Intestinal Longitudinal Smooth Muscle Research Articles

Cytokine-induced S-nitrosylation of soluble guanylyl cyclase and expression of phosphodiesterase 1A contribute to dysfunction of longitudinal smooth muscle relaxation.

The effect of proinflammatory cytokines on the expression and activity of soluble guanylyl cyclase (sGC) and cGMP-phosphodiesterases (PDEs) was determined in intestinal longitudinal smooth muscle. In control muscle cells, cGMP levels are regulated via activation of sGC and PDE5; the activity of the latter is regulated via feedback phosphorylation by cGMP-dependent protein kinase. In muscle cells isolated from muscle strips cultured with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) or obtained from the colon of TNBS (2,4,6-trinitrobenzene sulfonic acid)-treated mice, expression of inducible nitric oxide synthase (iNOS) was induced and sGC was S-nitrosylated, resulting in attenuation of nitric oxide (NO)-induced sGC activity and cGMP formation. The effect of cytokines on sGC S-nitrosylation and activity was blocked by the iNOS inhibitor 1400W [N-([3-(aminomethyl)phenyl]methyl)ethanimidamide dihydrochloride]. The effect of cytokines on cGMP levels measured in the absence of IBMX (3-isobutyl-1-methylxanthine), however, was partly reversed by 1400W or PDE1 inhibitor vinpocetine and completely reversed by a combination of 1400W and vinpocetine. Expression of PDE1A was induced and was accompanied by an increase in PDE1A activity in muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice; the effect of cytokines on PDE1 expression and activity was blocked by MG132 (benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), an inhibitor of nuclear factor κB activity. NO-induced muscle relaxation was inhibited in longitudinal muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice, and this inhibition was completely reversed by the combination of both 1400W and vinpocetine. Inhibition of smooth muscle relaxation during inflammation reflects the combined effects of decreased sGC activity via S-nitrosylation and increased cGMP hydrolysis via PDE1 expression.

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

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

Hypercontractility of Intestinal Longitudinal Smooth Muscle Induced by Cytokines Is Mediated by the Nuclear Factor-κB/AMP-Activated Kinase/Myosin Light Chain Kinase Pathway

Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca(2+)/calmodulin-dependent kinase kinase (CaMKKβ) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC20 phosphorylation and muscle contraction. Blockade of CaMKKβ with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1β or tumor necrosis factor-α, nuclear factor κB was activated as indicated by an increase in p65 phosphorylation and IκBα degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)-specific site (Ser(485)) that is distinct from the stimulatory CaMKKβ site (Thr(172)), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-κB activity with MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser(485) and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from IκBα complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor κB/PKA-dependent inhibition of AMPK and activation of MLCK.

Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of proteins best known for its role in neuronal survival, differentiation, migration, and synaptic plasticity in central and peripheral neurons. BDNF is also widely expressed in nonneuronal tissues including the gastrointestinal tract. The role of BDNF in intestinal smooth muscle contractility is not well defined. The aim of this study was to identify the role of BDNF in carbachol (CCh)- and substance P (SP)-induced contraction of intestinal longitudinal smooth muscle. BDNF, selective tropomyosin-related kinase B (TrkB) receptor agonists, and pharmacological inhibitors of signaling pathways were examined for their effects on contraction of rabbit intestinal longitudinal muscle strips induced by CCh and SP. BDNF activation of intracellular signaling pathways was examined by Western blot in homogenates of muscle strips and isolated muscle cells. One-hour preincubation with BDNF enhanced intestinal muscle contraction induced by CCh but not by SP. The selective synthetic TrkB agonists LM 22A4 and 7,8-dihydroxyflavone produced similar effects to BDNF. The Trk antagonist K-252a, a TrkB antibody but not p75NTR antibody, blocked the effect of BDNF. The enhancement of CCh-induced contraction by BDNF was blocked by the phospholipase C (PLC) antagonist U73122, but not by ERK1/2 or Akt antagonists. Direct measurement in muscle strips and isolated muscle cells showed that BDNF caused phosphorylation of TrkB receptors and PLC-γ, but not ERK1/2 or Akt. We conclude that exogenous BDNF augments the CCh-induced contraction of longitudinal muscle from rabbit intestine by activating TrkB receptors and subsequent PLC activation.

Brain‐derived neurotrophic factor augments the contraction of intestinal longitudinal muscle strips induced by Carbachol

BackgroundWe have demonstrated that intestinal longitudinal smooth muscle cells synthesize and secrete brain derived neurotrophic factor (BDNF) in response to neuropeptide transmitters. But the function of this BDNF in the gastrointestinal tract is undetermined.Aimto test the effect of BDNF on agonist‐induced contraction in intestinal longitudinal smooth muscle.Material and methodRabbit longitudinal muscle strips were prepared from the intestine. The contraction of the muscle strips was recorded in response to substance P 10–6M (SP 10–6) and Carbachol 10–5M (CCh 10–5). Muscle strips were preincubated with 10 nM exogenous BDNF and the changes in SP/CCh‐induced contraction were examined. A Trk antagonist and two Trk B specific agonists were used to investigate the role of the Trk B receptor in the effect of BDNF on contraction.ResultsBDNF significantly enhanced the contraction of longitudinal smooth muscle induced by CCh 10–5. However, contraction induced by SP 10–6 was not affected by pretreatment with BDNF. The pan Trk receptor inhibitor (K252a) inhibited the effect of BDNF on CCh‐induced contraction. Moreover, two Trk B agonists (LM 22A4 & 7, 8‐dihydroxyflavone) augmented the CCh‐induced contraction of the longitudinal muscle strips.ConclusionBDNF enhances the cholinergic‐induced longitudinal muscle contraction through the activation of its cognate Trk B receptor.

Substance P‐induced increase in Brain Derived Neurotrophic Factor expression and secretion by intestinal smooth muscle is mediated by a rise in intracellular Ca2+

BackgroundWe have previously shown that intestinal longitudinal smooth muscle cells express and secrete brain derived neurotrophic factor (BDNF) and addition of substance P (SP) to cultured smooth muscle cells induces further expression and secretion of BDNF above basal levels. It is not known, however, what cellular signaling pathways mediate SP‐induced expression and secretion of BDNF.AimTo identify the signaling pathways involved in BDNF synthesis and secretion by smooth muscle in response to SP.MethodCultured intestinal smooth muscle cells were treated with 10–100 nM SP for 24 hours in the presence and absence of 1,2 bis (o‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid (BAPTA) to chelate intracellular Ca2+. BDNF protein expression and secretion were measured by in‐cell western and ELISA, and BDNF mRNA expression was measured by qRT‐PCR.ResultsThe expression of BDNF protein was increased by 10–121% and BDNF secretion was increased by 50% by SP. Consistent with this, BDNF mRNA expression was increased by 161% by SP. Both BDNF protein and mRNA responses to SP were blocked by BAPTA.ConclusionSP stimulation of BDNF expression and secretion requires increased in intracellular Ca2+. The source of the intracellular Ca2+ increased remains to be determined. Supported by grants DK15564 and DK34153 from the National Institutes of Health.

Cytokine modulation of muscarinic receptors in the murine intestine

The extent to which gut motility and smooth muscle contractility are altered by intestinal inflammation depends on the nature of the underlying immune activation. The muscarinic receptor on smooth muscle plays a critical role in mediating acetylcholine-driven motor function. We examined the ability of cytokines to influence muscarinic receptor characteristics on intestinal longitudinal muscle and related the findings to studies on carbachol-induced contraction. Cells were isolated from longitudinal muscle myenteric plexus (LMMP). Cytokine receptor expression, muscle contractility, and muscarinic agonist receptor characteristics were examined by agonist displacement of [N-methyl-(3)H]scopolamine ([(3)H]NMS) binding. The TGF-beta1 receptor (543 bp) and the IFN-gamma receptor 1 (660 bp) were identified on smooth muscle cells. Scatchard analysis revealed dissociation constant and maximum binding values for [(3)H]NMS of 2.6 nM and 2.4 x 10(4) sites/cell, respectively, in control cells. Nematode infection was accompanied by a reduction in inhibitory constant of the high-affinity sites (K(H)), and this was independent of signal transduction and activator of transcription 6. Preincubation with TGF-beta1 enhanced longitudinal muscle contractility and decreased the K(H) to 2.2 pM (increased muscarinic receptor affinity), whereas preincubation with IFN-gamma increased the K(H) to 0.4 muM (decreased muscarinic receptor affinity) and decreased longitudinal muscle contractility. Preincubation of LMMP with IL-13 decreased the K(H) to 0.2 nM. Cytokines exert differential effects on the muscarinic receptor on intestinal longitudinal smooth muscle. These findings explain the basis for altered muscle contractility observed in Th1 and Th2 models of inflammation, as well as in the post-nematode-infected state.

Role of cADPR in sodium nitroprusside-induced opossum esophageal longitudinal smooth muscle contraction

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca(2+) mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC(50) of 1 nM and peak contraction of 57 +/- 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10(-6) M. The EC(50) of cADPR caused contraction (18 +/- 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 muM) induced contraction of LSM strips that averaged 67 +/- 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.

Contractile activity of mouse small intestinal longitudinal smooth muscle 1,2

Background Interest in genomic modulation experimentally often necessitates use of mouse models. Materials and methods AIM: To characterize and quantitate smooth muscle contractile activity of the mouse small intestine using in vitro techniques. Full-thickness jejunal and ileal muscle strips from mice were cut in the direction of longitudinal muscle, suspended in tissue baths (37°C), and connected to force transducers. Spontaneous contractility and two dose-response curves to the cholinergic agonist bethanechol and adrenergic agonist norepinephrine were quantitated for 6 h. Results Total contractile activity increased over 4 to 5 h in jejunum ( P < 0.01) but not in ileum. Frequency of contractions (counts/min) in jejunum increased from 16 to 33 ( P < 0.01) in the first 4 h, then remained stable; ileal frequency did not change. One hour of cold preservation had no major effect on contractile activity and frequency. Bethanechol increased and norepinephrine decreased contractile activity in dose-dependent fashion. The dose of bethanechol producing 50% increase in maximal response did not differ between the first and second dose-response; in contrast, the concentration of norepinephrine producing 50% decrease in activity for the second dose-response in jejunum was decreased compared to the first dose-response ( P < 0.01). Cold preservation had no substantive effect on agonist responses. Conclusion Experiments in murine jejunal but not ileal longitudinal muscle in vitro must consider early changes in contractile activity after tissue harvest. These experiments serve as a baseline for comparison of stimuli or genetic modifications on murine contractile activity of longitudinal muscle in vivo.

Ca 2+-recruitment in tachykinin-induced contractions of gut smooth muscle from African clawed frog, Xenopus laevis and rainbow trout, Oncorhynchus mykiss

Changes in intracellular Ca 2+ concentration control many essential cellular functions like the contraction of smooth muscle cells. The aim of this study was to investigate if the tachykinin substance P (SP) engages external Ca 2+-sources, internal Ca 2+-sources, or both in the contraction of the gastrointestinal smooth muscle of rainbow trout ( Oncorhynchus mykiss) and the African clawed frog ( Xenopus laevis). Strip preparations made of either longitudinal smooth muscle of proximal intestine or circular smooth muscle of cardiac stomach were mounted in organ baths and the tension was recorded via force transducers. Ca 2+-free Ringer’s solution containing the Ca 2+ chelating agent EGTA (2 mM) abolished all spontaneous contractions. Exposure to SP in Ca 2+-free solution decreased the response. Preparations were also treated with the Ca 2+-ATPase inhibitor thapsigargin (10 μM) during 30 min. Thapsigargin reduced the effect of SP on intestinal longitudinal smooth muscle in rainbow trout and on stomach circular smooth muscle in the African clawed frog and to a less extent in the intestinal longitudinal smooth muscle. The results show that external Ca 2+ is of great importance, but is not the only source of Ca 2+ recruitment in SP-activation of gastrointestinal smooth muscle in rainbow trout and the African clawed frog.

Contractile activity of mouse small intestinal longitudinal smooth muscle

Abstract Background Interest in genomic modulation experimentally often necessitates use of mouse models. Materials and methods AIM: To characterize and quantitate smooth muscle contractile activity of the mouse small intestine using in vitro techniques. Full-thickness jejunal and ileal muscle strips from mice were cut in the direction of longitudinal muscle, suspended in tissue baths (37°C), and connected to force transducers. Spontaneous contractility and two dose-response curves to the cholinergic agonist bethanechol and adrenergic agonist norepinephrine were quantitated for 6 h. Results Total contractile activity increased over 4 to 5 h in jejunum ( P P P Conclusion Experiments in murine jejunal but not ileal longitudinal muscle in vitro must consider early changes in contractile activity after tissue harvest. These experiments serve as a baseline for comparison of stimuli or genetic modifications on murine contractile activity of longitudinal muscle in vivo.

20] Radioimmunoassay for measuring endogenous levels of cyclic ADP-ribose in tissues

Cyclic ADP-ribose (cADPR) is a newly discovered cyclic nucleotide that functions by mobilizing intracellular Ca2+ stores. Unlike cAMP, cADPR is derived from NAD+ and its synthesis is catalyzed by ADP-ribosyl cyclase. Two modes of action are proposed for cADPR. First, cADPR can be a modulator constantly present in cells at some basal concentration that determines the Ca2+ sensitivity of the Ca2+-induced Ca2+ release mechanism. Substantial evidence from studies in sea urchin microsomes and Xenopus neurons supports this mode of action. Alternatively, cADPR can function as a Ca2+ messenger. The cADPR may play a messenger role in mediating the stimulatory action of glucose in pancreatic β cells and the Ca2+ mobilizing effect of a cholecystokinin octapeptide in intestinal longitudinal smooth muscle. The measurements of endogenous concentrations of cADPR are made by bioassays based on the Ca2+ release activity of cADPR. This chapter presents an improved radioimmunoassay (RIA) for cADPR using an antibody developed in chickens.

Agonist-mediated activation of PLA2 initiates Ca2+ mobilization in intestinal longitudinal smooth muscle.

Recent studies have shown that Ca2+ mobilization in longitudinal muscle is initiated by inositol 1,4,5-trisphosphate (IP3)-independent Ca2+ influx that acts as a trigger for Ca(2+)-induced Ca2R release. The present study examined whether arachidonic acid (AA) acts as mediator of the initial Ca2+ influx. Cholecystokinin octapeptide caused transient concentration-dependent increase in AA release in dispersed intestinal longitudinal but not circular muscle cells followed by sustained increase in both muscle cell types. The initial increase in AA release coincided with the initial Ca2+ transient and muscle contraction: all three events were abolished by guanosine 5'-O-(2-thiodiphosphate), pertussis toxin (PTX), and the phospholipase A2 (PLA2) inhibitor, dimethyleicosadienoic acid, but were not affected by calphostin C or neomycin. Exogenous AA caused concentration-dependent contraction and increase in cytosolic free Ca2+ ([Ca2+]i) in longitudinal but not circular muscle cells; both events were abolished by Ca2+ channel blockers. Depletion of Ca2+ stores with thapsigargin attenuated with thapsigargin attenuated agonist- and AA-mediated increase in [Ca2+]i and contraction in longitudinal muscle cells: the residual [Ca2+]i increase (35%) and contraction (25%) reflected the component of Ca2+ influx. We conclude that AA released by agonist-mediated G protein-dependent PTX-sensitive activation of PLA2 mediates Ca2+ influx, which then triggers Ca(2+)-induced Ca2+ release. The process is independent of phosphatidylinositol hydrolysis and occurs exclusively in longitudinal smooth muscle, in which Ca2+ release channels are highly sensitive to Ca2+, ryanodine, and cyclic ADP-ribose and insensitive to IP3.

Homologous histamine H 1 receptor desensitization results in reduction of H 1 receptor agonist efficacy

Prolonged exposure of the guinea-pig intestinal longitudinal smooth muscle to histamine caused homologous desensitization of the H 1 receptor, which led to reduced H 1 receptor-mediated production of [ 3H]inositol phosphates as well as to reduced Hj agonist-induced contractions. [ 3H]Mepyramine binding studies showed that desensitization affected neither the agonist affinity nor the number of H 1 receptors. Combining the data from the binding studies and the contraction measurements it was found that desensitization results in a selective reduction of agonist efficacy.

The motor effect of neuromedin U on rat stomach in vitro

A series of neuromedin U (NmU)-like peptides were found to evoke concentration-response contraction of rat fundic circular muscle in vitro. Isometric contraction of this tissue was induced by rat NmU, porcine NmU-8 and NmU-25, and frog NmU. Rat NmU was significantly more potent than frog NmU. The contractile action of rat NmU upon rat fundic strips was not affected by either atropine or tetrodotoxin indicating a direct effect. Maximal NmU-induced contractions were 10% of the maximal contraction induced by carbachol and ranged between those of the bradykinin and angiotensin II. None of the NmU peptides were active on the circular smooth muscle of the frog stomach or on the small and large intestinal longitudinal smooth muscle of either rat or frog. The results of this study demonstrate that members of the NmU peptide family all induce in vitro contraction of rat gastric circular smooth muscle independent of cholinergic or other neuronal mechanisms. Their activity, however, appears to be both tissue and species specific.

Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig

Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.