©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1 and Tumor Necrosis Factor- Stimulate the cat-2 Gene of the

L

-Arginine Transporter in Cultured Vascular Smooth Muscle Cells (*)

(Received for publication, November 13, 1995; and in revised form, January 30, 1996)

Denzil J. Gill Boon Chuan Low (§) Murray R. Grigor (¶)

From the Department of Biochemistry and Center for Gene Research, University of Otago, Dunedin, New Zealand 9001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The production of nitric oxide (NO) from L-arginine by nitric oxide synthase (NOS) in cytokine-stimulated vascular smooth muscle cells (VSMC) is thought to play an important role in the pathophysiology of several vascular disease states including septic shock. This study examines the relationship between cytokine-stimulated NO production and L-arginine transport in cultured VSMC. Cultured VSMC from rat aorta were stimulated with interleukin-1beta, tumor necrosis factor-alpha, and/or angiotensin II (Ang II); and the accumulation of nitrite, a stable product of NO metabolism, in the culture media and the rates of net L-arginine uptake were measured. Interleukin-1beta and tumor necrosis factor-alpha, alone or in combination, stimulated both the uptake of L-arginine and the accumulation of nitrite in the culture media in a dose-dependent manner. Inhibition of NOS activity by substituted analogues of L-arginine had no effect on cytokine-stimulated L-arginine transport. Ang II in the presence of cytokines up-regulated L-arginine transport while inhibiting nitrite accumulation. Two forms of the L-arginine transporter, cat-1b and cat-2, are expressed in VSMC. Northern analysis revealed that the cytokine-stimulated increase in L-arginine transport coincided with increased levels of cat-2 mRNA. In contrast, cat-1b does not appear to be regulated by cytokines at the mRNA level, although significant increases in response to Ang II were observed. These results show that, while cytokines can stimulate both NOS activity and L-arginine uptake, NO production is not required to signal the increase in L-arginine transport. Furthermore, Ang II and cytokine stimulation of L-arginine uptake involves the differential regulation of the cationic amino acid transporter (cat) genes.


INTRODUCTION

Nitric oxide (NO) (^1)is an important regulator of vascular tone(1) , and several studies have shown that excess production of NO may contribute to the hypotension and vascular hyporesponsiveness observed in septic shock. The activation of inflammatory cells during infection leads to the release of cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and -interferon. These cytokines, along with bacterial toxins such as lipopolysaccharides (LPS), can stimulate the expression of the inducible isoform of nitric oxide synthase (iNOS) in most vascular cells, including tissue macrophages, endothelial cells, and vascular smooth muscle cells (VSMC)(2, 3, 4, 5) . Expression of iNOS leads to the sustained production of NO as measured by the accumulation of its stable end product, nitrite. Recently, treatment of sepsis patients with NOS inhibitors was shown to produce a widespread increase in vascular tone and a rise in blood pressure(6) . This observation, along with the finding that mice lacking a functional inos gene have an increased survival rate over wild-type animals during LPS-induced septic shock(7) , strongly suggests that the overproduction of NO is an important mechanism in the vascular dysfunction and mortality associated with sepsis.

Because NO production requires free L-arginine as a substrate, the availability of L-arginine provides a potential regulatory site for NO production. Cationic amino acids are transported across mammalian cell membranes by a small number of well defined transport systems (y, b, yL)(8, 9, 10) . In VSMC, transport (influx and efflux) of cationic amino acids appears to be mediated by the Na-independent System y(11, 12) . Genes encoding three murine System y transporters (cat-1, cat-2, and cat-2a) have been cloned(10, 13, 14, 15, 16, 17, 19) and shown to infer System y transport activity when expressed in Xenopus oocytes(10, 13, 14, 17, 19) . Recently, genes for two rat homologues for cat-1 have been reported. The first was detected and cloned from a rat hepatoma genomic library (20) . Subsequently, we have reported the partial cloning of a cDNA for a second gene (cat-1b) from VSMC(12) . These cells also express a cat-2 homologue.

In murine macrophages LPS- and -interferon-induced NO production is absolutely dependent on the availability of extracellular L-arginine. When murine macrophages were treated with LPS, System y transport activity was shown to be increased more than 10-fold(21, 22) . The induction of L-arginine transport was sensitive to the protein synthesis inhibitor cycloheximide, implying that the LPS-stimulated activity of System y in these cells requires the de novo protein synthesis(23) . The aim of the present work was to determine whether the cytokine stimulation of NO production in VSMC was associated with changes in the System y transport activity and with specific changes in the expression of cat genes.


EXPERIMENTAL PROCEDURES

Cells and Cell Cultures

Cultures of rat aortic smooth muscle cells were established and maintained as described previously (24, 25, 26) . Cells were isolated from aortae of 15-20-week-old rats by enzymatic digestion (24) and passaged weekly as described previously (12, 26) . Cells were grown to confluence in 24-well plastic dishes with Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, made quiescent by removal of serum for 48 h, and then exposed to cytokines or hormones for periods of up to 24 h. Nitrite accumulation in the media was measured (27, 28) with the assay modified to a 300-µl reaction volume for use in 96-well microtiter plates.

L-Arginine Uptake

L-Arginine uptake was determined as described by Low and Grigor (12) in cells that had previously been depleted of intracellular free amino acids by incubation for 3 h in 0.5 ml of Na/HEPES buffer (140 mM NaCl, 5 mM KCl, 0.9 mM CaCl(2), 1 mM MgCl(2), 20 mMD-glucose, and 25 mM HEPES, pH 7.4). Buffer was removed by aspiration, and L-arginine uptake was measured by incubating the cells for 1 min at 37 °C with 250 µl of prewarmed Na/HEPES buffer containing L-[2,3,4,5-^3H]arginine (50 µM, 2 µCi/ml). To terminate the uptake, the medium was aspirated, and cell layers were rapidly rinsed twice with ice-cold Na/HEPES buffer and lysed in 0.5 ml of 5% (w/v) trichloroacetic acid, and total radioactivity was determined. To correct for nonspecific uptake and binding, cells were incubated in parallel wells with buffer containing L-[^3H]arginine (100 mM, 2 µCi/ml), the fraction of the radioactivity associated with the cells was determined, and this fraction was then subtracted from each data point. L-Arginine uptake was linear over the time period used.

Estimation of Protein Content

Cells washed with the Na/HEPES 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 (29) with bovine serum albumin as the standard.

RNA Isolation and Analysis

Total RNA was prepared from VSMC cultures using the acid guanidinium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (30) as modified (25) and quantified spectrophotometrically. Electrophoresis and transfer of RNA to Hybond-N membranes were performed as described previously (12) . Hybridization was carried out for at least 12 h with cDNA probes for rat cat-1b or mouse cat-2(14) that had been prepared as described earlier (12) or a 4.5-kilobase pair full-length murine macrophage inos (31). Probes were labeled with [alpha-P]deoxycytidine triphosphate using the random prime method and purified through Nick columns. Membranes were washed at 65 °C for the rat cat-1b cDNA and at 60 °C for the cat-2 and inos cDNAs and were exposed to Cronex x-ray films at -80 °C with double intensifying screens for different periods of time.

Materials

Cell culture media, amino acids (L-isomers) and analogues, angiotensin II, cycloheximide, lipopolysaccharide, and recombinant human cytokines were all obtained from Sigma. Culture dishes were supplied by Becton Dickinson Labware, and fetal calf serum was supplied by Life Technologies, Inc. Nucleotides were purchased from Boehringer Mannheim, and restriction enzymes were obtained from New England Biolabs Inc. DNA purification kits, all radioisotopes, Hybond-N membranes, and labeling kits were supplied by Amersham International plc, United Kingdom.

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(32) .


RESULTS

To investigate the relationship between cytokine-stimulated NO production and L-arginine transport in VSMC, cells were incubated for 24 h with IL-1beta and TNF-alpha alone, or in combination. Both cytokines stimulated nitric oxide synthase activity as determined by the recovery of nitrite in the media and by the initial rates of uptake of L-arginine into the cells (Fig. 1). The cytokines in combination produced results that appeared to be additive with respect to the effects observed when used alone. In contrast, while LPS and -interferon both stimulated nitrite production, no changes in the L-arginine uptake rates could be detected (data not shown). Dose-response curves showed that IL-1beta stimulated both processes with an EC of between 1 and 3 ng/ml (data not shown), suggesting that the same receptor population was controlling the induction in each case. When the time courses of IL-1beta stimulation of nitrite accumulation and L-arginine uptake activity were determined, detectable increases in nitrite concentration were observed from 6 h increasing linearly through to 48 h. In contrast, while the L-arginine uptake activity was enhanced from 6 h and increased through to 24 h, no further increase could be detected (data not shown). Both lysine and ornithine inhibited the cytokine-stimulated component of the L-arginine transport (data not shown), confirming that the System y activity was being stimulated.


Figure 1: TNF-alpha and IL-1beta stimulate nitrite accumulation and L-arginine transport in VSMC. Quiescent VSMC were incubated for 24 h in the absence (Control) or presence of TNF-alpha (100 ng/ml) or IL-1beta (10 ng/ml), alone or in combination. The amount of nitrite accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described under ``Experimental Procedures.'' Values are the means ± S.D. of three replicate determinations. Differences between values not sharing the same letter are statistically significant (p < 0.05).



While investigating whether NO production was required for the stimulation of L-arginine transport, inhibitors, or nitric oxide synthase, N^G-methyl-L-arginine and N-nitro-L-arginine methyl ester were both shown to largely block the IL-1beta/TNF-alpha stimulation of nitrite production without affecting the changes in L-arginine transport (Fig. 2). In a parallel experiment, dexamethasone treatment (1 mM for 24 h) was shown to reduce the cytokine-stimulated nitrite accumulation by over 70%, whereas there was no change in the cytokine stimulation of L-arginine uptake (data not shown). There was, however, a decrease in the basal L-arginine uptake rate in the absence of cytokines suggesting that dexamethasone inhibited the synthesis of some component in the L-arginine transport pathway. Further evidence for the requirement of new protein synthesis was obtained when cells were exposed to the protein synthesis inhibitor cycloheximide, which completely blocked the cytokine-stimulated nitrite accumulation and the increased L-arginine uptake rate. However, cycloheximide caused a significant decrease in the L-arginine uptake rate in the absence of cytokines (Fig. 3). These results suggest that new protein synthesis is required not only for the cytokine-stimulated nitrite production and increases in L-arginine uptake but is also necessary for the maintenance of the basal L-arginine uptake.


Figure 2: Inhibitors of NOS decrease nitrite accumulation but do not affect cytokine-stimulated L-arginine uptake in VSMC. Quiescent VSMC were treated for 24 h without NOS inhibitors (open bars), with 1 mMN^G-methyl-L-arginine (hatched bars), and with 1 mMN-nitro-L-arginine methyl ester (filled bars) in the absence (Control) or presence of a combination of IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml). The amount of nitrite accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described under ``Experimental Procedures.'' Values are the means ± S.D. of four replicate determinations. Differences between values not sharing the same letter are statistically significant (p < 0.05).




Figure 3: Cytokine-stimulated increases in nitrite accumulation and L-arginine uptake require de novo protein synthesis. Quiescent VSMC were treated for 24 h with the inhibitor of protein translation cycloheximide (10 µg/ml, hatched bars) or without cycloheximide (open bars) in the absence (Control) or presence of a combination of IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml). The amount of nitrite accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described under ``Experimental Procedures.'' Values are the means ± S.D. of three replicate determinations. Differences between values not sharing the same letter are statistically significant (p < 0.05).



Recently we reported that the vasoactive peptide angiotensin II (Ang II) could stimulate L-arginine transport in VSMC(12) . When tested in combination with the cytokines IL-1beta and TNF-alpha, Ang II was shown to partially inhibit the cytokine-induced accumulation of nitrite in the media while stimulating the L-arginine transport in a manner that was additive compared with that produced by the cytokines alone (Fig. 4). Ang II alone did not affect the control levels of nitrite production, and it appeared to delay the cytokine stimulation of nitrite production by about 6 h with the rates of nitrite production over the final 12 h of the experiment being similar in both the presence and absence of Ang II. These data confirm the observation of Nakayama et al.(33) that Ang II suppresses NOS activity in these cells and further indicate that Ang II is likely to stimulate L-arginine uptake by a mechanism that is independent of that of the cytokines.


Figure 4: Ang II increases cytokine-stimulated L-arginine uptake but decreases nitrite accumulation in VSMC. Quiescent VSMC were treated with a combination of IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml, filled squares), Ang II (100 nM, open circles), or a combination of IL-1beta, TNF-alpha, and Ang II (filled circles) for the indicated times. Control (untreated) VSMC were co-cultured in parallel (open squares). The amount of nitrite accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described under ``Experimental Procedures.'' Values are the means ± S.D. of four replicate determinations. Differences between 24-h values not sharing the same letter are statistically significant (p < 0.05).



Previous studies have shown that cultured VSMC express two different genes of the L-arginine transporter, cat-1b and cat-2 (12). To examine the induction of the cat and inos genes, cells were exposed to either Ang II or the cytokines TNF-alpha and IL-1beta for periods up to 24 h before harvesting and extraction of total RNA for Northern analysis. The cytokines in combination led to parallel increases in the abundance of inos and cat-2 transcripts without any effect on the level of cat-1b mRNA (Fig. 5). In contrast, Ang II was able to enhance cat-1b mRNA concentrations at early time points, 3 and 6 h, but not at longer time points, which was consistent with our previous results(12) .


Figure 5: Cytokines regulate inos and cat-2 mRNA levels in VSMC. Quiescent VSMC were treated with Ang II (100 nM) or a combination of IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml) for the times indicated with control (untreated) VSMC co-cultured in parallel. Total RNA was prepared and analyzed (20 µg/lane) for the expression of inos, cat-1b, and cat-2/mRNA as described under ``Experimental Procedures.'' The integrity of the RNA was determined by visualizing ethidium bromide-stained rRNA under ultraviolet light following electrophoresis on a formaldehyde gel.




DISCUSSION

This work has clearly demonstrated that the cytokines IL-1beta and TNF-alpha stimulate both NO production and the rate at which L-arginine is transported into VSMC. The increase in L-arginine transport is due to up-regulation of the System y family of cationic amino acid transporters, a result which is consistent with this family being the only transporter of L-arginine expressed in these cells(11, 12, 34) . The finding that the cytokine-stimulated increase in L-arginine transport in VSMC was inhibited by cycloheximide, and therefore required de novo protein synthesis suggested that regulation of L-arginine transport possibly required protein synthesis of the L-arginine transporter itself. However, the observation that cycloheximide caused a decrease in the non-stimulated rate of L-arginine transport suggests either that the transporter has a rapid turnover rate or that some other component was required for the regulation of the transporter activity.

The presence of messenger RNA for two cat genes has been demonstrated in VSMC(12) . However, other studies have shown that the CAT-1 and CAT-2 isoforms have identical kinetic properties and are indistinguishable at the cellular level(10, 13, 14, 17, 19) . Our study using transcript-specific probes has clearly demonstrated that IL-1beta and TNF-alpha not only regulate the expression of inos mRNA but specifically up-regulate the expression of the cat-2 mRNA transcript. The cytokine-stimulated expression of iNOS is at least partially mediated in other cells through the nuclear translocation of the cytoplasmic transcription factor, nuclear factor kappaB(35) . Recent analysis of the murine cat-2 gene has demonstrated the presence of multiple promoters, suggesting a complex regulation of this gene(36) .

Our observation that Ang II inhibits the cytokine stimulation of NO production in VSMC is consistent with that of Nakayama et al.(33) . In our experiment, this appears to be a result of the Ang II delaying the cytokine effect rather than blocking it completely. In contrast, Ang II stimulates the System y activity over and above that observed for the cytokines, suggesting that there are multiple signaling pathways regulating the expression of the cat genes.

The inos and cat genes are not the only genes involved in L-arginine metabolism that are regulated by cytokines(35) . Both the activity and the mRNA level of argininosuccinate synthetase are coinduced with inos in VSMC in response to cytokines(37) . Recently, the type II arginase activity in macrophages has been shown to be induced by LPS but not by -interferon(18) . These results suggest that there is a complex regulation of genes encoding proteins required for L-arginine metabolism that may ultimately affect the ability of cells to sustain NO production following chronic stimulation by cytokines.


FOOTNOTES

*
This work was supported by grants from the Health Research Council of New Zealand, the University of Otago, the Lee Foundation of Malaysia/Singapore, and Genesis Research and Development Corporation, Auckland, New Zealand. 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. for Molecular and Cellular 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 9001. Tel.: 64-3-479-7840; Fax: 64-3-479-7866; grigor{at}sanger.otago.ac.nz.

(^1)
The abbreviations used are: NO, nitric oxide; iNOS, inducible isoform of nitric oxide synthase; VSMC, vascular smooth muscle cells; Ang II, angiotensin II; IL-1beta, interleukin-1beta, TNF-alpha, tumor necrosis factor-alpha, LPS, lipopolysaccharide(s); NOS, nitric oxide synthase; CAT, cationic amino acid transporter.


ACKNOWLEDGEMENTS

We thank Dr. C. MacLeod for making available the MCAT-2 parental cDNA and Dr. J. Cunningham for the iNOS cDNA.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.