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(Received for publication, June 26,
1995; and in revised form, September 4, 1995) From the
The effect of angiotensin II (Ang II) on the transport of
cationic amino acids has been examined in vascular smooth muscle cells
(VSMC) isolated from rat aortae. Ang II stimulated the uptake rates of
radiolabeled arginine and lysine in a time- and concentration-dependent
manner. The stimulated arginine uptake could be blocked by
pretreatments with cycloheximide and actinomycin D or co-treatment with
valsartan, an antagonist specific for Ang II receptor subtype-1. The
modulation by Ang II was bidirectional as the efflux of arginine was
also stimulated, 5-fold over basal. Using reverse transcription-coupled
polymerase chain reaction methodology, a partial cDNA with 94% sequence
identity to that of cationic amino acid transporter subtype-1 (CAT-1) of mouse fibroblasts was obtained from VSMC. This
sequence also exhibited 14 base changes compared with the sequence of
ecotropic retrovirus receptor (ERR)/CAT-1 from rat
hepatoma. Northern analyses with this partial CAT-1 cDNA and CAT-2 cDNA of mouse T-lymphocytes showed that Ang II rapidly
stimulated the expression of both CAT-1 and CAT-2 in
VSMC. Both signals peaked at 2 h after exposure to Ang II. The CAT-1 signal decayed over the next 6 h to levels 3-fold above
basal, which are maintained up until 24 h. The induced CAT-2 mRNA concentration also decayed rapidly but increased again
between 16 and 24 h to levels comparable with those observed at 2 h.
Several studies have reported enhanced growth of vascular smooth
muscle cells (VSMC) ( A number of reports have described the cloning and expression of
cDNA for the System y This study was set
up to examine the regulation of the System y
2 µl of reverse transcription product were
amplified by polymerase chain reaction (PCR) using primer pairs
designed to bind to a portion of the mouse CAT-1 gene
corresponding to two hydrophobic putative trans-membrane
domains in the translated peptide sequence (Albritton et al.,
1989). The primer sequences were: 1F, CGGAATTCGCTTCATAGCGTACTTTGGCG-3`
(corresponding to sense strand base 1070-1090 of mouse CAT-1; added EcoRI site underlined) and 1R,
5`CGGGATCCGGGACGCTTCCTCACTGTGCC-3` (corresponding to antisense base
1997-2017 of mouse CAT-1; added BamHI site
underlined). The reaction was carried out in a 100-µl volume
containing 4 mM MgCl A second pair of primers, 2F, 5`-CGGAATTCGTCCATTGGCACTCTCCTGGC-3`
(corresponding to sense strand base 1416-1436 of mouse CAT-1; added EcoRI site underlined), and 2R,
5`-CGGGATCCCGTCATTTGCACTGGTCCA-3` (corresponding to antisense strand
base 2054-2072 of mouse CAT-1; added BamHI site
underlined), were used to amplify a 680-base pair fragment of the CAT-1 gene from the reverse transcription product generated
using this same reverse primer rather than the oligo(dT) as described
above. An unexpected product of 1.5 kilobase pairs was also obtained,
blunt-ended, and cloned into EcoRV site of pBluescript
SK
Figure 1:
Ang II stimulates arginine and lysine
uptake into VSMC. Quiescent VSMC were treated with (
Ang II can exert its effect
through two classes of receptors, AT-1 and AT-2. The Ang II stimulation
of arginine uptake was inhibited both by 100 nM
Sar To investigate whether the enhanced
uptake of arginine required ongoing protein synthesis, cells were
pretreated with cycloheximide, an inhibitor of protein synthesis, or
actinomycin D, an inhibitor for mRNA synthesis, before they were
challenged with Ang II. The stimulated uptake of arginine at 24 h of
treatment was completely abolished by these inhibitors (Fig. 2),
indicating that de novo protein synthesis, possibly of the
transporter molecule itself, was required.
Figure 2:
Cycloheximide and actinomycin D inhibit
Ang II-stimulated arginine uptake. Quiescent VSMC were preincubated
with or without 10 µg/ml cycloheximide (Chx) or 10
µg/ml actinomycin D (ActD) for 30 min followed by
treatment with 100 nM Ang II for a further 24 h. Uptake of
arginine was assayed as described under ``Materials and
Methods.'' Values are the means ± S.D. of four replicate
determinations. Differences between values not sharing the same letter
are statistically significant (p <
0.01).
Figure 3:
Stimulation of System y
Because System
y
Figure 4:
Ang II stimulates efflux of arginine from
VSMC. Quiescent VSMC were treated with (
Figure 5:
Sequence variations between CAT-1 of rat VSMC and rat hepatoma. A, the deduced peptide
sequence of CAT-1 partial cDNA from VSMC (cat1Rsmc)
was aligned to the corresponding regions of rat hepatoma (cat1Rliv; Wu et al.(1994)), mouse fibroblasts (cat1M; Albritton et al.(1989)), and human
lymphocytes (cat1H; Yoshimoto et al. (1991)).
Differences in the residues between sequences of rat VSMC and rat
hepatoma are highlighted in boxes, whereas differences between
sequences of rat VSMC and mouse fibroblasts are indicated by asterisks. Putative trans-membrane domains as
predicted for the mouse sequence by Albritton et al.(1989) are
depicted with Roman numerals. Residues are numbered from the
derived VSMC peptide sequence, with residue 1 (top)
corresponding to residue 291 of the mouse sequence (bottom). B, cDNA sequence alignment showing the base changes between CAT-1 of rat VSMC and rat hepatoma (boxes) that
translate into the cluster of three unique amino acid residues for rat
VSMC CAT-1 as indicated (residues 151, 155, and 161 as in A). Bases are numbered according to the rat VSMC sequence (top) or the mouse fibroblasts sequence (bottom).
Figure 6:
Ang II stimulates CAT-1 and CAT-2 expression in VSMC from normotensive and hypertensive
rats. Quiescent VSMC isolated from outbred normotensive Wistar-derived (N) rats or inbred hypertensive GH or SHR were treated with
100 nM Ang II for the times indicated before total RNA was
prepared and analyzed (20 µg/lane) for the expression of CAT-1, CAT-2, and TSP-1 by Northern blotting
as described under ``Materials and Methods.'' The same blots
were sequentially stripped and reprobed with different cDNA probes or
oligonucleotide probe for 28 S rRNA whose hybridization signals serve
as internal controls for unequal loading between samples. The intensity
of CAT-1 and CAT-2 mRNA signals were analyzed using
an imaging densitometer and were normalized to the signals of 28 S rRNA
within each sample. The ratio of each value (normalized to one for the
0 h) measures the relative expression of CAT-1 (
The filters were stripped and reprobed for
transcripts of rat thrombospondin-1 (TSP-1) encoding an
extracellular matrix component of VSMC (Majack et al., 1986,
1988) and glucose transporter isotype-1 (GLUT-1), both of
which increase after exposure to Ang II (Hahn et al., 1993;
Low et al. 1992). The TSP-1 probe detected a
transcript of 5.5 kb, equivalent in size to those reported earlier for
this gene (Laherty et al., 1992; Hahn et al., 1993).
The abundance of TSP-1 mRNA rapidly increased after Ang II
treatment in a manner that was very similar to that for CAT-1.
This was most obvious in the RNA samples obtained from the SHR cells.
In contrast, Ang II stimulated the expression of GLUT-1 mRNA
after 2 h to a level that was maintained throughout the remainder of
the 24-h period of treatment (data not shown), consistent with our
previous observation (Low et al., 1992). The early (2 h)
accumulation of CAT-1 mRNA occurred maximally at 100 nM Ang II, with a threshold response at 1 nM (data not
shown) similar to the observed dose-response of Ang II-stimulated
arginine uptake (Fig. 1C). Thrombin (1 unit/ml),
epidermal growth factor (100 ng/ml), fetal calf serum (10% v/v) (agents
known to increase glucose transporter activity in VSMC; Low et
al.(1992)), A23187 (6 µM),
12-O-tetradecanoylphorbol-13-acetate (100 ng/ml), all
increased the expression of CAT-1 and TSP-1 after 2 h
of treatment but to levels less than those obtained with Ang II. In
addition, down-regulation of PKC activity by prolonged treatment with
12-O-tetradecanoylphorbol-13-acetate (Taubman et al.,
1989) attenuated the maximal response to Ang II, suggesting that PKC
was required in Ang II activation of CAT-1 expression (data
not shown). This study shows clearly that Ang II can stimulate both the
System y The identification of the murine ERR gene as a CAT-1 gene followed from the expression of the cRNA in the Xenopus oocyte system (Kim et al., 1991; Wang et
al., 1991). Similarly the Tea/CAT-2 gene
stimulated System y The fact that we detected both CAT-1 and CAT-2 transcripts does not necessarily mean that both
gene products are active in the VSMC. Assuming that the rat homologues
have similar kinetics to the mouse CATs, it would not be
possible to distinguish between them on kinetic grounds, and our
kinetic data would be consistent with either. Several murine organs,
however, such as skeletal muscle, stomach, skin, lung, and uterus,
express both CAT-1 and CAT-2 (MacLeod et
al., 1994). Furthermore, there exist distinct patterns of
regulation for these CAT isotypes in different tissues. For
example, MacLeod et al.(1994) recently showed that quiescent
lymphocytes express only CAT-1 mRNA, whereas upon activation
by concanavalin A, these cells express both genes. This group also
reported that liver expression of CAT-2 mRNA is constitutive
and unaltered by partial hepatectomy or food deprivation. This is in
contrast to their findings for skeletal muscle where CAT-2 mRNA accumulated in response to surgical trauma and fasting but
without any changes in the basal level of CAT-1 transcripts.
These same treatments, however, did not modify the expression of either CAT-1 and CAT-2 in the uterus, which largely consists
of smooth muscle cells (MacLeod et al., 1994). The
co-stimulation of the expression of CAT-1 and CAT-2 genes in the smooth muscle cells of rat aorta by Ang II and fetal
calf serum as reported here provides the first example of parallel
hormonal regulation for System y The early and
transient expression of the CAT-1 and CAT-2 transcripts does not appear to match well the increased System
y
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27577-27583
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
and Cationic Amino Acid
Transporter Gene Expression in Cultured Vascular Smooth Muscle Cells (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)in culture in response to vasoactive
peptides and growth factors such as angiotensin II (Ang II) (Berk et al., 1989; Harris et al., 1990; Griendling et
al., 1986; Schelling et al., 1991; Scott-Burden et
al., 1992). A source of amino acids is necessary for the synthesis
of new proteins, and changes in the uptake rates of specific amino
acids may be important regulated steps in the growth response of these
cells. The transport of amino acids across the cell membrane is
catalyzed by a family of amino acid transporters that can be
distinguished by their substrate specificity and requirement for a
co-transport of Na (Christensen, 1990; Cheeseman,
1991). We have recently characterized two major transport systems in
VSMC that are responsible for the uptake of several nutritionally
essential amino acids (Low et al., 1993). In particular, the
uptake properties of the system mediating lysine and arginine transport
were shown to resemble System y
as described
previously in other cell types (Christensen, 1990; Cheeseman, 1991).
transporter in several cell
types. At least two forms for the cationic amino acid transporter (CAT) gene have been described. The first of these, CAT-1, was initially identified as the gene for an ecotropic
retrovirus receptor (ERR) in murine fibroblasts (Albritton et al., 1989). Its ability to encode the System y
transporter was established following expression studies in Xenopus oocytes (Kim et al., 1991; Wang et
al., 1991). A human homologue of this gene has also been cloned
from T-lymphocytes (Yoshimoto et al., 1991). A second form, CAT-2, was first detected as a murine T-cell early activated
gene (Tea) (MacLeod et al., 1990), where it was also
found to encode System y
activity with similar
properties to that encoded by CAT-1 (Kakuda et al.,
1993). Both are high affinity (K
=
0.1 mM), low capacity transporters of cationic amino
acids, and their activity can be stimulated by the presence of other
substrate amino acids on the opposite side of the membrane (trans-stimulation). Subsequently CAT-2a, a variant
of CAT-2, was cloned from mouse liver and was found to be
expressed only in these cells. In contrast to CAT-1 and CAT-2, this gene encodes a System y
transporter that possesses low affinity but high capacity (with K
between 2 and 5 mM) and lacks
the trans-stimulation property. It is believed to arise from
an alternative splicing of CAT-2 (Closs et al.,
1993a, 1993b). DNA elements controlling the expression of CAT-2 have been described (Finley et al., 1995), but equivalent
information is not available for CAT-1.
activity
in cultured VSMC and to investigate the corresponding changes in the CAT-1 and CAT-2 mRNA concentrations in these cells in
response to Ang II treatment. The results show that Ang II stimulates
transport of arginine via System y
that is accompanied
by increased levels of both CAT-1 and CAT-2 mRNA with
differential induction profiles.
Cell Culture
Rats from an outbred
Wistar-derived colony or rats from inbred New Zealand genetically
hypertensive (GH) and spontaneously hypertensive rat (SHR) strains were
obtained from the University of Otago Animal Breeding Station. Cells
were isolated from aortae of 15-20-week-old rats by enzymatic
digestion (Harris et al., 1990) and passaged weekly as
described previously (Low et al., 1992). Briefly, replicates
of VSMC subcultures, plated at approximately 2 
10
cells/ml, were grown to confluence in a humidified CO
incubator at 37 °C on 24-well plastic dishes containing
Dulbecco's modified Eagle's medium with 10% (v/v) fetal
calf serum. The cells were then made quiescent for 48 h in serum-free
Dulbecco's modified Eagle's medium supplemented with 0.05%
(w/v) fatty acid-free bovine serum albumin. The medium was changed
daily, and uptake assays were carried out at least 12 h after the last
medium change. Unless otherwise stated, all experiments were performed
with cell lines isolated from the outbred Wistar rats and at passages
4-12.Transport Assays
VSMC grown as monolayers
were washed twice with 1.0 ml of prewarmed HEPES buffer (140 mM NaCl, 5 mM KCl, 0.9 mM CaCl, 1
mM MgCl, 5.6 mMD-glucose, and
25 mM HEPES, pH 7.4). Net amino acid uptake was measured under
zero-trans conditions after cells had been incubated in the
uptake buffer containing 10 mM sucrose for 2 or 3 h to deplete
the intracellular amino acid pools. Cells were washed and then
incubated immediately in the same buffer containing 50 µML-[2,3,4,5-
H]arginine (1 min) or 50
µML-[4,5-H]lysine (2 min)
each at 1-2 µCi/ml at 37 °C. The times for these
measurements were chosen to be within linear portions of uptake curves
(data not shown). To terminate the uptake, the medium was aspirated,
and cell layers were rapidly rinsed twice with the appropriate ice-cold
buffer and lysed in 0.5 ml of 5% (w/v) trichloroacetic acid. Total
radioactivity was counted in 6 ml of a scintillant containing 4.5
g/liter diphenyloxazole in toluene/Triton X-100 (3:1 by volume). To
correct for nonspecific uptake or binding, cells were incubated in
parallel wells containing 100 mM arginine in the uptake
buffer, the fraction of the radioactivity associated with the cells
determined, and these values were subtracted from each data point.Efflux Studies
VSMC were preincubated in
HEPES buffer for 2 h with 10 mM sucrose. They were then washed
twice with prewarmed amino acid-free buffer and loaded with L-[H]arginine (8 min) at 50 µM (2 µCi/ml). Cells were washed once very quickly (<5 s) with
prewarmed buffer; 0.5 ml of this fresh buffer was then added for the
times indicated before it was removed by aspiration. Efflux was
terminated by one rapid wash (<5 s) in ice-cold buffer. Cells were
lysed in 0.5 ml of 5% (w/v) trichloroacetic acid, and the total
radioactivity associated with cells was counted in 6 ml of
toluene/Triton scintillant. The first order rate constant for the
efflux was estimated by linear regression of the plot of percentage cpm
remaining inside the cells to that at zero time.Estimation of Protein Content
Cells
washed with the appropriate buffer were solubilized with 0.2 M NaOH at 37 °C for 3 h, and aliquots were used for the
determination of protein content using the bicinchoninic acid method
(Smith et al., 1985) with bovine serum albumin as the
standard.RNA Isolation and Analysis
Total RNA was
prepared from VSMC cultures or rat tissues using the acid guanidinium
thiocyanate/phenol/chloroform method of Chomczynski and Sacchi(1987) as
modified (Low et al., 1992) and quantified
spectrophotometrically. For Northern blot analysis, total RNA (20
µg) was denatured with 2.2 M formaldehyde and separated by
1% (w/v) agarose gel electrophoresis in MOPS buffer (20 mM MOPS, 10 mM EDTA, and 50 mM sodium acetate, pH
7.0) as described by Sambrook et al.(1989). RNA was electro
blotted onto Hybond-N membranes in 1
TAE
buffer (40 mM Tris, 5 mM sodium acetate, and 1 mM EDTA, pH 7.8) and fixed with 50 mM NaOH followed by brief
rinsing in 2 SSC (1 SSC contains 150 mM NaCl,
and 15 mM sodium citrate, pH 7.0). Membranes were
prehybridized at 65 °C in 50 ml buffer containing 2 SSPE (1
SSPE contains 150 mM NaCl, 10 mM
NaHPO
, and 1 mM EDTA, pH 7.4), 0.1%
(w/v) sodium pyrophosphate, 0.5 mg/ml heparin, and 2.5% (w/v) SDS for 1
h. Hybridization was carried out in 5 ml of the fresh buffer containing
the desired probe for at least 12 h. Membranes were washed at 65 °C
for all probes used except for mouse CAT-2 cDNA (60 °C) in
50-ml solutions as follows: two 10-min washes in 2 SSPE and
0.5% (w/v) SDS, followed by one 15-min wash in 1 SSPE and 0.1%
(w/v) SDS, and then two 15-min washes in 0.7 SSPE and 0.1%
(w/v) SDS. Membranes probed with oligonucleotide for 28 S rRNA were
only washed once for 10 min in the initial step. Washed membranes were
exposed to Cronex x-ray films at -80 °C with double
intensifying screens for different periods of time. Films were
developed, washed, and fixed in an Allpro-100 automatic film developer.
The relative intensity of bands on autoradiograms was quantified with
an imaging densitometer. To correct for unequal loading of RNA samples,
concentrations of mRNA were expressed as the ratio of the transcript of
interest to that of 28 S rRNA (de Leeuw et al., 1989).Reverse Transcription-coupled PCR Cloning of VSMC
CAT-1 cDNA
Total RNA (10 µg) isolated from cells grown
in the presence of 10% fetal calf serum was reverse transcribed using
oligo(dT) (0.1 µg) and avian myeloblastosis virus reverse
transcriptase (21 units) in a 50-µl reaction volume containing 10
mM dithiothreitol, a mixture of dATP, dTTP, dGTP, and dCTP
(each at 1 mM), 50 units of RNAsin, a ribonuclease inhibitor,
and 0.1% (w/v) sodium pyrophosphate in 1 first strand buffer
(Boehringer Mannheim). Total RNA was heated to 80 °C for 10 min and
chilled on ice before being added to the reaction mixture. Reactions
were carried out for 1 h at 42 °C and terminated by heating at 90
°C for 5 min., 0.1 mg/ml bovine serum
albumin, a mixture of dATP, dTTP, dGTP, and dCTP (each at 0.2
mM), 50 pmol each primer, and 2 units of Taq DNA
polymerase in 1 Jeffreys buffer (Jeffreys et al.,
1988). The reaction mixture, layered with paraffin oil (40 µl), was
subjected to the following PCR running conditions: denaturation at 93
°C for 2 min (initial cycle) and 0.5 min (subsequent cycles),
annealing at 55 °C for 0.5 min, and extension at 72 °C for 1
min and 5 min (final cycle) (total, 35 cycles). A product of an
expected size (950 base pairs) was obtained and purified by gel
electrophoresis in 1% (w/v) low melting point agarose. It was
blunt-ended and ligated into the pBluescript SK vector
at the SmaI restriction site. Electroporation into Escherichia coli, strain DH5
, yielded two clones
containing inserts, the DNA sequences of which were determined in both
directions using an ABI 373 automated DNA sequencer. The DNA sequence
of these clones (pRCAT-1), and the deduced peptide sequence both share
94% identity with those of CAT-1 from mouse fibroblasts and
85% identity with those of CAT-1 from human T-lymphocytes. as above. Sequencing at the 5` and 3` termini of
three separate clones revealed high homology (91% identity) between
this partial cDNA and the thrombospondin-1 gene of mouse embryo kidney
(Laherty et al., 1992). This clone was named pRTSP-1 and was
subsequently used as a probe for rat TSP-1 mRNA.
Preparation of DNA Probes
The DNA probes
used in this study were: CAT-1 cDNA, a 0.95-kilobase pair SacI-HincII fragment from pRCAT-1; TSP-1 cDNA, a 1.5-kilobase pair SacI-HincII
fragment from pRTSP-1; GLUT-1 cDNA, a 1.6-kilobase pair BglII fragment from pSPGT-1 (Gould and Lienhard, 1989); and a
DNA oligonucleotide derived from the rat 28 S rRNA sequence:
5`-AACGATGAGAGTAGTGGTATTTCACC-3` (de Leeuw et al., 1989). A
mouse CAT-2 partial cDNA was prepared by PCR amplification of
the sequence between base 1036 and base 2397 of a MCAT-2 parental clone, 20.5.1 cDNA (MacLeod et al., 1990) using
2F as the forward primer and a T
promoter primer of the
vector for the reverse. Inserts for each gene were released from the
plasmids using appropriate restriction endonucleases, identified
following electrophoresis in 1.5% (w/v) low melting point agarose gels,
excised, and extracted into a final concentration of 5 ng/µl.
Purified PCR products of CAT-1 and CAT-2 cDNA were
also used directly for radiolabeling. DNA fragments (25 ng) were
labeled with [-P]dCTP to a specific
activity greater than 1
10
cpm/µg DNA using the
random priming method and purified through Nick columns.
The oligonucleotide of 28 S rRNA was labeled with
[
-
P]dATP by the kinase/end-labeling method
as described by Sambrook et al.(1989).
Materials
Cell culture media, amino acids (L-isomers), and analogues, angiotensin II, cycloheximide,
actinomycin D, calcium ionophore A 23187,
12-O-tetradecanoylphorbol-13-acetate, and
Sar,Ile-angiotensin II were all obtained from
Sigma. Valsartan was obtained from CIBA-Geigy Ag Basel. Culture dishes
were supplied by Becton Dickinson Labware, and fetal calf serum was
supplied by Life Technologies, Inc. Nucleotides, T DNA
ligase, T DNA polymerase, Taq DNA polymerase, calf
intestine alkaline phosphatase, and Klenow enzyme were purchased from
Boehringer Mannheim, and restriction enzymes were obtained from New
England Biolabs. DNA purification kits, avian myeloblastosis virus
reverse transcriptase, T polynucleotide kinase, and RNAsin
were from Promega Corporation, and all radioisotopes, Hybond
N membranes, and labeling kits were supplied by
Amersham International.
Statistical Analyses
The results were
expressed as the means ± S.D. of multiple determinations. Where
appropriate, statistical comparisons were made using analysis of
variance and the Newman-Keuls multiple range test (Zar, 1974) or the t test.
Angiotensin II Stimulates Uptake of Cationic Amino
Acids into Vascular Smooth Muscle Cells
The effect of Ang
II on System y was studied by measuring the uptake of
[
H]arginine and [H]lysine.
Confluent VSMC were made quiescent for 2 days in media devoid of fetal
calf serum before they were challenged with hormones. Cells were first
depleted of intracellular amino acids by incubation in an amino
acid-free buffer to enable the determination of the true influx rate of
arginine (Low et al., 1993). Ang II stimulated the uptake of
arginine and lysine in a time- and concentration-dependent manner. The
enhanced activity for arginine uptake was not apparent until after at
least 6 h, and it reached the maximum by 1.8-2-fold after 24 h of
treatment, whereas enhanced lysine uptake was detectable after 3 h (Fig. 1, A and B). After 24 h of exposure to
Ang II, the total amount of cellular protein/well had increased by 38
± 5% (p < 0.01, data not shown). Ang II stimulated
arginine uptake with a threshold concentration of 0.1 nM, a
maximal response occurred at 100 nM Ang II, and the EC was between 1 and 5 nM Ang II (Fig. 1C).
This response curve closely resembles that for the increase in total
cellular protein (data not shown) and the Ang II activation of glucose
transporter activity in these cells previously observed in this
laboratory (Low et al., 1992).
) or without
(
) 100 nM Ang II for the times indicated, and uptake of
arginine (A) or lysine (B) was measured as described
under ``Materials and Methods.'' Values are the means
± S.D. of three replicate determinations. C, quiescent
VSMC were treated with indicated concentrations of Ang II for 24 h, and
initial rates of arginine uptake were determined as described under
``Materials and Methods.'' Values are the means ± S.D.
of four replicate determinations.
,Ile-Ang II, a nonspecific antagonist that
binds both AT-1 and AT-2 receptors, and by 100 nM valsartan,
an AT-1-specific antagonist (data not shown). This result shows that
the Ang II stimulation of arginine transport in VSMC is mediated via
the AT-1 receptor subtype.
Ang II Stimulates Arginine Transport via System
y
Previously we argued that the only route
of transport for cationic amino acids in VSMC is via System
y (Low et al., 1993). In order to establish
that the enhanced transport of arginine due to Ang II treatment was via
System y
rather than through the activation of some
other system, the uptake of arginine was measured in the presence of
other test substrates in cis (Fig. 3). The results show
that the enhanced component of arginine uptake was inhibited completely
by other cationic substrates, such as lysine and ornithine, but not by
-(methylamino)isobutyric acid, a test substrate for System A, or
by 2-amino-2-norbornane-carboxylic acid, a test substrate for System L
(Low et al., 1994). The considerable inhibition of arginine
transport by leucine in the presence of Na was
consistent with the noncompetitive inhibition of System y
activity described earlier (Low et al., 1993). The
inhibition by lysine of arginine transport in stimulated cells was
competitive with an K
at 0.1 mM (data not shown). These results provide strong evidence for System
y
being stimulated by Ang II.
amino acid transport by Ang II. Quiescent VSMC were treated with (hatched bars) or without (open bars) 100 nM Ang II for 24 h, and uptake of 50 µM arginine was
measured as described under ``Materials and Methods'' in the
presence of 5 mM sucrose (control) or 5 mM amino acids or analogues as indicated. Values are the means
± S.D. of three replicate determinations. Orn, ornithine; BCH,
2-amino-2-norbornane-carboxylic acid; MeAIB,
-(methylamino)isobutyric acid.
transporter also exhibits exchanger properties where
it can mediate both the uptake and exodus of substrates into and from
the cell (Low et al., 1993), the efflux of arginine under
conditions that stimulated the influx of this substrate was also
examined. Control cells or cells treated with Ang II for 24 h were
pulsed with a similar amount of radioactive arginine for 8 min and
assayed for the amount of radioactivity left within the cells as a
function of time (Fig. 4). Ang II-stimulated cells released
arginine back into amino acid-free media with a first order rate
constant of 0.01 s
for the first 4 min compared with
0.002 s
for the control cells. This observation
shows that Ang II modulates the transport of arginine in both
directions and provides further evidence that the Ang II stimulation of
arginine transport results from activation of System
y
.
) or without () 100
nM Ang II for 24 h. Cells under zero-trans condition
were pulsed with 50 µM (2 µCi/ml) arginine, and the
release of this substrate was then determined at times indicated as
described under ``Materials and Methods.'' Values are the
means ± S.D. of three replicate
determinations.
Cloning of Partial Rat VMSC CAT-1 cDNA
In
order to investigate the correlation between enhanced uptake of
arginine via System y and the expression levels of CAT genes in VSMC, reverse transcription-coupled PCR was used
to amplify a portion of the gene for the rat CAT-1 in these
cells to be used as a probe for Northern analyses. Primers were
designed based on the sequences that encode hydrophobic, putative trans-membrane domains of mouse CAT-1 (Albritton et
al., 1989). The amplified product (950 base pairs) was ligated
into pBluescript SK
and cloned in DH5
, and two
separate clones were sequenced in both directions. Comparison of the
sequence obtained and its deduced peptide sequence showed that both
share 94% identity with those of CAT-1 from mouse fibroblasts
and 85% identity with those of CAT-1 from human T-lymphocytes.
This partial cDNA of VSMC CAT-1 also shows 14 base changes
compared with the sequence of ERR/CAT-1 from rat
hepatoma that was subsequently published (Wu et al., 1994).
These variations translate into five amino acid substitutions, with
three of them clustering between residues 440 and 453 of the deduced
murine CAT-1 peptide corresponding to a putative
extramembraneous loop in the translated gene product (Fig. 5).
Cultured Rat VSMC Express Both CAT-1 and CAT-2
mRNA
The partial CAT-1 cDNA, along with a cDNA for
the murine CAT-2, was used as a probe in Northern analyses. To
confirm the specificity of these probes for the detection of specific CAT mRNA species, total RNA were extracted from rat aortic
VSMC, three rat tissues (brain, liver, and kidney), and mouse 3T3
fibroblasts. All cells were cultured and maintained in media containing
10% fetal calf serum. When CAT-1 cDNA probe was used in
hybridization with high stringency, a discrete band at 7.9-8 kb
was clearly identified in samples from VSMC, 3T3 fibroblasts, rat
brain, and rat kidney, but it was completely absent from adult rat
liver. When similar blots were probed with CAT-2 cDNA, only
the VSMC and rat liver samples showed a strong transcript signal at
8 kb (data not shown). These results verified the specificity of
the probes used and showed that VSMC express both CAT-1 and CAT-2, thus validating the use of these two probes to study
the regulation of these CAT isotypes in VSMC.
Differential Stimulation of CAT-1 and CAT-2 Gene
Expression by Ang II
The effect of Ang II on the expression
of CAT-1 and CAT-2 in VSMC was then investigated.
Cells from three different isolates of VSMC prepared from the outbred
Wistar rats and inbred rats of the GH and SHR strains were made
quiescent by the removal of serum for 48 h and treated with Ang II (100
nM) for different lengths of time, and total RNA was isolated
and hybridized with cDNA probes of CAT-1 and CAT-2.
By normalizing the densitometric signals detected with these probes to
those obtained using an oligonucleotide probe for 28 S rRNA as the
internal control, a semiquantitative measure of the abundance of CAT-1 and CAT-2 mRNA in these samples was obtained.
In the control untreated cells, the levels of these two transcripts
were just detectable after prolonged exposure of autoradiograms. Ang II
rapidly induced the expression of both CAT-1 and CAT-2 in VSMC with unique induction profiles (Fig. 6). Both
signals peaked 2 h after exposure to Ang II, but the maximal induction
of CAT-1 was between 11- and 20-fold basal compared with
4-7-fold induction for CAT-2. For both the Wistar and
the GH cells, the CAT-1 signals decayed over the next 6 h to
levels only 3-fold above basal, which were maintained up until 24 h.
The induced CAT-2 signals also decayed rapidly, but the mRNA
reappeared between 16 and 24 h at levels comparable with those observed
at 2 h. A slightly different pattern was observed with the SHR cells
where the maximal induction of CAT-2 was delayed with respect
to CAT-1 and occurred after 4 h of Ang II treatment rather
than 2 h. In addition, no chronic stimulation of CAT-1 was
detected in these cells, whereas there was an apparent reactivaton of CAT-2 after 24 h (Fig. 6). With all cell lines, a minor
transcript of about 3.5 kb was also detected in samples treated with
Ang II when hybridized to CAT-1 cDNA. The induction profile
for this transcript, however, did not match that of major CAT-1 transcript. This minor hybridization signal, the identity of which
remains to be established, has also been observed previously in rat
hepatocytes (Closs et al., 1992) and rat hepatoma cells (Wu et al., 1994). No equivalent band was detected with the CAT-2 probe.
) or CAT-2 () in each isolate after treatment with 100 nM Ang II for the times indicated (graphs).
activity of cultured aortic smooth muscle
cells and the mRNA concentrations of both CAT-1 and CAT-2. Several lines of evidence argue that arginine uptake in
both the basal and Ang II-stimulated cells is mediated solely by the
System y
activity. These include the kinetic data and
inhibition data we report. The timing of the increase in arginine
transport activity is slower than that we reported for glucose
transporter activity (Low et al., 1992) or for leucine uptake. (
)This timing is also quite different from the changes in
the concentrations of the CAT-1 and CAT-2 mRNAs. This
raises several important questions. Firstly, do the CAT-1 and CAT-2 genes actually encode for the System y transporter? What is the advantage to the cell to express both,
and indeed are both coding for functionally active transporters in the
VSMC? What is the significance of the early transient expression of
these genes following Ang II treatment? What function might the very
large transcript have in regulating the expression at the functional
level?
activity when expressed in the
oocyte system (Kakuda et al., 1993). The deduced peptide
sequence for our partial CAT-1 clone is very similar to but
distinct from both the murine fibroblast CAT-1 (Albritton et al., 1989) and a rat hepatoma homologue recently reported
(Wu et al., 1994). The amino acid differences between the two
rat sequences all occur in putative extramembraneous loops of the
molecule and three of the five cluster between trans-membrane
helices X and XI. The nucleotide changes corresponding to this region
have been verified through the sequencing of two independent clones in
both directions. It is unlikely that the differences result from either
PCR or sequencing errors. Indeed independent analysis of an equivalent
cDNA derived from lactating rat mammary RNA shows an identical sequence
to that observed for the VSMC. (
)Both the transcript sizes
detected by the CAT-1 and CAT-2 probes and the
tissue-specific expression of these genes are similar to published data
for the murine clones. This argues strongly that the genes detected in
the VSMC are the rat homologues of those encoding System y in the mouse.
isotypes within a
homogenous cell system in vitro and suggests that smooth
muscle cells in different tissues or organs may be differentially
regulated. At this stage it is not possible to relate the different CAT isoforms to the physiology of any nonhepatic tissue. Other
transport systems, for example the glucose transporter, exist as
members of gene families (Gould and Bell, 1990), and more than one
isoform may be expressed in any one cell type.
activity detected following Ang II treatment,
suggesting that there may be additional translational or
post-translational regulation of the formation of the functional
protein. Both CAT-1 and CAT-2 have very large
transcript sizes (8 kb) relative to the coding sequence (3 kb). In
other genes large untranslated regions in the mRNA contain elements
controlling both the translation and stability of the RNA (Leibold and
Guo, 1993; Sachs, 1993). The function of the smaller 3.5-kb transcript
detected by the CAT-1 probe has yet to be established. Current
work in this laboratory is directed at obtaining the full cDNA sequence
for the VSMC CAT-1 gene, which we suggest be termed rat CAT-1b to distinguish it from the hepatoma gene.
)))
We thank Dr. Carol MacLeod (University of California,
San Diego) for making available the MCAT-2 parental clone and
information about CAT-2 expression prior to publication.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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