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

Abstract

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.