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A phorbol ester‐nonproliferative variant of Swiss 3T3 cells is deficient in Na + K + Cl − cotransport activity
Author(s) -
O'Brien Thomas G.,
Prettyman Ralph,
George Kenneth S.,
Herschman Harvey R.
Publication year - 1988
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041340219
Subject(s) - cotransporter , 3t3 cells , cell culture , membrane transport , microbiology and biotechnology , symporter , ion transporter , extracellular , furosemide , ouabain , dids , efflux , chemistry , intracellular , biology , biophysics , biochemistry , transporter , endocrinology , sodium , membrane , transfection , genetics , organic chemistry , gene
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 86 Rb + 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. 86 Rb + 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 86 Rb + 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 86 Rb + influx was quite sensitive to TPA (K 1/2 ≅ 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 by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of contransport 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 − contransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.