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

Atlan

1990

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In this study we investigated the correlation between the mitogenic effect and stimulation of Rb+ (K+) fluxes in human skin fibroblasts treated by purified growth factors. Both K+ transporters, bumetanide-sensitive and ouabain-sensitive, are stimulated 2-3-fold after addition of either fetal calf serum or purified recombinant growth factors to quiescent G0/G1 human skin fibroblasts. Three groups of mitogens were compared: i) the phorbol ester 2-O-tetradecanoyl-phorbol-13-acetate (TPA); ii) growth factors that stimulate inositol phosphate hydrolysis and subsequently activate protein kinase C--fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and alpha-thrombin; and iii) growth factors that do not activate kinase C--insulin-like growth factor-1 (IGF-1), and transforming like growth-factor-alpha (TGF-alpha). The three groups of mitogens stimulated human skin fibroblasts proliferation and Rb+ influxes in a similar dose-dependent fashion. The results indicate that both the bumetanide-sensitive and the ouabain-sensitive Rb+ fluxes are stimulated by protein kinase C-dependent and by the protein kinase C-independent pathways of the mitogenic signal.

Section: Discussionmentioning

confidence: 58%

Bumetanide‐sensitive Na+ /K+ /Cl− transporter is stimulated by phorbol ester and different mitogens in quiescent human skin fibroblasts

Atlan

1990

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“…Na+ ions are translocated through the cell membrane by the following transport systems: i) amiloridesensitive Na + /H + antiport (Frelin et al, 1984(Frelin et al, , 1985Kim and Smith, 1986; Piwnica-Worms et al, 19861, ii) Na + /Na + exchange (Ehrlich and Diamond, 1979), iii) tetrodotoxine-sensitive Na + influx (Pouyssegur et al, 1980;Davis et al, 1982), iv) Na+/Ca+ + antiport (Barry et al, 1985;Eisner and Lederer, 1985;, and v) the "loop" diuretic-sensitive Na+ in flux coupled to K + and C1-fluxes (Geck et al, 1980;Panet and Atlan, 1980; Aiton et al, 1981;Palfrey and Greengard, 1981;McRoberts et al, 1982;Panet et al, 1982Panet et al, , 1987Atlan et al, 1984;Panet, 1985; PiwnicaWorms et al, 1985;Frelin et al, 1986;Baumgarten and Duncan, 1987;Liu et al, 1987;Heller et al, 1988).…”

Section: Pumpmentioning

confidence: 96%

Role of the Na+ /K+ /Cl− transporter in the positive inotropic effect of ouabain in cardiac myocytes

Fixler

Snyder

et al. 1990

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In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.

“…Two minutes before the indicated time with PHA, 86 Rb (1 mCi/ ml) was added with ouabain in absence and presence of bumetanide. At the end of the incubation, the cells were centrifuged and washed with cold isotonic MgCl 2 , lysed with 0.2 N NaOH, and radioactivity was measured in a b-counter, and bumetanide-sensitive 86 Rb in¯ux rate was calculated as described before (Panet et al, 1982). The experiment presented in this ®gure was repeated three times, and the results illustrated here are of a representative experiment.…”

Section: /Clmentioning

confidence: 99%

“…Bumetanide-sensitive 86 Rb in¯ux was measured as reported previously (Panet et al, 1982). Measurements of 86 Rb in¯ux in the presence of ouabain (0.5 mM) and bumetanide (10 mM) were subtracted from ouabainresistant 86 Rb in¯ux.…”

Section: Rb In¯ux Measurementsmentioning

confidence: 99%

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

Eliash

Pick

et al. 2002

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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.