Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation.

Abstract

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...

Characteristics and Regulation of the Na/K/2CI Cotransporter

“…It was found that when cells were exposed to both amiloride and bumetanide, the mitogen-induced early increase in intracellular Na and DNA synthesis were both abolished, whereas nei ther inhibitor was completely effective by itself. The same workers reassessed the role of Na/K/2C1 cotransport in mitogenesis using primary cultures of normal diploid human skin fibroblasts [76]. In contrast with earlier work [68], bumetanide and furosemidc each caused a marked inhibition (50% or greater) o f mitogen-induced DNA synthesis, mea sured as pH]thymidine incorporation.…”

Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection

“…There are reports that mitogens stimulate functional NKCC activity (e.g., Refs. 8,47) and that overexpression of the NKCC protein will stimulate cell proliferation (48). In light of these findings, it is very interesting to note that HCMV infection stimulates the host cells to enter the cell cycle, but causes the cycle to halt at the G 1 /S interface (e.g., Refs.…”

Fate of hypertonicity-stressed corneal epithelial cells depends on differential MAPK activation and p38MAPK/Na–K–2Cl cotransporter1 interaction

“…The dependence of an increase in proliferation on NKCC1 activation is indicated by the finding that this response is suppressed by bumetanide (Bildin et al, 2000). This association was described in human skin fibroblasts, in which stimulation of bumetanide-sensitive NKCC by different mitogens was shown to be essential for cell proliferation (Panet and Atlan, 1991), and the NKCC inhibitors-bumetanide and furosemide-were shown to inhibit cell proliferation (Panet et al, 1994). Additional evidence for the dependence of proliferation on amount of NKCC1 comes from the finding that gene overexpression augments cell proliferation in human skin fibroblasts (Panet et al, 2000).…”