The 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 of Vm increase while the Km for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrynic 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.

Abstract. In this study, we examined the role of the bumetanide-sensitive Na+/K+/CI -cotransport in the mitogenic signal of human skin fibroblast proliferation . The Na+/K+/Cl -cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent Go/Gt human skin fibroblasts . The following mitogens, FGF, PDGF, a-thrombin, insulin-like growth factor-1, transforming growth factor-a, and the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl -cotransport . In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl -cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth NE of the earliest responses of quiescent cells to a mitogenic signal is activation of Na+ influx (Rozengurt, 1986;Rozengurt and Mendoza, 1986) . The bumetanide-sensitive Na+/K+/Cl -cotransport was shown to be dramatically stimulated by the addition of serum growth factors to quiescent cells (Tupper et al., 1977; Pallet et al., 1982 Pallet et al., , 1983Panet, 1985;Amsler et al., 1985;Paris and Pouyssegur, 1986; Panet et al., 1986aPanet et al., , 1989. The question of whether the Na+/K+/Cl-cotransport has an essential role in the mitogenic response is still unsolved . Several groups have observed that inhibition of the Na+/K+/Cl-cotransport affected only slightly the initiation of DNA synthesis (Owen and Prastein, 1985 ;Amsler et al., 1985;Paris and Pouyssegur, 1986) . It has therefore been proposed that the Na+/K+/Cl-cotransport does not play a major role in the mitogenic signal (Amsler et al ., 1985 ;Paris and Pouyssegur, 1986). Most ofthese studies, however, were carried out with immortal rodent cell lines. Nevertheless, the response of normal diploid human fibroblasts, rather than immortal cell lines to growth factors, may be different and more relevant to the normal control of cell proliferation . Recently, we have investigated the optimal conditions for thearrest ofhuman skin fibroblasts . Optimal cell density, du-0 The Rockefeller University Press, 0021-9525/91/07/337/6 $2 .00 The Journal of Cell Biology, Volume 114, Number 2, July 1991 337-342 factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the Go/G, phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis . In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect . This study clearly demonstrates that the growth facto...

Na(+)/K(+)/Cl(-) cotransporter activity is stimulated in early G(1) 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 G(0)/G(1) 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.

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl-cotransport in the mitogenic signal of vascular endothelial cell proliferation. The activity of the Na+/K+/Cl- cotransport is dramatically decreased in quiescent subconfluent cells, as compared to subconfluent cells growing in the presence of FGF. The Na+/K+/Cl- cotransport activity of quiescent subconfluent cultures deprived of FGF decreased to 6%, whereas that of quiescent cells grown to confluency was reduced to only 33% of the activity of subconfluent cells growing in the presence of FGF. The basal low activity of Na+/K+/Cl- cotransport in the quiescent subconfluent vascular endothelial cells was dramatically stimulated by FGF. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal of the endothelial cells, the effect of two specific inhibitors of the cotransport -furosemide and -bumetanide was tested on cell proliferation induced by FGF. Bumetanide and furosemide inhibited synchronized cell proliferation measured by direct counting of cells and by DNA synthesis. Inhibition by furosemide and bumetanide was reversible; removal of these compounds completely released the cells to proliferate. These results indicate that the effect of these drugs is specific and is not due to an indirect toxic effect. This study clearly demonstrates that the FGF-induced activation of the Na+/K+/Cl- cotransport plays a role in the mitogenic signal pathway of vascular endothelial cells.

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