Fluorescent derivatives of bile salts. III. Uptake of 7 beta-NBD-NCT into isolated hepatocytes by the transport systems for cholyltaurine.
Uptake of 7 beta-NBD-NCT ([N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) in isolated rat liver hepatocytes occurs by saturable transport without being superimposed by simple diffusion. The dependency of flux rate of uptake on the concentration of 7 beta-NBD-NCT in the presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems. Maximal flux rates of uptake Jn and half-saturation constants KT for 7 beta-NBD-NCT are in presence of Na+ for transport system 1 J1(Na+ 143) = 0.15 +/- 0.03 nmol/(min.mg protein) and KT1(Na+ 143) = 3.5 +/- 0.5 microM and for transport system 2 J2(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein) and KT2(Na+ 143) = 190 +/- 25 microM, and in case of Na+ depletion J1(Na+ 1) = 0.1 +/- 0.03 nmol/(min.mg protein), KT1(Na+ 1) = 3.0 +/- 0.5 microM, and J2(Na+ 1) = 0.85 +/- 0.9 nmol/(min.mg protein) and KT2(Na+ 1) = 195 +/- 27 microM. Uptake of 7 beta-NBD-NCT by both transport systems is competitively inhibited by cholyltaurine in the presence of Na+ and with Na+ depletion. Two transport systems are likewise involved in the uptake of cholyltaurine in the presence of Na+ as well as in case of Na+ depletion. Their kinetic parameters are in presence of Na+ J'1(Na+ 143) = 1.55 +/- 0.14 nmol/(min.mg protein) and K'T1(Na+ 143) = 16.1 +/- 3.0 microM, and J'2(Na+ 143) = 0.51 +/- 0.05 nmol/(min.mg protein) and K'T2(Na+ 143) = 38.0 +/- 4.1 microM, and in case of Na+ depletion J'1(Na+ 1) = 0.10 +/- 0.02 nmol/(min.mg protein), K'T1(Na+ 1) = 7.7 +/- 1.2 microM, and J'2(Na+ 1) = 0.40 +/- 0.03 nmol/(min.mg protein) and K'T2(Na+ 1) = 41.0 +/- 4.2 microM. Uptake of cholyltaurine by both transport systems is competitively inhibited by 7 beta-NBD-NCT in the presence of Na+ as well as in case of Na+ depletion. In both cases the inhibition constants are practically identical with the KT values for uptake of 7 beta-NBD-NCT. Photoaffinity labeling of isolated hepatocytes using 7,7-ACT (400 microM) resulted in the irreversible inhibition of uptake of both bile salts to similar extents, confirming the kinetic data that 7 beta-NBD-NCT is a true analogue of cholyltaurine.
Schramm
U
U
Institut für Organische Chemie und Biochemie, Universität Freiburg, Germany.
Fricker
G
G
Buscher
H P
HP
Gerok
W
W
Kutz
G
G
D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D001647:Bile Acids and Salts / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D004058:Diffusion; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010069:Oxadiazoles / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013656:Taurocholic Acid / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D001391:Azo Compounds; D001647:Bile Acids and Salts; D005456:Fluorescent Dyes; D010069:Oxadiazoles; C079985:(N-(7-(nitrobenz-2-oxa-1,3-diazol-4-yl))-7-amino-3alpha,12alpha-dihydroxycholan-24-oyl)-2-aminoethanesulfonate; D013656:Taurocholic Acid; C037641:(7,7-azo-3,12-dihydroxy-5-cholan-24-oyl)-2-aminoethanesulfonic acid