A phorbol ester-nonproliferative variant of Swiss 3T3 cells is deficient in Na+K+Cl- cotransport activity.
The identity of the genetic defect(s) in Swiss 3T3 TNR-2 and TNR-9 that confers nonresponsiveness to the proliferative effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide-sensitive Na+K+Cl- cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA-nonresponsive lines TNR-2 and TNR-9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide- and TPA-inhibitable component of efflux was clearly evident in parental and TNR-9 cells but was virtually absent in TNR-2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR-9 line of a substantial furosemide-sensitive flux that could be inhibited by TPA. In contrast, much less furosemide-sensitive influx was present in 3T3-TNR-2 cells and it was relatively unaffected by TPA. In both parental 3T3 and 3T3-TNR-2 cells, most of the furosemide-sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl-. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide-sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 approximately equal to 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume-regulatory role, inhibition of Na+K+Cl- cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR-2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3-TNR-2 cells (but not TNR-9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+K+Cl- cotransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.
O'Brien
T G
TG
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Prettyman
R
R
George
K S
KS
Herschman
H R
HR
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D005665:Furosemide / Q000494:pharmacology; D014644:Genetic Variation; D051379:Mice; D008815:Mice, Inbred Strains; D009994:Osmolar Concentration; D010703:Phorbol Esters / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002352:Carrier Proteins; D002712:Chlorides; D010703:Phorbol Esters; D028021:Sodium-Potassium-Chloride Symporters; D005665:Furosemide; D012964:Sodium; D012413:Rubidium; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium