Manabu Hanano

Comparison of the Hepatic Uptake Clearances of Fifteen Drugs with a Wide Range of Membrane Permeabilities in Isolated Rat Hepatocytes and Perfused Rat Livers

The hepatic uptake clearances of 15 ligands with a wide range of permeabilities were determined in rats using two techniques: centrifugal filtration with isolated hepatocytes and the multiple indicator dilution (MID) method with isolated perfused livers. Some of the uptake clearance values were taken from the literature. Uptake clearance values obtained from isolated hepatocytes were extrapolated to that per gram liver (PSinf, cell), assuming that 1 g of liver has 1.3 × 108 cells. The values of PSinf, cell varied from approximately 0.1 to 72 (mL/min/g liver). The values of PSinf, cell were similar to those (PSinf,MID) determined by the MID method for ligands with uptake clearances below approximately 1 mL/min/g liver. However, for the ligands with larger uptake clearances, the PSinf, MID values were lower than the PSinf,cell values and appeared to reach an upper limit (approx. 15–20 mL/min/g liver). The PSinf,cell values of 1-propranolol, tetraphenylphosphonium (TPP+), and diazepam were 72, 43, and 22 mL/min/g liver, respectively, whereas their uptake clearances (PSinf,MID) determined by the MID method were 4 to 10 times lower. One of the possible mechanisms for this discrepancy is that an unstirred water layer, which may exist in Disses space in isolated perfused livers (and probably under in vivo condition), limits the hepatic uptake rate of ligands with extremely high membrane permeabilities.

Correlation between the inhibitory effects of basic drugs on the uptake of cardiac glycosides and taurocholate by isolated rat hepatocytes

The role of the multispecific bile acid transporter for cardiac glycoside uptake is still controversial. This study was designed to examine the inhibitory effects of basic drugs (verapamil, dipyridamole, nifedipine, chlorpromazine, disopyramide, quinidine, propranolol, and lidocaine) on taurocholate uptake by isolated rat hepatocytes and to compare these effects with inhibition of ouabain uptake. Sodium-dependent taurocholate uptake was significantly reduced, to 50-70% of the control value, by 50 µM verapamil, dipyridamole, and nifedipine. Sodium-independent taurocholate uptake was more extensively inhibited, to 20-40%, by these basic drugs. The inhibition of ouabain uptake correlated better with sodium-independent taurocholate uptake (γ = 0.918) than with sodium-dependent taurocholate uptake (γ = 0.714). Taurocholate competitively inhibited ouabain uptake in the absence of sodium. These results indicate that the cardiac glycoside transport system is similar to the sodium-independent taurocholate transport system.

Kinetic Analysis of Transcytosis of Epidermal Growth Factor in Madin-Darby Canine Kidney Epithelial Cells

AbstractPurpose. The aim of this study is to clarify the intracellular fate and a rate limiting step in transcytosis of epidermal growth factor (EGF) in Madin-Darby Canine Kidney (MDCK) epithelial cells.nMethods. The kinetics of transcytosis of 125I-EGF was investigated. To examine the fate of EGF molecules bound to its receptor on the cell surface, 125I-EGF was prebound to the basal surface at 4°C, followed by extensive washing and subsequent incubation at 37°C in EGF-free medium.nResults. Saturable transport of 125I-EGF through the cell monolayer could only be observed from the basal to apical side. Most (~90%) of the EGF molecules bound to the surface receptor are internalized with a half-life of 1−3 min, followed by intracellular degradation with a half-life of 20−50 min. The exocytosis of internalized EGF into the apical medium is much slower with a half-life of 130−250 min. Even when 125I-EGF was incubated with MDCK cells at 37°C and washed with acid to remove cell-surface 125I-EGF, intact 125I-EGF appeared in the basal medium with a half life of 160−170 min.nConclusions. The exocytosis of internalized EGF into the apical medium is a rate limiting step in EGF transcytosis. At least a small amount of internalized EGF is recycled.