(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) ()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).
A number of reports have described the cloning and expression of
cDNA for the System y 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.
This study was set
up to examine the regulation of the System y 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.
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, 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.
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 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.
Figure 1:
Ang II stimulates arginine and lysine
uptake into VSMC. Quiescent VSMC were treated with () 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.
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,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.
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 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.
Because System
y 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
.
Figure 4:
Ang II stimulates efflux of arginine from
VSMC. Quiescent VSMC were treated with () 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.
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 () or CAT-2 (
) in each isolate after treatment with 100 nM Ang II for the times indicated (graphs).
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 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?
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 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.
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 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.
The early and
transient expression of the CAT-1 and CAT-2 transcripts does not appear to match well the increased System
y 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.