thromboxane-b2 and Cholecystitis

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.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Antibodies; Cells, Cultured; Cholecystitis; Cytochrome P-450 Enzyme System; Dinoprost; Dose-Response Relationship, Drug; Epoprostenol; Gallbladder; Immunoblotting; Indomethacin; Intramolecular Oxidoreductases; Isomerases; Platelet Activating Factor; Prostaglandin-Endoperoxide Synthases; Proteins; Rabbits; Thromboxane B2

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.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Bucladesine; Calcimycin; Cells, Cultured; Cholecystitis; Cytochrome P-450 Enzyme System; Dinoprostone; Eicosanoids; Enzyme Activation; Epoprostenol; Fibroblasts; Gallbladder; Intramolecular Oxidoreductases; Ionophores; Isomerases; Male; Phorbol Esters; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Rabbits; Signal Transduction; Thromboxane B2

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.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Cholecystitis; Dinoprostone; Gallbladder; Hydrostatic Pressure; Immunoenzyme Techniques; Indomethacin; Male; Perfusion; Rabbits; Thromboxane B2

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.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Bile Ducts; Bradykinin; Cells, Cultured; Cholecystitis; Cytochrome P-450 Enzyme System; Dinoprostone; Fibroblasts; Gallbladder; Intramolecular Oxidoreductases; Isomerases; Ligation; Male; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rabbits; Receptors, Bradykinin; Thromboxane B2