Regulation of the Na+-dependent and the Na+-independent polyamine transporters in renal epithelial cells (LLC-PK1)

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Abstract
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 < 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‐in‐duced 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+‐deperdent 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.