Colonic Longitudinal Muscle Research Articles

Characterization of Vabicaserin (SCA-136), a Selective 5-Hydroxytryptamine 2C Receptor Agonist

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor subtype has received considerable attention as a target for drug discovery, having been implicated in a wide variety of disorders. Here, we describe the in vitro pharmacological profile of the novel 5-HT(2C) receptor-selective agonist vabicaserin [(-)-4,5,6,7,9,9a,10,11,12,12a-decahydrocyclopenta[c] [1,4]diazepino[6,7,1-ij]quinoline hydrochloride] (SCA-136), including a comprehensive strategy to assess 5-HT(2B) receptor selectivity using diverse preparations and assays of receptor activation. Vabicaserin displaced (125)I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT(2C) receptor sites in Chinese hamster ovary cell membranes with a K(i) value of 3 nM and was >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT(2B) receptor subtype using [(3)H]5HT was 14 nM. Vabicaserin was a potent and full agonist (EC(50), 8 nM; E(max), 100%) in stimulating 5-HT(2C) receptor-coupled calcium mobilization and exhibited 5-HT(2A) receptor antagonism and 5-HT(2B) antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. In rat stomach fundus and human colonic longitudinal muscle endogenously expressing 5-HT(2B) receptors, vabicaserin failed to induce a 5-HT(2B) receptor-dependent contraction and produced a rightward shift of the 5-HT and α-methyl-5-HT concentration-response curves in these preparations, respectively, consistent with 5-HT(2B) competitive antagonism. Likewise, vabicaserin failed to induce a 5-HT(2B) receptor-mediated contraction in arteries from deoxycorticosterone acetate-salt-treated rats, a model of hypersensitized 5-HT(2B) receptor function, and produced a rightward shift in the 5-HT-induced response that was consistent with 5-HT(2B) receptor antagonism. In summary, vabicaserin is a novel, potent, and selective 5-HT(2C) receptor agonist.

Activation of Corticotropin-Releasing Factor Receptor 2 Mediates the Colonic Motor Coping Response to Acute Stress in Rodents

Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress. We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2). In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2). In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress.

Acetylene compound isolated from Atractylodes japonica stimulates the contractility of rat distal colon via inhibiting the nitrergic–purinergic relaxation

Our previous research has showed that rhizome of Atractylodes japonica Koidz (Compositae) exhibits an increase in the spontaneous contractility of distal colon in rats. The aims of this study are to identify the phytochemical(s), which stimulate(s) the colonic contractility, contained in Atractylodes japonica and to evaluate the pharmacological mechanism responsible for the colonic muscle contraction. Based on the stimulatory activity-guided fractionation on the isometric contraction of rat distal colonic strips, atractylodiol (ATD) and diacetyl-atractylodiol (DATD) were isolated from the CHCl(3) fractions of Atractylodes japonica. ATD and DATD dose-dependently increased both tension and amplitude of distal colon longitudinal muscle (DCLM), but they stimulated only amplitude in the distal colon circular muscle. The ED(50) values of ATD and DATD to stimulate the amplitude of DCLM were revealed as 9.1×10(-9)M and 1.8×10(-8)M, respectively. l-NAME (0.1mM) significantly increased the ADT (1μM)-induced contraction of DCLM, whereas SNAP (0.1mM) markedly reduced the stimulatory effects of ATD on DCLM contractility. The combined effects of SNAP and atropine (0.5μM) on the ATD-induced contraction of DCLM were similar to the inhibitory effects of SNAP alone. Suramin (0.1mM) significantly enhanced the increase of ATD-induced DCLM contraction, whereas ADPβS (0.1mM) markedly abolished the stimulatory effects of ATD on the spontaneous contractility of DCLM. The present results demonstrate that acetylene compounds, ATD and DATD, are the effective phytochemical of Atractylodes japonica to stimulate the motility of distal colon in rats, and ATD possibly enhances the spontaneous contractility of distal colon through inhibiting the mechanism of nitrergic-purinergic relaxation.

Differential recruitment of high affinity A 1 and A 2A adenosine receptors in the control of colonic neuromuscular function in experimental colitis

This study investigated the expression of A 1 and A 2A receptors in the rat colonic neuromuscular compartment, and characterized their roles in the control of motility during inflammation. Colitis was induced by 2,4-dinitrobenzenesulfonic acid. A 1, A 2A receptors, and ecto-5′-nucleotidase (CD73, adenosine producing enzyme) mRNA expression was examined by RT-PCR. The effects of DPCPX (A 1 receptor antagonist), CCPA (A 1 receptor agonist), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (A 2A receptor antagonist), 4-[2-[[6-amino-9-(N-ethyl-b- d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (A 2A receptor agonist), AOPCP (CD73 inhibitor) were tested on electrically or carbachol-evoked contractions in colonic longitudinal muscle preparations. In normal colon, RT-PCR revealed the presence of A 1 receptors, A 2A receptors and CD73, and an increased expression of A 2A receptors and CD73 was detected in inflamed tissues. In normal colon, DPCPX or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol enhanced electrically-induced contractions, while in inflamed preparations the effect of DPCPX no longer occurred. In normal colon, CCPA or 4-[2-[[6-amino-9-(N-ethyl-b- d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride decreased electrically-induced contractions. Under inflammation, 4-[2-[[6-amino-9-(N-ethyl-b- d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride reduced electrically evoked contractions with higher efficacy, while the inhibition by CCPA remained unchanged. A 1 and A 2A receptor ligands did not affect carbachol-induced contractions. AOPCP enhanced electrically-induced contractions and prevented the contractile effects of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, without interfering with DPCPX, both in normal and inflamed colons. These results indicate that, in normal colon, both A 1 and A 2A receptors contribute to the inhibitory control of motor functions at neuronal level. Under bowel inflammation, A 1 receptor loses its modulating actions, while the recruitment of A 2A receptor by CD73-dependent endogenous adenosine drives an enhanced inhibitory control of colonic neuromotility.

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.

Colon lengthening slows transit: is this the mechanism underlying redundant colon or slow transit constipation?

The study by Heredia et al. (2010) in a recent issue of The Journal of Physiology shows that elongation of colon longitudinal muscle results in slow colonic transit. This study was performed in mice, but the results are probably applicable to humans. The study by Heredia et al. presents a new mechanism that may explain the association of elongated colon and poor motility and might be manipulated to overcome bowel lengthening and stasis. Their study shows that elongation of the longitudinal muscle triggers inhibition of the colonic migrating motor complex (CMMC), resulting in slow colonic transit. In humans, slow colonic transit occurs in patients with chronic constipation and is known as slow transit constipation (STC) (Hutson, 2009). Interestingly, we have observed that many patients with slow transit constipation have an elongated transverse colon (Fig. 1). The results from Heredia et al. suggest this elongation would inhibit colonic motility. Differences in colon length in children with constipation A, normal length colon; B, elongated transverse colon. Both images are from radioisotope transit studies with gamma camera images taken 24 h after ingesting a radioactive milk drink (Southwell et al. 2009). Note the large loop of transverse colon in B. The elongated colon is also narrower than the colon in A. Image provided by Dr Ian Yik (Surgical Research Group, Murdoch Childrens Research Institute, Royal Childrens Hospital, Melbourne, Australia). Elongated (called redundant) colon often occurs in patients with constipation. These patients are notable during colonic endoscopy because it is very hard to get along the length of the colon (Rex et al. 2007). Both colonic transit time and constipation symptoms increase with the number of redundancies and these are the patients that are least likely to respond to treatments (Raahave et al. 2009). Redundant bowel is often removed surgically. Propagating contractions in the proximal colon are highly propulsive and are a major determinant of proximal colonic flow in normal human colon (Dinning et al. 2008). High amplitude propagating contractions (HAPCs) underlie mass movements along the colon and may be similar to CMMC. Children with STC have reduced numbers of propagating contractions in the proximal colon (King, 2008) and lack of HAPC in constipated patients is associated with poor outcomes (van den Berg et al. 2006). Heredia et al. show that lengthening the colon inhibits CMMC in mice. Possibly the same is occurring in humans with colonic lengthening inhibiting HAPCs and so reducing colonic motility and slowing transit. STC can be associated with abnormal recto/sigmoid function (outlet obstruction, anorectal retention or functional fecal retention) or occur with normal recto/sigmoid function (Southwell et al. 2009). There is a lot of discussion about whether the defect is primarily at the outlet (ano-rectum) producing slowing of motility upstream as a secondary effect or if a defect can occur in the proximal colon without an outlet defect. Heredia et al.'s study shows the effects of elongation are different in the proximal and distal colon, and provides evidence that the proximal colon can inhibit motility in the distal colon. Thus it is possible that elongation can inhibit motility in the proximal colon primarily and this can then affect the distal colon. Heredia et al. also investigated the mechanism and showed that nitric oxide was released and activated inhibitory neurons reducing pellet propulsion and amplitude of colonic migrating motor complex (CMMC). Ileus (lack of movement of the bowel) develops after surgery and has been shown to be due to release of nitric oxide that then activates inhibitory neurons. Removal of the tonic nitrergic inhibition is a potent stimulus for human proximal colonic propagating sequences (Dinning et al. 2006). STC often develops after childbirth and there may be a connection between surgery (anaesthesia and caesarean) inducing nitric oxide release within the bowel and then leading to stasis in the ileum and in the colon. This study reveals a previously unrecognised mechanism for control of colonic motility: lengthening of the longitudinal muscle. If this mechanism can be manipulated in people, we may have a treatment for a currently poorly treated condition. Further detailed physiological studies of the effects of longitudinal muscle lengthening on migrating motor complexes in proximal and distal colon in other species should be helpful.

Control of enteric neuromuscular functions by purinergic A3 receptors in normal rat distal colon and experimental bowel inflammation

Adenosine A(3) receptors mediate beneficial effects in experimental colitis, but their involvement in enteric neuromuscular functions during bowel inflammation is undetermined. This study investigated the regulatory role of A(3) receptors on colonic motility in the presence of experimental colitis. Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. A(3) receptors and adenosine deaminase (ADA, adenosine catabolic enzyme) mRNA were examined by RT-PCR. Tissue distribution of A(3) receptors was detected by confocal immunofluorescence. The effects of 2,3-ethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS1523) (MRS, A(3) receptor antagonist), 2-chloro-N(6) -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (2Cl-IB-MECA) (CIB, A(3) receptor agonist), dipyridamole (DIP, adenosine transport inhibitor) and ADA were assayed on contractile responses evoked by electrical stimulation (ES) or carbachol in colonic longitudinal muscle preparations (LMP). RT-PCR showed A(3) receptors and ADA mRNA in normal colon and their increased level in inflamed tissues. Immunofluorescence showed a predominant distribution of A(3) receptors in normal myenteric ganglia and an increased density during colitis. MRS enhanced ES-induced cholinergic contractions in normal LMP, but was less effective in inflamed tissues. After pretreatment with dipyridamole plus ADA, to reduce extracellular adenosine, CIB decreased cholinergic motor responses of normal LMP to ES, with enhanced efficacy in inflamed LMP. A(3) receptor ligands did not affect carbachol-induced contractions in LMP from normal or inflamed colon. Normally, adenosine modulated colonic cholinergic motility via activation of A(3) receptors in the myenteric plexus. A(3) receptor-mediated tonic inhibitory control by adenosine was impaired in inflamed bowel, despite increased density of functioning and pharmacologically recruitable A(3) receptors.

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

Effectiveness of trimebutine maleate on modulating intestinal hypercontractility in a mouse model of postinfectious irritable bowel syndrome

Trimebutine maleate, which modulates the calcium and potassium channels, relieves abdominal pain in patients with irritable bowel syndrome. However, its effect on postinfectious irritable bowel syndrome is not clarified. The aim of this study was to investigate the effectiveness of trimebutine maleate on modulating colonic hypercontractility in a mouse model of postinfectious irritable bowel syndrome. Mice infected up to 8 weeks with T. spiralis underwent abdominal withdrawal reflex to colorectal distention to evaluate the visceral sensitivity at different time points. Tissues were examined for histopathology scores. Colonic longitudinal muscle strips were prepared in the organ bath under basal condition or to be stimulated by acetylcholine and potassium chloride, and consecutive concentrations of trimebutine maleate were added to the bath to record the strip responses. Significant inflammation was observed in the intestines of the mice infected 2 weeks, and it resolved in 8 weeks after infection. Visceral hyperalgesia and colonic muscle hypercontractility emerged after infection, and trimebutine maleate could effectively reduce the colonic hyperreactivity. Hypercontractility of the colonic muscle stimulated by acetylcholine and high K(+) could be inhibited by trimebutine maleate in solution with Ca(2+), but not in Ca(2+) free solution. Compared with 8-week postinfectious irritable bowel syndrome group, 2-week acute infected strips were much more sensitive to the stimulators and the drug trimebutine maleate. Trimebutine maleate was effective in reducing the colonic muscle hypercontractility of postinfectious irritable bowel syndrome mice. The findings may provide evidence for trimebutine maleate to treat postinfectious irritable bowel syndrome patients effectively.

Activation of proteinase activated receptor‐2 (PAR‐2) inhibits spontaneous activity in rat colon

Proteolytic cleavage of proteinase activated receptor‐2 (PAR‐2) reveals a tethered ligand which binds to and activates the receptor. Rat colonic smooth muscle shows spontaneous contractile activity which is generated by pacemaker cells. This study investigated the effects of PAR‐2 activation on spontaneous contractile activity in the isolated rat colon.Strips of rat colon longitudinal muscle were suspended in organ baths and increasing concentrations of trypsin, or the mimetic peptide FLIGRL, were applied. In some experiments carbachol was used to increase smooth muscle tone.Trypsin and FLIGRL each induced concentration‐dependent inhibition of spontaneous contractile activity of rat colon. Trypsin‐evoked relaxations (66.6±3.3; n=8) were abolished by apamin, an inhibitor of small conductance Ca2+‐activated potassium channels (SKCa). Similar results were obtained with FLIGRL (n=5). Cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca2+‐ATPase, also abolished the effects of trypsin (n=7). Neither trypsin (n=4) nor FLIGRL (n=4) reduced carbachol‐induced tone.In conclusion, activation of PAR‐2 inhibits spontaneous contractile activity in the rat colon via release of Ca2+ from intracellular stores and activation of SKCa channels. PAR‐2 activation is limited to pacemaker cells since no effect was observed on carbachol stimulated smooth muscle.Supported by NSERC

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.

Effects and its possible mechanism of Radix Saposhnikoviae on rat colonic smooth muscle in vitro

Objective To determine the effect of different concentrations of Radix Saposhnikoviae (RS) on the contraction of smooth muscle strips and the Ca 2+ mobilization of cultured smooth muscle cells of rat colon and its possible mechanism of action. Methods Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca 2+ was assessed using fluorescent intensity (FI) of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results In the in vitro experiment, RS (0.02, 0.2, 2, 20 g/L) inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations ( P < 0.01) for both Peak (the maximal contraction amplitude) and Area (the area under curves). Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant ( P < 0.01), as was the inhibition of the Area values in all RS groups ( P < 0.05). Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect ( P < 0.01). In experiments using primary smooth muscle cell cultures in Ca 2+ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values ( P < 0.05), and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant ( P < 0.01). However, in Ca 2+-free buffer, FS had no significant effect on cell fluorescence. Conclusion RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α-adrenoceptors, but not β-adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca 2+ from extracellular sources, but may had no effect on the release of Ca 2+ from sarcoplasmic reticulum and endoplasmic reticulum.

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

Inhibition of cyclooxygenase-2 and EP 1 receptor antagonism reduces human colonic longitudinal muscle contractility in vitro

We investigated the contribution of cyclo-oxygenase enzyme inhibition and prostamide agonism on human colonic contractility in vitro. The effects of the non-specific COX inhibitor diclofenac were compared against selective COX-2 inhibition via nimesulide, the prostanoid EP 1 receptor antagonist SC19220 or the prostaglandin prodrug/prostamide receptor agonist bimatoprost, on potency of contraction to acetylcholine in human colonic circular and longitudinal muscle strips. Pre-treatment with either nimesulide (10 −5 M) or diclofenac (10 −6 M) caused a significant decrease in the potency of acetylcholine-evoked longitudinal muscle contraction, but did not inhibit acetylcholine-evoked circular muscle contraction. Pre-treatment with the EP 1 receptor antagonist SC19220 (10 −5 M) similarly decreased cholinergic potency in longitudinal muscle, without influence on circular muscle contraction. The prostamide agonist bimatoprost (10 −6 M) increased basal circular and longitudinal muscle tone, but did not alter cholinergic potency in either muscle layer. In conclusion, colonic longitudinal muscle contraction is augmented by COX-2 activity, most likely via PGE 2 acting at EP 1 receptors. While colonic contraction is tonically modulated by bimatoprost, it does not share the same functional properties attributed to other endogenous COX-2 metabolites on colonic contractile function.

Activation of P2Y receptors by ATP and by its analogue, ADPβS, triggers two calcium signal pathways in the longitudinal muscle of mouse distal colon

Our previous research showed that ATP and adenosine 5′-O-2-thiodiphosphate (ADPβS) induce contractile effects in the longitudinal muscle of mouse distal colon via activation of P2Y receptors which are not P2Y 1 or P2Y 12 subtypes. This study investigated the nature of the P2Y receptor subtype(s) and the mechanisms leading to the intracellular calcium concentration increase necessary to trigger muscular contraction. Motor responses of mouse colonic longitudinal muscle to P2Y receptor agonists were examined in vitro as changes in isometric tension. ATP or ADPβS induced muscular contraction, which was not affected by P2Y 11 or P2Y 13 selective antagonists. Calcium-free solution or the calcium channel blocker, nifedipine, failed to modify the contractile responses to ATP or ADPβS, which were virtually abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-free medium with addition of cyclopiazonic acid. Neomycin or U-73122, phospholipase C inhibitors, or 2-aminoethoxy-diphenylborate (2-APB), membrane-permeant IP 3 receptor inhibitor reduced the response to ATP, whilst ryanodine or ruthenium red, inhibiting calcium release from ryanodine-sensitive stores, abolished the response to ADPβS. Responses to maximally effective concentrations of ATP and ADPβS were not fully additive. Desensitisation with ADPβS antagonized the contractile effects of ATP, as desensitisation with ATP antagonized the response to ADPβS. In the longitudinal muscle of mouse distal colon, ATP and ADPβS induce muscular contraction via a P2Y receptor, coupled to differential signal pathways leading to intracellular calcium increase.

EFFECTS OF DIRECT HAEMOPERFUSION THROUGH FIBRES IMMOBILIZING POLYMYXIN B AND NAFAMOSTAT MESILATE ON ENDOTOXAEMIA IN CONSCIOUS GUINEA‐PIGS

1. Direct haemoperfusion through a network of fibres immobilizing polymyxin B (PMX-B) is used for the treatment of septic shock, but the mechanism underlying its clinical benefits remains unclear. The aims of the present study were to assess the actions of direct haemoperfusion through fibres immobilizing PMX-B (PMX-DHP) on the effects of exogenously administered endotoxin in conscious guinea-pigs and to examine the difference in the effects of heparin compared with nafamostat mesilate (NM) used as an anticoagulant. Although nafamostat is widely used in Japan, the agent cannot necessarily be used elsewhere in the world. Therefore, the study aimed to investigate and elucidate the effectiveness of NM compared with heparin. 2. Colonic motion was monitored continuously via telemetry using a force transducer attached to the taenia caecum, whereas blood pressure was monitored using a carotid artery catheter. To establish a haemoperfusion circuit in each freely moving and conscious guinea-pig, catheters were implanted in the carotid artery and the jugular vein, tunnelled subcutaneously, exteriorized at the back of the neck in contact with a lightweight tethering spring and attached to a swivel device at the top of the cage. On the day after the operation, lipopolysaccharide (LPS; Escherichia coli, O111:B4; 1 mg/kg) was administered i.v. and PMX-DHP was conducted for 2 h. Heparin (50 IU/h) or NM (0.4 mg/h) was used as the anticoagulant. Furthermore, guinea-pigs were administered a lethal dose of LPS (10 mg/kg) and the survival rate was examined for animals undergoing PMX-DHP compared with control animals. 3. In guinea-pigs treated with PMX-DHP, relaxation of colonic longitudinal muscle caused by LPS was significantly suppressed, as were decreases in blood pressure. Of the two anticoagulants used, NM was more effective than heparin. In addition, PMX-DHP significantly increased the survival rate of guinea-pigs that received potentially lethal doses of LPS. 4. In conscious and unrestrained guinea-pig endotoxaemia model, PMX-DHP significantly improved intestinal paralysis and decreases in blood pressure. These effects were augmented more by NM than by heparin when an anticoagulant was used in the perfusion process. These findings suggest that haemoperfusion using PMX and NM performed in the early stage of endotoxaemia is an effective treatment.

The endocannabinoids anandamide and 2-arachidonoylglycerol inhibit cholinergic contractility in the human colon

The effects of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) were determined on cholinergic contractility in strips of human colonic longitudinal muscle and circular muscle in vitro, in the presence of nitric oxide synthase blockade with N-nitro-l-arginine (10(-4) M). Anandamide and 2-AG inhibited longitudinal muscle and circular muscle contractile responses to acetylcholine (10(-9)-10(-4) M) in a concentration-dependent manner. This was unaltered following pretreatment with the cannabinoid CB(1) receptor-selective antagonist AM251 (10(-7) M), however in isolation AM251 elicited a significant rightward shift in the potency of acetylcholine-evoked contraction in both longitudinal muscle and circular muscle preparations. Pretreatment with an inhibitor of anandamide catabolism, arachidonoyl trifluoromethyl ketone (10(-5) M), alone caused a significant decrease in the potency of acetylcholine-evoked contraction in both longitudinal and circular muscle, but had no significant additional effect on the anandamide-induced (10(-5) M) suppression of contraction. Pretreatment with the cannabinoid CB(2) receptor inverse agonist JTE 907 (10(-6) M) neither influenced the potency of acetylcholine-evoked contraction alone nor prevented the potency shift in acetylcholine-evoked contraction in the presence of anandamide (10(-5) M). The findings of the present study indicate that the endocannabinoids anandamide and 2-arachidonoylglycerol suppress colonic cholinergic contractility via a non conventional cannabinoid or non-cannabinoid receptor-mediated pathway. Cholinergic contraction may be tonically modulated by endocannabinoids and/or products of arachidonate metabolism unrelated to endocannabinoid production. The extent of anandamide metabolism is not sufficient to influence the functional effects of its exogenous administration in human colonic tissue in vitro.

Nitrotyrosylation of Ca2+ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis

Basal levels of c-Src kinase are known to regulate smooth muscle Ca(2+) channels. Colonic inflammation results in attenuated Ca(2+) currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase in experimental colitis. Ca(2+)-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K(+). The E(max) to CaCl(2) was significantly less in inflamed tissues (38.4 +/- 7.6%) than controls, indicative of reduced Ca(2+) influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a selective Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD(2) from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/peroxynitrite combination, the maximal contraction to CaCl(2) was reduced by 46 +/- 7% in controls but unaffected in inflamed tissues (13 +/- 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca(2+) currents by 84.1 +/- 3.9% in normal but only 36.2 +/- 13% in inflamed tissues. Neither the Ca(2+) channel Ca(v)1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca(2+) channel protein expression but increased nitrotyrosylated-Ca(2+) channel proteins during inflammation. These data suggest that post-translational modification of Ca(2+) channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca(2+) influx.

Nitric oxide mediates the inhibitory effect of ethanol on the motility of isolated longitudinal muscle of proximal colon in rats

The aim of the present study was to investigate the effect of ethanol on colon motility in rats and to test the possibility that nitric oxide (NO) mediates this effect. Proximal colon longitudinal muscle strips (LM) (8 x 3 mm) cut parallel to the longitudinal muscle fibres of the colon were isolated and mounted in an organ bath. Ethanol (0.57, 0.87 and 1.30 mmol L(-1)) dose-dependently inhibited the motility of LM. Longitudinal muscle strips from female rats were more sensitive to the inhibitory effect of ethanol than that from male rats. L-NAME (N-nitro-L-arginine methyl ester) (100 micromol L(-1)), AG (aminoguanidine) (10 micromol L(-1)), ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) (10 micromol L(-1)) and PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) (200 micromol L(-1)) partly blocked the inhibitory effect of ethanol on LM. Pretreatment with L-NAME, AG, ODQ and PTIO abolished the sex difference of the inhibitory effect of ethanol on LM. Tetrodotoxin (TTX) (10 micromol L(-1)) partly blocked the inhibitory effect but did not influence the sex difference. The relaxation of LM induced by SNP (sodium nitroprusside) (0.1-10 micromol L(-1)) in female rats was greater than that in male rats. In conclusion, ethanol inhibited the colon motility in vitro. This inhibitory effect on LM was mediated by NO through the iNOS - NO - cGMP pathway.

In vitro characterization of the effects of endomorphin 1 and 2, endogenous ligands for μ-opioid receptors, on mouse colonic motility

The effects of endomorphin 1 (EM1) and 2 (EM2) in colonic motility remain unknown. We investigated the effects and mechanisms of these endomorphins (EMs) on the colonic motility in vitro by applying various neural blocking agents and various opioid receptor antagonists. EMs (10−9 to 10−6M) displayed significant stimulatory effects on the basal tonus or spontaneous activity of mouse colon but not of stomach and small intestine. It is noteworthy that the contractile actions of EMs varied slightly among different regions of colonic longitudinal muscle layers, whereas the contractile responses induced by EMs were significantly different among different regions of circular muscle layers. EMs-induced longitudinal or circular muscle contractions were not significantly affected by atropine, NG-nitro-l-arginine methyl ester, phentolamine, propranolol and methysergide. Tetrodotoxin, indomethacin and naloxone completely abolished the EMs-induced colonic contractions. Surprisingly, EMs (10−7M)-induced longitudinal muscle contractions were significantly attenuated by nor-binaltorphimine (3×10−6M). By contrast, pretreatment with naltrindole (10−6M) did not significantly affect EMs-induced longitudinal or circular muscle contractions. Interestingly, the circular muscle contractions in response to EM2 (10−7M) were not fully blocked by β-funaltrexamine (6×10−6M). Naloxonazine (10−6M) almost fully antagonized the EMs-induced longitudinal or circular muscle contractions, and these effects could be only partially reversed by extensive washing. All the results indicated that the mechanisms and sites of actions of EMs were region-specific. Furthermore, these findings showed that the activation of multiple subtypes of opioid receptors, possibly including μ1 (naloxonazine-sensitive), μ2 and even other forms of μORs (β-FNA-insensitive), was required for EMs-induced mouse colonic motility.