Impaired Interoception in a Preclinical Model of Functional Dyspepsia.

INTRODUCTION: The etiologies of functional dyspepsia symptoms, including postprandial distress syndrome, remain unknown. We tested the hypothesis that neonatal colon inflammation induces postprandial distress syndrome-like symptoms in adult life that associate with increased activation of vagal afferent pathways and forebrain limbic regions. RESULTS: These rats showed a significant decrease in nutrient meal consumption to satiety after an overnight fast, decrease in gastric emptying, decrease in total distance traveled, and decrease in percent distance traveled in midfield versus control rats in open field test, indicating postprandial anxiety- and depression-like behaviors. Adult naïve rats treated with oral iodoacetamide to induce H. pylori-like mild gastritis demonstrated similar postprandial effects as the above rats. CONCLUSIONS: We concluded that neonatal colon inflammation is a risk factor for the development of postprandial distress syndrome-like symptoms. While mild gastritis can induce symptoms similar to those of neonatal colon inflammation, gastritis in these rats does not worsen the symptoms.

Enhanced sympathetic nerve activity induced by neonatal colon inflammation induces gastric hypersensitivity and anxiety-like behavior in adult rats.

Gastric hypersensitivity (GHS) and anxiety are prevalent in functional dyspepsia patients; their underlying mechanisms remain unknown largely because of lack of availability of live visceral tissues from human subjects. Recently, we demonstrated in a preclinical model that rats subjected to neonatal colon inflammation show increased basal plasma norepinephrine (NE), which contributes to GHS through the upregulation of nerve growth factor (NGF) expression in the gastric fundus. We tested the hypothesis that neonatal colon inflammation increases anxiety-like behavior and sympathetic nervous system activity, which upregulates the expression of NGF to induce GHS in adult life. Chemical sympathectomy, but not adrenalectomy, suppressed the elevated NGF expression in the fundus muscularis externa and GHS. The measurement of heart rate variability showed a significant increase in the low frequency-to-high frequency ratio in GHS vs. the control rats. Stimulus-evoked release of NE from the fundus muscularis externa strips was significantly greater in GHS than in the control rats. Tyrosine hydroxylase expression was increased in the celiac ganglia of the GHS vs. the control rats. We found an increase in trait but not stress-induced anxiety-like behavior in GHS rats in an elevated plus maze. We concluded that neonatal programming triggered by colon inflammation upregulates tyrosine hydroxylase in the celiac ganglia, which upregulates the release of NE in the gastric fundus muscularis externa. The increase of NE release from the sympathetic nerve terminals concentration dependently upregulates NGF, which proportionately increases the visceromotor response to gastric distention. Neonatal programming concurrently increases anxiety-like behavior in GHS rats.

Noninflammatory upregulation of nerve growth factor underlies gastric hypersensitivity induced by neonatal colon inflammation.

Gastric hypersensitivity is one of the key contributors to the postprandial symptoms of epigastric pain/discomfort, satiety, and fullness in functional dyspepsia patients. Epidemiological studies found that adverse early-life experiences are risk factors for the development of gastric hypersensitivity. Preclinical studies found that neonatal colon inflammation elevates plasma norepinephrine (NE), which upregulates expression of nerve growth factor (NGF) in the muscularis externa of the gastric fundus. Our goal was to investigate the cellular mechanisms by which NE upregulates the expression of NGF in gastric hypersensitive (GHS) rats, which were subjected previously to neonatal colon inflammation. Neonatal colon inflammation upregulated NGF protein, but not mRNA, in the gastric fundus of GHS rats. Western blotting showed upregulation of p110γ of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K), phosphoinositide-dependent kinase-1 (PDK1), pAKT(Ser473), and phosphorylated 4E-binding protein (p4E-BP1)(Thr70), suggesting AKT activation and enhanced NGF protein translation. AKT inhibitor MK-2206 blocked the upregulation of NGF in the fundus of GHS rats. Matrix metalloproteinase 9 (MMP-9), the major NGF-degrading protease, was suppressed, indicating that NGF degradation was impeded. Incubation of fundus muscularis externa with NE upregulated NGF by modulating the protein translation and degradation pathways. Yohimbine, an α2-adrenergic receptor antagonist, upregulated plasma NE and NGF expression by activating the protein translation and degradation pathways in naive rats. In contrast, a cocktail of adrenergic receptor antagonists suppressed the upregulation of NGF by blocking the activation of the protein translation and degradation pathways. Our findings provide evidence that the elevation of plasma NE induces NGF expression in the gastric fundus.

Genesis of anxiety, depression, and ongoing abdominal discomfort in ulcerative colitis-like colon inflammation.

Psychological disorders are prevalent in patients with inflammatory bowel disease; the underlying mechanisms remain unknown. We tested the hypothesis that ulcerative colitis-like inflammation induced by dextran sodium sulfate (DSS) exacerbates the ongoing spontaneous activity in colon-projecting afferent neurons that induces abdominal discomfort and anxiety, and depressive-like behaviors in rats. In this study, we used the conditioned place preference and standard tests for anxiety- and depression-like behaviors. DSS rats developed anxiety- and depression-like behaviors 10 to 20 days after the start of inflammation. Single-fiber recordings showed an increase in the frequency of spontaneous activity in L6-S1 dorsal root ganglion (DRG) roots. Prolonged desensitization of transient receptor potential vanilloid 1 (TRPV1)-expressing colonic afferents by resiniferatoxin (RTX) suppressed the spontaneous activity, as well as the anxiety- and depressive-like behaviors. Reduction in spontaneous activity in colon afferents by intracolonic administration of lidocaine produced robust conditioned place preference (CPP) in DSS rats, but not in control rats. Patch-clamp studies demonstrated a significant decrease in the resting membrane potential, lower rheobase, and sensitization of colon-projecting L6-S1 DRG neurons to generate trains of action potentials in response to current injection in DSS rats. DSS inflammation upregulated the mRNA levels of transient receptor potential ankyrin 1 and TRPV1 channels and downregulated that of Kv1.1 and Kv1.4 channels. Ulcerative colitis-like inflammation in rats induces anxiety- and depression-like behaviors, as well as ongoing abdominal discomfort by exacerbating the spontaneous activity in the colon-projecting afferent neurons. Alterations in the expression of voltage- and ligand-gated channels are associated with the induction of mood disorders following colon inflammation.

Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.

OBJECTIVE: Chronic pain is a common neurological comorbidity of human immunodeficiency virus (HIV)-1 infection, but the etiological cause remains elusive. The objective of this study was to identify the HIV-1 causal factor that critically contributes to the pathogenesis of HIV-associated pain. METHODS: We first compared the levels of HIV-1 proteins in postmortem tissues of the spinal cord dorsal horn (SDH) from HIV-1/acquired immunodeficiency syndrome patients who developed chronic pain (pain-positive HIV-1 patients) and HIV-1 patients who did not develop chronic pain (pain-negative HIV-1 patients). Then we used the HIV-1 protein that was specifically increased in the pain-positive patients to generate mouse models. Finally, we performed comparative analyses on the pathological changes in the models and the HIV-1 patients. RESULTS: We found that HIV-1 gp120 was significantly higher in pain-positive HIV-1 patients (vs pain-negative HIV-1 patients). This finding suggested that gp120 was a potential causal factor of the HIV-associated pain. To test this hypothesis, we used a mouse model generated by intrathecal injection of gp120 and compared the pathologies of the model and the pain-positive human HIV-1 patients. The results showed that the mouse model and pain-positive human HIV-1 patients developed extensive similarities in their pathological phenotypes, including pain behaviors, peripheral neuropathy, glial reactivation, synapse degeneration, and aberrant activation of pain-related signaling pathways in the SDH. INTERPRETATION: Our findings suggest that gp120 may critically contribute to the pathogenesis of HIV-associated pain.

Developmental origins of functional dyspepsia-like gastric hypersensitivity in rats.

BACKGROUND & AIMS: Gastric hypersensitivity (GHS) contributes to epigastric pain in patients with functional dyspepsia (FD); the etiology and cellular mechanisms of this dysfunction remain unknown. We investigated whether inflammatory insult to the colons of neonatal rats induced GHS in adult life. METHODS: We used cellular, molecular, and in vivo approaches to investigate the mechanisms of GHS in adult rats subjected to neonatal colonic insult by intraluminal administration of trinitrobenzene sulfonic acid; controls received saline. Six to 8 weeks later, rats were evaluated for GHS and tissue was collected for molecular experiments. RESULTS: Inflammatory insult to the colon on post-natal day 10 caused an aberrant increase of corticosterone on post-natal day 15 and induced GHS in adult life. We called these FD-like rats. Inhibition of glucocorticoid receptors after neonatal insult blocked the induction of GHS in adult rats. The aberrant increase of plasma corticosterone in neonates increased the plasma concentration of norepinephrine, nerve growth factor in the gastric fundus muscularis externae, brain-derived neurotrophic factor in the thoracic dorsal root ganglia and spinal cord, and down-regulated K(v)1.1 messenger RNA in thoracic dorsal root ganglia without affecting the expression of K(v)1.4, Na(v)1.8, TrpA1, TrpV1, or P2X3 in FD-like rats. Inhibition of glucocorticoid receptors during neonatal insult or the inhibition of adrenergic receptors, nerve growth factor, or brain-derived neurotrophic factor in FD-like rats suppressed GHS. The intrathecal administration of small interfering RNAs against K(v)1.1 increased GHS in naive rats. CONCLUSIONS: Inflammatory insult to the colons of rat pups leads to GHS in adult life. GHS is caused by altered expression of genes encoding neurotrophins and ion channels, and altered activity of the sympathetic nervous system.

Cell culture retains contractile phenotype but epigenetically modulates cell-signaling proteins of excitation-contraction coupling in colon smooth muscle cells.

Smooth muscle cell cultures are used frequently to investigate the cellular mechanisms of contraction. We tested the hypothesis that cell culture alters the expression of select cell-signaling proteins of excitation-contraction coupling in colon smooth muscle cells without altering the contractile phenotype. We used muscularis externa (ME) tissues, freshly dispersed cells (FC), primary cell cultures (PC), and resuspensions of cell cultures (RC). Colon smooth muscle cells retained their phenotype in all states. We investigated expression of 10 cell-signaling proteins of excitation-contraction coupling in all four types of tissue. Expression of all these proteins did not differ between ME and FC (P > 0.05). However, expression of the α(1C)-subunit of Ca(v)1.2b, myosin light chain kinase, myosin phosphatase target subunit 1, and 17-kDa C kinase-potentiated protein phosphatase-1 inhibitor (CPI-17) decreased in PC and RC vs. ME and FC (all P < 0.05). Expression of Gα(i3), serine/threonine protein phosphatase-1 ß-catalytic subunit, and Rho kinase 1 increased in PC and RC vs. ME and FC (all P < 0.05). Cell culture and resuspension downregulated expression of α-actin and calponin, but not myosin heavy chain. The net effect of these molecular changes was suppression of cell reactivity to ACh in RC vs. FC. Overexpression of CPI-17 in PC partially reversed the suppression of contractility in resuspended cells. Methylation-specific PCR showed increased methylation of the Cpi-17 gene promoter in PC vs. ME (P < 0.05). We concluded that smooth muscle cells retain their contractile phenotype in culture. However, reactivity to ACh declines because of altered expression of specific cell-signaling proteins involved in excitation-contraction coupling. DNA methylation of the Cpi-17 promoter may contribute to its gene suppression.

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

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

Paradoxical regulation of ChAT and nNOS expression in animal models of Crohn's colitis and ulcerative colitis.

Morphological and functional changes in the enteric nervous system (ENS) have been reported in inflammatory bowel disease. We examined the effects of inflammation on the expression of choline acetyltransferase (ChAT) and nNOS in the muscularis externae of two models of colonic inflammation, trinitrobenzene sulfonic acid (TNBS)-induced colitis, which models Crohn's disease-like inflammation, and DSS-induced colitis, which models ulcerative Colitis-like inflammation. In TNBS colitis, we observed significant decline in ChAT, nNOS, and protein gene product (PGP) 9.5 protein and mRNA levels. In DSS colitis, ChAT and PGP9.5 were significantly upregulated while nNOS levels did not change. The nNOS dimer-to-monomer ratio decreased significantly in DSS- but not in TNBS-induced colitis. No differences were observed in the percentage of either ChAT (31 vs. 33%)- or nNOS (37 vs. 41%)-immunopositive neurons per ganglia or the mean number of neurons per ganglia (55 ± 5 vs. 59 ± 5, P > 0.05). Incubation of the distal colon muscularis externae in vitro with different types of inflammatory mediators showed that cytokines decreased ChAT and nNOS expression, whereas H2O2, a component of oxidative stress, increased their expression. NF-κB inhibitor MG-132 did not prevent the IL-1ß-induced decline in either ChAT or nNOS expression. These findings showed that TNBS- and DSS-induced inflammation differentially regulates the expression of two critical proteins expressed in the colonic myenteric neurons. These differences are likely due to the exposure of the myenteric plexus neurons to different combinations of Th1-type inflammatory mediators and H2O2 in each model.

Nitric oxide modifies chromatin to suppress ICAM-1 expression during colonic inflammation.

Nitric oxide (NO) is an established inflammatory mediator. However, it remains controversial whether NO enhances the inflammatory response in the colon or suppresses it. We investigated the epigenetic regulation of Icam-1 expression by NO following induction of colonic inflammation in rats by 2,4,6-trinitrobenzene sulfonic (TNBS) acid and obtaining colonic muscularis externae tissues 24 h later. TNBS inflammation induced intercellular adhesion molecule-1 (ICAM-1) expression by translocating NF-κB to the nucleus. The incubation of inflamed tissues with S-nitrosoglutathione (GSNO) did not affect the nuclear translocation of NF-κB; however, it suppressed the NF-κB binding to DNA. Chromatin immunoprecipitation analysis (ChIP)-qPCR assays showed that the increase in NF-κB/DNA interaction following inflammation is due to the transcriptional downregulation of global HDAC3 and a decrease in its interaction with the DNA on the Icam-1 promoter containing the binding motifs of NF-κB. The decrease in the association of histone deacetylase (HDAC) 3 with the Icam-1 promoter increased the acetylation of histone 4 lysine residue 12 (H4K12), which would favor chromatin relaxation and greater access of NF-κB to its DNA binding sites. HDAC3 dissociation from the DNA did not affect the acetylation levels of H4K8 and H4K16. The NO release by GSNO countered the upregulation of Icam-1 by increasing the transcription of global HDAC3 and its association with the Icam-1 promoter, and by suppressing H4K12 acetylation. We conclude that chromatin modification by transcriptional downregulation of HDAC3 plays a critical role in the induction of the inflammatory response. NO may serve as an anti-inflammatory mediator during the acute stage of inflammation by blunting the downregulation of global HDAC3, increasing HDAC3 interaction with the nucleosomes containing the binding moieties of NF-κB, reducing H4K12Ac to restrict the access of NF-κB to DNA, and suppressing ICAM-1 expression.

Prophylactic and therapeutic benefits of COX-2 inhibitor on motility dysfunction in bowel obstruction: roles of PGE2 and EP receptors.

We reported previously that mechanical stretch in rat colonic obstruction induces cyclooxygenase (COX)-2 expression in smooth muscle cells. The aims of the present study were to investigate whether in vivo treatment with COX-2 inhibitor has prophylactic and therapeutic effects on motility dysfunction in colon obstruction, and if so what are the underlying mechanisms. Partial colon obstruction was induced with a silicon band in the distal colon of 6-8-wk-old Sprague-Dawley rats; obstruction was maintained for 3 days or 7 days. Daily administration of COX-2 inhibitor NS-398 (5 mg/kg) or vehicle was started before or after the induction of obstruction to study its prophylactic and therapeutic effects, respectively. The smooth muscle contractility was significantly suppressed, and colonic transit rate was slower in colonic obstruction. Prophylactic treatment with NS-398 significantly prevented the impairments of colonic transit and smooth muscle contractility and attenuated fecal collection in the occluded colons. When NS-398 was administered therapeutically 3 days after the initiation of obstruction, the muscle contractility and colonic transit still improved on day 7. Obstruction led to marked increase of COX-2 expression and prostaglandin E(2) (PGE(2)) synthesis. Exogenous PGE(2) decreased colonic smooth muscle contractility. All four PGE(2) E-prostanoid receptor types (EP1 to EP4) were detected in rat colonic muscularis externa. Treatments with EP1 and EP3 antagonists suppressed muscle contractility in control tissue but did not improve contractility in obstruction tissue. On the contrary, the EP2 and EP4 antagonists did not affect control tissue but significantly restored muscle contractility in obstruction. We concluded that our study shows that COX-2 inhibitor has prophylactic and therapeutic benefits for motility dysfunction in bowel obstruction. PGE(2) and its receptors EP2 and EP4 are involved in the motility dysfunction in obstruction, whereas EP1 and EP3 mediate PGE(2) regulation of colonic smooth muscle contractile function in normal state.

Cellular mechanism of mechanotranscription in colonic smooth muscle cells.

Mechanical stretch in obstruction induces expression of cyclooxygenase-2 (COX-2) in gut smooth muscle cells (SMCs). The stretch-induced COX-2 plays a critical role in motility dysfunction in obstructive bowel disorders (OBDs). The aims of the present study were to investigate the intracellular mechanism of mechanotranscription of COX-2 in colonic SMCs and to determine whether inhibition of mechanotranscription has therapeutic benefits in OBDs. Static stretch was mimicked in vitro in primary culture of rat colonic circular SMCs (RCCSMCs) and in colonic circular muscle strips. Partial obstruction was surgically induced with a silicon band in the distal colon of rats and COX-2-deficient mice. Static stretch of RCCSMCs significantly induced expression of COX-2 mRNA and protein and activated MAP kinases ERKs, p38, and JNKs. ERKs inhibitor PD98059, p38 inhibitor SB203580, and JNKs inhibitor SP600125 significantly blocked stretch-induced COX-2 expression. Pharmacological and molecular inhibition of stretch-activated ion channels (SACs) and integrins significantly suppressed stretch-induced expression of COX-2. SAC blockers inhibited stretch-activated ERKs, p38, and JNKs, but inhibition of integrins attenuated p38 activation only. In colonic circular muscle strips, stretch led to activation of MAPKs, induction of COX-2, and suppression of contractility. Inhibition of p38 with SB203580 blocked COX-2 expression and restored muscle contractility. Administration of SB203580 in vivo inhibited obstruction-induced COX-2 and improved motility function. Stretch-induced expression of COX-2 in RCCSMCs depends on mechanosensors, SACs, and integrins and an intracellular signaling mechanism involving MAPKs ERKs, p38, and JNKs. Inhibitors of the mechanotranscription pathway have therapeutic potentials for OBDs.

Impaired integrity of DNA after recovery from inflammation causes persistent dysfunction of colonic smooth muscle.

BACKGROUND & AIMS: Patients with inflammatory bowel disease who are in remission and those who developed irritable bowel syndrome after enteric infection continue to have symptoms of diarrhea or constipation in the absence of overt inflammation, indicating motility dysfunction. We investigated whether oxidative stress during inflammation impairs integrity of the promoter of Cacna1c, which encodes the pore-forming α1C subunit of Ca(v)1.2b calcium channels. METHODS: We used long-extension polymerase chain reaction to evaluate DNA integrity in tissues from distal colons of rats; trinitrobenzene sulfonic acid was used to induce inflammation. RESULTS: The H2O2 increased in the muscularis externa 1-7 days after inflammation was induced with trinitrobenzene sulfonic acid. The oxidative stress significantly impaired DNA integrity in 2 specific segments of the Cacna1c promoter: -506 to -260 and -2193 to -1542. The impairment peaked at day 3 and recovered partially by day 7 after induction of inflammation; expression of the products of Cacna1c followed a similar time course. Oxidative stress suppressed the expression of nuclear factor-erythroid-2-related factor 2 (Nrf2), an important regulator of anti-oxidant proteins. Intraperitoneal administration of sulforaphane significantly reversed the suppression of Nrf2, oxidative damage in the promoter of Cacna1c, and suppression of Cacna1c on day 7 of inflammation. The inflammation subsided completely by 56 days after inflammation was induced; however, impairment of DNA integrity, expression of Nrf2 and Cacna1c, and smooth muscle reactivity to acetylcholine remained suppressed at this time point. CONCLUSIONS: Oxidative stress during inflammation impairs the integrity of the promoter of Cacna1c; impairment persists partially after inflammation has subsided. Reduced transcription of Cacna1c contributes to smooth muscle dysfunction in the absence of inflammation.

Chronic stress targets posttranscriptional mechanisms to rapidly upregulate α1C-subunit of Cav1.2b calcium channels in colonic smooth muscle cells.

Chronic stress elevates plasma norepinephrine, which enhances expression of the α(1C)-subunit of Ca(v)1.2b channels in colonic smooth muscle cells within 1 h. Transcriptional upregulation usually does not explain such rapid protein synthesis. We investigated whether chronic stress-induced release of norepinephrine utilizes posttranscriptional mechanisms to enhance the α(1C)-subunit. We performed experiments on colonic circular smooth muscle strips and in conscious rats, using a 9-day chronic intermittent stress protocol. Incubation of rat colonic muscularis externa with norepinephrine enhanced α(1C)-protein expression within 45 min, without a concomitant increase in α(1C) mRNA, indicating posttranscriptional regulation of α(1C)-protein by norepinephrine. We found that norepinephrine activates the PI3K/Akt/GSK-3ß pathway to concurrently enhance α(1C)-protein translation and block its polyubiquitination and proteasomal degradation. Incubation of colonic muscularis externa with norepinephrine or LiCl, which inhibits GSK-3ß, enhanced p-GSK-3ß and α(1C)-protein time dependently. Using enrichment of phosphoproteins and ubiquitinated proteins, we found that both norepinephrine and LiCl decrease α(1C) phosphorylation and polyubiquitination. Concurrently, they suppress eIF2α (Ser51) phosphorylation and 4E-BP1 expression, which stimulates gene-specific translation. The antagonism of two upstream kinases, PI3K and Akt, inhibits the induction of α(1C)-protein by norepinephrine. Cyanopindolol (ß(3)-AR-antagonist) almost completely suppresses and propranolol (ß(1/2)-AR antagonist) partially suppresses norepinephrine-induced α(1C)-protein expression, whereas phentolamine and prazosin (α-AR and α(1)-AR antagonist, respectively) have no significant effect. Experiments in conscious animals showed that chronic stress activates the PI3K/Akt/GSK-3ß signaling. We conclude that norepinephrine released by chronic stress rapidly enhances the protein expression of α(1C)-subunit of Ca(v)1.2b channels by concurrently suppressing its degradation and enhancing translation of existing transcripts to maintain homeostasis.

Differential immune and genetic responses in rat models of Crohn's colitis and ulcerative colitis.

Crohn's disease and ulcerative colitis are clinically, immunologically, and morphologically distinct forms of inflammatory bowel disease (IBD). However, smooth muscle function is impaired similarly in both diseases, resulting in diarrhea. We tested the hypothesis that differential cellular, genetic, and immunological mechanisms mediate smooth muscle dysfunction in two animal models believed to represent the two diseases. We used the rat models of trinitrobenzene sulfonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced colonic inflammations, which closely mimic the clinical and morphological features of Crohn's disease and ulcerative colitis, respectively. DSS inflammation induced oxidative stress initially in mucosa/submucosa, which then propagated to the muscularis externa to impair smooth muscle function. The muscularis externa showed no increase of cytokines/chemokines. On the other hand, TNBS inflammation almost simultaneously induced oxidative stress, recruited or activated immune cells, and generated cytokines/chemokines in both mucosa/submucosa and muscularis externa. The generation of cytokines/chemokines did not correlate with the recruitment and activation of immune cells. Consequently, the impairment of smooth muscle function in DSS inflammation was primarily due to oxidative stress, whereas that in TNBS inflammation was due to both oxidative stress and proinflammatory cytokines. The impairment of smooth muscle function in DSS inflammation was due to suppression of Gα(q) protein of the excitation-contraction coupling. In TNBS inflammation, it was due to suppression of the α(1C)1b subunit of Ca(v)1.2b channels, CPI-17 and Gα(q). TNBS inflammation increased IGF-1 and TGF-ß time dependently in the muscularis externa. IGF-1 induced smooth muscle hyperplasia; both IGF-1 and TGF-ß induced hypertrophy. In conclusion, both TNBS and DSS induce transmural inflammation, albeit with different types of inflammatory mediators. The recruitment or activation of immune cells does not correlate directly with the intensity of generation of inflammatory mediators. The inflammatory mediators in TNBS and DSS inflammations target different genes to impair smooth muscle function.

Pathophysiology of motility dysfunction in bowel obstruction: role of stretch-induced COX-2.

In gastrointestinal conditions such as bowel obstruction, pseudo-obstruction, and idiopathic megacolon, the lumen of affected bowel segments is distended and its motility function impaired. Our hypothesis is that mechanical stretch of the distended segments alters gene expression of cyclooxygenase-2 (COX-2), which impairs motility function. Partial obstruction was induced with a silicon band in the distal colon of rats for up to 7 days, and wild-type and COX-2 gene-deficient mice for 4 days. Mechanical stretch was mimicked in vitro in colonic circular muscle strips and in primary culture of colonic circular smooth muscle cells (SMC) with a Flexercell system. The rat colonic circular muscle contractility was significantly decreased in the distended segment oral to obstruction, but not in the aboral segment. This change started as early as day 1 and persisted for at least 7 days after obstruction. The expression of COX-2 mRNA and protein increased dramatically also in the oral, but not aboral, segment. The upregulation of COX-2 expression started at 12 h and the effect persisted for 7 days. At 24 h after obstruction, the COX-2 mRNA level in the oral segment increased 26-fold compared with controls. This was not accompanied by any significant increase of myeloperoxidase or inflammatory cytokines. Immunohistochemical studies showed that COX-2 was selectively induced in the colonic SMC. In vitro stretch of colonic muscle strips or cultured SMC drastically induced COX-2 expression. Incubation of circular muscle strips from obstructed segment with COX-2 inhibitor NS-398 restored the contractility. The impairment of muscle contractility in obstructed colon was attenuated in the COX-2 gene-deficient mice. In conclusion, mechanical stretch in obstruction induces marked expression of COX-2 in the colonic SMC, and stretch-induced COX-2 plays a critical role in the suppression of smooth muscle contractility in bowel obstruction.

Adrenergic stimulation mediates visceral hypersensitivity to colorectal distension following heterotypic chronic stress.

BACKGROUND & AIMS: Chronic stress exacerbates or causes relapse of symptoms such as abdominal pain and cramping in patients with irritable bowel syndrome. We investigated whether chronic stress increases plasma norepinephrine and sensitizes colon-specific dorsal root ganglion (DRG) neurons by increasing expression of nerve growth factor (NGF) in the colon wall. METHODS: Heterotypic chronic stress (HeCS) was applied to male Wistar rats and neurologic and molecular responses were analyzed. Tissues were analyzed for NGF expression. RESULTS: HeCS significantly increased visceromoter response to colorectal distension; expression of NGF increased in colonic muscularis externa and mucosa/submucosa. Rheobase decreased, resting membrane potential was depolarized, and electrogenesis of action potentials increased in colon-specific thoracolumbar DRG neurons. Luminal administration of resiniferatoxin in distal colon, systemic administration of anti-NGF antibody, or inhibition of the NGF receptor trkA by k252a or antisense oligonucleotides in thoracolumbar DRG blocked the chronic stress-induced visceral hypersensitivity to colorectal distension. Blockade of alpha1/alpha2- and beta1/beta2-adrenergic receptors prevented the stress-induced visceral hypersensitivity and increased expression of NGF in the colon wall. HeCS did not induce any inflammatory response in the colon wall. CONCLUSIONS: The peripheral stress mediator norepinephrine induces visceral hypersensitivity to colorectal distension in response to HeCS by increasing the expression of NGF in the colon wall, which sensitizes primary afferents in the absence of an inflammatory response.

Nuclear myosin II regulates the assembly of preinitiation complex for ICAM-1 gene transcription.

BACKGROUND & AIMS: Actin-myosin II motor converts chemical energy into force/motion in muscle and nonmuscle cells. The phosphorylation of 20-kilodalton regulatory myosin light chain (MLC(20)) is critical to the cytoplasmic functions of these motors. We do not know whether myosin II and actins in the nucleus function as motors to generate relative motion, such as that between RNA polymerase II holoenzyme and DNA, for assembly of the preinitiation complex. METHODS: The experiments were performed on primary cultures of human colonic circular smooth muscle cells and rat colonic circular muscle strips. RESULTS: We show that myosin II and alpha- and beta-actins are present in the nuclei of colonic smooth muscle cells. The nuclear myosin II is tethered to recognition sequence AGCTCC (-39/-34) in the intercellular adhesion molecule 1 (ICAM-1) core promoter region. The actins are known to complex with RNA polymerase II, and they are tethered to the nucleoskeleton. The dephosphorylation of MLC(20) increases the transcription of ICAM-1, whereas its phosphorylation decreases it. Colonic inflammation suppresses nuclear myosin light chain kinase, which increases the unphosphorylated form of nuclear MLC(20), resulting in enhanced transcription of ICAM-1. CONCLUSIONS: Myosin II is a core transcription factor. The phosphorylation/dephosphorylation of nuclear MLC(20) results in the sliding of myosin and actin molecules past each other, producing relative motion between DNA bound to the myosin II and RNA polymerase II holoenzyme bound to actins and nucleoskeleton.

Homeostatic and therapeutic roles of VIP in smooth muscle function: myo-neuroimmune interactions.

We tested the hypothesis that spontaneous release of vasoactive intestinal peptide (VIP) from enteric neurons maintains homeostasis in smooth muscle function in mild inflammatory insults and that infusion of exogenous VIP has therapeutic effects on colonic smooth muscle dysfunction in inflammation. In vitro experiments were performed on human colonic circular smooth muscle tissues and in vivo on rats. The incubation of human colonic circular smooth muscle strips with TNF-alpha suppressed their contractile response to ACh and the expression of the pore-forming alpha(1C) subunit of Ca(v)1.2 channels. VIP reversed both effects by blocking the translocation of NF-kappaB to the nucleus and its binding to the kappaB recognition sites on halpha(1C)1b promoter. The translocation of NF-kappaB was inhibited by blocking the degradation of IkappaBbeta. Induction of inflammation by a subthreshold dose of 17 mg/kg trinitrobenzene sulfonic acid (TNBS) in rats moderately decreased muscularis externa concentration of VIP, and it had little effect on the contractile response of circular smooth muscle strips to ACh. The blockade of VIP and pituitary adenylate cyclase-activating peptide receptors 1/2 during mild inflammatory insult significantly worsened the suppression of contractility and the inflammatory response. The induction of more severe inflammation by 68 mg/kg TNBS induced marked suppression of colonic circular muscle contractility and decrease in serum VIP. Exogenous infusion of VIP by an osmotic pump reversed these effects. We conclude that the spontaneous release of VIP from the enteric motor neurons maintains homeostasis in smooth muscle function in mild inflammation by blocking the activation of NF-kappaB. The infusion of exogenous VIP mitigates colonic inflammatory response and smooth muscle dysfunction.