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Exposure of esophageal mueosa to hydrochloric acid (HC1) is a crucial factor in the pathogenesis of reflux disease. We examined supernatant of HC1-exposed rabbit mucosa for inflammatory mediators enhancing migration of leukocytes and production of [H.sub.2][O.sub.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 HC1-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.sub.2][O.sub.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.sub.2][O.sub.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.sub.2][O.sub.2] by PBL. These findings provide a direct link between acid exposure and recruitment and activation of immune cells in esophageal mucosa. PAF; substance P; IL-8; gastroesophageal reflux doi: 10.1152/ajpgi.00160.2010.

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. cytokines; motility; stricture; stenosis doi: 10.1152/ajpgi.00454.2009.

The slow transit time of the colon in t 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. G proteins; vasoactive intestinal polypeptide doi:10.1152/ajpgi.00346.2009.

Ma J, Harnett KM, Behar J, Biancani P, Cao W. Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells. Am J Physiol Gastrointest Liver Physiol 298: G233-G240, 2010. First published December 3, 2009; doi:10.1152/ajpgi.00409.2009.--Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RTPCR 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 (cPLA2) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA2, p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and [cPLA.sub.2] by Western blot. Capsaicin induced phosphorylation of p38 and [cPLA.sub.2]. Capsaicininduced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced [cPLA.sub.2] phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and [cPLA.sub.2]. To investigate how p38 phosphorylation may result from TRPV 1-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 TRPV l-mediated calcium elevation. capsaicin; esophagus doi: 10.1152/ajpgi.00409.2009

Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating [G[alpha].sub.q/11] proteins that mediate contraction, by upregulating [G[alpha].sub.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.sub.2[alpha]] and[ PGE.sub.2]. Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the [PGF.sub.2[alpha]]-induced contraction, and PGE2-induced inhibition of contraction were examined in muscle strips and cells. The levels of [PGF.sub.2[alpha]] and [PGE.sub.2] were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of [PGF.sub.2[alpha], and [PGF.sub.2[alpha]]-induced contraction. P4 increased [PGE.sub.2] levels, and [PGE.sub.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.sub.2[alpha]] antagonist A1-1180 abolished basal MI and [PGF.sub.2[alpha]]-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on [PGF.sub.2[alpha]] and [PGE.sub.2] effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased [PGF.sub.2[alpha]] levels; GTP[gamma]S increased and GDP[beta]S decreased the levels of [PGF.sub.2[alpha]]. [G[alpha].sub.q/11] protein antibodies (Abs) reduced [PGF.sub.2[alpha]] levels, and [G[alpha].sub.i3] Abs blocked its motor actions. G[alpha]s Abs increased [PGF.sub.2[alpha]] but decreased [PGE.sub.2] levels. We concluded that P4 decreases basal MI by reducing [PGF.sub.2[alpha]] levels caused by downregulation of [G[alpha].sub.q/11] and that [PGF.sub.2[alpha]]-induced contraction was blocked by downregulating [G[alpha].sub.i3]. P4 also decreased the basal MI by increasing [PGE.sub.2] levels, and [PGE.sub.2] induced relaxation by upregulating [G[alpha].sub.s] proteins. colon motility; guinea pigs; [PGF.sub.2[alpha]]; [PGE.sub.2]; G proteins doi: 10.1152/ajpgi.00184.2009.

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.sup.-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. smooth muscle; signal transduction; platelet-activating factor

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 [sup.3]H-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-[gamma]S (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 [sup.3]H-P4 into caveolae. Treatment with GDP-[beta]S (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. muscle cells; caveolar 3 proteins; G proteins

Colon muscle strips and cells from female patients with slow-transit constipation (STC) exhibit impaired motility, signal transduction abnormalities characterized by downregulation of [G.sub.q/11] and upregulation of [G.sub.s] proteins, decreased cyclooxygenase (COX)-1 and thromboxane (Tx)[B.sub.2] levels, increased COX-2 and [PGE.sub.2] levels, and overexpression of progesterone receptors (PGR). Progesterone ([P.sub.4]) 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 [P.sub.4]. Cultured human colon muscle was transfected with a plasmid DNA expressing PGR-B. The mRNAs of PGR, COX-l, COX-2, and [G.sub.q/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.sup.-6] M [P.sub.4]. In contrast, muscle cells transfected with PGR-B responded to lower [P.sub.4] concentration ([10.sup.-7] M). This [P.sub.4] concentration reduced MLC phosphorylation induced by CCK-8 ([10.sup.-8] M), downregulated [G.sub.q/11], and decreased COX-1 and Tx[B.sub.2] levels. It upregulated [G.sub.s] proteins. It also increased COX-2 and [PGE.sub.2] levels. We conclude that overexpression of PGR-B renders the cells more sensitive to physiological concentrations of [P.sub.4]. 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 [P.sub.4]. G proteins; cyclooxygenase enzymes; prostaglandins

The tonic contraction of human and guinea pig gallbladder (GB) is dependent on basal levels of [PGE.sub.2] and thromboxane [A.sub.2] (Tx[A.sub.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.sub.2][O.sub.2] and [PGE.sub.2] levels. Apocynin also inhibited the tonic contraction. Antibodies against subunits of NADPH oxidase present in GB muscle cells lowered [H.sub.2][O.sub.2] and [PGE.sub.2] levels. NOX-1 siRNA transfection reduced the tonic contraction, NOX-1 expression, and levels of [H.sub.2][O.sub.2] and [PGE.sub.2]. Tiron and apocynin inhibited the expected increase in tension and [H.sub.2][O.sub.2] levels induced by stretching of muscle strips. [H.sub.2][O.sub.2] increased the levels of [PGE.sub.2] and Tx[A.sub.2] by increasing platelet-activating factor-like lipids that phosphorylate p38 and [cPLA.sub.2] sequentially. [H.sub.2][O.sub.2] generated by NADPH oxidase participates in a signal transduction pathway that maintains the GB tonic contraction by activating PAF, p38, and [cPLA.sub.2] to generate prostaglandins. muscle; NADPH oxidase; prostaglandins

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 G[[alpha].sub.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, G[[alpha].sub.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 G[[alpha].sub.i3] proteins. caveolin-3 proteins; cholecystokinin-1 receptors; G protein fragments doi:10.1152/ajpgi.00495.2006

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 PG[E.sub.2], thromboxane [A.sub.2] (Tx[A.su.b.2]), and PG[F.sub.2[alpha]] cause a dose-dependent contraction of muscle strips and cells. However, only Tx[A.sub.2] and PG[E.sub.2] (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and Tx[B.sub.2] (Tx[A.sub.2] stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PG[E.sub.2] 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 Tx[B.sub.2] levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PG[E.sub.2]. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, T[B.sub.2], and PG[E.sub.2] levels; a COX-1 inhibitor prevented the increase in tension and Tx[B.sub.2]; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PG[E.sub.2]. Indomethacin blocked the rise in tension and Tx[B.sub.2] and PG[E.sub.2] levels. We conclude that PG[E.sub.2] generated by COX-2 and Tx[A.sub.2] generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PG[E.sub. 2] and Tx[A.sub.2] levels. gallbladder; muscle tension; prostaglandin [E.sub.2]; thromboxane [A.sub.2]; COX-1; COX-2; siRNA

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-1[beta] that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1[beta]-induced reduction in LES tone. IL-1[beta] significantly reduced acetylcholine-induced [Ca.sup.2+] release in [Ca.sup.2+]-free medium, and this effect was partially reversed by catalase, demonstrating a role of [H.sub.2][O.sub.2] in these changes. IL-1[beta] significantly increased the production of [H.sub.2][O.sub.2], and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase [A.sub.2] ([cPLA.sub.2]) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1[beta] significantly increased the phosphorylation of p38 MAPK and [cPLA.sub.2]. IL-1[beta]-induced [cPLA.sub.2] phosphorylation was blocked by SB203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating [cPLA.sub.2] The IL-1[beta]-induced reduction in LES tone was partially reversed by AACOCF3 and by the [Ca.sup.2+]-insensitive [PLA.sub.2] inhibitor bromoenol lactone (BEL). IL-1[beta] significantly increased cyclooxygenase (COX)-2 and [PGE.sub.2] levels. The increase in [PGE.sub.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-1[beta] reduces LES tone by producing [H.sub.2][O.sub.2], which may affect [Ca.sup.2+]-release mechanisms and increase the synthesis of COX-2 and [PGE.sub.2]. Both [H.sub.2][O.sub.2] and [PGE.sub.2] production depend on sequential activation of p38 MAPK and [cPLA.sub.2]. [cPLA.sub.2] activates NADPH oxidases, producing [H.sub.2][O.sub.2], and may produce arachidonic acid, converted to [PGE.sub.2] via COX-2. acetylcholine; smooth muscle

Platelet-activating factor (PAF) and interleukin-6 (IL-6) are produced in the esophagus in response to HC1 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.sub.2][O.sub.2]. [H.sub.2][O.sub.2] causes formation of IL-1[beta] that may induce production of PAF in the muscle, possibly closing a self-sustaining cycle of production of inflammatory mediators. inflammation; smooth muscle contraction; reactive oxygen species; cytokines

Progesterone (PG) affects muscle cells by genomic mechanisms through nuclear receptors and by nongenomic mechanisms through unidentified pathways. This study aimed to determine the pathways mediating its nongenomic actions. Experiments were performed in dissociated muscle cells from guinea pig colons. Nongenomic actions were defined as those occurring within 10 min of PG exposure. PG blocked the contraction to CCK-8 and NKA ([10.sup.-7] M) but did not impair ACh ([10.sup.-7] M) and KC1 (2.5 x [10.sup.-2] M)-induced contraction. Both CCK-8 and NKA contract muscle cells by releasing calcium from intracellular stores, whereas ACh and KC1 can utilize extracellular calcium. PG also blocked the contraction induced by inositol 1,4,5-trisphosphate, thapsigargin, and caffeine, agents that contract muscle cells by releasing calcium from storage sites. The nongenomic actions of PG were transient because they were absent 1 h after the first PG dose, remaining unresponsive after a second PG dose was administered. Furthermore, PG had no effect on the contraction induced by CCK-8 and thapsigargin in muscle cells from animals pretreated with daily intramuscular PG for 4 days. Cytosolic incorporation experiments of [[sup.3]H]PG showed that pretreatment with unlabeled PG significantly reduced the radiolabeled PG incorporation in the cytosol. We conclude that the nongenomic actions of PG on colonic muscle cells transiently blocked calcium release from storage sites, and this response became rapidly desensitized. This effect does not appear to be specific to PG because other steroid hormones such as aldosterone and testosterone can also induce it. calcium release; desenitization of progesterone; colonic circlular muscle cells

We have shown that IL-1[beta] 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 [H.sub.2][O.sub.2] levels in the circular muscle layer but not in mucosa. Exposure of the mucosa to HCl caused IL-1[beta] 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-1[beta] and IL-6 but not [H.sub.2][O.sub.2]. IL-1[beta] appears to remain in the mucosa. In contrast, IL-6 is produced and released by the mucosa, eventually resulting in the production of [H.sub.2][O.sub.2] by the circular muscle, with this affecting circular muscle contraction. esophageal mucosa; inflammation; smooth muscle contraction; reactive oxygen species; cytokines

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-1[beta] and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle (12), we examined the role of IL-1[beta] and IL-6 in this motor dysfunction. IL-1[beta], IL-6 (12), [H.sub.2][O.sub.2], PG[E.sub.2], and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1[beta] and IL-6 had increases in [H.sub.2][O.sub.2], PG[E.sub.2], and PAF levels. [H.sub.2][O.sub.2] contributed to increased PG[E.sub.2] and PAF, as the increase was partially (60-80%) reversed by the [H.sub.2][O.sub.2] scavenger catalase. EFS-induced [[.sup.3]H]ACh release from muscle strips significantly (42%) decreased in esophagitis and after 2 h incubation in PG[E.sub.2] and in PAF C-16. Similarly, EFS-induced but not ACh-induced muscle contraction decreased in esophagitis and after incubation in PG[E.sub.2] and PAF C-16. Finally, in normal muscle strips treated with IL-1[beta] 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-1[beta]-induced production of [H.sub.2][O.sub.2] causes formation of PG[E.sub.2] and PAF that inhibit ACh release from esophageal cholinergic neurons without affecting ACh-induced contraction of esophageal circular muscle. IL-6 causes production of [H.sub.2][O.sub.2], PAF, and other unidentified inflammatory mediators. inflammation; smooth muscle contraction; reactive oxygen species

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular [Ca.sup.2+] stores. We therefore measured cytosolic [Ca.sup.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.sub.2][O.sub.2] to the reduction in [Ca.sup.2+] signal. In normal cells, the ACh-induced [Ca.sup.2+] increase was the same in normal-[Ca.sup.2+] and [Ca.sup.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.sup.2+] release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced [Ca.sup.2+] increase in normal-[Ca.sup.2+] medium was significantly lower in esophagitis than in normal cells and was further reduced (~70%) when the cells were incubated in [Ca.sup.2+]-free medium. This reduction was partially reversed by the [H.sub.2][O.sub.2] scavenger catalase. [H.sub.2][O.sub.2] measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with [H.sub.2][O.sub.2], the ACh-induced [Ca.sup.2+] increase was significantly reduced in normal[Ca.sup.2+] and [Ca.sup.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.sub.2][O.sub.2] [H.sub.2][O.sub.2], when applied to cells at sufficiently high concentration, produced a visible and prolonged [Ca.sup.2+] signal in normal cells. [H.sub.2][O.sub.2]-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, [H.sub.2][O.sub.2] reduced TG-induced contraction, suggesting that TG and [H.sub.2][O.sub.2] may operate through similar mechanisms. [Ca.sup.2+]ATPase activity measurement indicates that [H.sub.2][O.sub.2] and TG reduced [Ca.sup.2+]-ATPase activity, confirming similarity of mechanism of action. We conclude that [H.sub.2][O.sub.2] may be at least partly responsible for impairment of [Ca.sup.2+] release in acute experimental esophagitis by inhibiting [Ca.sup.2+] uptake and refilling [Ca.sup.2+] stores. acetylcholine; smooth muscle

Myogenic lower esophageal sphincter (LES) tone is maintained by arachidonic acid metabolites, such as [PGF.sub.2[alpha]] and thromboxane [A.sub.2]/[B.sub.2]. Experimental esophagitis in cat reduces LES in vivo pressure and in vitro tone. Because IL-1[beta] may mediate esophagitis-associated reduction in ACh release in esophagus, we examined whether IL-1[beta] 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-1[beta] levels were higher in esophagitis than in normal LES. IL-1[beta] reduced normal LES tone, and the reduction was reversed by catalase, suggesting a role of [H.sub.2][O.sub.2]. This was confirmed by IL-1[beta]-induced production of [H.sub.2][O.sub.2] in normal LES and elevated [H.sub.2][O.sub.2] levels in esophagitis. [H.sub.2][O.sub.2] by itself is sufficient to explain the changes that occur in the muscle, reducing its ability to contract. [H.sub.2][O.sub.2] increased [PGE.sub.2] in normal LES, and [PGE.sub.2] levels were elevated in esophagitis LES, whereas [PGF.sub.2[alpha]], levels were unchanged. [H.sub.2][O.sub.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.sub.2[alpha]] analog 8-iso-[PGF.sub.2[alpha]] caused little contraction of LES strips but reduced [PGF.sub.2[alpha]] binding and contraction of normal LES. In esophagitis, [PGF.sub.2[alpha]] 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-1[beta] causes production of [H.sub.2][O.sub.2]. [H.sub.2][O.sub.2] increases [PGE.sub.2], which relaxes the LES, and 8-iso-[F.sub.2[alpha]], which blocks [PGF.sub.2[alpha]]-mediated contraction. smooth muscle; contraction; tone; signal transduction; hydrogen peroxide; cytokines; lower esophageal sphincter

Cholinergic mechanisms are largely responsible tbr 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 peffusion 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 proinltammatory cytokines IL-1[beta] (100 U/ml), IL-6 (1 ng/ml), or TNF-[alpha] (1 ng/ml). IL-I[beta] and IL-6 levels, measured by Western blot analysis, increased in esophagitis compared with normal circular muscle. IL-1[beta] 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-1[beta] 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-I[beta] and IL-6 contribute to reduced esophageal contraction by inhibiting release of ACh from myenteric neurons. smooth muscle; signal transduction; hydrogen peroxide

Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high [Ca.sup.2+] concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic [Ca.sup.2+] levels. The link between activation of the [Ca.sup.2+]-independent protein kinase C[epsilon] (PKC[epsilon]) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, CaD W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281:G467-G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKC[epsilon]-dependent, [Ca.sup.2+]-independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKC[epsilon], but not antibodies to PKC[beta]II, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKC[epsilon] from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKC[epsilon], MEK, ERK1/2, and ILK. protein kinase C; myosin light chain kinase; phosphatase; integrinlinked kinase

[H.sub.2][O.sub.2] and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PG[E.sub.2] and damage functions of membrane proteins except for PG[E.sub.2] receptors. The aim of this study was to examine whether the preserved PG[E.sub.2] actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PG[E.sub.2] prevented the inhibition of [H.sub.2][O.sub.2] or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by [H.sub.2][O.sub.2] and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PG[E.sub.2] pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of [H.sub.2][O.sub.2] and TCDC on agonists-induced contraction, including that of PG[E.sub.2]. Pretreatment with PG[E.sub.2] before [H.sub.2][O.sub.2] and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PG[E.sub.2]. We conclude that pretreatment with PG[E.sub.2] prevents the muscle cell damage caused by [H.sub.2][O.sub.2] and TCDC due to the resistance of PG[E.sub.2] receptors to agonist-induced desensitization. The preservation of PG[E.sub.2] receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane. reactive oxygen species; free radical scavengers; bile acids; receptor desensitization

[H.sub.2][O.sub.2] stimulates gallbladder muscle contraction and scavengers of free radicals through the generation of PG[E.sub.2]. Oxidative stress causes lipid peroxidation and generation of platelet-activating factor (PAF) or PAF-like lipids. The present studies therefore were aimed at determining whether either one induced by [H.sub.2][O.sub.2] mediates the increased generation of PG[E.sub.2]. Dissociated muscle cells of guinea pig gallbladder were obtained by enzymatic digestion. Both PAF-like lipids and PAF-induced muscle contraction was blocked by the PAF receptor antagonist CV-3988. This antagonist also blocked the increased PG[E.sub.2] production caused by PAF-like lipids or PAF. Actions of PAF-like lipids were completely inhibited by indomethacin, but those of PAF were only partially reduced by indomethacin or by nordihydroguaiaretic acid and completely blocked by their combination. PAF-like lipids-induced contraction was inhibited by AACOC[F.sub.3] (cystolic phospholipase [A.sub.2] inhibitor), whereas the actions of PAF were blocked by M[J.sub.33] (secretory phospholipase [A.sub.2] inhibitor). Receptor protection studies showed that pretreatment with PAF-like lipids before N-ethylmaleimide protected the contraction induced by a second dose of PAF-like lipids or PG[E.sub.2] but not by PAF. In contrast, pretreatment with PAF protected the actions of PAF and PG[E.sub.2] but not that of PAF-like lipids. Both PAF-like lipids and PAF-induced contractions were inhibited by anti-G[[alpha].sub.q/11] antibody and by inhibitors of MAPK and PKC. In conclusion, PAF-like lipids seem to activate a pathway different from that of PAF probably by stimulating a different PAF receptor subtype. oxidative stress; PG[E.sub.2] production; receptor protection

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone, p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-1inked p38 MAPK. second messenger system; signal transduction; mitogen-activated protein kinase; protein kinase C; lower esophageal sphincter

Lower esophageal sphincter (LES) tone depends on PG[F.sub.2[alpha]] and thromboxane [A.sub.2] acting on receptors linked to [G.sub.i3] and [G.sub.q] to activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PG[F.sub.2[alpha]] signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PG[F.sub.2[alpha]]-induced contraction was inhibited by antibodies against the [alpha]-subunit of [G.sub.13] and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [[sup.35]S]GTP[gamma]S-binding assay confirmed that [G.sub.13], RhoA, and ARF1 were activated by PG[F.sub.2[alpha]]. Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PG[F.sub.2[alpha]]-induced contraction was inhibited by antibodies against the [alpha]-subunit of [G.sub.q] and [G.sub.i3], and a [[sup.35]S]GTP[gamma]/S-binding assay confirmed that [G.sub.q] and [G.sub.i3] were activated by PG[F.sub.2[alpha]]. PG[F.sub.2[alpha]]-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PG[F.sub.2[alpha]] concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PG[F.sub.2[alpha]] activates a PLD- and protein kinase C (PKC)-dependent pathway through [G.sub.13], RhoA, and ARF1. In LES, PG[F.sub.2[alpha]] receptors are coupled to [G.sub.q] and [G.sub.13], activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PG[F.sub.2[alpha]] activates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway. smooth muscle contraction; prostaglandins; G proteins; phospholipases

Effects of bile acids on the muscle functions of guinea pig gallbladder. Am J Physiol Gastrointest Liver Physiol 283: G87-G94, 2002. First published March 28, 2002; 10. l152/ajpgi.00536.2001.--Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused [H.sub.2][O.sub.2] production, lipid peroxidation, and increased PG[E.sub.2] synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase [A.sub.2] (cPL[A.sub.2]), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of [H.sub.2][O.sub.2] via activation of NADPH and xanthine oxidase. [H.sub.2][O.sub.2] causes lipid peroxidation and activates cPL[A.sub.2] to increase PG[E.sub.2] production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway. [H.sub.2][O.sub.2] production; oxidase inhibitors; smooth muscle

Reactive oxygen species ([H.sub.2][O.sub.2]): effects on the gallbladder muscle of guinea pigs. Am J Physiol Gastrointest Liver Physiol 282: G300-G306, 2002. First published October 24, 2001; 10.1152/ajpgi.00241.2001.--Reactive oxygen species (ROS) have been implicated in the pathogenesis of muscle dysfunction in acute inflammatory processes. The aim of these studies was to determine the effects of ROS on gallbladder muscle function in vitro. Single muscle cells were obtained by enzymatic digestion. [H.sub.2][O.sub.2] (70 [micro]M) caused maximal contraction of up to 14% and blocked the response to CCK-8, ACh, and KCl. It did not affect the contractions induced by guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and inositol 1,4,5-trisphosphate that circumvent membrane receptors. The contraction induced by [H.sub.2][O.sub.2] was inhibited by AACOC[F.sub.3] [cytosolic phospholipase [A.sub.2] (cPL[A.sub.2]) inhibitor], indomethacin (cyclooxygenase inhibitor), chelerythrine [protein kinase C (PKC) inhibitor], or PD98059 [mitogen-activated protein kinase (MAPK) inhibitor]. [H.sub.2][O.sub.2] also reduced the CCK receptor binding capacity from 0.36 [+ or -] 0.05 pmol/mg protein (controls) to 0.17 [+ or -] 0.03 pmol/mg protein. The level of lipid peroxidation as well as the PG[E.sub.2] content was significantly increased after [H.sub.2][O.sub.2] pretreatment. Unlike superoxide dismutase, the free radical scavenger catalase prevented the [H.sub.2][O.sub.2] induced contraction, and its inhibition of the CCK-8 induced contraction. It is concluded that ROS cause damage to the plasma membrane of the gallbladder muscle and contraction through the generation of PG[E.sub.2] induced by cPL[A.sub.2]-cyclooxygenase and probably mediated by the PKC-MAPK pathway. lipid peroxidation; prostaglandin [E.sub.2] content; smooth muscle

Human gallbladders with cholesterol stones show impaired muscle contraction caused by agonists that act on transmembrane receptors, increased membrane cholesterol content and abnormal cholesterol-to-phospholipid ratio compared to those with pigment stones. The functions of CCK receptor of gallbladder membranes were studied by radioreceptor assay and cross-linking. Results showed that human gallbladders with cholesterol stones have impaired CCK receptor binding compared to those with pigment stones.

The mechanisms underlying antral and colonic hypomotility in guinea pigs during pregnancy have been investigated to test whether pregnancy has an inhibitory effect on the motility of the gastrointestinal tract. Circular smooth muscle cells from the left colon and antrum were synthesized by enzymatic digestion with collagenase from pregnant and nonpregnant guinea pigs. Results show that the contraction of antral and colonic circular muscle from pregnant guinea pigs was inhibited in response to cholecystokinin-8 and to ((super 35)S)guanosine 5'-O-(3-thiotriphosphate) but was normal in response to KCl and D-myo-inositol 1,4,5-triphosphate compared with nonpregnant subjects.

The signal transduction that controls CCK-induced contraction of gallbladder muscle was examined in the cat. The contraction induced by CCK was inhibited phospholipase C (PLC) inhibitor U-73122, anti-PLC-beta3 antibody and the D-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin. However, it was not inhibited by the phospholipase D inhibitor propanolol or antibodies against PLC-beta1 or PLC-beta2. Findings indicated that CCK contracts cat gallbladder muscle by stimulating pertussin toxin-sensitive G13 protein coupled wiht PLC-beta3, producing IP3 and sn-1,2-diacylglycerol.

Gallbladder smooth muscle cells from progesterone-treated guinea pigs exhibited impaired contraction with cholecystokinin octapeptide, guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride. An analysis of the types of G-binding protein and an assessment of the G binding properties of crude membranes revealed the presence of a number of G-binding proteins and the impaired activity of the Gi3 protein. It is postulated that progesterone produces hypomotility in gallbladder smooth muscle cells by downregulating this G protein.

The role of inositol 1,4,5-triphosphate (IP3) on the muscular tone and contraction of the lower esophageal sphincter (LES) was analyzed during experimental esophagitis. Analysis of esophageal contraction during IP3-induced release of Ca2+ indicated the direct effects of IP3 on the mobilization of Ca2+. Alterations in the signal transduction of LES smooth muscles were also characterized by reductions in intracellular Ca2+ stores.

The mechanisms that mediate impaired muscle response to cholecystokinin (CCK) were analyzed in prairie dog and human gallbladders with cholesterol stones. Analysis of gallbladder muscle cells isolated from prairie dogs and humans with cholelithiasis indicated the presence of impaired muscle contraction in response to CCK administration. The CCK-induced contraction was also dependent on protein kinase C activity. Furthermore, altered CCK-response in gallbladder muscles cells during cholelithiasis was induced by defective membrane receptors and receptor-G protein coupling.

The effect of a high-cholesterol diet on cholesterol incorporation in plasma membranes of gallbladder muscle and on gallbladder muscle contractility were investigated using prairie dogs. A high-cholesterol diet increased cholesterol content in the plasma membranes by 90% and decreased muscle contraction by 58%. Similar results were obtained when plasma membranes were incubated with cholesterol-rich liposomes for 2 hr. However, these effects were reversed when the membranes were incubated with cholesterol-free liposomes for 2-6 hrs, suggesting that a high-cholesterol diet increases cholesterol incorporation but decreases muscle contractility.

A study was conducted to examine the disorders in the signal transduction pathways that cause muscle contraction in human gallbladders in which cholesterol stones or cholesterol crystals have developed. Single cells isolated from gallbladder muscle were treated with inositol 1,4,5-triphosphate and diacylglycerol. Cholecystokinin stimulation of the muscle revealed decreased muscle contraction. Two G protein activators, aluminum fluoride and guanosine 5'-O-(3-thiotriphosphate), has been shown to have the same effect, suggesting that these activators reverses the effects of impaired signal transduction pathways.

Ion exchange chromatography, immunoprecipitation and western blot analysis of Ach-initiated circular muscle contraction in the esophageal muscle of cat show that 100-kDa phospholipase A2 generates arachidonic acid to participate in the muscular contraction process of esophageal and not low esophageal sphincter cells. Arachidonic acid reacts with the PKC-dependent pathway that enhances the esophageal muscle contraction. PKC inhibitor H-7 suppresses DAG-induced muscle contraction while calmodulin inhibitor and indomethacin have no such inhibitory effects on DAG-induced contraction.

An analysis of pathways involved in the formation of cat gall bladder tone, using samples of smooth muscle, suggested a role for the synergistically activated secretion of diacylglycerol (DAG) and inositol-1, 4,5-trisphosphate (IP3)-calcium in directing gall bladder tone formation. Contraction of gall bladder tone is associated with the activation of protein kinase C, mediated by IP3-DAG interaction. H-7 inhibited the IP3-induced contraction at low doses of IP3 and failed to affect high doses of IP3 which was inactivated by active calmodulin.

Muscle strips from experimental acute cholecystitis (AC) exhibit a defective contraction. The mechanisms responsible for this impaired contraction are not known. The present studies investigated the nature of these abnormalities. AC was induced by ligating the common bile duct of guinea pigs for 3 days. Contraction was studied in enzymatic dissociated muscle cells. Cholecystokinin (CCK) and prostaglandin E2 ([PGE.sub.2]) receptor binding studies were performed by radio-receptor assay. The levels of lipid peroxidation, cholesterol, phospholipid, and [H.sub.2][O.sub.2] as well as the catalase and superoxide dismutase (SOD) activities were determined. [PGE.sub.2] content was measured by radioimmunoassay. Muscle contraction induced by CCK, ACh, or KCl was significantly reduced in AC, but [PGE.sub.2]-induced contraction remained normal. GTP[Gamma]S, diacyglycerol (DAG), and 1,4,5-trisphosphate ([IP.sub.3]), which bypass the plasma membrane, caused a normal contraction in AC. The number of functional receptors for CCK was significantly decreased, whereas those for [PGE.sub.2] remained unchanged in AC. There was a reduction in the phospholipid content and increase in the level of lipid peroxidation as well as [H.sub.2][O.sub.2] content in the plasma membrane in AC. The [PGE.sub.2] content and the activities of catalase and SOD were also elevated. These data suggest that AC cause damage to the constituents of the plasma membrane of muscle cells. The preservation of the [PGE.sub.2] receptors may be the result of muscle cytoprotection. lipid peroxidation; free radical scavengers; cytoprotection

Xiao, Zuo-Liang, Qian Chen, Joseph Amaral, Piero Biancani, and Jose Behar. Defect of receptor-G protein coupling in human gallbladder with cholesterol stones. Am. J. Physiol. Gastrointest. Liver Physiol. 278: G251-G258, 2000.--Human gallbladders with cholesterol stones (ChS) exhibit an impaired muscle contraction and relaxation and a lower CCK receptor-binding capacity compared with those with pigment; stones (PS). This study was designed to determine whether there is an abnormal receptor-G protein coupling in human gallbladders with ChS using [sup.35]S-labeled guanosine 5'-O-(3-thiotriphosphate) ([[sup.35]S]GTP [Gamma] S) binding, [sup.125]I-labeled CCK-8 autoradiography, immunoblotting, and G protein quantitation. CCK and vasoactive intestinal peptide caused significant increases in ([[sup.35]S]GTP [Gamma] S binding to [G [Alpha].sub.i-3] and [G.sub.s] [Alpha], respectively. The binding was lower in ChS than in PS (P [is less than] 0.01). The reduced [[sup.35]S]GTP [Gamma] S binding in ChS was normalized after the muscles were treated with cholesterol-free, liposomes (P [is less than] 0.01). Autoradiography and immunoblots showed a decreased optical density (OD) for CCK receptors, an even lower OD value for receptor-G protein coupling, and a higher OD for uncoupled receptors or G [[Alpha].sub.i-3] protein in ChS compared with PS (P [is less than] 0.001). G protein quantitation also showed that there were no significant differences in the [G [Alpha].sub.i-3] and [G.sub.s] [Alpha] content in ChS and PS. We conclude that, in addition to an impaired CCK receptor-binding capacity, there is a defect in receptor-G protein coupling in muscle cells from gallbladder with ChS. These changes may be normalized after removal of excess cholesterol from the plasma membrane. immunoblotting; [sup.35]S-labeled guanosine 5'-O-(3-thiotriphosphate) binding; autoradiography; G protein quantitation; smooth muscle.