Cystine and glutamate transport in renal epithelial cells transfected with human system x(-) (c)

Abstract

Background: System x(-) (c) is a heterodimeric transporter, comprised of a light chain, xCT, and heavy chain, 4F2hc, which mediates the sodium-independent exchange of cystine and glutamate at the plasma membrane. In the current study we tested the hypothesis that stable transfection of Madin-Darby canine kidney (MDCK) cells with human xCT and 4F2hc results in the expression of functional system x(-) (c).

Methods: MDCK cells were transfected stably with human clones for xCT and 4F2hc. Analyses of time- and temperature-dependence, saturation kinetics, and substrate specificity of l-cystine and l-glutamate transport were carried out in control and xCT-4F2hc-transfected MDCK cells. We also measured the uptake of l-cystine in Xenopus oocytes expressing human xCT and/or 4F2hc or xCT and/or rBAT (a heavy chain homologous to 4F2hc).

Results: All of the different sets of data revealed that transport of l-cystine and l-glutamate increased significantly (twofold to threefold) in the MDCK cells subsequent to transfection with xCT-4F2hc. Moreover, uptake of l-cystine also increased (about tenfold) in Xenopus oocytes expressing hxCT and h4F2hc. Biochemical analyses of l-cystine uptake in oocytes verified our findings in the transfected MDCK cells. Interestingly, in oocytes injected with rBAT with or without xCT, uptake of l-cystine was significantly greater than that in water-injected oocytes.

Conclusion: Our findings indicate that stable transfection of MDCK cells with xCT and 4F2hc results in a cell-line expressing a functional system x(-) (c) transporter that can utilize l-cystine and l-glutamate as substrates. This study apparently represents the first stable transfection of a mammalian cell line with system x(-) (c).

Figure 1

Photographic presentation of RT-PCR analyses for human and canine xCT (hxCT; cxCT), human and canine 4F2hc (h4F2hc; cxCT), and canine GAPDH (cGAPDH) in control and xCT-4F2hc transfected MDCK cells. The expected sizes of the RT-PCR products, as predicted from the positions of the primers, were 724 bp for hxCT, 603 bp for h4F2hc, and 415 bp for cGAPDH.

Figure 2

Uptake of [³⁵S]-L-cystine or [³H]-L-glutamate in control and xCT-4F2hc-transfected MDCK cells. Cells were exposed to 5 μM [³⁵S]-L-cystine (A) or [³H]-L-glutamate (B) at 37°C for time periods ranging from 5 to 90 minutes. Samples were taken for determinations at indicated times. Data are presented as mean ± SE for n=4 samples. Each experiment was performed at least twice for the purpose of validation. * = significantly different (p < 0.05) from the mean for the corresponding group of control cells. + = significantly different from the mean for the same cell-type at the initial time point. ++, +++, ++++, +++++, or ++++++ = significantly different from the mean for the same cell-type at each of the preceding time points.

Figure 3

Saturation kinetics for the transport of [³⁵S]-L-cystine or [³H]-L-glutamate in control and xCT-4F2hc transfected-MDCK cells. Cells were incubated for 15 minutes at 37°C with 5 μM [³⁵S]- L-cystine (A) or [³H]-L-glutamate (B) in the presence of unlabeled L-cystine or L-glutamate, respectively. Each inset represents an Eadie-Hofstee plot of the data. Results are presented as mean ± SE for n=4 samples. Each experiment was performed twice for the purpose of validation. *, significantly different (p < 0.05) from the mean for the corresponding group of control cells. +, significantly different from the mean for the same cell-type at the initial concentration. ++ or +++, significantly different from the mean for the same cell-type at each of the preceding concentrations.

Figure 4

Effect of temperature on the uptake of cystine or glutamate in control and xCT-rBAT transfected-MDCK cells. Cells were exposed to 5 μM [³⁵S]-L-cystine (A) or [³H]-L-glutamate (B) for 15 min at 37°C or 4°C in the presence of unlabeled L-cystine or L-glutamate, respectively. Data are presented as mean ± SE for n=4 samples. Each experiment was performed at least twice for the purpose of validation. * = significantly different (p < 0.05) from the mean for the corresponding group of control cells.

Figure 5

Substrate specificity analyses of the sodium-independent transport of [³⁵S]-L-cystine or [³H]-L-glutamate in control and xCT-4F2hc-transfected MDCK cells. Cells were incubated for 15 minutes at 37°C with 5 μM [³⁵S]-L-cystine (A) or [³H]-L-glutamate (B) in the presence of various unlabeled amino acids (3 mM; unlabeled L-cystine and DL-homocystine = 1 mM). Data are presented as mean ± SE for n=4 samples. Each experiment was performed at least twice for the purpose of validation. * = significantly different (p < 0.05) from the mean for the corresponding group of control cells. + = significantly different from the mean for the control group of the corresponding cell-type.

Figure 6

Uptake of cystine in Xenopus oocytes injected simultaneously with human xCT (hxCT) and human 4F2hc (h4F2hc) or water. Oocytes were exposed to 5 μM [³⁵S]- L-cystine at room temperature for time periods ranging from 5 − 60 minutes. Samples were taken for estimation at indicated time points. Data are presented as mean ± SE of n=3 samples, with each sample obtained from 10 oocytes per well. Each experiment was also performed at least twice for the purpose of validation. * = significantly different (p < 0.05) from the mean for the corresponding group of water-injected oocytes. + = significantly different from the mean for the same set of oocytes at the initial time point. ++ or +++, significantly different from the mean for the same set of oocytes at each of the preceding time points.

Figure 7

Saturation kinetics of cystine uptake in Xenopus oocytes injected simultaneously with human xCT (hxCT) and human 4F2hc (h4F2hc) or water. Oocytes were exposed to 5 μM [³⁵S]-L-cystine for 30 minutes at room temperature in the presence of unlabeled cystine. (Inset: Eadie-Hofstee plot). Data are presented as mean ± SE of n=3, with each sample obtained from 10 oocytes per well. Each experiment was also performed at least twice for the purpose of validation. * = significantly different (p < 0.05) from the mean for the corresponding group of water-injected oocytes. + = significantly different from the mean for the same set of oocytes at the initial concentration. ++ or +++, significantly different from the mean for the same set of oocytes at each of the preceding concentrations.

Figure 8

Analyses of sodium-independent, substrate specificity of L-cystine uptake in Xenopus oocytes injected simultaneously with human xCT (hxCT) and human 4F2hc (h4F2hc) or water. Oocytes were exposed to 5 μM [³⁵S]-L-cystine for 30 minutes at room temperature in the presence of unlabeled amino acids (3 mM; L-cystine, DL-homocystine = 1 mM). Data are presented as mean ± SE of n=3, with each sample obtained from 10 oocytes per well. Each experiment was also performed at least twice for the purpose of validation. * = significantly different (p < 0.05) from the mean for the corresponding group of water-injected oocytes. + = significantly different from the mean for the control group of corresponding oocytes.

Figure 9

Uptake of L-cystine in Xenopus oocytes injected with various cDNA constructs. (A) Oocytes were injected with either water, human xCT (hxCT), human 4F2hc (h4F2hc), human rBAT (hrBAT) or simultaneously with hxCT and h4F2hc or hxCT and hrBAT and exposed to 5 μM [³⁵S]- L-cystine for 30 minutes at room temperature. (B) Oocytes were injected with either water, hxCT and h4F2hc, or hxCT and hrBAT and were exposed to 5 μM [³⁵S]-L-cystine for 30 minutes at room temperature in the presence of 3 mM unlabeled arginine or glutamate. Data are presented as mean ± SE of n=3, with each sample obtained from 10 oocytes per well. Each experiment was also performed at least twice for the purpose of validation. *, significantly different (p < 0.05) from the mean for the water-injected oocytes. +, significantly different from all other groups of oocytes. ++, significantly different from the mean for the control oocytes injected with the same RNA transcripts.