Regulation of the Na(+)-dependent and the Na(+)-independent polyamine transporters in renal epithelial cells (LLC-PK1).
We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.
Parys
J B
JB
Laboratorium voor Fysiologie, Campus Gasthuisberg, Leuven, Belgium.
De Smedt
H
H
Van Den Bosch
L
L
Geuns
J
J
Borghgraef
R
R
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D000518:Eflornithine / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D007668:Kidney / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D008935:Mitoguazone / Q000494:pharmacology; D011073:Polyamines / Q000378:metabolism / Q000493:pharmacokinetics*; D011700:Putrescine / Q000493:pharmacokinetics / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D012964:Sodium / Q000502:physiology*; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013552:Swine
D002352:Carrier Proteins; D011073:Polyamines; D003609:Dactinomycin; D012710:Serum Albumin, Bovine; D013096:Spermine; D003513:Cycloheximide; D012964:Sodium; D008935:Mitoguazone; D013095:Spermidine; D011700:Putrescine; D000518:Eflornithine