Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and L-arginine transport.

The cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) plays an important role in the immune response to some pathogens. Within the heart, increased activity of NOS2 in cardiac microvascular endothelial cells (CMEC) also can diminish the contractile function of adjacent cardiac myocytes. Glucocorticoids, which are known to suppress cytokine induction of NOS2 in many cell types, caused only a moderate (approximately 20%) decline in NOS2 protein content and maximal activity measured in homogenates of cytokine-treated CMEC, but almost completely inhibited synthesis of nitrogen oxides (NOx) by intact cells. To determine whether glucocorticoids were inhibiting cellular NOx production by limiting the availability of NOS co-factors or substrate, the effect of dexamethasone on tetrahydrobiopterin (BH4) and L-arginine availability in cytokine-treated CMEC was examined. Dexamethasone prevented the coordinate induction of GTP cyclohydrolase I with NOS2 after exposure to interleukin-1beta and interferon-gamma and also the increase in intracellular BH4 content in cytokine-treated CMEC. Addition of BH4 overcame dexamethasone-mediated suppression of nitrite production. Dexamethasone also prevented a cytokine-mediated increase in L-arginine uptake into CMEC by suppressing the induction of the high affinity cationic amino acid transporters CAT-1 and CAT-2B and the low affinity CAT-2A transporter. In addition, dexamethasone also inhibited cytokine induction in CMEC of argininosuccinate synthase, the rate-limiting enzyme for the de novo synthesis of arginine from citrulline. Thus, glucocorticoids regulate NOx production following cytokine exposure in cardiac microvascular endothelial cells primarily by limiting BH4 and L-arginine availability.

microvascular endothelial cells (CMEC) 1 and cardiac myocytes, express the inducible isoform of nitric oxide synthase (iNOS or NOS2) after exposure to soluble inflammatory mediators in vitro and in vivo in animal models of systemic sepsis or myocardial inflammation (1)(2)(3)(4)(5)(6)(7)(8). As part of the rapidly mobilized nonselective innate or natural immune response, NOS2 expression in the microvascular endothelium may regulate blood flow, platelet adhesion, endothelial activation, and microvascular permeability, as well as directly limiting the growth of some pathogens (9 -13). However, in the heart, high levels of nitrogen oxides (NO x ), produced either by cardiac myocytes or by CMEC following induction of NOS2, cause impaired myocyte contractile function that may contribute to the cardiac dysfunction characteristic of the systemic inflammatory response syndrome or of the reversible early form of cardiac allograft rejection (8, 14 -16). Glucocorticoids are efficacious in the management of cardiac allograft rejection, and this may relate in part to their ability to suppress NOS2 activity in cardiac muscle with concomitant improvement in contractile function (5,17). In cardiac myocytes, glucocorticoids suppress NOS2 activity, largely by decreasing NOS2 mRNA and protein abundance (5). In contrast, we have recently demonstrated that glucocorticoids suppress NO x production by CMEC with only modest declines in NOS2 mRNA, protein levels, and maximal enzyme activity measured in cell homogenates (4). These data suggested that glucocorticoids were affecting CMEC NO x generation by mechanisms other than suppression of the induction of NOS2 itself.
Potential additional sites for regulating cellular NO x production include the intracellular availability of the NOS co-factor tetrahydrobiopterin (BH4) and substrate L-arginine. BH4 is a necessary cofactor for maximal activity of all NOS isoforms (18 -20). The precise role for BH4 in the NOS-catalyzed reaction is incompletely resolved (21,22), but there is evidence that it may function both as an allosteric cofactor, to promote assembly of NOS2 into its active dimeric form (23), as well as a redox-active cofactor (21). Another potential catalytic role for BH4 may be to prevent NO x -mediated inactivation of NOS (24). GTP cyclohydrolase I, the rate-limiting enzyme for the de novo synthesis of BH4 (25), is co-induced by cytokines in a variety of cell types, including cardiac myocytes (5, 26 -30). Subsequent to GTP cyclohydrolase I, sepiapterin reductase catalyzes the terminal step in the de novo biosynthetic pathway for BH4 and also catalyzes the initial reaction of the salvage pathway for BH4 re-synthesis (25).
Other potential sites for regulation of cellular NO production by glucocorticoids include L-arginine availability that, along with oxygen, is a substrate for the NOS reaction. NO x production by NOS2 is dependent on extracellular arginine, both in vitro and in vivo (31)(32)(33)(34)(35). Arginine is obtained from exogenous sources via a plasma membrane cationic amino acid transport system termed "system y ϩ " (36), and arginine transport activity has been reported to be increased following exposure to inflammatory mediators (35,37,38). The transport activity of system y ϩ is characterized by high affinity for cationic amino acids, sodium independence, and stimulation of transport by substrate on the opposite (trans) side of the membrane (36). System y ϩ transport activity has previously been detected in large vessel endothelial cells (39,40). Two transporters exhibiting y ϩ system properties were cloned from the mouse and were termed cationic amino acid transporter-1 and -2B (CAT-1 and CAT-2B) (41)(42)(43)(44)(45). CAT-1 has widespread tissue distribution (42), including the vascular endothelium (46), whereas CAT-2B was originally identified only in activated murine macrophages and lymphocytes (43,45). The third member of the cationic amino acid transporter family, cloned from murine hepatocytes (CAT-2A), was distinguished by its lower affinity for cationic amino acids and insensitivity to trans-stimulation (i.e. absence of the "y ϩ " phenotype) (47). Changes in the activity of these transporters could potentially alter NO x production. Recently, we reported that both the neonatal and adult phenotypes of rat ventricular myocytes in vitro express low levels of CAT-1 mRNA but respond to inflammatory cytokines with a marked increase in CAT-1 as well as CAT-2A and CAT-2B mRNA and arginine uptake activity (35).
Arginine can also be endogenously synthesized within cells (48). Many NO x -producing cell types, including murine macrophages and bovine aortic endothelial cells, are capable of synthesizing arginine from citrulline by the sequential actions of argininosuccinate synthetase and argininosuccinate lyase (49,50). Furthermore, expression of argininosuccinate synthetase is increased along with NOS2 in a variety of cell types including cardiac myocytes following exposure to soluble inflammatory mediators (35,51,52).
In this report, we demonstrate that glucocorticoids suppress NO x production by decreasing GTP cyclohydrolase I mRNA abundance and BH4 synthesis in cytokine-treated CMEC. Furthermore, we demonstrate that both arginine transport into CMEC and mRNA for the rate-limiting enzyme of de novo synthesis of arginine are suppressed by glucocorticoids in these cells. The data support the conclusion that glucocorticoids inhibit cytokine-induced NO production in CMEC by mechanisms that are largely independent of the NOS2 enzyme and relate predominantly to the effect of glucocorticoids to suppress BH4 synthesis.

EXPERIMENTAL PROCEDURES
Cell Isolation in Culture-CMEC were isolated from adult male Sprague-Dawley rats (225-275 g) as described previously (53). All studies were performed on confluent monolayers of primary isolates that were serum-starved for 24 h and had been documented to contain greater than 90% endothelial cells (53). Where indicated, cell number was determined by Coulter counter following separation of the cells from the plates by incubation at 37°C for 2 min in Hanks' balanced salt solution containing 0.25% trypsin and 1 mM EDTA.
In some studies, non-myocyte cell fractions were obtained from freshly isolated hearts of rats that had been injected intraperitoneally with lipopolysaccharide (LPS, from Salmonella typhimurium, 4 mg/kg, Sigma) and/or dexamethasone (1.2 mg/kg), while control animals received only phosphate-buffered saline (PBS, Sigma) injections. When animals received both reagents, the LPS was injected 1 h after dexamethasone. Animals were sacrificed 16 h following injections.
Measurement of NO Synthase (NOS) Activity-NOS activity in car-diac microvascular endothelial homogenates was quantified as described previously (5,35)  Nitrite Assay-Nitrite accumulation in the medium was used as an indicator of cellular NO x synthesis and was determined as described previously (35,54) using the Griess reaction. Nitrite concentration was calculated from the standard curve constructed over the linear range of the assay (0.1-50 M).
Intracellular Biopterin Levels-Confluent monolayers of CMEC in 100-mm plates (2.4 Ϯ 0.06 ϫ 10 6 cells/plate, mean Ϯ S.E.) were placed on ice, media were aspirated, and the cells were washed three times with ice-cold PBS. The final wash buffer was aspirated, and cell extracts were prepared by scraping cells free of the culture plate with a rubber policeman in 1 ml of 0.1 N perchloric acid. Cell extracts were rapidly frozen on a dry ice/ethanol mixture and stored at Ϫ70°C. Extracts were then oxidized with acidic or basic iodine as described (25,55). Acidic iodine quantitatively converted BH4, dihydrobiopterin, and biopterin to biopterin; basic iodine converted dihydrobiopterin and biopterin to biopterin and destroyed BH4. Total biopterin levels in the oxidized extracts were then determined by high performance liquid chromatography with fluorescence detection, and the percent of total biopterin that was presented as BH4 in the extracts prior to oxidation was determined from the difference between acidic and basic iodine-generated biopterin.
Northern Hybridization-Total RNA was isolated as described previously (56), and 15 g of RNA was separated by electrophoresis on a 1% formaldehyde-agarose gel. Following transfer to nylon membranes (GeneScreen Plus, DuPont NEN) and fixation by UV cross-linking, hybridizations were performed overnight at 42°C in 50% formamide with 32 P-radiolabeled cDNAs for rat liver GTP cyclohydrolase I (57), rat liver sepiapterin reductase (58), rat ecotropic retrovirus receptor (i.e. CAT-1) (59), argininosuccinate synthetase (60), and rat argininosuccinate lyase (61). Hybridized blots were sequentially washed as described (35), and autoradiography was performed at Ϫ70°C for 24 h. Relative mRNA abundance was quantified by measuring the density of the exposed film with a laser densitometer (Ultrascan 2202, LKB). mRNA levels were normalized to 18 S ribosomal RNA following re-hybridization of blots with 32 P-labeled oligonucleotide complementary to rat 18 S ribosomal RNA, with repeat washing and autoradiography.
Ribonuclease Protection Analysis-The MCAT-2A RNase protection probe was generated from a 127-bp fragment of MCAT-2A cDNA (bp 1095-1222) (43,47), and the MCAT-2B RNase protection probe was generated from a 151-bp template of MCAT-2B cDNA (bp 1074 -1225) (43), both cDNAs generously provided by Dr. James M. Cunningham. Plasmid vectors were linearized and antisense riboprobes transcribed in the presence of [␣ 32 P]UTP (DuPont NEN, specific activity 800 Ci/ mmol) as described (35). The 18 S ribosomal RNase protection probe was generated from the 80-bp antisense template of a highly conserved region of the human 18 S ribosomal RNA gene, obtained commercially (Ambion, pT7 18 S), and linearized with HindIII. A lower specific activity probe was generated by using T7 RNA polymerase in the presence of a 1:1000 dilution of [␣ 32 P]UTP with the balance being unlabeled UTP (3.3 mM).
Total RNA was isolated from cardiac microvascular endothelial cells using the method of Chomczynski and Sacchi (56). RNase protection analyses were performed as described (63). Following gel purification of the probes, hybridization reactions were performed with 20 g of total RNA in 50% formamide for 12 h at 50°C using 2 ϫ 10 5 cpm/reaction of the radiolabeled antisense RNA transcripts, except for the 18 S riboprobe where 10 4 cpm/reaction was co-hybridized with all samples. Samples were then digested with ribonuclease A and T1 (Boehringer Mannheim) and analyzed on 8% denaturing polyacrylamide gels, with adjacent RNA size markers (Ambion), followed by autoradiography. Total RNA from murine macrophages and liver was also analyzed by RNase protection on initial gels that verified the presence of protected fragments of the predicted size for the rat.

Effects of IL-1␤/IFN␥ and Dexamethasone on NOS Activity
and Nitrite Production by CMEC-Incubation of confluent serum-starved CMEC with the combination of IL-1␤ and IFN␥ for 24 h produced a marked increase in maximal NOS activity in cellular homogenates compared with control, as measured by the conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline in the presence of excess substrate and co-factors (Fig. 1A). Pretreatment and co-incubation of dexamethasone with cytokines for 24 h produced a small (approximately 20%) decrease in maximal NOS activity in CMEC homogenates that did not reach statistical significance. This observation is consistent with prior studies from this laboratory that have demonstrated only a modest effect of dexamethasone on NOS2 protein content and activity in cytokine-pretreated CMEC (4).
Despite the minimal effect on NOS2 activity in CMEC, dexamethasone markedly reduced nitrite accumulation in media surrounding cytokine-pretreated cells by approximately 80% (Fig. 1B). Nitrite accumulation was undetectable in medium conditioned by CMEC in the absence of cytokine stimulation. This suggested that dexamethasone regulates cellular NO x production by cytokine-pretreated CMEC by a mechanism(s) that is(are) independent of NOS2 enzyme levels. Potential additional sites for regulation of cellular NO x production include the availability of the NOS co-factor BH4 and intracellular arginine.
Effect of Dexamethasone on Intracellular Biopterin Content of CMEC-To determine whether dexamethasone decreased biopterin levels in cytokine-pretreated CMEC, the intracellular biopterin content was assayed. As shown in Fig. 2, basal levels of biopterin in control CMEC primary isolates were below the limit of assay detection (less than 2 pmol). However, following exposure to IL-1␤ plus IFN␥ for 24 h, cellular biopterin increased to 15.4 Ϯ 0.9 pmol/10 6 cells. This cytokine-induced increase in cellular biopterin was reduced 12-fold when cells were co-incubated with dexamethasone. In these experimental groups, BH4 was approximately 70% of the total biopterin content (data not shown). Concomitant addition for 24 h of exogenous BH4 to the culture media of cells treated with cytokines, or the combination of dexamethasone with cytokines, increased intracellular biopterin levels 7-fold as compared with treatment with cytokines alone.
Effect of Exogenous BH4 on Nitrite Production-Exogenous BH4 enhanced nitrite accumulation by 30% when added to cytokine-pretreated CMEC, suggesting that the intracellular BH4 content was below that necessary for maximal NOS2 activity in these cells. The addition of BH4 to the medium of cells treated with the combination of dexamethasone and cytokines restored nitrite production to approximately 70% of that following treatment with cytokines in the absence of dexamethasone (Fig. 3). To examine further the dependence of nitrite production by cytokine-pretreated CMEC on endogenous BH4 synthesis, cells were co-incubated with DAHP, a selective inhibitor of GTP cyclohydrase I, the rate-limiting enzyme in the de novo BH4 synthetic pathway. DAHP reduced cytokine-induced nitrite production to control levels (Fig. 3), and this effect could be completely overcome by the co-administration of exogenous BH4.
GTP Cyclohydrolase I mRNA Expression by CMEC in Vitro and in Vivo: Regulation by Dexamethasone-To examine whether the cytokine-and dexamethasone-mediated changes in intracellular biopterin content were paralleled by similar directional changes in GTP cyclohydrolase I mRNA abundance, Northern hybridizations were performed on total RNA from CMEC with the full-length cDNA of rat GTP cyclohydrolase I (57). As shown in Fig. 4A, confluent cultures of CMEC expressed the two transcripts for GTP cyclohydrolase I (1.1 and 3.5 kilobase pairs) (5, 28) only following exposure to cytokines, with peak mRNA levels observed 18 -24 h following cytokine treatment. This time course for induction of GTP cyclohydrolase I mRNA by cytokines parallels that of NOS2 mRNA in this cell type (4).
As shown in Fig. 4B, either IL-1␤ or LPS, but not IFN␥ alone, were sufficient to induce GTP cyclohydrolase I mRNA. However, IFN␥ acted synergistically with IL-1␤ to increase GTP cyclohydrolase I mRNA abundance. This pattern of response to cytokines and LPS is identical for the induction of NOS2 in this cell type (4). The addition of dexamethasone to IL-1␤ and IFN␥ reduced steady-state GTP cyclohydrolase I mRNA levels by 10-fold as compared with treatment with cytokines alone. The two transcripts were regulated concordantly in response to either cytokines alone or the combination of dexamethasone and cytokines. Thus, GTP cyclohydrolase I mRNA expression paralleled the levels for intracellular biopterin in control, cytokine alone, or cytokine plus dexamethasone-treated cells.
To determine whether similar changes in GTP cyclohydrolase I mRNA abundance could be detected in vivo, adult rats were injected intraperitoneally with LPS, dexamethasone, or both. Total RNA was isolated from the non-myocyte fraction of freshly isolated hearts and analyzed by Northern hybridization. GTP cyclohydrolase I mRNA was detected 16 h following injection of LPS, but was absent in sham-injected control animals (Fig. 4C). The co-injection of dexamethasone with LPS diminished the extent of induction of GTP cyclohydrolase I mRNA by 3-fold compared with LPS alone.
In contrast to the cytokine-mediated regulation of GTP cyclohydrolase I mRNA expression, steady-state mRNA levels for sepiapterin reductase remained unchanged from control following treatment with IL-1␤ plus IFN␥ (Fig. 5). The addition of dexamethasone to cytokines did, however, produce a small reduction (50% decrease) in sepiapterin reductase mRNA abundance and again is consistent with the dexamethasonemediated changes in intracellular biopterin content.
Dexamethasone and GTP Cyclohydrolase I mRNA Stability-To determine the effect of dexamethasone on GTP cyclohydrolase I mRNA stability, confluent CMEC were treated with actinomycin D (10 g/ml, Sigma) after 24 h of cytokine or cytokine plus dexamethasone pretreatment. The results in Fig.  6 show that the half-life of GTP cyclohydrolase I mRNA in cytokine-pretreated CMEC was approximately 4.8 h. The halflife for GTP cyclohydrolase I mRNA was reduced by 50%, to 2.4 h, in cells pretreated with the combination of dexamethasone and cytokines. Thus, one mechanism by which dexamethasone regulates GTP cyclohydrolase I mRNA abundance in cytokine-pretreated cells appears to be at the post-transcriptional level. However, the principal mechanism by which glucocorticoids regulate GTP cyclohydrolase I mRNA abundance appears to be at the transcriptional level, as the effect of dexamethasone on steady-state mRNA levels (i.e. a 10-fold decline) was far in excess of that which could be explained by a 50% reduction in mRNA half-life.
Dependence of Nitrite Production on L-Arginine Transport: Effects of Dexamethasone and IL-1␤/IFN␥-The failure of exogenous BH4 to completely overcome dexamethasone-mediated suppression of cytokine-induced nitrite production, in contrast to the inhibition caused by DAHP, suggested that additional mechanisms may be limiting cellular NO x production that are independent of BH4 availability and maximal NOS activity as assayed in cell homogenates. One such mechanism is the regulation of intracellular arginine availability by changes in arginine uptake.
The dependence of nitrite production by intact cells on Larginine transport was studied by incubating CMEC for 24 h with IL-1␤ and IFN␥ in the presence and absence of L-lysine, a cationic amino acid that competes with L-arginine for transmembrane transport (36). The combination of IL-1␤ and IFN␥ increased L-[ 3 H]arginine uptake 2-fold compared with control (Fig. 7A) at a concentration of this amino acid (100 M) that FIG. 4. Regulation of GTP cyclohydrolase I mRNA abundance by cytokines and dexamethasone in CMEC. Northern blot analyses of total RNA from confluent serum-starved CMEC demonstrating the time course for GTP cyclohydrolase I mRNA induction in cells exposed to rhIL-1␤ (4 ng/ml) plus rmIFN␥ (500 units/ml) for the indicated times (A) and the effect of dexamethasone on GTP cyclohydrolase I mRNA expression (B). Cells were incubated in control medium or preincubated for 1 h with dexamethasone (DEX, 3 M) prior to treatment for 24 h with lipopolysaccharide (LPS, from S. typhimurium, 10 g/ml), IL-1␤ (4 ng/ml), or IFN␥ (500 units/ml), or the combination of IL-1␤ and IFN␥. C, regulation of GTP cyclohydrolase I mRNA abundance in non-myocyte fractions of hearts from adult rats injected in vivo with dexamethasone and/or LPS. Animals were injected intraperitoneally with saline (Control) or with either dexamethasone (DEX, 1.2 mg/kg) or LPS (4 mg/kg), or both reagents. Animals were sacrificed 16 h later to prepare nonmyocyte fractions from which total RNA was isolated. In all experiments, hybridizations were performed with 32 P-labeled cDNA for rat liver GTP cyclohydrolase I and oligonucleotide for 18 S ribosomal RNA to correct for loading differences. The experiments were performed three or more times in each case with similar results.
FIG. 5. Sepiapterin reductase mRNA abundance in response to cytokines and dexamethasone. Northern hybridization was performed on total RNA prepared from confluent serum-starved CMEC with 32 P-labeled cDNA for rat liver sepiapterin reductase and oligonucleotide for 18 S ribosomal RNA. Cells were preincubated for 1 h with control medium or medium supplemented with dexamethasone (DEX, 3 M) prior to treatment for 24 h with lipopolysaccharide (LPS, from S. typhimurium, 10 g/ml), IL-1␤ (4 ng/ml), or IFN␥ (500 units/ml) or the combination of IL-1␤ and IFN␥. This blot is representative of three independent experiments.
FIG. 6. Effect of dexamethasone on GTP cyclohydrolase I mRNA stability. Confluent serum-starved CMEC were preincubated for 1 h with control medium (solid circles) or medium containing dexamethasone (3 M) (open circles) prior to treatment for 24 h with the combination of IL-1␤ (4 ng/ml) and IFN␥ (500 units/ml) after which actinomycin D (10 g/ml) was added and total RNA prepared at the indicated times. Northern hybridizations were performed using 32 Plabeled rat liver GTP cyclohydrolase I cDNA and 18 S probes. The signal density of the 1.1-kilobase pair transcript of GTP cyclohydrolase I was divided by that of 18 S ribosomal RNA, and data were expressed as the natural logarithm of this ratio at successive time points (R t ) normalized to the maximal level at time 0 (R 0 ). By linear regression analysis, the rate of GTP cyclohydrolase I mRNA decrease was significantly more rapid in cytokine-treated CMEC in the presence of dexamethasone (T1 ⁄2 ϭ 2.4 h, slope ϭ Ϫ0.28 Ϯ 0.009, mean Ϯ S.E.) than in its absence (T1 ⁄2 ϭ 4.8 h, slope ϭ Ϫ0.14 Ϯ 0.007, p Ͻ 0.001; n ϭ 5 per time point for each treatment group from five separate experiments).
approximates the mammalian plasma concentration (65). At a concentration of L-lysine (10 mM) that inhibited L-arginine uptake by greater than 95%, nitrite production by cytokine-pretreated cells declined by 30% (Fig. 7B). This concentration of L-lysine has not been observed to directly affect maximal NOS activity assayed in cell homogenates (35).
The time course of arginine uptake into CMEC and its regulation by dexamethasone and cytokines are shown in Fig. 8. Transport of L-arginine was approximately linear over 5 min. There was no detectable metabolism of L-arginine to L-citrulline in these cells during this portion of the time course for L-arginine uptake. IL-1␤ and IFN␥ treatment for 24 h increased the rate of uptake of 100 M L-[ 3 H]arginine at least 2-fold compared with control during the linear range of uptake (212.0 Ϯ 35.2 versus 104.4 Ϯ 32.7 pmol/well/min for cytokinetreated and control cells, respectively, at t ϭ 1 min; mean Ϯ S.E.; n ϭ 6 in both groups; p Ͻ 0.001). Addition of dexamethasone to IL-1␤ and IFN␥ reduced L-arginine uptake to rates comparable with those of control cells. Also, a small reduction (ϳ30%) in L-arginine uptake was observed following treatment with dexamethasone alone, as compared with control, and this was statistically significant at all time points beyond t ϭ 1 min Effects of Dexamethasone and IL-1␤/IFN␥ on Cationic Amino Acid Transporter mRNA Abundance-To determine whether the observed cytokine-and dexamethasone-induced changes in L-arginine uptake were associated with similar directional changes in levels of mRNA for the CAT family of transporters, total RNA was analyzed by Northern hybridization and RNase protection assays. The mRNA for the high affinity arginine transporter, CAT-1, was detectable in confluent control CMEC cultures as shown in Fig. 9A. Treatment of CMEC with IL-1␤ and IFN␥ for 24 h increased steady-state CAT-1 mRNA levels normalized to 18 S mRNA by 2-fold, as determined by densitometry from four separate experiments. Dexamethasone treatment alone decreased the abundance of CAT-1 mRNA by 4-fold as compared with control, and combined with cytokines, it reduced CAT-1 mRNA abundance by 5-fold when compared with cytokines alone. Similar changes in CAT-1 mRNA could be detected in vivo in adult rats injected intraperitoneally with LPS, dexamethasone, or both (Fig. 9B). LPS increased CAT-1 mRNA abundance by 10-fold in the nonmyocyte fraction of freshly isolated hearts as compared with controls. Co-injection of dexamethasone with LPS prevented this increase in CAT-1 mRNA (Fig. 9B).
The mRNA coding for CAT-2B (Fig. 10A), a high affinity cationic amino acid transporter originally described in activated macrophages and lymphocytes (43,45), could be detected in control CMEC and increased 10-fold following a 24-h exposure to IL-1␤ and IFN␥. Preincubation with dexamethasone prevented the induction of CAT-2B mRNA by these cytokines. The expression of a low affinity cationic amino acid transporter, CAT-2A, originally identified only in hepatocytes (47), was also observed in CMEC, but only following 24-h treatment with IL-1␤ and IFN␥ (Fig. 10B). As was noted for CAT-2B, dexamethasone abolished the cytokine induction of CAT-2A mRNA.
Effects of IL-1␤/IFN␥ and Dexamethasone on Argininosuccinate Synthetase and Argininosuccinate Lyase mRNA Levels-An additional possible site for regulating arginine supply, and hence cellular NO x production, is through changes in the endogenous synthesis of arginine from citrulline by arginino- FIG. 7. Dependence of cytokine-induced nitrite production on L-arginine uptake. Following 24 h incubation of confluent serumstarved CMEC with either control medium or medium supplemented with the combination of IL-1␤ (4 ng/ml) and IFN␥ (500 units/ml), uptake of L-[ 3 H]arginine (100 M) was measured for 1 min in the absence and presence of 10 mM L-lysine (A). B, 24-h nitrite accumulation was measured in the absence and presence of 10 mM L-lysine in control and IL-1␤ plus IFN␥-treated CMEC. Each point is the mean Ϯ S.E. from six repetitions and is from a single experiment that was representative of three separate experiments (* ϭ p Ͻ 0.001 versus other groups; ** ϭ p Ͻ 0.001 versus controls).
FIG. 8. Time course of L-arginine uptake and the effects of cytokines and dexamethasone in CMEC. Confluent serum-starved CMEC in 24-well cluster trays (7.9 Ϯ 0.2 ϫ 10 4 cells/well; mean Ϯ S.E.) were exposed for 24 h to control medium (q), control medium containing 4 ng/ml rhIL-1␤ and 500 units/ml IFN␥ (f), medium supplemented with 3 M dexamethasone (E), or were preincubated for 1 h with dexamethasone prior to treatment with the combination of cytokines (Ⅺ). The uptake of 100 M L-[ 3 H]arginine was then initiated as described under "Experimental Procedures" for the times indicated. The data are from a representative experiment (repeated three times with similar results). Each point represents the mean Ϯ S.E. from six replicates. The S.E. was less than 8.0 pmol/well on those data points where the bars are not visible.
succinate synthetase and argininosuccinate lyase. Steady-state mRNA levels for these two enzymes were determined by Northern hybridizations performed on total mRNA from confluent CMEC. As shown in Fig. 11A, mRNA argininosuccinate synthetase, the rate-limiting enzyme for the de novo synthesis of arginine, was undetectable in control cells and was induced by cytokines, as has been described previously in other cell types (51,52). Dexamethasone reduced argininosuccinate synthetase mRNA levels by 10-fold in cytokine-pretreated cells. Argininosuccinate lyase mRNA was detected in control CMEC (Fig.  11B), and its abundance increased 1.5-fold following exposure to cytokines, but was not decreased by the addition of dexamethasone to cytokines. DISCUSSION Glucocorticoids have previously been identified to affect NO x production by directly inhibiting NOS2 activity or protein levels (5, 66, 67), with or without a concomitant reduction in NOS2 mRNA expression (5, 66 -70). The results presented here identify two additional mechanisms whereby glucocorticoids inhibit cytokine-induced NO x production by the cardiac microvascular endothelium that are independent of their effect on NOS2 transcription. First, dexamethasone suppressed a cytokine-mediated increase in BH4 content, probably through its effect both in vitro and in vivo to inhibit GTP cyclohydrolase I mRNA abundance and, to a lesser extent, its modest effect to decrease sepiapterin reductase mRNA abundance. Approximately twothirds of the suppression of NO x production (measured as nitrite release) by dexamethasone was dependent on its action to reduce BH4 content, as determined by the ability of exogenous BH4 to partially restore nitrite production. Second, dexamethasone inhibited the cytokine-induced increases in L-arginine transport and the expression of mRNA for the cationic amino acid family of transporters CAT-1, CAT-2A, and CAT-2B. Finally, dexamethasone inhibited the induction by cytokines of mRNA for argininosuccinate synthetase, the rate-limiting enzyme in the de novo synthesis of arginine.
The present study found that in the absence of inflammatory mediators, CMEC contain neither detectable quantities of BH4 in vitro nor mRNA in vitro or in vivo for GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis. Following FIG. 9. Regulation of CAT-1 mRNA abundance by cytokines and dexamethasone in CMEC. A, total RNA from confluent serumstarved CMEC was isolated following a 24-h incubation in control medium alone, medium supplemented with IL-1␤ (4 ng/ml) and IFN␥ (500 units/ml), medium supplemented with dexamethasone alone (DEX, 3 M), or from cells preincubated for 1 h with dexamethasone prior to the addition of cytokines. B, regulation of CAT-1 mRNA abundance in non-myocyte fractions of hearts from adult rats injected in vivo with dexamethasone and/or LPS. Animals were injected intraperitoneally with saline (control) or with either dexamethasone (DEX, 1.2 mg/ kg), or LPS (4 mg/kg), or both reagents. Animals were sacrificed 16 h later to prepare non-myocyte fractions and prepare total RNA. In all cases, Northern hybridizations were performed with 32 P-labeled cDNA for the rat ecotropic retrovirus receptor (i.e. CAT-1) and oligonucleotide to 18 S ribosomal RNA to correct for differences in loading. These blots are representative of four independent experiments. exposure to IL-1␤ plus IFN␥, BH4 accumulation coincides with the induction of GTP cyclohydrolase I mRNA in CMEC in vitro. This enzyme was also induced in vivo in the non-myocyte fraction of hearts isolated from rats injected intraperitoneally with LPS, an inflammatory stimulus that has been shown to induce NOS2 in the microvascular endothelium and ventricular muscle of these animals (4,5). A recent report indicates that BH4 availability in this in vivo model can limit LPS-induced NO synthesis (71). A qualitatively similar regulation of BH4 content and GTP cyclohydrolase I activity (26,29,72,73) or mRNA expression (27,28) by inflammatory mediators has been documented in a variety of cell types, both in vitro and in vivo (74).
The suppression of the cytokine-induced increases in GTP cyclohydrolase I mRNA expression and BH4 content by glucocorticoids has not previously been documented. This may represent a cell type-specific response, as others have reported either a stimulatory effect (75) or lack of effect (27) of dexamethasone on the induction of GTP cyclohydrolase I activity and expression in other cell types. In CMEC, dexamethasone reduced GTP cyclohydrolase I mRNA abundance at least in part by abbreviating mRNA half-life by 50%.
However, the principal mechanism by which glucocorticoids regulate GTP cyclohydrolase I mRNA abundance in CMEC appears to be at the level of transcription, as steady-state mRNA levels were reduced to a much greater degree (Ͼ10-fold) compared with the reduction in mRNA half-life. Recently, the promoter region of the rat GTP cyclohydrolase I gene was characterized, and it shares several regulatory elements with the human and murine NOS2 genes (76 -79), including sites potentially responsive to NF-B, IFN␥, NF-IL6, and AP-1, among others (86). Glucocorticoids could inhibit GTP cyclohydrolase I transcriptional activation in CMEC after treatment with IL-1␤ plus IFN␥ either by inhibiting the action of NF-B (80,81) or by repressing AP-1-mediated transcriptional activation (82).
As has been demonstrated in aortic smooth muscle cells (83), supplementation of CMEC with BH4 completely restored cytokine-induced nitrite production in the presence of the GTP cyclohydrolase I inhibitor, DAHP. In contrast, addition of BH4 was able to overcome only two-thirds of dexamethasone-mediated suppression of nitrite production in CMEC. Thus, although suppression of CMEC BH4 content is one major mechanism by which cytokine-induced nitrite production is inhibited by this agent, this observation suggested that additional mechanisms were also responsible for dexamethasone's reduction in cytokine-induced nitrite production. These include changes in the availability of intracellular arginine, either from changes in uptake and/or de novo synthesis. In separate experiments, 2 total intracellular arginine levels in CMEC decreased following treatment with dexamethasone and cytokines by 90 and 50%, respectively. This raises the possibility that the CMEC intracellular arginine concentration may become limiting for the NOS2 enzyme despite a relatively low K m for arginine (ϳ10 M) for the isolated NOS enzyme (22). Prior studies demonstrating the much greater K m for L-arginine for nitrite production by intact cells (73-150 M) (31, 32) and the dependence of cytokine-induced nitrite production on extracellular arginine (33)(34)(35) support this conclusion.
In CMEC, the basal uptake of L-arginine is associated with the constitutive expression of the high affinity transporters CAT-1 and, to a lesser extent, CAT-2B. Treatment of cells with IL-1␤ and IFN␥ increased arginine uptake 2-fold, compared with control, and was associated with increased expression of mRNA for both transporters, CAT-1 (2-fold) and CAT-2B (10fold). The much greater increase in CAT-2B mRNA abundance in vitro as compared with CAT-1 suggests that this transporter may mediate most of the cytokine-induced increase in arginine uptake. In addition, CMEC also exhibit de novo expression of mRNA for the low affinity transporter, CAT-2A, following exposure to cytokines. However, expression of the low affinity transporter, CAT-2A, in response to cytokines in both CMEC and myocytes may be of little physiologic significance given that the high affinity CAT-1 and CAT-2B transporters would mediate most of the cellular arginine uptake at typical plasma concentrations of this amino acid.
In this study, we demonstrate that dexamethasone inhibits the stimulation by cytokines of arginine uptake in CMEC, along with the expression of mRNA for CAT-1, CAT-2B, and CAT-2A. Dexamethasone also inhibited the LPS-mediated induction of CAT-1 mRNA in vivo in a non-myocyte fraction of ventricular muscle. This contrasts with a prior report in a murine monocyte/macrophage cell line that found no effect of dexamethasone on the LPS-mediated induction of arginine uptake (38) and demonstrates that the potential sites for regulating NO x production by intact cells may vary for different cell types. In the absence of cytokines, dexamethasone also produced a small reduction in arginine uptake (approximately a 2 W. W. Simmons, W. E. Mitch, and R. A. Kelly, unpublished data. FIG. 11. Effect of cytokines and dexamethasone on mRNA levels for argininosuccinate synthetase and argininosuccinate lyase. Total RNA from confluent serum-starved CMEC was isolated following preincubation of cells for 1 h with control medium or medium supplemented with dexamethasone (DEX, 3 M) and treatment for 24 h with lipopolysaccharide (LPS, from S. typhimurium, 10 g/ml), IL-1␤ (4 ng/ml), or IFN␥ (500 units/ml), or a combination of IL-1␤ and IFN␥. Northern hybridizations were performed using 32 P-labeled cDNA for argininosuccinate synthetase (A) and argininosuccinate lyase (B). Equal loading of samples was confirmed by hybridizing the same membrane to a labeled oligonucleotide probe for 18 S rRNA. These blots are representative of four separate experiments. 30% decrease) and in CAT-1 mRNA abundance when compared with controls in vitro. The mechanism(s) by which glucocorticoids inhibit the cytokine induction of CAT family of transporters is unknown, although the CAT-2B promoter is known to contain a putative AP-1 site (84) that may be repressed by glucocorticoids (82). Only one-third of cytokine-induced nitrite production by CMEC is arginine uptake-dependent (i.e. lysineinhibitable) and thus potentially subject to this regulatory action of dexamethasone on arginine transport. This dependence on arginine uptake for cellular NO x production by the cardiac microvascular endothelium is significantly less than for cardiac muscle (35) and probably reflects the greater capacity of these cells for de novo arginine synthesis. Therefore, glucocorticoids could also limit intracellular arginine availability by suppressing the de novo synthesis of arginine within CMEC. The expression of mRNA for argininosuccinate synthetase, the rate-limiting enzyme of arginine synthesis, was induced in CMEC by IL-1␤ and IFN␥, as has previously been reported for other cell types including cardiac myocytes (35,51,52), and recently in vivo in LPS-injected rats (85). Addition of dexamethasone to cytokines reduced steadystate levels of mRNA for argininosuccinate synthetase by 10fold, and a qualitatively similar response to dexamethasone has been reported for rat aortic smooth muscle cells (52). In contrast, argininosuccinate lyase mRNA is constitutively expressed in CMEC and increases 1.5-fold following cytokine treatment, but is unaffected by the addition of dexamethasone to cytokines. Therefore, glucocorticoid inhibition of argininosuccinate synthetase expression may be a third mechanism whereby cytokine-induced NO x production could be limited independent of the number of NOS2 enzyme copies in CMEC.
In summary, we present two previously undescribed mechanisms whereby glucocorticoids could regulate cellular NO x production by cardiac microvascular endothelium following induction of NOS2 by inflammatory mediators. The predominant effect of glucocorticoids on NO production by CMEC appears to be through their suppression of cytokine-induced increases in GTP cyclohydrolase I expression and intracellular BH4 content. Glucocorticoids may also limit arginine availability by blocking the increase in L-arginine transport and induction of CAT-1, CAT-2B, and CAT-2A following cytokine exposure, as well as the induction of argininosuccinate synthetase, which is necessary for the de novo synthesis of arginine. Therefore, cellular NO x production by cytokine-pretreated CMEC is determined not only by the extent of transcriptional induction of NOS2, but also by substrate and co-factor availability exemplified by the effects of glucocorticoids in this study.