk2 is involved in the process of colonic smooth muscle contraction through the RhoA/ROCK pathway. These pathways may provide very important targets for investigating GI motility disorders.

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.

The potential role of thyrotropin-releasing hormone in colonic dysmotility induced by water avoidance stress in rats

ObjectiveThis study sought to investigate the effect and underlying mechanism of thyrotrophin releasing hormone (TRH) on colonic contractile disorders induced by chronic water avoidance stress (WAS). MethodsMale SD rats were exposed to daily 1-h WAS or sham WAS for 10 consecutive days. The presence of TRH in the serum and colonic mucosa were determined using enzyme immunoassay kits. Immunohistochemistry and western blotting were performed to detect the expression of TRH receptor 1 (TRH-R1). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record the currents of both L-type calcium currents (ICa,L) and large conductance Ca2+-activated K+ (BKCa) channels in colonic smooth muscle cells (SMCs) isolated from adult rats. ResultsEnzyme immunoassay revealed that TRH was present in both serum and colonic mucosa and that this expression increased in the WAS group. Immunohistochemistry revealed that the TRH-R1 level increased in colons devoid of mucosa and submucosa from the stressed rats as compared with the control group. TRH increased the spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner in vitro. The effect was also confirmed in an vivo experiment, where an intraperitoneal injection of TRH in rats significantly increased fecal pellet output during a 24-h period as compared with the control group. Furthermore, intraperitoneal injection of a non-specific TRH receptor antagonist, chlordiazepoxide and a TRH-R1 antibody, partially decreased the fecal pellets of WAS rats during the 10-day stress period. Furthermore, TRH increased the peak current of L-type channels in colonic smooth muscle cells (SMCs) at a membrane potential of 0 mV, while the current of large conductance Ca2+-activated K+ (BKCa) channels was not changed following the addition of TRH. ConclusionTRH may be involved in the dysmotility induced by chronic stress and may have some potential clinical therapeutic use in regulating gut motility.

Uncovering the Mechanisms of Chinese Herbal Medicine (MaZiRenWan) for Functional Constipation by Focused Network Pharmacology Approach.

MaZiRenWan (MZRW, also known as Hemp Seed Pill) is a Chinese Herbal Medicine which has been demonstrated to safely and effectively alleviate functional constipation (FC) in a randomized, placebo-controlled clinical study with 120 subjects. However, the underlying pharmacological actions of MZRW for FC, are still largely unknown. We systematically analyzed the bioactive compounds of MZRW and mechanism-of-action biological targets through a novel approach called “focused network pharmacology.” Among the 97 compounds identified by UPLC-QTOF-MS/MS in MZRW extract, 34 were found in rat plasma, while 10 were found in rat feces. Hierarchical clustering analysis suggest that these compounds can be classified into component groups, in which compounds are highly similar to each other and most of them are from the same herb. Emodin, amygdalin, albiflorin, honokiol, and naringin were selected as representative compounds of corresponding component groups. All of them were shown to induce spontaneous contractions of rat colonic smooth muscle in vitro. Network analysis revealed that biological targets in acetylcholine-, estrogen-, prostaglandin-, cannabinoid-, and purine signaling pathways are able to explain the prokinetic effects of representative compounds and corresponding component groups. In conclusion, MZRW active components enhance colonic motility, possibly by acting on multiple targets and pathways.

Comparison of In Vitro Effects of Opioid Analgesics on Spontaneous Proximal and Distal Colon Contractions in Healthy Rats and Rats with Peritonitis.

The goal of this study was to investigate and compare the effects of opioids on proximal and distal colon contractions in normal rats and rats with peritonitis, with and without the presence of naloxone in the environment. The study was approved by Cumhuriyet University Ethics committee. In this study, 16 Wistar Albino male rats were used. Rats were divided into two groups. Peritonitis was induced using a cecum ligation and perforation method, 24 h before the tissues of rats in the peritonitis group were collected, and sham surgery was performed 24 h before the tissues of rats in the control group were collected. Twenty-four hours after the surgery, rats' organs were harvested and hung in organ baths. Concentration-dependent inhibitory effects of morphine and meperidine on spontaneous intestinal movements were observed. Any differences between the groups were tested using the Kruskal-Wallis test, and any differences between the groups were tested using the Tukey test. No significant difference was observed between the proximal and distal colon smooth muscle contraction responses in both groups after 80 mM Potassium Chloride (KCl) injection (p>0.005). In the peritonitis group, amplitudes and frequencies of spontaneous contractions in proximal and distal colon significantly increased (p<0.05). Drugs decreased the amplitude and frequency responses in the control group (p<0.05). In the peritonitis group, whereas morphine decreased the amplitude and frequency responses in comparison with the control group (p<0.05), meperidine did not cause any significant changes (p>0.05). In both groups, adding naloxone to the organ baths before adding opioids completely blocked the morphine's inhibitory effect on the amplitude and frequency (p<0.05), but it could not completely block the inhibition caused by meperidine. Morphine and meperidine exhibit an inhibitory effect on the intestinal motility in both groups. This effect can be blocked by naloxone completely in morphine, and partially in meperidine.

Tu1590 - Advanced Glycation End Products Accumulated with Aging Inhibit Colonic Smooth Muscle Contraction by Upregulation of BK Channel Î'1-Subunit

Tu1590 - Advanced Glycation End Products Accumulated with Aging Inhibit Colonic Smooth Muscle Contraction by Upregulation of BK Channel Î'1-Subunit

Potential role of fecal microbiota from patients with slow transit constipation in the regulation of gastrointestinal motility

The gut microbiota is involved in various physiological functions, and disturbances in the host-microbiome have been proven to contribute to the dysfunction of gut; however, whether microbiota participates in the pathogenesis of constipation remains unclear. In this study, we extracted and analyzed microbiota in feces from constipated donors who had undergone effective therapy with fecal microbiota transplantation, transplanted microbiota into pseudo-germ-free mice, and measured gut motility. These mice presented with lower pellet frequency and water percentage, smaller pellet size, delayed gastrointestinal transit time, and weaker spontaneous contractions of colonic smooth muscle. To determine the mechanism underlying delayed gut motility, microbial metabolites were measured. Short chain fatty acids and secondary bile acids were decreased in mice receiving microbiota from constipated donors. Moreover, the compositional changes of gut microbiota in constipated patients were identified, including the operational taxonomic unit, and the species richness and α diversity were much greater than those in healthy volunteers. These findings suggest that alterations of the microbiome might affect gut motility via altered microbial-derived metabolites in the development of constipation, and the restoration of disturbed microbiota might improve the clinical phenotype. This study indicates that regulating the intestinal environment may be a novel therapy strategy for constipation.