Bile duct ligation induced acute inflammation up-regulates cyclooxygenase-2 content and PGE2 release in guinea pig gallbladder smooth muscle cell cultures.

INTRODUCTION: This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE2 release by up-regulation of COX-2. METHODS: BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto-PGF(1alpha), PGE2 and TxB2 by EIA. RESULTS: BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE2 and 6-keto-PGF(1alpha) by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. CONCLUSIONS: BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.

Effect of indomethacin on gallbladder inflammation and contractility during acute cholecystitis.

OBJECTIVE: The aim of this study was to determine whether the prostaglandin synthase inhibitor indomethacin reverses the inflammation and abnormal gallbladder contractility that occur after common bile duct ligation (CBDL), a model of acute cholecystitis. METHODS: Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Animals were treated with saline or indomethacin in vivo. Acetylcholine (ACh) was used to directly contract the muscle and electric field stimulation (EFS) to activate intrinsic nerves. Hematoxylin and eosin-stained slides of muscle strips were scored for inflammation. RESULTS: CBDL in saline-treated animals increased the inflammation score and decreased gallbladder muscle contractility to ACh and EFS. Indomethacin decreased the inflammation score and partly reversed the smooth muscle contractile response to ACh 6 and 24 h after CBDL, but not at 48 h. Indomethacin did not reverse the CBDL-induced decrease in nerve-evoked contractions. CONCLUSION: Gallbladder inflammation and contractile dysfunction after CBDL are partly reversed with indomethacin at 6 and 24 h, but not at 48 h. This suggests that, early in the course of CBDL, the inflammation and contractile dysfunction are, in part, prostaglandin-mediated.

Effect of acalculous cholecystitis on gallbladder neuromuscular transmission and contractility.

BACKGROUND: Impaired smooth muscle contractility is important in the pathophysiology of acalculous cholecystitis. Common bile duct ligation (CBDL) is a model of acalculous cholecystitis, producing acute inflammatory changes and decrease in gallbladder smooth muscle contractility. The aim of this study was to determine whether there is coexistent dysfunction of neural efferent motor pathways of the gallbladder after CBDL. MATERIALS AND METHODS: Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Electric field stimulation (EFS; 2-16 Hz) was used to activate intrinsic nerves and exogenous acetylcholine (ACh) was used to directly stimulate the muscle. H&E-stained slides of muscle strips were scored for inflammatory changes. RESULTS: After CBDL, there was a progressive increase in the inflammation score and decrease in gallbladder muscle contractility to ACh. There was also a progressive decline in EFS-induced contractility when expressed as absolute force or normalized to the maximal muscle contractile response to ACh. The nitric oxide synthase inhibitor l-NNA (10 microM) increased EFS-induced contractions by 50 +/- 25% (P = 0.05) in CBDL animals but had no effect in sham surgical controls. CONCLUSIONS: CBDL with its acute gallbladder inflammation affects gallbladder contractility by two mechanisms: (1) decreased smooth muscle contractility, and (2) decreased neurally mediated contractions. The neurally mediated alterations result from dysfunction of cholinergic excitatory nerves and upregulation of nitric-oxide-mediated inhibition of smooth muscle contractility.

Effect of experimental acalculous cholecystitis on gallbladder smooth muscle contractility.

Gallbladder motility is impaired in chronic cholelithiasis but has not been studied in acute acalculous cholecystitis. The aim of this study was to determine the effects of acute acalculous inflammation on gallbladder contractility using the common bile duct ligation (CBDL) model in guinea pigs. Three groups of guinea pigs were studied: CBDL, normal, and sham surgical controls. Gallbladder dimensions were measured, and muscle strips were used for histology and in vitro contractility studies. CBDL resulted in progressive gallbladder distension, increased serum bilirubin, and gallbladder inflammation. There was a progressive decline in muscle contractility in the CBDL group as evidenced by a decrease in the contractile response to potassium and bethanechol with the duration of CBDL. In conclusion, CBDL in the guinea pig produces acute gallbladder inflammation and decreased gallbladder muscle contractility. Direct inhibition of muscle function is indicated by impaired contractile responses to potassium depolarization and bethanechol stimulation. Although the mechanism of the decrease in contractility with CBDL is unknown, we speculate that impaired muscle contractility is secondary to inflammation and may play a role in the clinicopathology of acute acalculous cholecystitis.

Enhanced bradykinin-stimulated prostaglandin release in the acutely inflamed guinea pig gallbladder is due to new synthesis of cyclooxygenase 1 and prostacyclin synthase.

BACKGROUND: Our previous studies have shown that acute gallbladder (GB) inflammation increases endogenous bradykinin (BK)-stimulated prostaglandin (PG) release and inhibits guinea pig (GP) GB contractility. This study examines the hypothesis that exaggerated PG release following BK stimulation in the inflamed guinea pig GB is due to new protein synthesis of cyclooxygenase 1 (COX-1) and prostacyclin synthase (PS). MATERIALS AND METHODS: Male Hartley GPs (450-550 g) were anesthetized and underwent common bile duct ligation (BDL, a model of acute inflammation). GBs were harvested after 3 days from BDL and control groups. Tissue slices were prepared and placed in oxygenated tissue culture medium at 37 degrees C for 1 h (basal) and for a second hour in medium alone (carrier, Car), medium plus 10(-6) M BK, or medium plus 10(-6) M BK plus cycloheximide 100 microgram/ml (BK + CX). The medium was assayed for net release of 6-keto-PGF1alpha (PGI2 metabolite), thromboxane B2 (TxB2), PGE2, leukotriene B4 (LTB4), and C4 (LTC4) by enzyme immunoassay and data are reported as nanograms per milligram of protein. GB tissue from control and BDL groups was examined for COX-1, COX-2, PS, and inducible nitric oxide synthase (iNOS) content by Western blot analysis, analyzed by densitometry, and reported as densitometry units. RESULTS: All data were analyzed by ANOVA and t test and reported as means +/- SEM, N >/= 5.BK increased the release of PGI2 and PGE2 from the control group and markedly exaggerated release of PGI2 and PGE2 from the BDL GP gallbladder. This exaggerated PGI2 and PGE2 release was greatly diminished by inhibition of new protein synthesis with cycloheximide. TxB2, LTB4, and LTC4 showed no significant differences between any groups. COX-1 and PS contents were significantly elevated in the BDL group compared with control. COX-2 and iNOS were not present in control or BDL GBs. CONCLUSIONS: These data suggest that the enhanced BK-stimulated PG release seen in the acutely inflamed GP gallbladder is due to the synthesis of new COX-1 and PS enzymes.

Cocaine-induced alterations in prostaglandin production in rabbit aorta.

To determine if alterations in endothelial prostaglandin production occur after long-term cocaine use, 26 New Zealand White rabbits were randomized to a low fat diet with (n = 12) or without (n = 14) daily intravenous cocaine (2 mg/kg body weight). Rabbits were killed at 6 or 12 weeks. Segments of aorta were examined in blinded manner for histologic changes. Additional slices were incubated in oxygenated Krebs buffer and release of 6-keto-prostaglandin F1 alpha, thromboxane B2 and prostaglandin E2 was assayed by radioimmunoassay. Minimal intimal histologic changes were seen in the aorta of three cocaine-treated rabbits. At 12 weeks 6-keto-prostaglandin F1 alpha was increased in the cocaine group (p = 0.063) as compared with levels in the control group. When rabbits killed at 6 and 12 weeks were considered together, increases in thromboxane B2 (p = 0.044) and a trend to increased prostaglandin E2 (p = 0.083) were seen in the cocaine group. The ratio of thromboxane B2 to 6-keto-prostaglandin F1 alpha was increased in the cocaine group compared with that in the control group (p less than 0.02). These data suggest that an increase in prostaglandin production occurs in the vascular endothelium of rabbits ingesting cocaine before gross histologic changes are evident. In addition, thromboxane B2 increases disproportionately with respect to 6-keto-prostaglandin F1 alpha, suggesting that a milieu for thrombosis may exist in users of cocaine.

Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis.

This study evaluated the effects of 25% ethanol, a mild irritant, on endogenous prostanoid synthesis in the rat stomach before and after exposure to oral 100% ethanol. Rats received water or 25% ethanol orally. After 15 min, a portion of each group was sacrificed and the remaining animals treated with 100% ethanol prior to sacrifice one minute later. Microsomal membrane fractions were prepared from the glandular gastric mucosa in all groups and incubated with 14C arachidonic acid in the presence of cofactors. Endogenous mucosal prostanoid synthesis was analyzed by radiochromatography and results correlated with the presence or absence of gastric injury macroscopically. Prostanoids measured included PGI2, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane A2. Additional experiments were performed in like manner to those just described with the exception that indomethacin (5 mg/kg intraperitoneally) pretreatment was rendered. Stomachs exposed to water or 25% ethanol alone demonstrated a modest and equivalent level of synthesis of all prostanoids measured. Exposure to 100% ethanol (with and without mild irritant pretreatment) significantly increased prostanoid synthesis (especially PGI2, PGF2 alpha, and PGE2) compared with stomachs exposed to water or 25% ethanol alone; only mild irritant treated mucosa was protected from injury by 100% ethanol. Indomethacin pretreatment reversed the increased prostanoid synthesis in mucosa exposed to 100% ethanol, with or without mild irritant pretreatment, and partially reversed the protective effect of 25% ethanol. Other experiments using tissue slices in which perturbations in mucosal levels of prostanoids were measured by radioimmunoassay under identical experimental conditions exhibited similar results. These data dispute the notion that adaptive cytoprotection is mediated by increased endogenous prostanoid synthesis. The partial reversal of this process by indomethacin was most likely secondary to some other action of this agent, such as a reduction in gastric blood flow, rather than direct effects on prostanoid synthesis.

Sex differences in gallbladder prostaglandin synthesis mediated by acute inflammation.

The influences of sex and acute inflammation on prostaglandin biosynthesis in rabbit gallbladder were examined by radiochromatography. Male rabbit gallbladder microsomes converted small amounts of labelled arachidonate to total prostaglandin synthesis with PGE2, 6-keto PGF1 alpha (stable metabolite of PGI2) and PGF2 alpha as the major products synthesized. Microsomes from the male rabbit gallbladder inflamed by bile duct ligation for 3 days increased total prostaglandin synthesis five-fold with 6-keto PGF1 alpha being the major prostaglandin produced. Female rabbit gallbladder microsomes converted three times more arachidonate to total prostaglandin synthesis than did microsomes from the male rabbit. Bile duct ligation did not alter total prostaglandin biosynthesis in the female rabbit gallbladder, but significantly decreased synthesis of PGE2, thromboxane B2 and PGF2 alpha and increased synthesis of 6-keto PGF1 alpha. These data suggest that although bile duct ligation had different effects on male and female gallbladder total prostaglandin synthesis, 6-keto PGF1 alpha is the major product induced by this stimulus for acute inflammation.

Common bile ligation in the rabbit: an appropriate model for investigating the relationship of endogenous gallbladder prostanoid synthesis with evolving acute inflammation.

Gallbladder prostanoid (PG) synthesis and histologic inflammatory changes were compared after 6, 24, and 72 hours of bile duct ligation (BDL) or cystic duct ligation (CDL) in the male rabbit. At each time interval the gallbladder was scored for degree of acute inflammation, examined by radiochromatography for endogenous PG synthesis and analyzed by ANOVA. BDL induced progressive increases in acute inflammation whereas prostanoid synthesis significantly increased only after the 6 and 72 hour groups. Indomethacin treatment inhibited PG synthesis in all BDL groups but only decreased the inflammation score in the 6 and 24 hour BDL groups. CDL did not induce progressive gallbladder inflammatory changes or prostanoid synthesis. These data show that prostanoids are intimately involved with the development of early acute gallbladder inflammation following BDL. Inhibition of PG synthesis could attenuate or retard the progression of early acute gallbladder inflammation if started prior to development of established disease.