Rivka Panet

Overexpression of the Na+/K+/Cl− cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Na+/K+/Cl− cotransporter activity is stimulated in early G1 phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na+/K+/Cl− cotransporter in cell proliferation, we overexpressed the gene encoding the Na+/K+/Cl− cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na+/K+/Cl− cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c‐NKCC1 cells). The transfected cells exhibited up to 10‐fold greater bumetanide‐sensitive Rb+ influx compared to the control cells. The Balb/c‐NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na+/K+/Cl− cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c‐NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin‐resistant clones, but rather with the overexpression of the Na+/K+/Cl− cotransporter gene. In order to ascertain that the regulated and normal expression of the Na+/K+/Cl− cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G0/G1 arrest and entering S‐phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes. J. Cell. Physiol. 182:109–118, 2000.

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

SummaryThe addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofVm increase while theKm for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO3−, CO3− and CH3COO− resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl−-dependent.

Synthesis of ATP coupled to Ca2+ release from sarcoplasmic reticulum vesicles

Abstract Ca 2+ release from sarcoplasmic reticulum vesicles was enhanced by the addition of low concentrations of ADP and P i . The effect of ADP and P i was abolished in the presence of Mg 2+ . These findings lead to a study of ATP synthesis coupled with Ca 2+ release by ethyleneglyco-bis-(β-aminoethyl ether)- N , N ′-tetraacetic acid. It was demonstrated that one molecule of ATP is synthesized per two Ca 2+ released from the sarcoplasmic reticulum. The high ratio of ATP synthesized per each Ca 2+ released indicates that most of the Ca 2+ leaves the vesicles through the pump system.

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.

Serum-induced net K+ influx performed by the diuretic-sensitive transport system in quiescent NIH 3T3 mouse fibroblasts

In serum deprived NIH 3T3 mouse cells the diuretic-sensitive transport system performs K+ self-exchange. The addition of serum which stimulates cell proliferation induces a net influx of K+, carried out by the diuretic-sensitive transport system. Thus, serum growth factors appear to induce a change in the mechanism of action of the diuretic-sensitive transporter from K+ self-exchange to an uphill transport pumping K+ into the cell. I propose here that this uphill uptake of K+ contributes to the increase of intracellular K+ content, found in the early G1 phase of the cell cycle.

Na+/K+/Cl− cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes

In a previous work, we have shown that overexpression of the Na+/K+/Cl− cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c‐NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP‐ERK). Furthermore, the level of DP‐ERK was reduced by 50–80% following the addition of bumetanide, a specific inhibitor of the Na+/K+/Cl− cotransporter, in quiescent as well as in proliferating cultures of the Balb/c‐NKCC1 clone. In order to explore further the role of the Na+/K+/Cl− cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP‐ERK level in immortalized non‐transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50–60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K+ influx mediated by the Na+/K+/Cl− cotransporter in these cells. To analyze whether the Na+/K+/Cl− cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na+/K+/Cl− cotransporter controls the ERK/MAPK (mitogen‐activated protein kinase) signal transduction pathway, support our hypothesis that Na+ and K+ influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na+/K+/Cl− cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery. J. Cell. Physiol. 190: 227–237, 2002.

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK.

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl− cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet‐derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl− cotransporter, is downstream of tyrosine kinase receptors, as well as of the G‐protein‐coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl− cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

SummaryIt is shown that the ouabain-resistant (OR) furosemide-sensitive K+(Rb+) transport system performs a net efflux of K+ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K+(Rb+) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K+(Rb+) influx. The oubain-resistant furosemide-sensitive influxes of both22Na and86Rb appear to be Cl− dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na+, K+ and Cl− with a ratio of 1 ∶ 1 ∶ 2. However, the net efflux of K+ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na+ since the unidirectional ouabain-resistant efflux of Na+ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na+ efflux appears to be ouabainsensitive and sufficient to balance the Na+ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K+ and Na+, driven by their respective concentration gradients: a net K+ efflux and a net Na+ influx.

Characterization of a potassium carrier in rabbit reticulocyte cell membrane.

SummaryIn this study we present evidence that high ouabain-resistant Rb influx, carried out by the rabbit reticulocyte membrane, is composed of carrier-mediated Rb influx and passive diffusion across the cell membrane. To meet this end, an assay was developed by which the two ouabain-resistant Rb influxes could be measured separately.Whereas theKm for Rb of the carrier (12.5mm) did not change by increasing the pH, theVm was markedly reduced. At the optimal pH (6.0–6.5) theVm was 6–8 mmol h−1 liter−1 and fell to zero at pH 8.0. This may indicate a possible role of H+ ions in this transport mechanism.The carrier is inhibited by furosemide and ethacrynic acid, similarly to “pump II” in the erythrocyte and kidney. In addition, its activity is dependent upon the ionic content of the medium. The K(Rb) carrier appeared not to be involved in an active transport since depletion of ATP had no effect on the carrier activity. The carrier activity was also measured in rabbit erythrocytes and was found to be 10 times lower than that of rabbit reticulocytes. TheKm for Rb, optimal pH, and high sensitivity to furosemide and ethacrynic acid of the erythrocyte and the reticulocyte carrier are similar.Our study suggests that maturation of reticulocytes to erythrocytes is accompanied by a loss or inactivation of most of a K (or Rb) carrier very active in the reticulocyte cell.

Rb+ influxes differentiate between growth arrest of cells by different agents

SummaryThe effect of cell cycle on Rb+ (K+) fluxes was studied in NIH 3T3 mouse fibroblasts. Serum starvation or isoleucine deprivation resulted in cell arrest at an earlyG1/G0 phase, accompanied by a marked decrease in both ouabainsensitive and ouabain-resistant Rb+ influx. On the other hand, cells arrested at lateG1/G0 phase by hydroxyurea treatment have high ouabain-sensitive and ouabain-resistant Rb+ influx. Butyric acid treatment resulted in cell arrest at an earlyG1/G0 phase, but in contrast to serum or isoleucine starvation did not decrease Rb+ influxes. It is thus shown that quiescent cells may have Rb+ influx rates as high as that of logarithmically growing cells. The results are consistent with the hypothesis that an increased ion permeability of the cell is initiated at a critical stage inG1/G0 phase, and that butyric acid may arrest the cell beyond that stage.