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

Panet, Rivka; Atlan, Henri

doi:10.1002/jcp.1041450106

Cited by 20 publications

(14 citation statements)

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“…Serum regulates membrane channels (25) and transporters (26). In the present study, we found that serum factors were important to maintain the basal activity of K-Cl COT in VSMCs because serum deprivation for 24 h abolished this activity (Fig.…”

Section: Discussionsupporting

confidence: 51%

“…Serum plays multiple roles in the regulation of the activity of ion channels and transporters in cultured cells (26,29). To examine whether serum has an effect on basal K-Cl COT activity in primary cultures of rat VSMCs, cells were serum deprived for 24 h and K-Cl COT activity was subsequently measured.…”

Section: Resultsmentioning

confidence: 99%

“…Serum plays multiple roles in cultured cells, including regulation of membrane transporters (26,29). Serum was reported to stimulate the Na/H exchanger through phosphorylation in resting hamster fibroblasts and A431 human epidermoid cells (29).…”

mentioning

confidence: 99%

“…Serum was reported to stimulate the Na/H exchanger through phosphorylation in resting hamster fibroblasts and A431 human epidermoid cells (29). In quiescent human fibroblasts, serum and growth factors stimulated the bumetanide-sensitive Na-K-2Cl cotransporter in a dose-dependent manner (26). However, there is little knowledge on the regulation of K-Cl COT by these effectors.…”

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confidence: 99%

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Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Zhang¹,

Lauf

Adragna³

2003

American Journal of Physiology-Cell Physiology

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Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

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Section: Discussionsupporting

confidence: 51%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Zhang¹,

Lauf

Adragna³

2003

American Journal of Physiology-Cell Physiology

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“…ration of serum deprivation, and the serum concentration in the arrest medium, were analyzed . We have shown that in cultures of human skin fibroblasts arrested under optimal conditions, the bumetanide-sensitive Na+/K+/Cl-cotransport is stimulated by two-to threefold after the addition of fetal calf serum, or purified recombinant growth factors (Panet and Allan, 1990) . In the present study, we analyzed, with the use of specific inhibitors furosemide and bumetanide, whether stimulation of the Na+/K+/Cl -cotransporter by serum growth factors is essential for human fibroblast proliferation .…”

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confidence: 99%

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

Panet

Atlan

1991

The Journal of Cell Biology

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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...

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

Panet

Eliash

Pick

et al. 2002

Journal Cellular Physiology

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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.

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

Cited by 20 publications

References 39 publications

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

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

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

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

Cited by 20 publications

References 39 publications

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

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

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

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