Membrane transport mechanisms for amphipathic cations in the liver

THE EFFECT OF HOMOLOGOUS EXTRACELLULAR MATRIX (ECM) ON THE VIABILITY~ MORPHOLOGY AND SOMEFUNCTIONAL ASPECTS DF ADULT HUMAN HEPATDCYTES IN PRIMARY CULTURES. 231 H.3. Moshage, P.J.M. R i j n t j e s , J.F.G.M. Meis*, 3.C.M. Hafkenscheid° , S.H. Yap. Oiv. of G.I. and Liver diseases, *Dept. of parasitology and °Dept. of Clinical Chemistry, St. Radboud Univ. Hospital, Nijmegen, The Netherlands. Cultured human hepatocytes (H.H) provide an useful model for specific metabolic studies of the liver in man. Under classical culture conditions, adult H.H exhibit rapid phenotypic changes and die. By coculturing H.H with rat epithelial cells, prolonged maintainance of cell functions and survival have been observed. Since epithelial cells from non-human origin were used, it has been subjected for criticism in studying specific metabolic functions of H.H. In this study, we report the effect of ECM on the survival, morphology and some functional aspects of adult H.H in primary cultures. H.H were isolated using collagenase ) to 9 hrs after tissue removal. The cell yield was ranging from 4 to 37x10 o with a viability of 75 to 92% (trypan blue exclusion). H.H were seeded (175x10)cells per cm 2) onto tissue culture plastic or plastic coated with ECM from human liver prepared according to the method of Rojkind et al (I). Fetal calf serum supplemented medium was used throughout. ECM increased the efficiency of cell seeding. H.H attached to substrata within 6 hrs and maintained their cuboidal shape and cell polarity. Electronmicroscopy showed typical fine structures with well developed desmosomes and structures similar to bile canaliculi. In cultures using ECM, these typical fine structures were maintained for at least 4 weeks (on plastic I-2 weeks) and the cell survival period was extended to at least ) months. These H.H retained the ability to secrete high levels of albumin for at least ) weeks (8~g/ pgDNA). Enhanced secretion of transaminases and lactate dehydrogenase at the2n°t0 )rdweek as observed under classical conditions were delayed. Conclusions: ECM increased the efficiency of cell seeding and prolonged the survival of H.H in primary cultures. They retained their typical fine structures and the ability to secrete high levels of albumin for at least ) weeks.(1): Rojkind et al. 3 Cell Biol (1980) 87: 255.

MEMBRANE TRANSPORT MECHANISMS FOR AMPNIPATHIC CATIONS IN THE LIVER M. MUller, W.E.M. Mol t , D.K.Fo Meijer #, W. Gerok §, G. Kurz, Institut fur Organische Chemie und Biochemie, Freiburg, FRG, tDepartment of Pharmacology and Pharmacotherapeutics, University of Groningen, The Netherlands, §Medizinische Klinik de Universitat, Freiburg, FRG. Many basic drugs contain both a positively charged tertiary or quaternary amine group as well as hydrophobic, aromatic, or heterocyclic ring structures and are therefore amphipathic cations. The hepatic membrane transport systems for such organic cations were determined by combined kinetic and photoaffinity labeling studies using N-alkyl-deoxyajmalinium model compounds which are monoquaternary cations under physiological conditions. N-(n-Propyl)-deoxyajmalinium exhibits a rapid (flow limited) initial clearance of 35 ml/min by the isolated perfused rat liver and is subsequently rapidly excreted into bile, partly as metabolites. Uptake was not inhibited by non-amphipathic organic cations such as tributyl-methylammonium and eth~ procainamide but in contrast strongly affected by the uncharged steroidal compound K-strophar thoside. Kinetic studies of uptake of N-(n-Propyl)-deoxyajmalinium in isolated hepatocytes r( vealed that two different transport systems are involved, the first with an apparent k m of about ! ~M and a Vma x of 0.15 nmole/min/mg of protein and the second with an apparent km of 80 ~M and a Vma x of 4.0 nmole/min/mg of protein. Uptake by both transport systems was competl tively inhibited by uncharged compounds such as K-strophanthoside and 5-8-cholestane-3~,7o, 12~-triol as well as the amphipathic cations quinidine and d-tubocurarine. Photoaffinity lab~ ling of a plasmamembrane subfraction enriched with sinusoidal surfaces and of isolated hepat¢ cytes with N-(4,4-azo-n-pentyl)-deoxy-3H ajmalinium disclosed two membrane polypeptides with apparent molecular weights of 54000 and 48000 as components of different transport systems. Photoaffinity labeling of isolated hepatocytes resulted not only in the labeling of these tw( polypeptides but also in a partially irreversible inhibition of the uptake by amphipathic cat ions and neutral compounds mentioned before. Both the differential photoaffinity labeling an~ kinetic studies indicate that amphipathic mono- and bisquaternary cations and uncharged stere dal compounds share a common carrier-mediated system for hepatic uptake. 232

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