From the San Diego Cancer Center and Department of
Medicine, University of California, La Jolla, California 92093-0684 and
¶ Department of Pharmacology, Faculty of Medicine, Kyoto
University, Kyoto 606, Japan
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ABSTRACT |
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Arginine transport is important for a number of
biological processes in vertebrates, and its transport may be
rate-limiting for the production of nitric oxide. The majority of
L-Arg transport is mediated by System y+,
although several other carriers have been kinetically defined. System
y+ cationic amino acid transport is mediated by proteins
encoded by a family of genes, Cat1, Cat2, and
Cat3. High affinity L-arginine transport was
investigated in embryonic fibroblast cells derived from
Cat1 knockout mice that lack functional Cat1. Both wild
type and knockout cells transport arginine with comparable
Km and Vmax. However, the
apparent affinity for lysine transport was 2.4 times lower in
Cat1/
cells when compared with wild type
cells, a property characteristic of Cat3-mediated transport. Northern
analysis-documented Cat2 mRNA increased 2-fold, whereas
Cat3 mRNA levels increased 11-fold in
Cat1
/
relative to
Cat1+/+ cells. The low affinity
Cat2a mRNA was not detectably expressed in these cells.
Even though Cat3 expression is normally limited to adult
brain, there was a large increase in the amount of Cat3 protein present
at the plasma membrane of Cat1
/
embryonic
fibroblast cells. These results suggest that Cat3 compensates for the
loss of functional Cat1 in cells derived from Cat1 knockout mice and mediates the majority of high affinity arginine transport.
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INTRODUCTION |
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Since first kinetically characterized by Christensen in the 1960s, L-Arg uptake has been found to be mediated by a number of competing systems such as System y+ (1-3), bo,+ (4), and y+L (5) (for reviews see Refs. 6-9). System y+ is a sodium-independent transporter with a high affinity for the L-isomers of cationic amino acids and is subject to trans-stimulation (2). System y+ is widely believed to be the major carrier of cationic amino acids in adult tissues (6).
Three high affinity cationic amino acid transporters, Cat1, Cat2, and Cat3, were cloned serendipitously in 1989 (10), 1990 (11), and 1997 (12), respectively. Whereas these transporters share only about 61% amino acid sequence identity they were found to be kinetically indistinguishable with very similar Km values for L-arginine in oocytes (13, 14). Cat11 appears to have a greater capacity than Cat2 for trans-stimulation (14), whereas an alternately spliced transcript of Cat2 encodes a kinetically distinct low affinity form of Cat2a (15). Cat3 was cloned from mouse (12) and rat (16). In both species, Cat3 expression was localized to the brain although it was widely expressed in the 13.5-day postcoital mouse embryo. Unlike Cat1 or Cat2, Cat3 has different Km values for L-arginine (40-60 µM) and L-lysine (115-165 µM) uptake in Xenopus oocytes (12) and COS7 cells (L-Arg, 103 ± 12 µM; L-Lys, 147 ± 22 µM) (16). Hence, Cat3 can be kinetically distinguished from Cat1, Cat2, and Cat2a by their differential affinity to L-Lys. Similarly, a difference in the Km values for L-Arg and L-Lys transport in HTC hepatoma cells was previously reported (3) but never assigned to a particular Cat family member. The importance of Cat1-mediated transport was recently underscored by the production of knockout mice (17). Mice homozygous for this mutation die within 12 h of birth; they are severely anemic and runted. The knockout embryos express a truncated, non-functional Cat1 mRNA (17). Despite the apparent functional similarity of the Cat transporters, Cat1 function is essential for survival past parturition.
As with the glutamate transporters (for review, see Ref. 18) the
Cat gene family appears to encode functionally redundant arginine transporter proteins (19). It is impossible to kinetically distinguish the contribution of one member of the Cat family by assessing arginine transport in the presence of competing members. Taking advantage of the differential affinity for lysine and a cell
line established from the Cat1/
embryonic
fibroblasts, we investigated arginine and lysine uptake into
Cat1
/
MEF cells. Results described in this
paper will reveal that the ablation of Cat1 appears to be
mainly compensated for by an enhanced expression of
Cat3.
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EXPERIMENTAL PROCEDURES |
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Materials--
L-[2,3,4,5-3H]Arginine
monohydrochloride, L-[4,5-3H] lysine
monohydrochloride, [-32P]dCTP, and the multiprime DNA
labeling system were all from Amersham Pharmacia Biotech. Tissue
culture reagents and Trizol were purchased from Life Technologies, Inc.
Magnacharge nylon membranes were obtained from Micron Separations Inc.
Titermax was from CytRx Co., and the rhodamine-conjugated goat
anti-rabbit IgG was purchased from Jackson Laboratories Inc.
Cat1
/
MEF cells were kindly provided by Dr.
Chris Perkins (17), and the Cat1+/+ MEF cells
were the gift of Dr. Daniel Fink (20). The Cat3 cDNA was
obtained from Dr. Ken Ito (12) via Dr. Daniel Feldman (Medical College
of Georgia). The glyceraldehyde-3-phosphate dehydrogenase (gapdh) cDNA was a kind gift of Dr. Erich Weber
(UCSD).
Cell Culture--
Both the Cat1/
and
Cat1+/+ mouse embryonic fibroblasts were
cultured in high glucose Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) fetal bovine serum, 2 mM
L-glutamine, 100 units/ml penicillin, 100 units/ml
streptomycin sulfate, and 10 µg/ml gentamicin sulfate. Four days
prior to transport studies or RNA extraction, similar numbers of cells
were plated into 35-mm tissue culture dishes and had reached confluence
at the time of the experiments.
Transport Measurements-- To avoid the effects of trans-stimulation (2) cells were washed once in HEPES buffer (140 mM choline chloride, 5 mM KCl, 0.9 mM CaCl2, 1 mM MgSO4, 5.6 mM D-glucose, and 25 mM HEPES, pH 7.4) and incubated in HEPES buffer for 2 h at 37 °C. Na+-independent transport experiments were performed essentially as described (21) with the following modifications. First, cells were washed (4 × 1.25 ml) with transport buffer (137 mM choline chloride, 5.4 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 10 mM HEPES, adjusted to pH 7.4 with Tris). Amino acid transport was instigated by the addition of 1 ml of transport buffer supplemented with L-arginine and 5 mM L-Leu. 38 nM L-[2,3,4,5-3H]arginine monohydrochloride was mixed to a final concentration of 250 µM L-Arg (unless stated otherwise) with unlabeled L-Arg in the supplemented transport buffer. Competition analysis revealed significant y+L and b0,+ activity in the embryonic fibroblasts that was inhibited with 5 mM L-leu. Unless stated otherwise all transport experiments were performed over 3 min at 24 °C. The transport was terminated by rapidly washing (4 × 1.25 ml) with ice-cold 137 mM NaCl, 10 mM Tris, 10 mM HEPES, pH 7.4. Non-saturable binding plus uptake was measured in parallel in the presence of 38 nM L-[2,3,4,5-3H]arginine, 5 mM L-Arg, and 5 mM L-Leu. Above 3 mM L-Arg, no significant saturable uptake of L-Arg occurred. Therefore, the disintegrations per min (dpm) in the cells following washing represented the non-saturable and binding components of arginine uptake into mouse embryonic fibroblasts. The dpm at 5 mM were normalized for the protein content and subtracted from the dpm/mg at lower arginine concentrations to reveal the saturable uptake of L-arginine into the mouse embryonic fibroblasts (22). L-Lysine transport studies were identical except 3H-labeled L-Arg and unlabeled L-Arg were replaced with L-[4,5-3H]lysine monohydrochloride and unlabeled L-Lys, respectively. In experiments measuring trans-stimulation of transport, cells were preincubated with 10 mM L-arginine for 2 h in HEPES buffer as described above.
RNA Extraction and Northern Blot Analysis-- RNA was isolated from cells previously washed and cultured for 2 h in HEPES buffer using Trizol according to the manufacturer's instructions. 10 µg of total cellular RNA was resolved on a 0.8% (w/v) agarose gel under denaturing conditions, transferred overnight to Magnacharge blotting membrane in 20× SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), and cross-linked by baking for 2 h at 80 °C. Blots were prehybridized at 42 °C in 50% (v/v) formamide, 10% (w/v) dextran sulfate, 5× standard sodium phosphate-EDTA (0.6 M NaCl, 40 mM NaH2PO4, 4 mM EDTA, pH 7.4), 5× Denhardt's solution (1 g/liter bovine serum albumin, 1 g/liter polyvinylpyrrolidone 40, 1 g/liter Ficoll), 0.1 mg/ml denatured sonicated salmon sperm DNA, and 0.5% (w/v) SDS. 106 cpm/ml of individual cDNA probes labeled with [32P]dCTP using the multiprime DNA labeling system were hybridized to blots overnight at 42 °C. Blots were washed twice for 20 min at 42 °C with 1× SSC, 0.1% (w/v) SDS and once at 42 °C with 0.1× SSC, 0.1% (w/v) SDS before exposure to PhosphorImager cassettes. Densitometry was performed using a PhosphorImager (Molecular Dynamics). When required the blot was stripped with 0.1% (w/v) SDS at 100 °C.
Generation of Cat3-specific Antisera--
A synthetic peptide
corresponding to amino acids 218-238 of mouse and rat Cat3 (12, 16)
was synthesized using mitogen-activated protein resin as a solid phase.
One mg of the peptide was emulsified with 0.2 ml of Titermax and
injected intradermally in the interscapulovertebral region of New
Zealand White rabbits. Four immunizations were carried out at 2-week
intervals. The sera were collected by centrifugation and stored at
20 °C.
Immunofluorescence-- Embryonic fibroblasts were cultured on poly-D-lysine-coated coverslips. Cat3 protein expression was assessed by immunofluorescence essentially as described (23). Cat3 antisera was diluted 1:40 for use in these assays. The anti-rabbit-rhodamine conjugate was used at 1:200. The slides were visualized on an Axiophot epifluorescence microscope.
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RESULTS AND DISCUSSION |
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System y+ Transport Activity in Cat1/
and Cat1+/+ MEF Cells--
Cationic amino acid transport
was evaluated in Cat1
/
embryonic fibroblasts
by measuring the initial rate of 250 µM L-Arg
uptake in the presence and absence of other amino acids (Fig.
1A). Two substrates of System
y+, L-Lys and L-ornithine, both
inhibited L-arginine uptake by greater than 90% consistent
with System y+ activity (2). L-Arginine uptake
was also measurably inhibited by a weaker System y+
substrate, L-His (24), that is partially cationic at pH 7.0 (13, 25). As expected, the neutral (L-alanine and
L-glutamine) and acidic (L-aspartate) amino
acids had no effect on L-arginine uptake. Interestingly,
the capacity of D-arginine to partially inhibit
L-Arg uptake into Cat1
/
cells is
characteristic of Cat3 transport (12, 16). These data indicate that
both Cat1+/+ and
Cat1
/
MEF cells possess System
y+ transport activity.
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Cat3 Is Induced in Cat1/
MEF Cells--
The
relative steady state mRNA expression levels of all three Cat high
affinity transporters were assessed in Cat1
/
and Cat1+/+ cells by Northern analysis (Fig.
3, A and B).
Although a small amount of Cat1 RNA was observed in the
Cat1
/
cells, it is non-functional (17). Fig.
3B shows Cat3 mRNA was increased 11.1 ± 0.5-fold and Cat2 mRNA 2.0 ± 0.3-fold in
Cat1
/
cells as assessed by PhosphorImager
analysis (Fig. 3B). No detectable low affinity transporter
Cat2a mRNA was detectable in either cell type (data not
shown). It is valid to relate the expression of individual
Cats on Northern blots between the two genotypes, but a
direct comparison of the mRNA levels does not necessarily reflect the amount or activity of the transport proteins themselves.
Nevertheless, the data presented in Fig. 3, A and
B provide a good indication of the steady state mRNA
levels of the high affinity arginine transporters. Hence, at the
mRNA level it appears that Cat3 compensates for the
ablation of Cat1.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Chris Perkins for the
Cat1/
cell line and to Drs. Lon Van Winkle
and John McGivan for careful guidance and helpful advice in designing
the transport experiments.
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FOOTNOTES |
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* This research was supported by the Susan G. Komen Breast Cancer Foundation and was conducted in part by the Clayton Foundation for Research, California Division.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Postdoctoral fellow of the Susan G. Komen Breast Cancer Foundation.
Clayton Foundation Investigator. To whom correspondence should
be addressed: UCSD Cancer Center, 9500 Gilman Dr., 0684, La Jolla, CA
92093-0684. Tel.: 619-534-7251; Fax: 619-534-7340; E-mail: cmacleod{at}ucsd.edu.
1 The abbreviations used are: Cat, cationic amino acid transporter; MEF, mouse embryonic fibroblast; gapdh, glyceraldehyde-3-phosphate dehydrogenase.
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REFERENCES |
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