Phorbol ester inhibits furosemide-sensitive potassium transport in BALB/c 3T3 preadipose cells.

Abstract

The tumor promoter phorbol 12-myristate 13-acetate (PMA) rapidly decreased the rate of "Rb+ uptake into BALB/c 3T3 preadipose cells. The component of total 86Rb+ influx affected by PMA is insensitive to ouabain but sensitive to the diuretic furosemide. Experiments designed to investigate the characteristics of the K+ transport system sensitive to PMA revealed that: (i) 86Rb+ uptake is highly dependent on external Na+, (ii) "Rb+ uptake is highly dependent on external ClP, (iii) 22Na+ uptake is dependent on external K+, and (iv) a major component of 86Rb+ efflux that is sensitive to PMA and furosemide is not dependent on extracellular KV. These features strongly implicate a Na+K+/Cl-cotransport system as the target of PMA and furosemide in these experiments. PMA caused a net intracellular accumulation of K+ within 15 min in these cells, presumably via its inhibitory effect on furosemide-sensitive K+ transport. Within 30 min after PMA treatment, the mean cell volume was significantly reduced in treated compared to control cells, with a maximum decrease of 21% attained at 4 hr after PMA. The significance of these findings for biologic changes induced by PMA is discussed.In addition to their role in multistage carcinogenesis, the phorbol ester tumor promoters, exemplified by phorbol 12-myristate 13-acetate (PMA), cause a number of biologic and functional changes in diverse cell types in culture (for reviews, see refs. 1 and 2). To date, however, no experimental results to explain any of the actions of tumor promoters in (11), and inhibition of cell differentiation (12). Of particular importance to this study is the observation that PMA is mitogenic in quiescent cultures of this cell line (12), as it is for other 3T3 cell lines (8, 9). In contrast to the earlier work of others (8,9) showing an increased rate of 'Rb+ uptake caused by PMA, we found in these cells that inhibition of 86Rb+ uptake was an early event after PMA treatment. The transport system inhibited by PMA in these cells is insensitive to ouabain and sensitive to the diuretic furosemide, and it apparently catalyzes the cotransport of Na+, K+, and Cl-.MATERIALS AND METHODS Cells. BALB/c 3T3 cells (clone A3IT) were obtained from Leila Diamond (Wistar Institute) and cultured as described (12). The medium used was Eagle Auto-Pow minimal essential medium (Flow Laboratories) supplemented with 10% fetal bovine serum. For flux and ion content experiments, cells were plated from stocks into 35-or 60-mm plastic Petri dishes and used when growing logarithmically or just after reaching confluence. Routinely, cultures were refed with fresh medium every 2-3 days and always on the day prior to experiments.Flux Experiments. The uptake of 'Rb+ was measured in cells maintained in complete culture medium at 37°C in a humidified atmosphere of 95% air/5% CO2. Cells in 35-mm dishes were washed with warm phosphate-buffered saline (unless otherwise stated) and uptake was started by the addition of 2 ml of fresh medium containing 10% fetal bovine serum (K+, 5.4 mM)...

Restoration of responsiveness to phorbol ester by reconstitution of a functional Na/K/Cl cotransporter in cotransporter-deficient BALB/c 3T3 cells

“…Clues to the answer to this important question may lie in the similarities of early cellular responses to two diverse stimuli: phorbol ester and osmotic stress, especially hyperosmolar stress. Both stimuli alter net ion fluxes [1,18], cause rapid cell shrinkage followed by subsequent recovery, and stimulate phosphorylation of several proteins [1,. In fact, Lytle and Forbush [22] have shown that the same shark NKCCl used in these studies is phosphorylated on two threonine residues by hyperosmolar conditions.…”

“…In previous studies, we have developed circumstantial evidence that a membrane transporter, the Na/ K/Cl cotransporter (NKCCI), is a critical signal-generating substrate for PKC in BALB/c 3T3 cells [1][2][3][4]. The activity of this protein, which transports Na+, K' , and C1-in a bidirectional, electroneutral, and diuretic-(i.e., bumetanide, furosemide) sensitive manner in many cell types, is rapidly inhibited by phorbol-ester treatment [1,5,6].…”

“…The activity of this protein, which transports Na+, K' , and C1-in a bidirectional, electroneutral, and diuretic-(i.e., bumetanide, furosemide) sensitive manner in many cell types, is rapidly inhibited by phorbol-ester treatment [1,5,6]. Because this system is involved in volume regulation, phorbol estertreated cells rapidly shrink [l].…”

Modulation of cell communication and carcinogenesis.

“…Intracellular content of Na+ and K+ is mainly regulated by the Na+/K+ ATPase, and in some cases the Na+/H+, antiport in cell membranes, whose activities have been shown to be activated by phorbol ester tumor promoters and growth factors such as platelet-derived growth factor [6,22,37,38,44,56,60]. There is evidence that Na+/K+ ATPase activation is linked to the enhancement of cell growth and of TPA-mediated transformation in C3HIOT1/2 cells, and that a tumor promotion inhibitor, retinoic acid, inhibits TPA-mediated activation of the Na+/K+ ATPase in C3H10T1/2 [3].…”

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

“…TPA and dther mitogens normally induce a rapid change in ion transport properties. One such TPA-modulatable system which is rapidly inhibited in 3T3 cells is the sodium-potassium-chloride transport system (14). The BALB/c 3T3 variant cell line A31T6E12A was isolated by seletting for a defect in this TPA-sensitive transport system (25).…”