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.

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.

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.

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.

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.

NK2 receptor-mediated spontaneous phasic contractions in normal and ulcerative colitis human sigmoid colon.

Human colonic circular muscle produces spontaneous phasic contractions that are reduced in ulcerative colitis. How the spontaneous phasic contractions develop and why they decrease in ulcerative colitis are not known. We found that spontaneous phasic contractions of normal sigmoid circular muscle strips were significantly reduced by 90-min incubation with tetrodotoxin (10(-5) M), which blocked neurokinin A release in basal conditions and in response to electrical stimulation. In addition, spontaneous contraction of human sigmoid colon was significantly decreased by the NK2 receptor antagonists MEN10376 (Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH2) and NK2ra (Bz-Ala-Ala-D-Trp-Phe-D-pro-Pro-Nle-NH2) but not by atropine or by the NK1 antagonist FK888 (N2-[(4R)-4-hydroxyl-1-(1-methyl-1H-indol-3-yl)carbonyl-l-prolyl]-N-methyl-N-phenylmethyl-3-(2-naphthyl)-l-alaninamide), suggesting that NK2 receptors are involved in their development. The spontaneous phasic contractions were abolished by thapsigargin and cyclopiazonic acid and significantly decreased by the protein kinase C inhibitor chelerythrine and by the calmodulin inhibitor CGS9343B (1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate), suggesting that spontaneous phasic contractions may be mediated by Ca2+ release from intracellular stores and by a protein kinase C- and calmodulin-dependent pathway. In strips from patients with ulcerative colitis, spontaneous contractions were significantly reduced, and this reduction was partially restored by the hydrogen peroxide scavenger catalase. Neurokinin A release, however, was not affected. We conclude that spontaneous phasic contractions of human sigmoid circular smooth muscle may be mediated by activation of NK2 receptors, calcium release from intracellular stores, and activation of calmodulin and protein kinase C. In ulcerative colitis patients, spontaneous phasic contractions are decreased, and this decrease may be in part due to overproduction of hydrogen peroxide affecting sigmoid circular muscle.

HCl-induced inflammatory mediators in cat esophageal mucosa and inflammatory mediators in esophageal circular muscle in an in vitro model of esophagitis.

Platelet-activating factor (PAF) and interleukin-6 (IL-6) are produced in the esophagus in response to HCl and affect ACh release, causing changes in esophageal motor function similar to esophagitis (Cheng L, Cao W, Fiocchi C, Behar J, Biancani P, and Harnett KM. Am J Physiol Gastrointest Liver Physiol 289: G418-G428, 2005). We therefore examined HCl-activated mechanisms for production of PAF and IL-6 in cat esophageal mucosa and circular muscle. A segment of normal mucosa was tied at both ends, forming a mucosal sac (Cheng L, Cao W, Fiocchi C, Behar J, Biancani P, and Harnett KM. Am J Physiol Gastrointest Liver Physiol 289: G860-G869, 2005) that was filled with acidic Krebs buffer (pH 5.8) or normal Krebs buffer (pH 7.0) as control and kept in oxygenated Krebs buffer for 3 h. The supernatant of the acidic sac (MS-HCl) abolished contraction of normal muscle strips in response to electric field stimulation. The inhibition was reversed by the PAF antagonist CV3988 and by IL-6 antibodies. PAF and IL-6 levels in MS-HCl and mucosa were significantly elevated over control. IL-6 levels in mucosa and supernatant were reduced by CV3988, suggesting that formation of IL-6 depends on PAF. PAF-receptor mRNA levels were not detected by RT-PCR in normal mucosa, but were significantly elevated after exposure to HCl, indicating that HCl causes production of PAF and expression of PAF receptors in esophageal mucosa and that PAF causes production of IL-6. PAF and IL-6, produced in the mucosa, are released to affect the circular muscle layer. In the circular muscle, PAF causes production of additional IL-6 that activates NADPH oxidase to induce production of H(2)O(2). H(2)O(2) causes formation of IL-1beta that may induce production of PAF in the muscle, possibly closing a self-sustaining cycle of production of inflammatory mediators.

Hydrogen peroxide reduces lower esophageal sphincter tone in human esophagitis.

BACKGROUND & AIMS: We have previously used the normal lower esophageal sphincter (N-LES) of human organ donors to examine the physiologic signal transduction of lower esophageal sphincter (LES) circular muscle. Now, for the first time, we have obtained a human LES specimen with esophagitis (E-LES) and characterized its pathophysiologic mechanical and inflammatory profiles. METHODS: E-LES was examined histologically, and its in vitro circular muscle contraction and production of inflammatory mediators were compared with those of N-LES. RESULTS: E-LES exhibited scattered erosions and displayed inflammatory cells in the epithelial layer, basal zone hyperplasia, and elongation of lamina propria papillae, characteristic of chronic reflux esophagitis. E-LES muscle strips developed lower in vitro tone (0.78 g) than N-LES (3.3 +/- 0.2 g). E-LES tone was essentially restored to normal by the H2O2 scavenger catalase, suggesting that H2O2 was responsible for reduction of tone. NOX5 cDNA was higher and H2O2 levels were 4 times higher in E-LES circular muscle (0.85 nmol/mg protein) than in N-LES (0.19 +/- 0.05 nmol/mg protein). When N-LES smooth muscle was incubated in H2O2 (70 micromol/L, 2 hours), platelet activating factor (PAF), prostaglandin E2 (PGE2), and F2-isoprostane increased 2.5, 5.2, and 36 times, respectively. In E-LES, levels of PAF, PGE2, and F2-isoprostane were 4, 6, and 40 times, respectively, higher than in N-LES. PAF, PGE2, and F2 isoprostane produced dose-dependent reductions in tone of N-LES muscle strips. CONCLUSIONS: We conclude that an excessive production of H2O2 triggers an increased production of PAF, PGE2, and F2-isoprostane, which are responsible for reducing LES tone in human esophagitis.

In vitro model of acute esophagitis in the cat.

We have shown that IL-1beta and IL-6, possibly originating from the mucosa in response to injury, inhibit neurally mediated contraction of esophageal circular muscle but do not affect ACh-induced contraction, reproducing the effect of experimental esophagitis on esophageal contraction. To examine the interaction of mucosa and circular muscle in inflammation, we examined the effect of HCl on in vitro esophageal mucosa and circular muscle. Circular muscle strips, when directly exposed to HCl, contracted normally. However, when circular muscle strips were exposed to supernatants of mucosa incubated in HCl (2-3 h, pH 5.8), contraction decreased, and the inhibition was partially reversed by an IL-6 antibody. Supernatants from the mucosa of animals with in vivo-induced acute esophagitis (AE) similarly reduced contraction. IL-6 levels were higher in mucosal tissue from AE animals than in control mucosa and in AE mucosa supernatants than in normal mucosa supernatants. IL-6 levels increased significantly in normal mucosa and supernatants in response to HCl, suggesting increased production and release of IL-6 by the mucosa. IL-6 increased H2O2 levels in the circular muscle layer but not in mucosa. Exposure of the mucosa to HCl caused IL-1beta to increase only in the mucosa and not in the supernatant. These data suggest that HCl-induced damage occurs first in the mucosa, leading to the production of IL-1beta and IL-6 but not H2O2. IL-1beta appears to remain in the mucosa. In contrast, IL-6 is produced and released by the mucosa, eventually resulting in the production of H2O2 by the circular muscle, with this affecting circular muscle contraction.

Platelet-activating factor and prostaglandin E2 impair esophageal ACh release in experimental esophagitis.

ACh is a neurotransmitter in cat esophageal circular muscle, as atropine nearly abolishes contraction of in vitro circular muscle strips in response to electric field stimulation (EFS) (5, 12). Experimental esophagitis reduced EFS- but not ACh-induced contraction of esophageal circular muscle, suggesting that esophagitis impairs neurotransmitter release. Because IL-1beta and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle (12), we examined the role of IL-1beta and IL-6 in this motor dysfunction. IL-1beta, IL-6 (12), H2O2, PGE2, and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1beta and IL-6 had increases in H2O2, PGE2, and PAF levels. H2O2 contributed to increased PGE2 and PAF, as the increase was partially (60-80%) reversed by the H2O2 scavenger catalase. EFS-induced [3H]ACh release from muscle strips significantly (42%) decreased in esophagitis and after 2 h incubation in PGE2 and in PAF C-16. Similarly, EFS-induced but not ACh-induced muscle contraction decreased in esophagitis and after incubation in PGE2 and PAF C-16. Finally, in normal muscle strips treated with IL-1beta electrical field stimulation (EFS)-induced contraction was partially restored by indomethacin or by the PAF antagonist CV3988 and was completely restored by the combination of CV3988 and indomethacin, whereas in strips treated with IL-6, EFS-induced contraction was partially restored by the PAF antagonist CV3988 and not affected by indomethacin. We conclude that IL-1beta-induced production of H2O2 causes formation of PGE2 and PAF that inhibit ACh release from esophageal cholinergic neurons without affecting ACh-induced contraction of esophageal circular muscle. IL-6 causes production of H2O2, PAF, and other unidentified inflammatory mediators.

Signal-transduction pathways that regulate smooth muscle function I. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles.

Contraction of esophageal (Eso) and lower esophageal sphincter (LES) circular muscle depends on distinct signal-transduction pathways. ACh-induced contraction of Eso muscle is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid (AA), and activation of the Ca(2+)-insensitive PKCepsilon. Although PKCepsilon does not require Ca(2+) for activation, either influx of extracellular Ca(2+) or release of Ca(2+) from stores is needed to activate the phospholipases responsible for hydrolysis of membrane phospholipids and production of second messengers, which activate PKCepsilon. In contrast, the LES uses two distinct intracellular pathways: 1) a PKC-dependent pathway activated by low doses of agonists or during maintenance of spontaneous tone, and 2) a Ca(2+)-calmodulin-myosin light chain kinase (MLCK)-dependent pathway activated in response to maximally effective doses of agonists during the initial phase of contraction. The Ca(2+) levels, released by agonist-induced activity of phospholipase C, determine which contractile pathway is activated in the LES. The Ca(2+)-calmodulin-MLCK-dependent contractile pathway has been well characterized in a variety of smooth muscles. The steps linking activation of PKC to myosin light chain (MLC20) phosphorylation and contraction, however, have not been clearly defined for LES, Eso, or other smooth muscles. In addition, in LES circular muscle, a low-molecular weight pancreatic-like phospholipase A2 (group I PLA2) causes production of AA, which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to heterotrimeric G proteins to induce activation of phospholipases and production of second messengers to maintain contraction of LES circular muscle. We have examined the signal-transduction pathways activated by PGF(2alpha) and by thromboxane analogs during the initial contractile phase and found that these pathways are the same as those activated by other agonists. In response to low doses of agonists or during maintenance of tone, presumably due to low levels of calcium release, a PKC-dependent pathway is activated, whereas at high doses of PGF(2alpha) and thromboxane analogs, in the initial phase of contraction, calmodulin is activated, PKC activity is reduced, and contraction is mediated, in part, through a Ca(2+)-calmodulin-MLCK-dependent pathway. The PKC-dependent signaling pathways activated by PGF(2alpha) and by thromboxanes during sustained LES contraction, however, remain to be examined, but preliminary data indicate that a distinct PKC-dependent pathway may be activated during maintenance of tonic contraction, which is different from the one activated during the initial contractile response. The initial contractile response to low levels of agonists depends on activation of G(q). Sustained contraction in response to PGF(2alpha) may involve activation of the monomeric G protein RhoA, because the contraction is inhibited by the RhoA-kinase antagonist Y27632. This shift in signal-transduction pathways between initial and sustained contraction has been recently reported in intestinal smooth muscle.

H(2)O(2): a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter.

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca(2+) stores. We therefore measured cytosolic Ca(2+) in response to a maximally effective dose of ACh in fura 2-AM-loaded lower esophageal sphincter (LES) circular muscle cells and examined the contribution of H(2)O(2) to the reduction in Ca(2+) signal. In normal cells, the ACh-induced Ca(2+) increase was the same in normal-Ca(2+) and Ca(2+)-free medium and was abolished by the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C inhibitor U-73122, confirming that the initial ACh-induced contraction depends on Ca(2+) release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca(2+) increase in normal-Ca(2+) medium was significantly lower in esophagitis than in normal cells and was further reduced ( approximately 70%) when the cells were incubated in Ca(2+)-free medium. This reduction was partially reversed by the H(2)O(2) scavenger catalase. H(2)O(2) measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H(2)O(2), the ACh-induced Ca(2+) increase was significantly reduced in normal-Ca(2+) and Ca(2+)-free medium and was similar to that observed in animals with esophagitis. The initial ACh-induced contraction was also reduced in normal cells incubated with H(2)O(2). H(2)O(2), when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca(2+) signal in normal cells. H(2)O(2)-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H(2)O(2) reduced TG-induced contraction, suggesting that TG and H(2)O(2) may operate through similar mechanisms. Ca(2+)-ATPase activity measurement indicates that H(2)O(2) and TG reduced Ca(2+)-ATPase activity, confirming similarity of mechanism of action. We conclude that H(2)O(2) may be at least partly responsible for impairment of Ca(2+) release in acute experimental esophagitis by inhibiting Ca(2+) uptake and refilling Ca(2+) stores.

Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.

Myogenic lower esophageal sphincter (LES) tone is maintained by arachidonic acid metabolites, such as PGF(2alpha) and thromboxane A(2)/B(2). Experimental esophagitis in cat reduces LES in vivo pressure and in vitro tone. Because IL-1beta may mediate esophagitis-associated reduction in ACh release in esophagus, we examined whether IL-1beta may also play a role in esophagitis-induced reduction of LES tone. A cat model of experimental esophagitis was obtained by repeated esophageal perfusion with HCl (Biancani P, Barwick K, Selling J, and McCallum R. Gastreonterology 87: 8-16, 1984 and Sohn UD, Harnett KM, Cao W, Rich H, Kim N, Behar J, and Biancani P. J Pharmacol Exp Ther 283: 1293-1304, 1997.). LES circular muscle strips were examined in muscle chambers as previously described (Biancani P, Billett G, Hillemeier C, Nissenshon M, Rhim BY, Sweczack S, and Behar J. Gastroenterology 103: 1199-1206, 1992). Levels of inflammatory mediators were measured. IL-1beta levels were higher in esophagitis than in normal LES. IL-1beta reduced normal LES tone, and the reduction was reversed by catalase, suggesting a role of H(2)O(2). This was confirmed by IL-1beta-induced production of H(2)O(2) in normal LES and elevated H(2)O(2) levels in esophagitis. H(2)O(2) by itself is sufficient to explain the changes that occur in the muscle, reducing its ability to contract. H(2)O(2) increased PGE(2) in normal LES, and PGE(2) levels were elevated in esophagitis LES, whereas PGF(2alpha) levels were unchanged. H(2)O(2) also increased levels of 8-isoprostanes, stable prostaglandin-like compounds formed by free radical-induced peroxidation of arachidonic acid, and 8-isoprostane levels were elevated in esophagitis. The PGF(2alpha) analog 8-iso-PGF(2alpha) caused little contraction of LES strips but reduced PGF(2alpha) binding and contraction of normal LES. In esophagitis, PGF(2alpha) binding and contraction were reduced in LES, suggesting that isoprostanes may contribute to reduction in tone in esophagitis. The data suggest that, in esophagitis, IL-1beta causes production of H(2)O(2). H(2)O(2) increases PGE(2), which relaxes the LES, and 8-iso-F(2alpha), which blocks PGF(2alpha)-mediated contraction.

Proinflammatory cytokines alter/reduce esophageal circular muscle contraction in experimental cat esophagitis.

Cholinergic mechanisms are largely responsible for esophageal contraction in response to swallowing or to in vitro electrical field stimulation (EFS). After induction of experimental esophagitis by repeated acid perfusion, the responses to swallowing and to EFS were significantly reduced but contraction in response to ACh was not affected, suggesting that cholinergic mechanisms are damaged by acid perfusion but that myogenic mechanisms are not. Measurements of ACh release in response to EFS confirmed that release of ACh was reduced in esophagitis compared with normal controls. To examine factors contributing to this neuropathy, normal esophageal strips were incubated for 1-2 h with the proinflammatory cytokines IL-1beta (100 U/ml), IL-6 (1 ng/ml), or TNF-alpha (1 ng/ml). IL-1beta and IL-6 levels, measured by Western blot analysis, increased in esophagitis compared with normal circular muscle. IL-1beta and IL-6 reduced contraction in response to EFS (2-10 Hz, 0.2 ms) but did not affect ACh-induced contraction, suggesting that these cytokines inhibit ACh release without affecting myogenic contractile mechanisms. EFS-induced ACh release was significantly reduced in normal esophageal strips by incubation in IL-1beta or IL-6, suggesting that they may contribute to the contractility changes. TNF-alpha at 1 ng/ml, however, did not affect the response to ACh or to electrical stimulation but inhibited both at higher concentrations. TNF-alpha levels were low in normal muscle and did not increase with esophagitis. The data suggest that the proinflammatory cytokines IL-1beta and IL-6 contribute to reduced esophageal contraction by inhibiting release of ACh from myenteric neurons.

Interleukin 1beta-induced production of H2O2 contributes to reduced sigmoid colonic circular smooth muscle contractility in ulcerative colitis.

We have shown that neurokinin A-induced contraction of human sigmoid circular muscle (HSCM) is reduced in patients with ulcerative colitis and that interleukin (IL)-1beta may play a role in this change. We now examine changes in the signal transduction pathway mediating neurokinin A-induced contraction of HSCM and explore the role of IL-1beta and of H(2)O(2) in these changes. In Fura 2-AM-loaded ulcerative colitis HSCM cells, neurokinin A- and caffeine-induced peak Ca(2+) increase and cell shortening were significantly reduced. In normal cells, neurokinin A-induced contraction was decreased by protein kinase C inhibitor chelerythrine and by calmodulin inhibitor CGS9343B [1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate]. In ulcerative colitis muscle cells, contraction was inhibited only by chelerythrine but not by CGS9343B. IL-1beta treatment of normal HSCM strips and cells reproduced the changes observed in ulcerative colitis. IL-1beta-induced reduction in caffeine-induced peak Ca(2+) increase and contraction was reversed by catalase, suggesting a role of H(2)O(2). IL-1beta-induced H(2)O(2) production was inhibited by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) and by cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3 (arachidonyltrifluoromethyl ketone), but neither by p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] nor by nuclear factor-kappaB (NF-kappaB) inhibitory peptide NF-kappaB SN50 (H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). IL-1beta significantly increased the phosphorylation of extracellular signal-regulated kinase 1 (ERK1)/ERK2 MAPKs and cPLA(2) and IL-1beta-induced cPLA(2) phosphorylation was blocked by PD98059. We conclude that Ca(2+) stores of HSCM cells may be reduced in ulcerative colitis and that the signal transduction pathway of neurokinin A-induced contraction switches from calmodulin- and protein kinase C-dependent in normal cells to protein kinase C-dependent in ulcerative colitis cells. IL-1beta reproduces these changes, possibly by production of H(2)O(2) via sequential activation of MAPKs (ERK1/ERK2) and cPLA(2).