Biphasic response of Na+-dependent amino acid transport to tumor promoting phorbol esters in cultured renal epithelial cells, LLC-PK1.

Confluent cultures of LLC-PK1 pig kidney epithelial cells respond to tumor promoting phorbol esters and certain analogs of diacylglycerol with a 2-3 fold increase in Na -dependent amino acid transport via the A-system transporter. 12-O-tetra-decanoyl phorbol 13-acetate (TPA) activates the protein phosphorylating enzyme protein kinase C. That the A-system transporter also responds vigorously to TPA suggests that the A-system may be regulated in relation to growth control. We find that the transport increase requires 45 to 90 min to develop after exposure to TPA. If cells are transferred to an amino acid serum-free medium prior to exposure to TPA an additional rapid transient response is seen during which the A-system transport increases by 1.8 to 3 times in 5 to 7 min before returning to near control levels by 20 min. After 30 min or more of continued exposure to TPA the transport rate begins to rise in a manner similar to that seen in cells which have not been pre-incubated in the amino acid-serum-free medium. The rapid transient phase of the transport response correlates with an increase in TPA-stimulated membrane protein phosphorylation which is sensitive to the protein kinase C inhibitor sphingosine. The return of the transport rate tomore » control levels can be delayed by inhibitors of phosphatase activity. The later phase of TPA-stimulated A-system activity is associated with an increase in labeling of membrane proteins with (35S)methionine and is sensitive to cycloherimide. 7 refs., 6 figs.« less