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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 SIC 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. [ABSTRACT FROM AUTHOR]

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°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. [ABSTRACT FROM AUTHOR]

Colon muscle strips and cells from female patients with slow-transit consti- pation (STC) exhibit impaired motility, signal transduction abnormal- ities characterized by downregulation of Gqii i and upregulation of G. 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 abnor- malities. The purpose of the study was to examine whether overex- pression 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-l, COX-2, and Gqiii 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 pheno- typic 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 (l0 M). This P4 concentration reduced MLC phosphorylation induced by CCK-8 (l0 M), down- regulated Gqii i, and decreased COX- 1 and TxB2 levels. It upregulated G 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. [ABSTRACT FROM AUTHOR]

Plate- let-activating factor (PAF) and interleukin-6 (IL-6) are produced in the esophagus in response to HCI 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 HCI-activated mechanisms for production of PAF and lL-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-HCI) 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. lL-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 HCI, indicating that HCI 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 lL-6 that activates NADPH oxidase to induce production of H2O2. H2O2 causes formation of IL-1β that may induce production of PAF in the muscle, possibly closing a self-sustaining cycle of production of inflammatory mediators. [ABSTRACT FROM AUTHOR]

We have shown that IL-1β 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 HCI, 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-1β 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-1β and IL-6 but not H2O2. IL-1β 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. [ABSTRACT FROM AUTHOR]

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-1β and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle (12), we examined the role of IL-1β and IL-6 in this motor dysfunction. IL-1β, IL-6 (12), H2O2, PGE2, and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1β 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-1β 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-1β-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. [ABSTRACT FROM AUTHOR]

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca2+ stores. We therefore measured cytosolic Ca2+ 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 H2O2 to the reduction in Ca2+ signal. In normal cells, the ACh-induced Ca2+ increase was the same in normal-Ca2+ and Ca2+-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 Ca2+ release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca2+ increase in normal-Ca2+ medium was significantly lower in esophagitis than in normal cells and was further reduced (∼70%) when the cells were incubated in Ca2+-free medium. This reduction was partially reversed by the H2O2 scavenger catalase. H2O2 measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H2O2, the ACh-induced Ca2+ increase was significantly reduced in normalCa2+ and Ca2+-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 H2O2. H2O2, when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca2+ signal in normal cells. H2O2-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H2O2 reduced TG-induced contraction, suggesting that TG and H2O2 may operate through similar mechanisms. Ca2+ATPase activity measurement indicates that H2O2 and TG reduced Ca2+-ATPase activity, confirming similarity of mechanism of action. We conclude that H2O2 may be at least partly responsible for impairment of Ca2+ release in acute experimental esophagitis by inhibiting Ca2+ uptake and refilling Ca2+ stores. [ABSTRACT FROM AUTHOR]

Myoganic lower esophageal sphincter (LES) tone is maintained by arachidonic acid metabolites, such as PGF2α and thromboxane A2/B2. Experimental esoph- agitis in cat reduces LES in vivo pressure and in vitro tone. Because IL-1β may mediate esophagitis-associated reduction in ACh release in esophagus, we examined whether IL-1β 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-1β levels were higher in esophagitis than in normal LES. IL-1β reduced normal LES tone, and the reduction was reversed by catalase, suggesting a role of H2O2. This was confirmed by IL-lβ-induced production of H2O2 in normal LES and elevated H2O2 levels in esophagitis. H2O2 by itself is sufficient to explain the changes that occur in the muscle, reducing its ability to contract. H2O2 increased PGE2 in normal LES, and PGE2 levels were elevated in esophagitis LES, whereas PGF2α levels were unchanged. H2O2 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 PGF2α analog 8-iso-PGF2α caused little contraction of LES strips but reduced PGF2α binding and contraction of normal LES. In esophagitis, PGF2α 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-1β causes production of H2O2 H2O2 increases PGE2, which relaxes the LES, and 8-iso-F2α which blocks PGF2α-mediated contraction. [ABSTRACT FROM AUTHOR]

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-1β (100 U/ml), IL-6 (1 ng/ml), or TNF-α (1 ng/ml). IL-1β and IL-6 levels, measured by Western blot analysis, increased in esophagitis compared with normal circular muscle. IL-1β 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-1β or IL-6, suggesting that they may contribute to the contractility changes. TNF-α at 1 ng/ml, however, did not affect the response to ACh or to electrical stimulation but inhibited both at higher concentrations. TNF-α levels were low in normal muscle and did not increase with esophagitis. The data suggest that the proinflammatory cytokines IL-1β and IL-6 contribute to reduced esophageal contraction by inhibiting release of ACh from myenteric neurons. [ABSTRACT FROM AUTHOR]