T-helper 2 cytokines, transforming growth factor ß1, and eosinophil products induce fibrogenesis and alter muscle motility in patients with eosinophilic esophagitis.

BACKGROUND & AIMS: Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEFs), human esophageal muscle cells (HEMCs), and esophageal muscle strips to eosinophil-derived products. METHODS: Biopsy specimens were collected via endoscopy from the upper, middle, and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls). Primary cultures of esophageal fibroblasts and muscle cells were derived from 12 freshly resected human esophagectomy specimens. Eosinophil distribution was investigated by histologic analyses of full-thickness esophageal tissue. Active secretion of EoE-related mediators was assessed from medium underlying mucosal biopsy cultures. We quantified production of fibronectin and collagen I by HEF and HEMC in response to eosinophil products. We also measured the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by, and adhesion of human eosinophils to, HEFs and HEMCs. Eosinophil products were tested in an esophageal muscle contraction assay. RESULTS: Activated eosinophils were present in all esophageal layers. Significantly higher concentrations of eosinophil-related mediators were secreted spontaneously in mucosal biopsy specimens from patients with EoE than controls. Exposure of HEFs and HEMCs to increasing concentrations of eosinophil products or co-culture with eosinophils caused HEFs and HEMCs to increase secretion of fibronectin and collagen I; this was inhibited by blocking transforming growth factor ß1 and p38 mitogen-activated protein kinase signaling. Eosinophil binding to HEFs and HEMCs increased after incubation of mesenchymal cells with eosinophil-derived products, and decreased after blockade of transforming growth factor ß1 and p38 mitogen-activated protein kinase blockade. Eosinophil products reduced electrical field-induced contraction of esophageal muscle strips, but not acetylcholine-induced contraction. CONCLUSIONS: In an analysis of tissues samples from patients with EoE, we linked the presence and activation state of eosinophils in EoE with altered fibrogenesis and motility of esophageal fibroblasts and muscle cells. This process might contribute to the development of dysphagia.

HCl-induced and ATP-dependent upregulation of TRPV1 receptor expression and cytokine production by human esophageal epithelial cells.

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.

ATP: a mediator for HCl-induced TRPV1 activation in esophageal mucosa.

In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells.

Esophageal disease: updated information on inflammation.

The following on esophageal disease provides updated information the mucosal defense against acid and acid-pepsin injury; the roles of platelet activating factor, mast cells, proinflammatory cytokines, and chemokines in inflammation; differences and similarities in erosive and nonerosive esophagitis; acid and vanilloid receptors in esophageal mucosa; and bile acid receptors in esophageal epithelium.

Viewpoints on Acid-induced inflammatory mediators in esophageal mucosa.

We have focused on understanding the onset of gastroesophageal reflux disease by examining the mucosal response to the presence of acid in the esophageal lumen. Upon exposure to HCl, inflammation of the esophagus begins with activation of the transient receptor potential channel vanilloid subfamily member-1 (TRPV1) in the mucosa, and production of IL-8, substance P (SP), calcitonin gene related peptide (CGRP) and platelet activating factor (PAF). Production of SP and CGRP, but not PAF, is abolished by the neural blocker tetrodotoxin suggesting that SP and CGRP are neurally released and that PAF arises from non neural pathways. Epithelial cells contain TRPV1 receptor mRNA and protein and respond to HCl and to the TRPV1 agonist capsaicin with production of PAF. PAF, SP and IL-8 act as chemokines, inducing migration of peripheral blood leukocytes. PAF and SP activate peripheral blood leukocytes inducing the production of H(2)O(2). In circular muscle, PAF causes production of IL-6, and IL-6 causes production of additional H(2)O(2), through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Among these, NADPH oxidase 5 cDNA is significantly up-regulated by exposure to PAF; H(2)O(2) content of esophageal and lower esophageal sphincter circular muscle is elevated in human esophagitis, causing dysfunction of esophageal circular muscle contraction and reduction in esophageal sphincter tone. Thus esophageal keratinocytes, that constitute the first barrier to the refluxate, may also serve as the initiating cell type in esophageal inflammation, secreting inflammatory mediators and pro-inflammatory cytokines and affecting leukocyte recruitment and activity.

HCl-induced inflammatory mediators in esophageal mucosa increase migration and production of H2O2 by peripheral blood leukocytes.

Exposure of esophageal mucosa to hydrochloric acid (HCl) is a crucial factor in the pathogenesis of reflux disease. We examined supernatant of HCl-exposed rabbit mucosa for inflammatory mediators enhancing migration of leukocytes and production of H(2)O(2) as an indicator of leukocyte activation. A tubular segment of rabbit esophageal mucosa was tied at both ends to form a sac, which was filled with HCl-acidified Krebs buffer at pH 5 (or plain Krebs buffer as control) and kept oxygenated at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Rabbit peripheral blood leukocytes (PBL) were isolated, and sac supernatant was used to investigate PBL migration and H(2)O(2) production. HCl-exposed esophageal mucosa released substance P (SP), CGRP, platelet-activating factor (PAF), and IL-8 into the supernatant. PBL migration increased in response to IL-8 or to supernatant of the HCl-filled mucosal sac. Supernatant-induced PBL migration was inhibited by IL-8 antibodies and by antagonists for PAF (CV3988) or neurokinin 1 (i.e., SP), but not by a CGRP antagonist. Supernatant of the HCl-filled mucosal sac increased H(2)O(2) release by PBL that was significantly reduced by CV3988 and by a SP antagonist but was not affected by IL-8 antibodies or by a CGRP antagonist. We conclude that IL-8, PAF, and SP are important inflammatory mediators released by esophageal mucosa in response to acid that promote PBL migration. In addition, PAF and SP induce production of H(2)O(2) by PBL. These findings provide a direct link between acid exposure and recruitment and activation of immune cells in esophageal mucosa.

Inflammatory mediators in gastroesophageal reflux disease: impact on esophageal motility, fibrosis, and carcinogenesis.

Gastroesophageal reflux disease (GERD) is one of the most common problems in clinical practice today. It is widely believed that functional and structural abnormalities of the gastroesophageal junction as well as an abnormal exposure to gastroduodenal contents are the main contributors to its pathogenesis. Novel findings of the inflammatory process in GERD suggest a far more complex process involving multifaceted inflammatory mechanisms. This review summarizes knowledge about the expression of inflammatory mediators in GERD and their potential cellular sources and provides an integrated concept of disease pathogenesis. In addition we evaluate the contribution of inflammatory mediators to well-known complications of GERD, namely motility abnormalities, fibrosis, and carcinogenesis. Novel findings regarding the pathophysiology of esophageal inflammation should enhance our understanding of GERD and its complications and provide new treatment insights.

Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells.

Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA(2), p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA(2) by Western blot. Capsaicin induced phosphorylation of p38 and cPLA(2). Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA(2) phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA(2). To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.

Progesterone receptor A mediates VIP inhibition of contraction.

The slow transit time of the colon in females with constipation is due to impairment of agonist-induced contraction. The impairment is associated with downregulation of G proteins that mediate contraction and upregulation of Gs proteins that mediate relaxation. These changes are caused by overexpression of progesterone (P4) receptors in the colon, rendering its muscle cells sensitive to physiological P4 concentrations. Downregulation of Gq/11 is mediated by P4 receptor B (PR-B). We examined whether upregulation of Gs proteins increased the inhibition of contraction and whether the increase is mediated by the P4 receptor A (PR-A). These studies were conducted in colon-isolated colon muscle cells from human control and slow-transit constipation (STC) females and from guinea pigs. Muscle cell contraction was induced by CCK-8. Inhibition of contraction was induced by vasoactive intestinal polypeptide (VIP), and 8'bromo-c'AMP (8B-c'AMP) G protein levels were determined by Western blot. VIP-induced inhibition of contraction was greater in muscle cells from STC and P4-treated muscle cells. There were no differences in the inhibition induced by 8B-c'AMP between muscle cells from STC and P4-treated controls. The increased VIP-induced inhibition of muscle cells treated with P4 was blocked by pretreatment with PR-A antibodies and unaffected by PR-B antibodies. These antibodies had no effect on 8B-c'AMP induced-inhibition. The P4 upregulation of Gs proteins was blocked by PR-A antibodies and unaffected by PR-B antibodies. Similar results were obtained in muscle cells from guinea pig colons. We concluded that P4 upregulation of Gs proteins increases VIP-induced inhibition of contraction mediated by PR-A.

HCl-activated neural and epithelial vanilloid receptors (TRPV1) in cat esophageal mucosa.

To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10(-6) M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.

High levels of caveolar cholesterol inhibit progesterone-induced genomic actions in human and guinea pig gallbladder muscle.

Gallbladder disease is prevalent during pregnancy. It has been suggested that this complication of pregnancy is attributable to increased bile cholesterol (Ch) induced by estrogens and to gallbladder hypomotility caused by increasing levels of progesterone (P4). Studies on nonpregnant gallbladders have shown that increased levels of bile Ch contribute to both gallstone formation and bile stasis. These studies investigated the effects of high levels of plasma membrane Ch on P4 on gallbladder muscle cells from human and guinea pigs. Contraction was studied in intact and permeabilized muscle cells. G proteins were determined by Western blot, and 3H-P4 incorporation by muscle cells was measured in the beta-scintillation counter. High levels of caveolar Ch blocked the effects induced by P4 treatment for 6 h. They suppressed the expected P4 inhibition of GTP-gammaS (a G protein activator)-induced contraction and changes in G proteins by downregulating Gi3 and upregulating Gs protein levels. Ch inhibited these P4 actions at the caveolar 3 (CAV-3) level, since the P4 effects were antagonized by treatment with CAV-3 antibody, by reducing CAV-3 expression through CAV-3 siRNA. CAV-3 antibody and siRNA reduced caveolar Ch levels. High caveolar levels of Ch and CAV-3 antibody blocked the incorporation of 3H-P4 into caveolae. Treatment with GDP-betaS (a G protein antagonist) had no effect on P4 actions. High caveolar Ch levels blocked the P4 effects on muscle contraction and G protein changes probably because both Ch and P4 require CAV-3 proteins for their transport across the plasma membrane.

Effects of progesterone on motility and prostaglandin levels in the distal guinea pig colon.

Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating Galpha(q/11) proteins that mediate contraction, by upregulating Galpha(s) proteins that mediate relaxation, and by altering the pattern of cyclooxygenase (COX) enzymes and prostaglandins. We aimed to examine whether P4 treatment of guinea pigs in vivo affects basal colon motility [basal motility index (MI)] by altering the levels and actions of PGF(2alpha) and PGE(2). Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the PGF(2alpha)-induced contraction, and PGE(2)-induced inhibition of contraction were examined in muscle strips and cells. The levels of PGF(2alpha) and PGE(2) were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of PGF(2alpha), and PGF(2alpha)-induced contraction. P4 increased PGE(2) levels, and PGE(2) induced relaxation. Pretreatment with IM RU-486 (10 mg/kg per day), a P4 receptor antagonist, 1 h before P4 blocked the actions of P4. The PGF(2alpha) antagonist Al-1180 abolished basal MI and PGF(2alpha)-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on PGF(2alpha) and PGE(2) effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased PGF(2alpha) levels; GTPgammaS increased and GDPbetaS decreased the levels of PGF(2alpha). Galpha(q/11) protein antibodies (Abs) reduced PGF(2alpha) levels, and Galpha(i3) Abs blocked its motor actions. Galphas Abs increased PGF(2alpha) but decreased PGE(2) levels. We concluded that P4 decreases basal MI by reducing PGF(2alpha) levels caused by downregulation of Galpha(q/11) and that PGF(2alpha)-induced contraction was blocked by downregulating Galpha(i3). P4 also decreased the basal MI by increasing PGE(2) levels, and PGE(2) induced relaxation by upregulating Galpha(s) proteins.

Overexpression of progesterone receptor B increases sensitivity of human colon muscle cells to progesterone.

Colon muscle strips and cells from female patients with slow-transit constipation (STC) exhibit impaired motility, signal transduction abnormalities characterized by downregulation of Gq/11 and upregulation of Gs proteins, decreased cyclooxygenase (COX)-1 and thromboxane (Tx)B2 levels, increased COX-2 and PGE2 levels, and overexpression of progesterone receptors (PGR). Progesterone (P4) treatment of normal cells reproduced these motility and signal transduction abnormalities. The purpose of the study was to examine whether overexpression of PGR-B reproduces these abnormalities by rendering the cells more sensitive to physiological concentrations of P4. Cultured human colon muscle was transfected with a plasmid DNA expressing PGR-B. The mRNAs of PGR, COX-1, COX-2, and Gq/11 were determined by quantitative real-time PCR. Their protein expression was determined by Western blot, and prostaglandins were measured by radioimmunoassay. Cultured muscle cells maintained their phenotypic features determined with myosin light chain (MLC) and h-caldesmon antibodies. Control and transfected muscle cells responded to 10(-6) M P4. In contrast, muscle cells transfected with PGR-B responded to lower P4 concentration (10(-7) M). This P4 concentration reduced MLC phosphorylation induced by CCK-8 (10(-8) M), downregulated Gq/11, and decreased COX-1 and TxB2 levels. It upregulated Gs proteins. It also increased COX-2 and PGE2 levels. We conclude that overexpression of PGR-B renders the cells more sensitive to physiological concentrations of P4. These results are consistent with the hypothesis that overexpression of PGR-B contributes to the motility and signal transduction abnormalities observed in female patients with STC and normal serum levels of P4.

Reactive oxygen species are messengers in maintenance of human and guinea pig gallbladder tonic contraction.

The tonic contraction of human and guinea pig gallbladder (GB) is dependent on basal levels of PGE(2) and thromboxane A(2) (TxA(2)). The pathway involved in the genesis of these prostaglandins has not been elucidated. We aimed to examine the source of reactive oxygen species (ROS) and whether they contribute to the genesis of GB tonic contraction by generating basal prostaglandin levels. Tonic contraction was studied in human and guinea pig GB muscle strips treated with ROS scavengers (Tiron and catalase), apocynin (an inhibitor of NADPH oxidase), and NOX-1 small interference RNA (siRNA). The subunits of NADPH oxidase and their functional roles were determined with specific antibodies in GB muscle cells. ROS scavengers reduced the GB tonic contraction and H(2)O(2) and PGE(2) levels. Apocynin also inhibited the tonic contraction. Antibodies against subunits of NADPH oxidase present in GB muscle cells lowered H(2)O(2) and PGE(2) levels. NOX-1 siRNA transfection reduced the tonic contraction, NOX-1 expression, and levels of H(2)O(2) and PGE(2). Tiron and apocynin inhibited the expected increase in tension and H(2)O(2) levels induced by stretching of muscle strips. H(2)O(2) increased the levels of PGE(2) and TxA(2) by increasing platelet-activating factor-like lipids that phosphorylate p38 and cPLA(2) sequentially. H(2)O(2) generated by NADPH oxidase participates in a signal transduction pathway that maintains the GB tonic contraction by activating PAF, p38, and cPLA(2) to generate prostaglandins.

Abnormalities of prostaglandins and cyclooxygenase enzymes in female patients with slow-transit constipation.

BACKGROUND AND AIMS: Chronic constipation due to slow transit (STC) is more common in female than in male patients. We have previously shown that these gender differences may be due to over expression of progesterone (PG) receptors that alter G protein patterns. We sought to elucidate the mechanisms responsible for the impaired basal colonic motility in female patients with STC. METHODS: Muscle tissues from females with STC and controls with adeno-carcinoma of the colon were studied. Prostaglandins were determined by immunoassay, COX enzymes by Western blot and COX enzymes and progesterone receptors mRNA by RT-PCR. RESULTS: STC patients had impaired colonic motility index, lower TxA(2) and PGF(2) and higher PGE(2) levels than controls. STC had lower COX-1 protein and mRNA levels and higher COX-2 protein and mRNA levels than controls. These abnormalities were reproduced in normal colonic muscle cells treated with PG for 6 h. STC patients had higher PG receptor protein expression and mRNA levels than controls suggesting over expression of these receptors. CONCLUSIONS: These findings suggest that the impaired motility index of STC is due to abnormal levels of prostaglandin and COX enzymes, probably caused by an over expression of PG receptors that make muscle cells more sensitive to circulating levels of PG.

Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility.

Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane, and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (Cav-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of Cav-3 in the caveolae compared with that in GB with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB with ChS and PS, but they were increased in the caveolae in muscle cells from GB with ChS. Treatment of muscle cells from GB with ChS with a Galpha(i3) protein fragment increased the total binding of CCK-1 receptors (from 8.3 to 11.2%) and muscle contraction induced by CCK-8 (from 11.2 to 17.3% shortening). However, Galpha(q/11) protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of Cav-3 that results in the sequestration of CCK-1 receptors in the caveolae, probably by inhibiting the functions of Galpha(i3) proteins.

Gastroesophageal reflux disease-associated esophagitis induces endogenous cytokine production leading to motor abnormalities.

BACKGROUND & AIMS: Gastroesophageal reflux disease is a condition frequently associated with esophagitis and motor abnormalities. Recent evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1beta and IL-6, may be implicated because they reduce esophageal muscle contractility, but these results derive from in vitro or animal models of esophagitis. This study used human esophageal cells and tissues to identify the cellular source of cytokines in human esophagitis investigate whether cytokines can be induced by gastric refluxate, and examine whether esophageal tissue- or cell-derived mediators affect muscle contractility. METHODS: Endoscopic mucosal biopsy specimens were obtained from patients with and without esophagitis, organ-cultured, and undernatants were assessed for cytokine content. The cytokine profile of esophageal epithelial, fibroblast, and muscle cells was analyzed, and esophageal mucosa and cell products were tested in an esophageal circular muscle contraction assay. RESULTS: The mucosa of esophagitis patients produced significantly greater amounts of IL-1beta and IL-6 compared with those of control patients. Cultured esophageal epithelial cells produced IL-6, as did fibroblasts and muscle cells. Epithelial cells exposed to buffered, but not denatured, gastric juice produced IL-6. Undernatants of mucosal biopsy cultures from esophagitis patients reduced esophageal muscle contraction, as did supernatants from esophageal epithelial cell cultures. CONCLUSIONS: The human esophagus produces cytokines capable of reducing contractility of esophageal muscle cells. Exposure to gastric juice is sufficient to stimulate esophageal epithelial cells to produce IL-6, a cytokine able to alter esophageal contractility. These results indicate that classic cytokines are important mediators of the motor disturbances associated with human esophageal inflammation.

Prostaglandins mediate tonic contraction of the guinea pig and human gallbladder.

The gallbladder (GB) maintains tonic contraction modulated by neurohormonal inputs but generated by myogenic mechanisms. The aim of these studies was to examine the role of prostaglandins in the genesis of GB myogenic tension. Muscle strips and cells were treated with prostaglandin agonists, antagonists, cyclooxygenase (COX) inhibitors, and small interference RNA (siRNA). The results show that PGE2, thromboxane A2 (TxA2), and PGF(2alpha) cause a dose-dependent contraction of muscle strips and cells. However, only TxA2 and PGE2 (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and TxB2 (TxA2 stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PGE2 levels. Both inhibitors and the nonselective COX inhibitor indomethacin abolished the tonic contraction. Transfection of human GB muscle strips with COX-1 siRNA partially lowered the tonic contraction and reduced COX-1 protein expression and TxB2 levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PGE2. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, TxB2, and PGE2 levels; a COX-1 inhibitor prevented the increase in tension and TxB2; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PGE2. Indomethacin blocked the rise in tension and TxB2 and PGE2 levels. We conclude that PGE2 generated by COX-2 and TxA2 generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PGE2 and TxA2 levels.

Acid-induced release of platelet-activating factor by human esophageal mucosa induces inflammatory mediators in circular smooth muscle.

In a human in vitro model of esophagitis, we investigated the genesis of esophagitis-associated dysmotility by examining HCl-induced production of inflammatory mediators in the mucosa and investigating their effect on esophageal circular muscle. Muscularis propria was removed from organ donors' esophagi, leaving the mucosal tube intact. The tube was tied at both ends, forming a sac, and filled with HCl at pH 4. After 3 h of incubation, the supernatant surrounding the sac was analyzed or applied to circular muscle strips. HCl alone did not affect circular muscle contraction in response to electrical field stimulation (EFS), but supernatant of HCl-treated mucosa abolished contraction. The inhibition was reversed by the platelet-activating factor (PAF) antagonist CV3988 [(+/-)-3-(N-octadecylcarbamoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate], whereas the PAF analog 2-O-methyl platelet-activating factor C-16 (PAF-16) inhibited EFS-induced contraction and acetylcholine (ACh) release in circular muscle strips. The hydrogen peroxide scavenger catalase reversed the inhibition in contraction, to the same extent as CV3988. We therefore measured PAF and hydrogen peroxide (H(2)O(2)) in mucosa, mucosa supernatant, and circular muscle. HCl increased PAF and interleukin (IL)-1beta (but not IL-6, prostaglandin E(2), or H(2)O(2)) in mucosa, and only PAF was released into the supernatant, presumably to affect circular muscle. In circular muscle, exogenous PAF induced sequential formation of IL-6, H(2)O(2), IL-1beta, and PAF. Release of PAF by the mucosa inhibits ACh release from circular muscle layer neurons and initiates sequential formation of inflammatory mediators in muscle, resulting in production of PAF by the muscle itself, possibly initiating in a self-sustaining cycle.

IL-1beta signaling in cat lower esophageal sphincter circular muscle.

In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1beta that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1beta-induced reduction in LES tone. IL-1beta significantly reduced acetylcholine-induced Ca(2+) release in Ca(2+)-free medium, and this effect was partially reversed by catalase, demonstrating a role of H(2)O(2) in these changes. IL-1beta significantly increased the production of H(2)O(2), and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA(2). IL-1beta-induced cPLA(2) phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA(2). The IL-1beta-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca(2+)-insensitive PLA(2) inhibitor bromoenol lactone (BEL). IL-1beta significantly increased cyclooxygenase (COX)-2 and PGE(2) levels. The increase in PGE(2) was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1beta reduces LES tone by producing H(2)O(2), which may affect Ca(2+)-release mechanisms and increase the synthesis of COX-2 and PGE(2). Both H(2)O(2) and PGE(2) production depend on sequential activation of p38 MAPK and cPLA(2). cPLA(2) activates NADPH oxidases, producing H(2)O(2), and may produce arachidonic acid, converted to PGE(2) via COX-2.