Extensive Hepatic Uptake of Pz-peptide, a Hydrophilic Proline-Containing Pentapeptide, into Isolated Hepatocytes Compared with Colonocytes and Caco-2 Cells

  • Shin, Tae-Ha (College of Pharmacy, Chungbuk National University) ;
  • Lee, Pung-Sok (College of Pharmacy, Chungbuk National University) ;
  • Kwon, Oh-Seung (Toxicology Lab., Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology) ;
  • Chung, Youn-Bok (College of Pharmacy, Chungbuk National University)
  • 발행 : 2003.01.01

초록

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 routes 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 ($V_{max}$) and the Michaelis constant ($K_m$) were 0.190$\pm$0.020 $nmol/min/10^6$ cells and 12.1$\pm$3.23 $\mu$M, respectively. The permeability-surface area product ($PS{influx}$) was calculated to be 0.0157 ml/min/10^6$ cells. $V_{max}$ and $K_m$ values for Caco-2 cells were calculated to be 6.22$\pm$0.930 pmol/min/10^6$ cells and 82.8$\pm$8.37 $\mu$M, respectively, being comparable with those of colonocytes (6.04$\pm$1.03 pmol/min/10^6$ cells and 87.8$\pm$13.2 $\mu$M, respectively). $PS_{influx}$ values for Caco-2 cells and colonocytes were calculated to be 0.0751 $\mu$l/min/10^6$ cells and 0.0688 $\mu$l/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.

키워드

참고문헌

  1. Baur, H., Kasperek, S. and Pfaff, E., Criteria of viability of isolated liver cells. Heppe-Seyler's Z. Physiol. Chem., 356, 827-838 (1975) https://doi.org/10.1515/bchm2.1975.356.s1.827
  2. Brown, S. S. and Spudich, J. A., Mechanism of action of cytochalasin: Evidence that it binds to actin filament end. J. Cell Biol., 46, 163-197 (1990)
  3. Chung, Y. B., Han, K., Nishiura, A. and Lee, V. H. L., Ocular absorption of Pz-peptide and its effect on the ocular systemic pharmacokinetics of topically applied drugs in the rabbit. Pharm. Res., 15, 1882-1887 (1998) https://doi.org/10.1023/A:1011914324720
  4. Gibson-D'Ambrosio, R. E., Samuel, M. and D'Ambrosio S. M., A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment. In Vitro Cellular & Developmental Biology, 22, 529-34 (1986) https://doi.org/10.1007/BF02621139
  5. Iga, T., Eaton, D. L. and Klaassen, C. D., Uptake of unconjugated bilirubin by isolated hepatocytes. Am. J. Hepatol., 236, C9-14 (1979)
  6. Kompella, U. B., Kim K. -J., and Lee, V. H. L., Active chloride transport in the pigmented rabbit conjunctiva. Curr. Eye Res., 12, 1041-1048 (1993) https://doi.org/10.3109/02713689309033501
  7. Lin, J. H., Sugiyama, Y., Awazu, S. and Hanano, M., Physiological Pharmacokinetics of ethoxybenzamide based on biochemical data obtained in vitro. J. Pharmacokinet. Biopharm., 10, 649-661 (1982) https://doi.org/10.1007/BF01062546
  8. Yamaoka, K., Tanigawara, Y., Nakagawa, Y. and Uno, T., A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobio-Dyn., 4, 879-885 (1981) https://doi.org/10.1248/bpb1978.4.879
  9. Yamazaki, M., Suzuki, H., Iga, T. and Hanano, M., Uptake of organic anions by isolated rat hepatocytes: a classification in terms of ATP-dependency. J. Hepatol., 14, 41-47 (1992) https://doi.org/10.1016/0168-8278(92)90129-D
  10. Yamazaki, M., Suzuki, H. and Hanano, M., $Na^+$-independent multispecific anion transporter mediates active transport of pravastatin into rat liver. Am. J. Phsiol., 264, G36-G44 (1993)
  11. Yen W.-C. and Lee, V. H. L., Paracellular transport of a proteolytically labile pentapeptide across the colonic and other intestinal segments of the albino rabbit: implications for peptide drug design. J. Contr. Rel., 28, 97-109 (1994) https://doi.org/10.1016/0168-3659(94)90157-0
  12. Yen W.-C. and Lee, V.H.L., Penetration enhancement effect of Pz-peptide, a paracelullarly transported peptide, in rabbit intestinal segments and Caco-2 cell monolayers. J. Contr. Rel., 36, 25-37 (1995) https://doi.org/10.1016/0168-3659(95)00055-D
  13. Yen W. -C. and Lee, V. H. L., Role of $Na^+$ in the asymmetric paracellular transport of 4-phenylazobenzyloxycarbonyl-LPro-L-Leu-Gly-L-Pro-D-Arg across rabbit colonic segments and Caco-2 cell monolayers. J. Pharmacol. Exp. Therap., 275, 114-119 (1995)