©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Angiotensin II Stimulates System y and Cationic Amino Acid Transporter Gene Expression in Cultured Vascular Smooth Muscle Cells (*)

(Received for publication, June 26, 1995; and in revised form, September 4, 1995)

Boon Chuan Low (§) Murray R. Grigor (¶)

From the Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Several studies have reported enhanced growth of vascular smooth muscle cells (VSMC) (^1)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.


MATERIALS AND METHODS

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 times 10^5 cells/ml, were grown to confluence in a humidified CO(2) 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(2), 1 mM MgCl(2), 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-^3H]arginine (1 min) or 50 µML-[4,5-^3H]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-[^3H]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 times 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 times SSC (1 times 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 times SSPE (1 times SSPE contains 150 mM NaCl, 10 mM NaH(2)PO(4), 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 times SSPE and 0.5% (w/v) SDS, followed by one 15-min wash in 1 times SSPE and 0.1% (w/v) SDS, and then two 15-min washes in 0.7 times 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 times 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.

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(2), 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 times 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 DH5alpha, 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.

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(7) 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 [alpha-P]dCTP to a specific activity greater than 1 times 10^8 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^1,Ile^8-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(4) DNA ligase, T(4) 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(4) 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.


RESULTS

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 [^3H]arginine and [^3H]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).


Figure 1: Ang II stimulates arginine and lysine uptake into VSMC. Quiescent VSMC were treated with (bullet) or without (circle) 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^1,Ile^8-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).



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 alpha-(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(i) at 0.1 mM (data not shown). These results provide strong evidence for System y being stimulated by Ang II.


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, alpha-(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 (bullet) or without (circle) 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 DH5alpha, 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).


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).



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.


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 (bullet) or CAT-2 (circle) 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).


DISCUSSION

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. (^2)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. (^3)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.


FOOTNOTES

*
This work was supported by grants from the Health Research Council of New Zealand, the University of Otago, and the Lee Foundation of Malaysia/Singapore. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Inst. of Molecular and Cell Biology, National University of Singapore, Singapore.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand. Tel.: 64-4-479-7840; Fax: 64-4-479-7866.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cells; Ang II, angiotensin II; MOPS, 3-(N-morpholino)propanesulfonic acid; PCR, polymerase chain reaction; kb, kilobase(s).

(^2)
B. C. Low and M. R. Grigor, unpublished observations.

(^3)
M. Kiaei, B. C. Low, and M. R. Grigor, unpublished observations.


ACKNOWLEDGEMENTS

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.


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