Acta Physiologica Sinica, August 25, 2003, 55(4): 481

Acta Physiologica Sinica, August 25, 2003, 55(4): 481－486

Received 2002-11-02 Accepted 2003-02-06

This study was supported by Uniformed Services University (Maryland,USA) Grant RO83KA.

*Corresponding author.

Tel: +86-25-2618385;

Fax: +86-25-6798187;

E-mail: ying2000@yahoo.com

Research Paper

Role of transferrin in the stimulation of Na,K-ATPase induced by low K+ in Madin Darby canine kidney cells

YIN Wu1,*, ZHOU Xiao-Ming2, CAI Bao-Chang1

1Department of Pharmacy, Nanjing Univeristy of Traditional Chinese Medicine, Nanjing, Jiangsu 210029, China;

2Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20814, USA

Abstract: The presence of serum in a culture medium makes it impossible to identify whether changed cellular functions are directly caused by a manipulation itself or mediated by a component in serum. Madin Darby canine kidney cells can survive in a serum-free medium for about 48 h. We took this advantage to examine whether low K+-induced up-regulation of Na,K-ATPase requires serum. We found that serum was essential for low K+ to induce an increase in Na,K-ATPase binding sites as quantified by ouabain factor binding assays. In an attempt to identify which component was critical, we screened EGF, IGF1, PGE1 and transferrin to identify which one can replace serum. We discovered that transferrin was the single most important factor that mimicked about 80% to 90% of the effect of serum. Transferrin potentiated the effect of low K+ on the Na,K-ATPase binding sites in a time- and dose-dependent manner. Furthermore, transferrin was also required for low K+-induced increase in α1-promoter activity, α1- and β1-subunit protein abundance of the Na,K-ATPase. In the presence of transferrin, low K+ enhanced cellular uptake of iron approximately by 70%. Inhibition of intracellular iron activity by deferoxamine (30 μmol/L) abrogated the effect of low K+. We conclude that stimulation of the Na,K-ATPase by low K+ is critically dependent on transferrin. The effect of transferrin is mediated by increased iron transport.

Key words: Na,K-ATPase; low potassium; transferrin; iron; MDCK cells

转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

殷武1,*, 周晓明2, 蔡宝昌1

1南京中医药大学药学系, 南京 210029;

2美国军医大学, 贝塞斯达, 马里兰 20814

摘要: 体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48 h这一特征, 研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子, 观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(transferrin)在这一过程中的作用。结果表明, 在无血清培养液中低钾并不能刺激细胞膜钠-钾ATP酶, 而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点, 对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中, 细胞膜钠-钾ATP酶 α1亚基启动子活性增强, α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明, 低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(59Fe), 该作用可被铁螯合剂(deferoxamine, DFO; 35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多, α1亚基启动子活性增强, α1与β1亚基蛋白质表达增加。以上结果表明, 低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。

关键词: Na,K-ATP酶; 低钾; 转铁蛋白; MDCK细胞

中图分类号: Q274

In kidneys, the transmembrane enzyme Na,K-ATPase plays a pivotal role in cell metabolism and transepithelial electrolyte and fluid movement. By pumping three Na+ out of the cell in exchange for two K+, the enzyme maintains the transmembrance electrochemical potential that is essential for cell function. The Na+ gradient drives Na+ to enter cells passively through the apical membrane, while the role of K+ gradient in the renal conservation of K+ largely remains unknown. A number of cases have been described in which mammalian cells in culture respond to deprivation of an essential metabolite by an enhanced capacity for uptake of the metabolite. Frequently, this is considered as an induction of transport system. Such response is well known for glucose deprivation and low K+ starved whole animals[1]. It has also been reported that chronic inhibition of Na,K-ATPase activity by incubation of various cells in medium containing low concentration of K+ is associated with a subsequent “adaptive” increase in Na,K-ATPase content[2], however, the signaling pathway that transduces the effect of low K+ on the Na,K-ATPase remains to be elucidated.

Iron serves as a metal cofactor for many enzymes, therefore, it is involved in a broad spectrum of crucial biologic functions, including oxygen binding and metabolism, electron transfer and energy metabolism, and DNA synthesis[3]. Transferrin is a single-chain glycoprotein with molecular weight of near 80000, bearing two structurally similar but functionally distinct iron-binding sites. The transferrin receptor is a disulfide-linked homodimer present in the plasma membrane with two subtypes. Receptor-mediated endocytosis of transferrin-bound iron (diferric transferrin) is possibly the principal mechanism of iron uptake by intestinal cells. Binding of transferrin to transferrin receptor induces rapid formation of clathrin-coated pits and vesicles. After endocytosis of the transferrin-transferrin receptor complex, iron is released from transferrin due to a decrease in endosomal pH and enters the chelatable intracellular iron pool, the apo-transferrin-transferrin receptor complex is recycled back to the cell surface to mediate further rounds of endocytosis[4]. In kidneys, transferrin receptors show an uneven distribution, the highest level of expression being in the convoluted parts of the distal tubules of the cortex and in the collecting ducts of the medulla.

Serum is often included in a cell culture medium because it is important to cell growth. However, the presence of serum in a culture medium has complicated studies concerning regulation of cellular functions in vitro. Specifically, it is impossible to identify whether changed cellular functions are directly caused by a manipulation itself or mediated by a component in serum. Madin Darby canine kidney (MDCK) cells originate from the distal tubule that is the final tuning sites for Na+ excretion. Bowen and McDonough demonstrated that low K+ induced up-regulation of the Na,K-ATPase in this type of cells[5]. MDCK cells can survive in serum-free medium for approximately 48 h. We took this advantage to identify which component in serum is indispensable for the stimulation of the Na,K-ATPase and its underlying mechanism as well.

1 MATERIALS AND METHODS

1.1 Cell culture．

A subclone of MDCK cells, A4, was used throughout the study. MDCK cells were purchased from the American Type Culture Collection (Manassas, VA). The cells were kept in Dulbecco's Modified Essential Medium (DMEM) with 10% fetal bovine serum at 37℃ in an atmosphere containing 5% CO2. The cells were placed down with the complete growth medium overnight prior to experiment. The confluent cells were then switched to either the control or low K+ medium for additional 24 h before experiment. The control medium was regular DMEM (US Biological, Swampscott, MA). The low K+ medium was identical to the control medium except partial K+ was substituted by Na+ for osmolarity compensation. In experiments that required serum, the media were added with 7.5% horse serum and 2.5% fetal bovine serum which had been dialyzed against Ca2+, K+-free Hanks' solution (mmol/L: NaCl 150, MgCl2 0.5, Na2HPO4 0.2, NaH2PO4 0.4). Gentamycin (25 μg/ml, GIBCO BRL, Grand Island, NY) was included in all culture media.

1.2 Ouabain binding assay.

After treatment, the confluent cells were washed twice with Ca2+, K+-free Hanks' solution and incubated in the same solution plus 2 mmol/L EGTA at 37℃ for 15 min to disrupt tight junctions of the cells. The cells were then incubated in ouabain binding media containing 0.4 μmol/L [3H] ouabain in Ca2+, K+-free Hanks' solution at 37℃ for additional 15 min. After incubation, the cells were washed four times with ice-cold Ca2+, K+-free Hanks' solution and solubilized in 0.4 mol/L NaOH. Radioactivity remaining in the cells was quantified by scintillation counting. The concentration of ouabain in total binding medium was demonstrated to be sufficient to saturate binding sites. Non-specific binding was measured in the presence of 100 μmol/L unlabeled ouabain. Non-specific binding was less than 2% of total binding. Specific ouabain binding was defined as the difference between total binding and non-specific binding.

1.3 Western analysis.

After treatment, the confluent cells were rinsed with ice-cold phosphate buffered saline (PBS), scraped with a rubber policeman in a loading buffer supplemented with 5% β-mercaptoethanol (β-ME) and 0.1 mg/ml phenylmethysulfonyl fluoride (PMSF) and 0.04 μg/ml aprotinin. After they were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the samples were electrophoretically blotted onto PVDF membranes. The membranes were first hybridized with primary antibodies and then with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (Sigma, St. Louis, MO). The polyclonal antibody against the α1-subunit of the Na, K-ATPase was a generous gift from Dr. Thomas A. Pressley[6]. Monoclonal antibodies against β1-subunit, transferrin receptors and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Upstate Biotechnology (Lake Placid, NY).

1.4 Transfections and chloramphenical acetyl transferase (CAT) assays．

MDCK cells were transfected with plasmid DNA mixed with lipofectAMINE (GIBCO BRL, Grand Island, NY) according to manufacturer's instructions. The plasmid DNA was constructed with the proximal 96 bp of

α1-subunit promoter and CAT reporter gene. Stable cell lines were selected under 600 μg/ml G418. After treatment, the cells were transferred into an eppendorff tube with the lysis buffer included in the assay kit. Freezing and thawing method was used to rupture the cells. CAT assays were performed according to manufacturer's procedures (Promega, Madison, WI).

1.5 Iron uptake assay．

After treatment, the cells were incubated with Ca2+-free plus 2 mmol/L EGTA medium to break tight junction of the cells. The cells were then incubated with the uptake medium at 37℃ for 6 min, washed with ice-cold balanced Hanks' solution four times, solubolized in 0.4 mol/L NaOH, and counted for radioactivity with a scintillation counter. The uptake medium contained 3.3 μmol/L59Fe, 10 μmol/L nitrilotriacetic acid, and 4 μg/ml transferrin in balanced Hanks' solution.

1.6 Statistical analyses．

Data are expressed as means±SD. Statistical analyses were performed using analysis of variance or Student's t test as appropriate. Post hoc comparisons were made by Dunnett test. The null hypothesis was rejected at the 0.05 level of significance.

2 RESULTS

Our previous analyses demonstrated a maximum response at 0.1 mmol/L K+[7]. Therefore, this concentration was used. The cells were serum-deprived for 16 h before low K+ was introduced. We found that withdrawal of serum abolished the effect of low K+ on the Na,K-ATPase binding sites as quantified by ouabain binding assays (Fig.1). In an attempt to identify which component was indispensable, we screened a number of factors and observed that transferrin was the single most important factor that mimicked about 80% to 90% of the effect of serum (Fig.1).

To further examine the role of transferrin in the effect of low K+, we performed dose-response and time-course studies on the effect of transferrin on low K+-induced increase in the Na,K-ATPase binding sites. Our dose-response analyses revealed that transferrin showed maximal response between 2 μg/ml and 6 μg/ml (Fig.2). The time-course of the effect of low K+ in the presence of

Fig.1.

Effect of transferrin on the stimulation of ouabain binding sites induced by low K+.

The cells were treated with serum-free DMEM control medium or low K+ medium for 24 h after deprivation of serum for 16 to 20 h. Ouabain binding data from each control were arbitrated as 100%, therefore, standard deviation was zero. Each experiment was performed in duplicate or triplicate (n=3).

□, control.

■, low K+.

S, serum;

SF, serum free;

Tf, transferrin, 5 μg/ml ;

DFO, deferoxamine, a chelator of Fe, 35 μmol/L;

EGF, 0.01 μmol/L;

IGF1, 500 ng/ml;

PGE1, 25 ng/ml.

transferrin was similar to that in the presence of serum. Low K+ increased the Na,K-ATPase binding sites after a lag of more than 12 h and reached a plateau by 20 h. During this interval the number of binding sites increased by approximately 40% (Fig.3). Transferrin was also required for low K+ to increase CAT activity (Fig.4). Western analyses showed that transferrin was needed for the increased α1 and β1 subunit abundance induced by low K+ (Fig.5).

One of the major functions of transferrin is to facilitate cellular uptake of iron via transferrin receptors. We found that low K+ markedly increased transferrin-dependent 59Fe transport 45 min after incubation with serum or serum-free culture medium supplemented with transferrin (Fig.6). Deferoxamine, a chelator of Fe, abrogated the effect of low K+ on the Na,K-ATPase binding sites (Fig.1), α1-promoter activity (Fig.4), and α1- and β1-subunit protein abundance (Fig.5).

Fig.2.

Dose-response effect of transferrin on the stimulation of ouabain binding sites induced by low K+.

The cells were deprived of serum for 16 to 20 h prior to introduction of low K+ in serum-free DMEM medium. Ouabain binding data from DMEM control was arbitrated as 100%. (aP<0.01 as compared with serum-free 5.25 mmol/L[K+], ANOVA). X axis represents transferrin concentration (μg/ml).

Fig.3.

Time-course effect of transferrin (4 μg/ml) on the stimulation of ouabain binding sites induced by low K+. The cells were deprived with serum for 16 to 20 h prior to introduction of low K+ in serum-free DMEM medium. (aP<0.01 as compared with serum-free 5.25 mmol/L [K+], ANOVA).

Fig.4.

Transferrin (Tf) was required for low K+ to induce up-regulation of α1-subunit promoter activity.

Promoter activity was measured by chloramphenical acetyl transferase (CAT) assays (n=4). (aP<0.01 as compared with serum-free 5.25 mmol/L [K+], ANOVA).Each experiment was performed in duplicate or triplicate (n=3).

□, control.

■, low K+.

Tf, transferrin (4 μg/ml);

DFO, deferoxamine (35 μmol/L).

Fig.5.

Effect of transferrin on α1- and β1-subunit abundance of Na,K-ATPase.

Western analyses of α1- and β1-subunit protein expression in the absence of serum but supplemented with 4 μg/ml transferrin.

This is a representative of four independent experiments.

S, serum;

SF, serum free; SFT, serum free+transferrin; Tf, transferrin (4 μg/ml);

DFO, deferoxamine (35 μmol/L).

Fig.6.

Low K+ increased iron uptake in the presence of serum or transferrin.

The cells were treated with low K+ medium for 45 min after deprivation of serum for 16 to 20 h.

Low K+ increased iron uptake in the presence of serum or transferrin (Tf).

Each experiment was performed in duplicate or triplicate (n=3).

□, control. ■, low K+.

SF, serum free; Tf, transferrin (4 μg/ml).

(aP<0.01 as compared with serum or serum-free 5.25 mmol/L[K+], ANOVA).

3 DISCUSSION

Serum-free and chemically-defined medium has been used commonly not only for dissection of the mechanism underlying a specific hormone action, but also for determination of the role of this hormone in altered cellular functions induced by another manipulation. We took the advantage that MDCK cells survived in a serum-free medium for a couple of days and examined whether serum was indispensable for the stimulation of Na,K-ATPase induced by low K+. The results showed that transferrin was required for low K+ to induce an up-regulation of α1-promoter activity, α1- and β1-subunit protein abundance and ouabain binding sites.

Iron uptake is a fundamental requirement for many aspects of life, which participates in three general reactions: oxidation-reduction, hydrolysis and polynuclear complex formation. The main function of transferrin is to transport nonheme iron and facilitate cellular uptake of iron. In the presence of transferrin, low K+ increased iron uptake. Inhibition of iron activity by deferoxamine abrogated the effect of low K+ on the Na,K-ATPase. These data suggested that the effect of transferrin was mediated by increased iron uptake. However, excessive amount of iron may result in over formation of free radicals that damage cellular constituents, for instance, iron-overload may cause tubulointerstitial injury[8]. Therefore, the amount of iron within cells is carefully regulated. A major mechanism for the regulation of iron homeostasis relies on the post-transcriptional control of ferritin and transferrin receptor mRNAs. Increased intracellular iron activity stimulates synthesis of ferritin and down-regulates transferrin receptors, while decreased intracellular iron activity has an opposite effect. In view of transferrin receptor is the limiting factor determining transferrin-dependent iron transport, we examined whether low K+-induced increase in iron uptake in the presence of transferrin is mediated by increased abundance of transferrin receptor protein. However, our western blot data do not support this possibility (data not shown).

Besides post-transcriptional regulation of transferrin receptors, the number of transferrin receptors on cell surface can also be regulated by exocytosis and endocytosis. In some types of cells only 20% of the transferrin receptors are on the cell surface with 80% residing in cytosol[9]. PMA, EGF and IGE increase surface receptor number by stimulating exocytosis of the receptors[10－12], whereas ROS and iron decrease surface receptor number by stimulating endocytosis of receptors[12,13]. In the absence of serum or transferrin, transferrin-dependent iron uptake is diminished, which results in a decreased intracellular iron activity. Iron deficiency increases not only de novo synthesis of transferrin receptors[14], but also externalization rate of the receptors[13]. Therefore, it is likely that low K+ in the presence of serum or transferrin stimulates membrane insertion of transferrin receptors thus increasing iron uptake. This mechanism, however, needs to be examined. In addition, since distal tubules have the highest expression of transferrin receptors in kidneys[3] and also is the final tuning sites for Na+ and K+ excretion, the present study suggests that transferrin receptors may be regulated by Na+ or K+ balance.

In the A431 epidermoid carcinoma cells, EGF or IGF-1 increases the rate of transferrin receptor exocytosis. Whether a similar effect is present in MDCK cells is unknown. However, even EGF or IGF-1 stimulated externalization of the receptors in MDCK cells, EGF or IGF-1 could not increase transferrin-mediated iron transport, because there was no transferrin in the culture medium. Therefore, it is not surprising that EGF or IGF-1 failed to potentiate the effect of low K+ on the Na,K-ATPase binding sites (Fig.1).

Iron is needed for the function of NADH/NADPH oxidase that generates reactive oxygen species (ROS). Moreover, iron also catalyzes hydrogen peroxide to hydroxyl radical, the most powerful oxidant in biological system, through Fenton reaction. During the past decade ROS have been identified as the important second messengers in biological signaling cascades. Besides interaction with other signaling pathways, ROS have been shown to mediate ouabain-induced gene expressions that are related to cardiac hypertrophy[15]. Our previous study has suggested the involvement of ROS in the effect of low K+ on the Na,K-ATPase. Because of the critical role of iron in producing ROS and because ROS are involved in the effect of low K+, it is plausible to speculate that in the presence of transferrin low K+ increases iron transport thereby increasing generation of ROS signal, which mediates the effect of low K+ on the Na,K-ATPase, however, this speculation needs to be further investigated.

In sum, the upregulation of the Na,K-ATPase by low K+ is dependent on transferrin. The effect of transferrin is mediated by increased iron transport, the mechanism of which still needs to be further investigated.

***

Acknowledgement: The authors thank Dr. Jesse Bowen (University of Missouri, USA) for the low K+ medium. The author would also thank for generous assistance from Fangzhou Yin in the preparation and process of this manuscript.

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