Polyamine transport systems in the LLC-PK1 renal epithelial established cell line.
LLC-PK1 cells were brought to a quiescent state by treatment with DL-2-difluoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamine synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous putrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependent and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vmax values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent Km = 4.7 +/- 0.7 microM) than the Na+-independent transporter (apparent Km = 29.8 +/- 3.5 microM). As a consequence, although the latter transporter had a higher Vmax, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the antileukemic agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+-dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK1 are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.
De Smedt
H
H
Laboratorium voor Fysiologie, Campus Gasthuisberg O/N, Leuven, Belgium.
Van den Bosch
L
L
Geuns
J
J
Borghgraef
R
R
D000818:Animals; D015317:Biogenic Polyamines / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D000518:Eflornithine; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D011700:Putrescine / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000502:physiology*; D013095:Spermidine / Q000378:metabolism; D013096:Spermine / Q000378:metabolism
D015317:Biogenic Polyamines; D013096:Spermine; D012964:Sodium; D013095:Spermidine; D011700:Putrescine; D000518:Eflornithine