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.

Downstream effects of splanchnic ischemia-reperfusion injury on renal function and eicosanoid release.

This study examines the hypothesis that intestinal ischemia-reperfusion (I/R) injury contributes to renal dysfunction by altered renal eicosanoid release. Anesthetized Sprague-Dawley rats underwent 60 min of sham or superior mesenteric artery (SMA) occlusion with 60 min of reperfusion. The I/R groups received either allopurinol, pentoxifylline, 1-benzylimidazole, or carrier before SMA occlusion. In vivo renal artery blood flow was measured by Transonic flow probes, the kidneys were then perfused in vitro for 30 min, and the effluent was analyzed for eicosanoid release and renal function. Intestinal I/R caused a twofold increase in the ratio of renal release of thromboxane B2 to prostaglandin E2 and to 6-ketoprostaglandin F1alpha compared with the sham level, with a corresponding 25% decrease in renal sodium and inulin clearance and renal blood flow. Pentoxifylline or allopurinol pretreatment restored renal eicosanoid release and renal sodium and inulin clearance to the sham level but did not alter renal blood flow. Pretreatment with 1-benzylimidazole restored renal function, eicosanoid release, and renal blood flow to sham levels. These data suggest that severe intestinal I/R contributes to the downregulation of renal function. The decrease in renal function is due in part to toxic oxygen metabolites, which occur in the milieu of altered renal eicosanoid release, reflecting a decrease in vasodilator and an increase in vasoconstrictor eicosanoids.

Endotoxic shock after long-term resuscitation of hemorrhage/reperfusion injury decreased splanchnic blood flow and eicosanoid release.

OBJECTIVE: The authors examine the hypothesis that hemorrhage/reperfusion injury predisposes the splanchnic bed to decreased prostacyclin (PGl2) release and blood flow after subsequent endotoxin challenge. SUMMARY BACKGROUND DATA: Prostacyclin is a potent vasodilator that has been demonstrated to be an important regulator of splanchnic blood flow. Previous studies have demonstrated that during resuscitation from severe hemorrhage, there is a marked reduction in intestinal PGl2 levels, which is associated with reduced splanchnic perfusion. METHODS: Anesthetized Sprague-Dawley rats underwent hemorrhage to a mean arterial pressure of 30 mmHg for 30 minutes followed by the reinfusion of shed blood. Then the animals were maintained on total parenteral nutrition (TPN) for 10 days, after which time they received 20 mg/kg Escherichia coli endotoxin intraperitoneally. Aortic and superior mesenteric artery (SMA) blood flow was monitored with a Doppler flow probe. The splanchnic bed was excised and perfused in vitro for measurement of venous effluent eicosanoid concentrations. Controls consisted of animals that received TPN and endotoxin but did not undergo hemorrhage and resuscitation (sham). RESULTS: Total parenteral nutrition support of sham animals followed by endotoxin challenge did not alter splanchnic eicosanoid release or blood flow. Hemorrhage/reperfusion animals supported by long-term TPN and challenged with endotoxin demonstrated a threefold decrease in splanchnic prostacyclin metabolite (6-keto-PGF1 alpha) release and a 50% decrease in SMA blood flow. CONCLUSIONS: Hemorrhage/reperfusion injury predisposes the splanchnic bed from rats sustained with long-term TPN to decreased release of PGl2 and SMA blood flow when challenged with endotoxin as a second injury.

Estrogen increases male rat aortic endothelial cell (RAEC) PGI2 release.

Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI2. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4-10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of beta-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF1 alpha (6-keto, PGI2 metabolite), PGE2 and thromboxane B2 (TXB2) by EIA (hormone stimulated-basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF1 alpha release, whereas estrogen increased RAEC 6-keto-PGF1 alpha release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI2 release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI2.

Regulation of eicosanoid synthesis in fibroblasts from inflamed gallbladders.

Gallbladder cell cultures obtained from rabbits subjected to sham or 72 h of bile duct ligation (72 h BDL, cholecystitis model) were incubated with calcium ionophore (A23187), dibutyryl cAMP (cAMP), and phorbol 12,13-diacetate (phorbol) to determine the intracellular signal transduction mechanisms responsible for increased inflamed gallbladder eicosanoid synthesis. Incubation of sham and 72 h BDL cell cultures with A23187 or phorbol significantly increased, whereas cAMP decreased, release of 6-keto-PGF1 alpha, PGE2, thromboxane B2 (measured by enzyme immunoassay) in a dose-related manner. Seventy-two-hour BDL cell cultures contained a specific 2-fold increased level of prostacyclin synthase compared to sham cell cultures which was not altered by preincubation with A23187, phorbol or cAMP. These findings suggest that increased PGI2 release in the sham and inflamed cell cultures following A23187 and phorbol stimulation was mediated in part via the inositol triphosphate pathway and protein kinase C activation and was not associated with altered cyclooxygenase or prostacyclin synthase content.

Intestinal reperfusion up-regulates inducible nitric oxide synthase activity within the lung.

BACKGROUND: This study examines the hypothesis that pulmonary inducible nitric oxide synthase (iNOS) activity is up-regulated during intestinal reperfusion and that inhibition of NO generation exacerbates pulmonary microvascular dysfunction. METHODS: Sprague-Dawley rats underwent intestinal ischemia and reperfusion (IIR) or sham operation (SHAM). Pulmonary iNOS activity was measured by quantitating the conversion of L-arginine (L-Arg) to L-citrulline. Another set of animals undergoing IIR or SHAM received an inhibitor of NOS (NG-nitro-L-arginine methylester; L-NAME; 20 mg/kg intravenously), substrate for NO generation (L-Arg; 300 mg/kg intravenously), or vehicle (normal saline solution; 3 ml). Pulmonary microvascular dysfunction was then quantitated by measuring the extravasation of Evans blue dye (EBD) into the lung. RESULTS: Inducible NOS activity was six times greater in the lungs of animals sustaining IIR when compared with SHAM (p = 0.0005). The concentration of EBD within the lungs of animals sustaining IIR was 30% greater than SHAM (p < 0.05). Inhibiting NOS with L-NAME significantly increased pulmonary EBD concentration of both IIR and SHAM groups when compared with normal saline solution-treated animals (p < 0.0001). Treatment with L-Arg prevented this IIR-induced increase in pulmonary dye extravasation. CONCLUSIONS: These data suggest that pulmonary iNOS activity is up-regulated in animals sustaining IIR and that this may serve as a compensatory protective response to remote organ injury.

Neutrophil regulation of splanchnic blood flow after hemorrhagic shock.

OBJECTIVE: This study examines the hypothesis that neutrophils impair splanchnic blood flow during resuscitation from hemorrhage by inhibiting the release of the compensatory vasodilator PGI2 from the bowel. SUMMARY BACKGROUND DATA: Resuscitation from hemorrhagic shock is associated with neutrophil infiltration into the intestine, reduced splanchnic perfusion, and reduced release of PGI2 from the intestine. METHODS: Sprague-Dawley rats received either vinblastine (VIN) to deplete circulating neutrophils or normal saline (NS). These animals then underwent either hemorrhage and resuscitation (SK + R) or sham operation (SHAM). Superior mesenteric artery flow and splanchnic 6-keto PGF1a (metabolite of PGI2) release were measured. RESULTS: Superior mesenteric artery blood flow was significantly greater in VIN-treated animals sustaining SK + R than in those treated with NS (p < 0.05). Neutrophil depletion preserved 6-keto PGF1a release after SK + R, whereas 6-keto PGF1a release in the NS-treated, SK + R group was significantly reduced (p < 0.05). CONCLUSION: These data are compatible with the hypothesis that neutrophils may influence splanchnic perfusion after SK + R by inhibiting splanchnic PGI2 release.

Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures.

This study examines the hypothesis that PAF stimulates release of PGI2 from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF1 alpha from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF1 alpha was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF1 alpha release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI2 release.

Resuscitation of ischemia/reperfusion with hyperalimentation increases intestinal PGI2 synthase (PS) content and PGI2 release.

Normal rat splanchnic blood flow and eicosanoid synthesis were compared to sham (Sham) and hemorrhage/reperfusion (SK + R) animals treated with 5 days of total parenteral nutrition (TPN). After in vivo measurement of superior mesenteric artery (SMA) blood flow, the splanchnic bed was perfused in vitro and venous effluent assayed for eicosanoid release by EIA. Aortic, SMA, ileal muscularis/serosa, and mucosa homogenates were analyzed for cyclooxygenase (COX) and prostacyclin synthase (PS) content by Western blot and for COX activity by radiochromatography. SK + R + TPN decreased SMA blood flow 33% and increased splanchnic PGI2 release twofold (p < .05) compared with the Sham +TPN and normal groups. groups SK + R + TPN did not alter COX activity in any of the tissues but Western blot analysis showed a twofold increase in COX and PS content in ileum muscularis/serosa (p < .05). These data show that SK + R + TPN induced a decrease in SMA blood flow and a compensatory increase in release of splanchnic PGI2. The mechanism for increased splanchnic PGI2 release after SK + R + TPN was an increase in PS and COX content (not activity) in the ileal muscularis/serosa.

Pulmonary thromboxane release following intestinal reperfusion.

Microvascular dysfunction is a prominent feature of the lung injury associated with intestinal reperfusion (IR). This study examines the hypothesis that IR induces pulmonary thromboxane A2 (TxA2) release, which contributes to pulmonary microvascular dysfunction. Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 min of reperfusion (IR). Sham-operated animals served as controls (SHAM). Following IR or SHAM, the lungs were perfused in vitro with a modified Krebs buffer and ventilated with room air. Eicosanoid levels within the pulmonary venous effluent and bronchoalveolar lavage (BAL) fluid were determined (TxB2, 6-keto-PGF1a, and PGE2). Pulmonary artery pressure (PAP) was measured continuously and expressed as change from baseline in mm Hg. The dominant eicosanoid generated by the lungs in response to IR was TxB2. TxB2 levels in the pulmonary venous effluent of IR lungs were 75% greater than controls (P = 0.005). Similarly, TxB2 levels in the BAL were more than 2.5 times controls (P = 0.001). The change in PAP of lungs from IR animals was significantly greater than that of controls (4.1 +/- 1.5 vs 0.3 +/- 0.54 mm Hg, IR vs SHAM, P = 0.01). The increased PAP associated with IR lungs was prevented by cyclooxygenase inhibition with indomethacin (-1.28 +/- 0.29 mm Hg, P < 0.05) and thromboxane synthetase inhibition with imidazole (-1.75 +/- 0.95 mm Hg, P < 0.05). These experiments support the hypothesis that IR up-regulates endogenous pulmonary TxA2 release. Furthermore, the local release of TxA2 by the lung may contribute to the microvascular dysfunction characteristic of IR-induced lung injury.

Splanchnic PGI2 release and "no reflow" following intestinal reperfusion.

This study examines the hypothesis that reduced splanchnic blood flow during intestinal reperfusion (IR) is associated with impaired release of the vasodilatory prostanoid PGI2. Sprague-Dawley rats underwent occlusion of the superior mesenteric artery (SMA) for 120 min and reperfusion for up to 60 min. SMA blood flow was measured by transonic flow probe and radiolabeled microspheres (141Ce and 103Ru). Sham-operated animals served as controls (SHAM). Splanchnic eicosanoid release was quantitated by measuring thromboxane B2 (TxB2, stable metabolite of TxA2), 6-keto-PGF1a (6-keto, stable metabolite of PGI2), and PGE2 within the portal vein (PV) and inferior vena cava (IVC) of animals sustaining IR and SHAM. SMA flow in IR animals was < 10% of baseline and 27% of SHAM when measured by transonic flow probe (8 +/- 2% and 29 +/- 3%, IR and SHAM, respectively, P < 0.05). Similar results were obtained when intestinal blood flow was measured with microspheres (0.33 +/- 0.12 vs 1.34 +/- 0.13 ml/min/g, IR vs SHAM, P < 0.05). The greatest change in IR-induced splanchnic eicosanoid release occurred with 6-keto. Following ischemia, 6-keto levels in the PV were twice those of SHAM (P < 0.05). Five minutes after reperfusion, PV 6-keto levels were 22 times those of controls (P < 0.05) and 4 times greater than those of the IVC (P < 0.05). By 60 min of reperfusion, levels of 6-keto were reduced to those in the IVC. These data support the hypothesis that splanchnic blood flow is critically reduced by severe IR.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurodeoxycholic acid stimulates rabbit gallbladder eicosanoid release.

Rabbit common bile duct ligation has been shown to concomitantly increase levels of gallbladder taurodeoxycholic acid and gallbladder eicosanoid release. This study examines the hypothesis that taurodeoxycholic acid, a known chemical mediator of gallbladder inflammation, stimulates endogenous gallbladder eicosanoid release. Male New Zealand white rabbits were anesthetized, gallbladders removed and perfused in vitro with Krebs-Henseleit buffer (pH 7.4, 37 degrees C) at 1 ml/min with increasing doses of taurodeoxycholic acid (0, 10, 30 and 100 mM) added to the perfusate. The effluent was collected at 15, 30, 60 and 120 min of perfusion and assayed for 6-keto-PGF1 alpha (PGI2 metabolite), PGE2, and thromboxane B2 (TXB2) by enzyme immunoassay. Taurodeoxycholic acid increased gallbladder eicosanoid release in a dose-related manner with 6-keto-PGF1 alpha and PGE2 release 10-fold higher than TXB2. Indomethacin (1.5 mM) decreased gallbladder eicosanoid release by 50% in the gallbladders perfused with 30 mM taurodeoxycholic acid, demonstrating that the increased gallbladder eicosanoid release was due to de novo synthesis. These findings suggest that the increased release of gallbladder PGI2 and PGE2 described in animal models of cholecystitis may, in part, be related to increased gallbladder bile levels of taurodeoxycholic acid.

Increased splanchnic prostacyclin synthase and cyclooxygenase content and activity during ischemia is due to new protein synthesis.

BACKGROUND: This study examines the hypothesis that the exaggerated splanchnic release of prostacyclin is due to new synthesis of both cyclooxygenase and prostacyclin synthase (PS) in the ileum muscularis/serosa. METHODS: Sprague-Dawley rats were anesthetized and subjected to acute hemorrhage to 30 mm Hg for 30 minutes (shock) or sham shock. The superior mesenteric artery (SMA) was cannulated and removed with its end-organ intestine and perfused in vitro with Krebs-Henseleit buffer with and without cycloheximide (50 micrograms/ml) or indomethacin (20 micrograms/ml). Venous effluent was analyzed for eicosanoids by radioimmunoassay. The SMA, aorta and ileal mucosa, and muscularis/serosa were analyzed for PS and cyclooxygenase content by immunoblot analysis. RESULTS: The sham splanchnic bed released threefold more 6-keto-PGF1 alpha than prostaglandin E2 and thromboxane. Acute ischemia increased splanchnic release of 6-keto-PGF1 alpha threefold compared with sham, which was abolished by cycloheximide or indomethacin treatment. Acute ischemia increased content of PS and cyclooxygenase in the ileal muscularis/serosa twofold and PS in the aorta and SMA by 50%. CONCLUSIONS: Acute ischemia increased release of 6-keto-PGF1 alpha, which was dependent on new protein synthesis. The immunoblot data suggest that the location of the increased enzymes responsible for increased 6-keto-PGF1 alpha release is the ileal muscularis/serosa and in the aorta and SMA.

Increased intragallbladder pressure stimulates gallbladder eicosanoid release.

The stimulus for increased gallbladder eicosanoid synthesis during cholecystitis is unknown. This study examines the hypothesis that increased intragallbladder pressure stimulates endogenous gallbladder eicosanoid release. Rabbit gallbladders were perfused in vitro at 1 ml/minute with oxygenated Krebs-Henseleit buffer and subjected to 0, 12 or 24 mm Hg of intraluminal gallbladder pressure. Release of 6-keto-PGF1 alpha infinity PGE2 and thromboxane B2 were measured in all groups after 15 and 30 and 60 minutes of perfusion by enzyme immunoassay and gallbladders were examined histologically. Increasing intraluminal gallbladder pressure concomitantly increased gallbladder 6-keto-PGF1 alpha release 2 fold or more at all time of perfusion and altered gallbladder mucosal architecture by increasing basolateral edema in the submucosal space. Infusion of indomethacin (10 micrograms/ml in the perfusion media) decreased 6-keto-PGF1 alpha release from the in vitro perfused gallbladders subjected to 24 mm Hg by 70%. Increased gallbladder eicosanoid release during early cholecystitis may in part be related to the physical force of increased gallbladder intraluminal pressure on the gallbladder mucosa.

Acute cholecystitis potentiates bradykinin stimulated fibroblast prostanoid release in the rabbit.

Gallbladder explants from control rabbits and rabbits subjected to bile duct ligation (BDL) for 24 and 72 h (cholecystitis model) were placed in cell culture to determine the source for increased gallbladder prostanoid synthesis during cholecystitis. Cultures from control and 24 h BDL gallbladders grew spindle shaped fibroblasts which did not exhibit increased prostanoid synthesis. 72 h BDL gallbladder cell cultures grew large polygonal shaped cells which appeared to be 'stimulated fibroblasts' by light and electron microscopy and were associated with increased basal and bradykinin stimulated 6-keto-PGF1 alpha release and increased content of prostacyclin synthase when measured by enzyme immunoassay and protein immunoblot analysis respectively. Use of bradykinin antagonists showed that the bradykinin BK2 subtype receptor was the most prominent in the 72 h BDL cell cultures. The 'stimulated fibroblasts' were the source of bradykinin stimulated gallbladder 6-keto-PGF1 alpha synthesis in the inflamed rabbit gallbladder which was mediated by the bradykinin B2 subtype receptor.

Long-term hyperalimentation following hemorrhage/reperfusion injury induces intestinal prostanoid synthesis.

Intestinal mucosa and muscularis/serosa prostaglandin (PG) biosynthesis was compared in sham control female rats and rats subjected to mild hemorrhage (hemorrhage to 80 mm Hg for 60 min), blood reperfusion, and maintenance on hyperalimentation (TPN) for 5 days. Tissue PG synthesis was analyzed by radiochromatographic analysis of microsomal membrane fractions prepared from mucosa and muscularis/serosa removed from the duodenum, jejunum, and ileum. Individual PG synthesis in the mucosa and muscularis/serosa sham group was modest, with low levels of synthesis of 6-keto-PGF1 alpha, PGE2, PGF2 alpha, and thromboxane B2. Hemorrhage, reperfusion, and maintenance on TPN for 5 days did not alter PG synthesis in the mucosa, whereas muscularis/serosa synthesis of 6-keto-PGF1 alpha and PGE2 was increased five-fold or more in each tissue studied. These data suggested that the muscularis/serosa could serve as a major source for the elevated PG, previously shown to occur following long-term resuscitation after acute hemorrhage.