Kinetic analysis about the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) by isolated rat hepatocytes.

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a Km of 29.1 +/- 3.2 microM and Vmax of 2.9 +/- 0.1 mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was 2.2 +/- 0.1 microL/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at 4 degrees C, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The Vmax and Km values were 0.54 mmol/min/mg protein, and 10.0 microM, respectively. Based on the comparison of the ratios of Vmax to Km (Vmax/Km) corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.

Extensive hepatic uptake of Pz-peptide, a hydrophilic proline-containing pentapeptide, into isolated hepatocytes compared with colonocytes and Caco-2 cells.

The objective of the present study was to investigate the uptake process of 4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide), a hydrophilic and collagenase-labile pentapeptide, by isolated hepatocytes. For comparison, the uptake of Pz-peptide by Caco-2 cells and colonic cells, two known paracellular mutes of Pz-peptide, was also evaluated. A simple and sensitive reversed-phase HPLC assay method using UV detection has been developed. The coefficient of variation for all the criteria of validation were less than 15%. The method was, therefore, considered to be sutable for measuring the concentration of Pz-peptide in the biological cells. Pz-peptide was extensively uptaked into hepatocytes. The initial velocity of Pz-peptide uptake assessed from the initial slope of the curve was plotted as Eadie-Hofstee plots. The maximum velocity (Vmax) and the Michaelis constant (Km) were 0.190+/-0.020 nmol/min/10(6) cells and 12.1+/-3.23 microM, respectively. The permeability-surface area product (PS(influx)) was calculated to be 0.0157 ml/min/10(6) cells. Vmax and Km values for Caco-2 cells were calculated to be 6.22+/-0.930 pmol/min/10(6) cells and 82.8+/-8.37 microM, respectively, being comparable with those of colonocytes (6.04+/-1.03 pmol/min/10(6) cells and 87.8+/-13.2 microM, respectively). PS(influx) values for Caco-2 cells and colonocytes were calculated to be 0.0751 microl/min/10(6) cells and 0.0688 microl/min/10(6) cells, respectively. The more pronounced uptake of Pz-peptide by hepatocytes, when compared with Caco-2 cells and colonocytes, is probably due to its specific transporter. In conclusion, Pz-peptide, a paracellularly transported pentapeptide in the intestine and ocular epithelia, was uptaked into hepatocytes extensively. Although Pz-peptide is able to be uptaked into the Caco-2 cells and colonocytes, it is less pronounced when compared with hepatocytes. PS(influx) values of Caco-2 cells and colonocytes for unbound Pz-peptide under linear conditions were less than 0.4% when compared with that of hepatocytes.