Acta pharmacol. et toxicol. 1976, 39,449455.
From the Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland
Biliary Excretion of Ouabain in Isolated Perfused Rat Liver after Treatment with Microsomal Enzyme Inducers' BY
Kaisu Nevasaari, Birgitta Alakare and N. T. Kiirki (Received December 22, 1975; Accepted March 5, 1976)
Abstruct: The effects of pretreatment with spironolactone, phenobarbital and 3,4-benzpyrene on biliary excretion of ouabain was studied in isolated perfused rat liver system after a single dose of 3H-ouabain. Spironolactone pretreatment (100 mg/kg intraperitoneally for 4 days) changed the time course of the excretion, thus accelerating the transport of ouabain into the bile. Phenobarbital pretreatment (75 mglkg intraperitoneally for 4 days) enhanced bile flow and increased biliary excretion of ouabain only after 15 min. At longer time periods the increase in bile flow diluted the bile level of ouabain there being no difference in the amounts excreted into the bile between the treated and untreated groups. 3,4-benzpyrene pretreatment (20 mglkg intraperitoneally for 4 days) was without effect on biliary excretion of ouabain. The results suggest that spironolactone differs from phenobarbital in its enhancing effect on biliary excretion of ouabain, possibly through a specific effect on a n unknown hepatic transport mechanism. Key-words: Ouabain - biliary excretion - enzyme induction - rat.
Ouabain is known to be actively transported into the bile in rat liver (KUPFERBERG & SCHANKER 1968). It has been shown that biliary excretion in vivo increases after pretreatment with hepatic microsomal enzyme inducers, phenobarbital (phenemalum NFN) and spironolactone, but not with 3-methylcholanthrene (KLAASSEN1970 & 1974). Since ouabain is not appreciably metabolized by rat liver (Cox et al. 1959), the mechanism of the enhanced biliary excretion does not involve induction of microsomal drug metabolism. It was the aim of this study to elucidate the mechanism of increased biliary excretion of ouabain by comparing its excretion in the 1
Presented in part at the Sixth International Congress of Pharmacology, July 1975, Helsinki, Finland.
K. NEVASAARI, B. ALAKARE AND N. T. U R K I
450
isolated perfused liver system after pretreatment with three different microsoma1 enzyme inducers. Materials and Methods l’relrratmeni of animals. The livers were obtained from non-fasted male rats (Sprague-Dawley) weighing 190-270 g. They were given intraperitoneally once a day for 4 days, phenobarbital sodium (E. Merck AG, Darmstadt) 75 mg/kg dissolved in saline, spironolactone (Laake Oy, Finland) 100 mg/kg suspended in 0.5 5% methyl cellulose saline solution, or 3,4-benzpyrene (Fluka AG, Switzerland) 20 mg/kg in sesame oil. The volume of all injections was 1 mltkg, and the controls received the same amount of the vehicle. The last drug dose was given at least 24 hours before the removal of the liver. Perjusion medium and technique. The perfusion medium and the perfusion method were the same as described 1976). The volume of perfusion medium was 50 ml in all perbefore (NEVASAARI fusions, the pressure was 16-17 cm H,O and the temperature 37”. Drugs siiidied and geiieraf procediire of iize perfusion. Randomly tritiated ouabain, with an initial specific activity of 12-16 cilmmol, was purchased from Radiochemical Center, Amersham (England). After a 30 min. equilibration period 290 pg ouabain (ouabain octahydrate, Medica Oy, Finland), mixed with SH-ouabain to give a final specific activity of 0.4 mci/mmol, was administered into the perfusion medium. Thereafter the bile was collected at 15 min. periods for 45 min. The livers were weighed after the perfusion. Deteririinatioti of the radioactivity. Tritium level in the bile was determined with a liquid scintillation spectrometer (Packard Ind., model 3320). The efficiency of the spectrometer, quenching and the specific activities were determined by the usual standards. 50 PI aliquots of bile were added to 20 ml of scintillation fluid (Instagel, Packard Ind.) and counted for 5 minutes. Statistical treatnzttrt. Student’s t-test, applied to an unpaired comparison, was used to evaluate the significance of the difference between mean values, the criterion of which was taken at P 0.05. The values represent means of 6 perfusions.
From the Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland
Biliary Excretion of Ouabain in Isolated Perfused Rat Liver after Treatment with Microsomal Enzyme Inducers' BY
Kaisu Nevasaari, Birgitta Alakare and N. T. Kiirki (Received December 22, 1975; Accepted March 5, 1976)
Abstruct: The effects of pretreatment with spironolactone, phenobarbital and 3,4-benzpyrene on biliary excretion of ouabain was studied in isolated perfused rat liver system after a single dose of 3H-ouabain. Spironolactone pretreatment (100 mg/kg intraperitoneally for 4 days) changed the time course of the excretion, thus accelerating the transport of ouabain into the bile. Phenobarbital pretreatment (75 mglkg intraperitoneally for 4 days) enhanced bile flow and increased biliary excretion of ouabain only after 15 min. At longer time periods the increase in bile flow diluted the bile level of ouabain there being no difference in the amounts excreted into the bile between the treated and untreated groups. 3,4-benzpyrene pretreatment (20 mglkg intraperitoneally for 4 days) was without effect on biliary excretion of ouabain. The results suggest that spironolactone differs from phenobarbital in its enhancing effect on biliary excretion of ouabain, possibly through a specific effect on a n unknown hepatic transport mechanism. Key-words: Ouabain - biliary excretion - enzyme induction - rat.
Ouabain is known to be actively transported into the bile in rat liver (KUPFERBERG & SCHANKER 1968). It has been shown that biliary excretion in vivo increases after pretreatment with hepatic microsomal enzyme inducers, phenobarbital (phenemalum NFN) and spironolactone, but not with 3-methylcholanthrene (KLAASSEN1970 & 1974). Since ouabain is not appreciably metabolized by rat liver (Cox et al. 1959), the mechanism of the enhanced biliary excretion does not involve induction of microsomal drug metabolism. It was the aim of this study to elucidate the mechanism of increased biliary excretion of ouabain by comparing its excretion in the 1
Presented in part at the Sixth International Congress of Pharmacology, July 1975, Helsinki, Finland.
K. NEVASAARI, B. ALAKARE AND N. T. U R K I
450
isolated perfused liver system after pretreatment with three different microsoma1 enzyme inducers. Materials and Methods l’relrratmeni of animals. The livers were obtained from non-fasted male rats (Sprague-Dawley) weighing 190-270 g. They were given intraperitoneally once a day for 4 days, phenobarbital sodium (E. Merck AG, Darmstadt) 75 mg/kg dissolved in saline, spironolactone (Laake Oy, Finland) 100 mg/kg suspended in 0.5 5% methyl cellulose saline solution, or 3,4-benzpyrene (Fluka AG, Switzerland) 20 mg/kg in sesame oil. The volume of all injections was 1 mltkg, and the controls received the same amount of the vehicle. The last drug dose was given at least 24 hours before the removal of the liver. Perjusion medium and technique. The perfusion medium and the perfusion method were the same as described 1976). The volume of perfusion medium was 50 ml in all perbefore (NEVASAARI fusions, the pressure was 16-17 cm H,O and the temperature 37”. Drugs siiidied and geiieraf procediire of iize perfusion. Randomly tritiated ouabain, with an initial specific activity of 12-16 cilmmol, was purchased from Radiochemical Center, Amersham (England). After a 30 min. equilibration period 290 pg ouabain (ouabain octahydrate, Medica Oy, Finland), mixed with SH-ouabain to give a final specific activity of 0.4 mci/mmol, was administered into the perfusion medium. Thereafter the bile was collected at 15 min. periods for 45 min. The livers were weighed after the perfusion. Deteririinatioti of the radioactivity. Tritium level in the bile was determined with a liquid scintillation spectrometer (Packard Ind., model 3320). The efficiency of the spectrometer, quenching and the specific activities were determined by the usual standards. 50 PI aliquots of bile were added to 20 ml of scintillation fluid (Instagel, Packard Ind.) and counted for 5 minutes. Statistical treatnzttrt. Student’s t-test, applied to an unpaired comparison, was used to evaluate the significance of the difference between mean values, the criterion of which was taken at P 0.05. The values represent means of 6 perfusions.
