Role of tetrahydrobiopterin availability in the regulation of nitric-oxide synthase expression in human mesangial cells.

Human mesangial cells express an inducible form of nitric-oxide synthase (iNOS) after treatment with cytokines. Tetrahydrobiopterin (BH4), an essential cofactor for NOS, is required for cytokine-induced NO generation. We report here that BH4 is necessary not only for the activity but also for the expression of iNOS in human mesangial cells. Inhibition of de novo BH4 synthesis with 2,4-diamino-6-hydroxypyrimidine (DAHP) significantly attenuated iNOS activity as well as mRNA and protein expression in response to interleukin 1beta plus tumor necrosis factor alpha (IL-1beta/TNF-alpha). In contrast, sepiapterin, which provides BH4 through the pterin salvage pathway, strongly potentiated IL-1beta/TNF-alpha-induced iNOS expression and abrogated the inhibitory effect of DAHP. Inhibition of the pterin salvage pathway with methotrexate abolished sepiapterin potentiation of iNOS induction but did not alter the effect of IL-1beta/TNF-alpha. Determination of intracellular pteridines confirmed that sepiapterin markedly raised BH4 content, an effect that was blocked by methotrexate. These results suggest that BH4 availability plays an important role in the regulation of iNOS expression. The effect of BH4 appears to be mediated, at least in part, by an increase in mRNA stability, as indicated by the observation that DAHP shortened, whereas sepiapterin prolonged the half-life of IL-1beta/TNF-alpha-induced iNOS mRNA. Taken together, our results suggest that the biosynthesis of BH4 contributes to cytokine induction of iNOS expression in human mesangial cells through the stabilization of iNOS mRNA.

Human mesangial cells express an inducible form of nitric-oxide synthase (iNOS) after treatment with cytokines. Tetrahydrobiopterin (BH 4 ), an essential cofactor for NOS, is required for cytokine-induced NO generation. We report here that BH 4 is necessary not only for the activity but also for the expression of iNOS in human mesangial cells. Inhibition of de novo BH 4 synthesis with 2,4-diamino-6-hydroxypyrimidine (DAHP) significantly attenuated iNOS activity as well as mRNA and protein expression in response to interleukin 1␤ plus tumor necrosis factor ␣ (IL-1␤/TNF-␣). In contrast, sepiapterin, which provides BH 4 through the pterin salvage pathway, strongly potentiated IL-1␤/TNF-␣-induced iNOS expression and abrogated the inhibitory effect of DAHP. Inhibition of the pterin salvage pathway with methotrexate abolished sepiapterin potentiation of iNOS induction but did not alter the effect of IL-1␤/TNF-␣. Determination of intracellular pteridines confirmed that sepiapterin markedly raised BH 4 content, an effect that was blocked by methotrexate. These results suggest that BH 4 availability plays an important role in the regulation of iNOS expression. The effect of BH 4 appears to be mediated, at least in part, by an increase in mRNA stability, as indicated by the observation that DAHP shortened, whereas sepiapterin prolonged the half-life of IL-1␤/TNF-␣-induced iNOS mRNA. Taken together, our results suggest that the biosynthesis of BH 4

contributes to cytokine induction of iNOS expression in human mesangial cells through the stabilization of iNOS mRNA.
Nitric oxide (NO) 1 is a biological messenger that is involved in many physiological processes such as the regulation of vascular tone, neurotransmission, and cell-mediated cytotoxicity (1,2). In addition, an altered NO generation is involved in several pathophysiological situations, including chronic and acute inflammation, atherosclerosis, glomerulonephritis, and renal failure (3)(4)(5). The synthesis of NO is accomplished by the NO synthases (NOS), a family of enzymes from which three isoforms are presently known. The neuronal and endothelial NOS are considered constitutive, and their activity is regulated mainly posttranslationally. The inducible isoform (iNOS) is expressed in various cell types, including macrophages and vascular smooth muscle cells, in response to certain stimuli, such as cytokines or bacterial cell products, and its regulation occurs mostly at the level of transcription (6,7). NOS require several cofactors for activity, namely, FAD, FMN, heme, and tetrahydrobiopterin (BH 4 ) (8,9). Heme and BH 4 have been shown to be required for dimerization and acquisition of NOgenerating capacity by macrophage iNOS (8). In addition, BH 4 availability has been demonstrated to be a limiting factor for iNOS activity in many cell types (10 -13). The intracellular levels of BH 4 are determined by the activity of two different biosynthetic pathways: the de novo synthesis from GTP and the regeneration of BH 4 from dihydropterins through a pterin salvage pathway (14). The first enzyme in the de novo pathway is GTP cyclohydrolase I. Cytokines have been reported to stimulate potently the de novo synthesis of BH 4 in several cell types through the induction of GTP cyclohydrolase I (15)(16)(17). In fact, expression of GTP cyclohydrolase I and iNOS appears to be regulated coordinately (16,18). This phenomenon has been interpreted previously as a mechanism to ensure an adequate supply of BH 4 for the activity of cytokine-induced NOS (10 -13, 18). In addition to its catalytic role, BH 4 has been reported to protect NOS from feedback inhibition by NO in vitro (19,20), and to stabilize the structure of both the macrophage and the neuronal NOS proteins (8,21). We were interested in studying the possibility that BH 4 could also be modulating iNOS protein or mRNA expression. To test this hypothesis we have used human mesangial cells (HMC) as an experimental model. Mesangial cells are specialized vascular smooth muscle cells that are disposed around the capillaries of the renal glomerulus serving functions of structural support and regulation of glomerular filtration rate (22). These cells express iNOS in response to cytokines (23)(24)(25). In contrast to rat mesangial cells, which can be induced with single stimulants (24,25), HMC require multiple cytokines to produce NO (26). In this article, we have studied the effect of BH 4 availability on the expression of iNOS by HMC elicited by a combination of interleukin 1␤ plus tumor necrosis factor ␣ (IL-1␤/TNF-␣). The results herein reported suggest that BH 4 modulates iNOS mRNA expression, thus identifying a new role for this cofactor in the complex regulation of cytokine-induced NO generation.
[␣-32 P]dCTP (3,000 Ci/mmol) was from Amersham Corp. Antibodies for cell characterization were obtained from local sources. L-Sepiapterin was from Alexis Co. (Laü felfingen, Switzerland). All other reagents used were of the highest purity available from Sigma. * This work was supported by Grants PB93-0044 (to S. L.) and PB93-0127 (to F. J. C.) from the Dirección General de Investigación Científica y Técnica (DGICYT). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from Fundación Renal Iñ igo Alvarez de Toledo.
Cell Culture-HMC were obtained from normal cadaver kidneys that were unsuitable for transplantation. Primary cultures were obtained using a technique of graded sieving. Groups of cells with mesangial morphology were subcloned and expanded. Mesangial cells displayed positive staining with anti-vimentin, anti-Thy 1.1, and anti-myosin and absence of staining with anti-factor VIII. The cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 24 g/ml gentamycin. For experiments, passages 7-12 were used. Confluent HMC were washed twice with RPMI without phenol red and incubated in this medium with the indicated agents in the absence of serum. TNF-␣ was used at 100 ng/ml, and IL-1␤ was used at 2 ng/ml, final concentrations. Several doses of sepiapterin (0.1-200 M), DAHP (0.5-2 mM), and methotrexate (5-20 M) were tested, and those found optimal for potentiation or inhibition of iNOS activity in HMC, respectively (100 M sepiapterin, 1.5 mM DAHP, 10 M methotrexate), were used for further experimentation. Potential toxicity of the reagents used on HMC was evaluated by trypan blue exclusion. According to these criteria, cell viability was above 90% under all experimental conditions studied.
Nitrite Determination-The accumulation of nitrite in the cell culture supernatant of HMC was taken as an index of iNOS activity. After treatment with the various agents for 24 h, nitrite was determined in the supernatants of HMC by the Griess reaction as described previously (27), using sodium nitrite as a standard.
RNA Isolation and Northern Blot Analysis-Total cellular RNA was isolated from HMC using the guanidinium thiocyanate-phenol-chloroform method (28). 10 g of total RNA was separated on 1% agarose, 0.66 M formaldehyde gels, transferred to Hybond hybridization transfer membranes (Amersham), and UV cross-linked before hybridization as described previously (27). A 1.8-kilobase fragment of the human hepatic inducible NOS isoform cDNA, a gift of Dr. D. Geller (29), was labeled with [␣-32 P]dCTP using a commercial kit for random hexamer labeling (Boehringer Mannheim) and used as probe for Northern analysis. Hybridization was performed at 42°C for 12-16 h. Membranes were then washed at final stringency conditions of 55°C, 0.2 ϫ SSC, 0.1% SDS, and exposed to XAR Kodak film at Ϫ80°C using an intensifying screen. To identify differences in mRNA expression specific for iNOS, blots were denuded and rehybridized with a ␤-actin probe (30). Densitometric analysis was performed on a computing densitometer or on a Phos-phorImager (Molecular Dynamics, Sunnyvale, CA). Results are expressed in arbitrary units as the ratio of iNOS to ␤-actin expression.
Determination of mRNA Half-life-HMC were stimulated with IL-1␤/TNF-␣ for 24 h in the presence or absence of DAHP or sepiapterin. For determination of mRNA half-life, actinomycin D (10 g/ml) was added subsequently, and cells were then harvested for RNA isolation and Northern blot analysis every 2 h for 8 h as described above.
Determination of Intracellular Biopterin-HMC treated for 24 h with the indicated agents were harvested with a cell scraper and washed twice with phosphate-buffered saline. Pellets were resuspended in 250 l of distilled water and homogenized by sonication with an ultrasonic cell disrupter. Cell extracts were centrifuged at 14,000 ϫ g for 3 min, and the supernatants were split into two aliquots that were subjected to oxidation with iodine at alkaline or at acidic pH, essentially as described (31). During alkaline oxidation 7,8-dihydrobiopterin is converted into biopterin (90% conversion), and BH 4 is converted into pterin (approximately 80% conversion), whereas during acidic oxidation both pteridines are quantitatively converted into biopterin (32). After partial purification on Dowex 50 ion exchange columns (31), pteridines were analyzed by reverse-phase HPLC on a Vydac RP C-18 column (The Separations Group, Hesperia, CA) using 5% methanol, 15 mM ammonium acetate, 0.05% triethylamine adjusted to pH 5.7 with acetic acid as eluant, at a flow rate of 0.75 ml/min. 6-Methylpterine (200 pmol) was added to all samples as internal standard (32). Quantification was performed using a Hewlett Packard 1046A fluorescence detector programmed at 271 nm, excitation wavelength, and 441 nm, emission wavelength. The concentration of BH 4 in every sample was calculated from the difference between the amount of biopterin measured in the aliquot subjected to acidic oxidation (which corresponds to total biopterin: biopterin plus 7,8-dihydrobiopterin plus BH 4 ) and the amount of biopterin measured in the aliquot subjected to alkaline oxidation (which corresponds to 7,8-dihydrobiopterin plus biopterin) (32).
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-For SDS-polyacrylamide gel electrophoresis, HMC were homogenized by sonication in 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM ␤-mercaptoethanol, containing 2 g/ml each of the protease inhibitors: leupeptin, pepstatin, trypsin inhibitor, and aprotinin. Aliquots from each experimental condition containing 10 g of protein were electrophoresed on 8% polyacrylamide gels and transferred to Immo-bilon-P membranes (Millipore, Bedford, MA) using a semidry electroblotting system (Hoefer Scientific Instruments, San Francisco). Blots were probed with anti-iNOS mouse monoclonal antibody from Affinity Research Products (Nottingham, U. K.), and the iNOS protein was visualized using an enhanced chemiluminescence detection system from Amersham. A lysate from mouse macrophages stimulated with interferon-␥ and lipopolysaccharide provided by Affinity Research Products was used as positive control.
Statistical Analysis-Results are expressed as means Ϯ S.E. Statistical analysis was performed with the use of the unpaired two-tailed Student's t test. When the number of observations was insufficient to perform adequate parametric tests, nonparametric tests were employed (Kruskal-Wallis). Comparisons were considered statistically significant at the p Ͻ 0.05 level.

BH 4 Synthesis Is Required for
Cytokine-induced NO Generation by HMC-Treatment of HMC with IL-1␤/TNF-␣ for 24 h resulted in the induction of NO generation as indicated by the accumulation of nitrite in the incubation medium ( Fig. 1). NO generation in response to cytokines was L-arginine-dependent since it was inhibited potently by the NOS antagonist L-nitroarginine methyl ester (L-NAME). The addition of 500 M L-NAME during cytokine stimulation produced an 80 -85% inhibition of NO synthesis. To determine the importance of BH 4 supply for cytokine-induced NOS activity, we explored the effect of both donors and inhibitors of BH 4 generation on nitrite formation by IL-1␤/TNF-␣-stimulated HMC. Inhibition of the de novo synthesis of BH 4 with DAHP, an inhibitor of GTP cyclohydrolase I (33), significantly reduced IL-1␤/TNF-␣-induced nitrite production. In contrast, addition of sepiapterin, which provides BH 4 via the dihydrofolate reductase-dependent pterin salvage pathway (14), produced a strong potentiation of cytokine-induced NO generation and circumvented the inhibitory effect of DAHP, although it did not elicit nitrite formation by uninduced cells (Fig. 1). Sepiapterin potentiation of cytokine-induced nitrite accumulation was dose-dependent, 1 M being the lowest concentration that induced a significant (2fold) increase over the values obtained by stimulation with IL-1␤/TNF-␣ alone (results not shown). At 100 M, sepiapterin produced a 6 -8-fold potentiation of cytokine-elicited NO generation (Fig. 1). This effect, which was also L-arginine-dependent, was unaffected by DAHP. However, inhibition of dihydrofolate reductase with methotrexate (14) completely prevented sepiapterin potentiation of cytokine-induced NO generation. In fact, when sepiapterin was given in combination with methotrexate, a slight inhibition of cytokine-induced NO synthesis could be observed. This effect could be attributed to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I (34), which can be unveiled after blocking its conversion to BH 4 (11). In contrast, the addition of methotrexate to IL-1␤/TNF-␣treated HMC did not affect the accumulation of nitrite in the incubation medium of HMC (Fig. 1), suggesting that generation of BH 4 through the salvage pathway is quantitatively less important during cytokine stimulation of HMC. Taken together, these results indicate that BH 4 biosynthesis is a necessary event during cytokine-induced NO generation by HMC.
BH 4 Modulates the Steady-state mRNA and Protein Levels of iNOS-The potentiation of cytokine-induced nitrite accumulation described above could be due to an enhancement of the catalytic activity of iNOS by BH 4 . In addition, BH 4 could play a regulatory role at the level of protein or mRNA expression. To test this hypothesis, we examined the levels of iNOS mRNA in HMC in the conditions under which we had observed changes in iNOS activity. Treatment of HMC with IL-1␤/TNF-␣ for 24 h induced the appearance of the iNOS transcript, which was undetectable in untreated cells as well as in cells treated with sepiapterin alone (Fig. 2). The addition of DAHP significantly diminished the levels of iNOS mRNA in response to IL-1␤/ TNF-␣. Conversely, supplementing the incubation medium with sepiapterin resulted in a 6 -8-fold potentiation of cytokine-induced iNOS mRNA expression. Sepiapterin potentiation of iNOS mRNA expression was unaffected by DAHP, but it was completely prevented by methotrexate. Quantitation of these results by densitometric analysis revealed the existence of a close correlation between iNOS activity (Fig. 1) and mRNA levels ( Fig. 2B) under the various conditions studied. This correlation could also be evidenced when iNOS expression was studied at the protein level by Western blot analysis (Fig. 3).
These results suggested that BH 4 biosynthesis was required not only for iNOS activity but also for its expression. To study the relationship between BH 4 availability and the observed effects on iNOS activity and expression, changes in BH 4 levels in response to the various agents used were monitored. BH 4 levels in control HMC were below 0.5 pmol/mg protein. Although no significant changes in intracellular BH 4 levels could be evidenced after 24-h treatment with either IL-1␤/TNF-␣ or IL-1␤/TNF-␣ plus DAHP, the addition of sepiapterin to the incubation medium did result in a potent increase in total intracellular biopterin levels (sum of biopterin plus 7,8-dihydrobiopterin plus BH 4 ), up to 53.5 Ϯ 8.5 pmol/mg protein. Approximately 65% of this increase was due to the generation of BH 4 . The conversion of sepiapterin into BH 4 was totally and selectively prevented by methotrexate, without significantly reducing total biopterin levels (45.4 Ϯ 9.6 pmol/mg protein). This indicates that sepiapterin potentiation of iNOS expression requires its conversion into BH 4 .
Effect of BH 4 Availability on iNOS mRNA Half-life-To gain insight into the mechanism of the effect of BH 4 on iNOS expression, we evaluated its potential contribution to iNOS mRNA stability. For this purpose, the half-life of iNOS mRNA from HMC that had been treated with IL-1␤/TNF-␣ alone or in combination with sepiapterin or DAHP as in Fig. 2 was estimated after the addition of actinomycin D (Fig. 4). We observed that DAHP significantly shortened iNOS mRNA half-life, from 6.1 Ϯ 1.0 to 2.8 Ϯ 0.1 h (p Ͻ 0.05), whereas the addition of sepiapterin to cytokine-treated HMC resulted in a marked stabilization of iNOS mRNA, the half-life of which increased to 14.4 Ϯ 3.2 h (p Ͻ 0.05). These effects contribute to generate the differences in iNOS mRNA levels illustrated in Fig. 2.
Effect of Donors and Inhibitors of NO Generation on iNOS mRNA Levels-To assess the potential role of NO as a mediator of the effect of BH 4 on iNOS expression, we first tested whether limiting NO generation with L-NAME would inhibit sepiapterin potentiation of cytokine-induced iNOS mRNA expression. We observed that in conditions in which L-NAME markedly reduced the accumulation of nitrite in the supernatant of cells treated with sepiapterin plus cytokines (Fig. 5A), the ability of sepiapterin to potentiate cytokine-induced expression of iNOS mRNA was not affected significantly (Fig. 5, B and C). We investigated next whether an exogenous supply of NO could mimic the effect of sepiapterin. As shown in Fig. 5, supplementing the incubation medium with sodium nitroprusside during treatment with cytokines did not induce any further increase in iNOS mRNA levels. DISCUSSION BH 4 biosynthesis appears to be an essential requirement for the induction of NOS activity in a variety of experimental models (11,13,35). Evidence for this has arisen mainly from the use of inhibitors of the BH 4 biosynthetic pathway, including inhibitors of GTP cyclohydrolase I, such as DAHP (33), or of sepiapterin reductase, such as N-acetylserotonin, phenprocoumon, or dicumarol (36,37), which cause a depletion of intracellular BH 4 levels, both in resting and in stimulated cells (15,17,38). Since the amount of NO generated seems to be limited by the amount of BH 4 present in cells, the inhibition of BH 4 biosynthesis has been envisaged as a target for the design of therapeutic tools to be used in the pathological conditions associated with an abnormally increased NO production (12,13,35). It is assumed that the main role of the increased pteridine synthesis observed during cytokine stimulation in a variety of cell types is to provide a cofactor for the BH 4 -dependent generation of NO by the cytokine-induced NOS. Our results indicate, however, that BH 4 biosynthesis also contributes to the regulation of the expression of iNOS mRNA and protein in response to cytokine stimulation of HMC. We have observed that the GTP cyclohydrolase I inhibitor DAHP notably diminishes cytokine-induced iNOS mRNA accumulation. In contrast, increasing BH 4 availability via the salvage pathway by the addition of sepiapterin not only circumvents the inhibitory effect of DAHP, but it strongly potentiates the effect of cytokines on iNOS mRNA and protein induction. Thus, in HMC, the mechanism of DAHP inhibition of iNOS expression appears to be due to the limitation of BH 4 biosynthesis, although a BH 4 -independent effect of DAHP on iNOS expression, analogous to that reported in primary murine macrophages (39), cannot be ruled out. Therefore, our results reinforce the significance of the frequently observed coinduction of GTP cyclohydrolase and iNOS by inflammatory stimuli (16,17). Although cytokines have been reported to stimulate the activity of GTP cyclohydrolase I strongly in a variety of systems, the overall stimulation of BH 4 biosynthesis and its impact on iNOS induction appear to be highly variable depending on the cell type, the stimuli employed, and the basal BH 4 levels (18,39). The BH 4 content of untreated HMC is remarkably low. The cytokine combination used throughout this study, IL-1␤/TNF-␣, elicited a submaximal induction of iNOS in these cells, which was not paralleled by a significant increase in intracellular BH 4 levels, but it was nevertheless susceptible to modulation by the inhi- bition of BH 4 biosynthesis or by the addition of an exogenous BH 4 source. This suggests that BH 4 availability is indeed playing a role in the regulation of iNOS expression in HMC. On the other hand, discrepancies between nitrite accumulation and cytosolic BH 4 concentrations during iNOS induction have been documented and interpreted previously on the basis that the newly synthesized iNOS protein, which has high affinity for BH 4 (40), depletes BH 4 from the cytosol rapidly (12). In this way, BH 4 would become a limiting factor for full iNOS activity probably because of a relatively lesser induction of BH 4 biosynthesis (12).
The effect of BH 4 on iNOS expression in HMC appears to be mediated, at least in part, by a contribution to mRNA stabilization since DAHP significantly reduced iNOS mRNA half-life, whereas sepiapterin prolonged it. This implies that in HMC, iNOS expression can be subjected to regulation at the posttranscriptional level. In addition, preliminary results from our laboratory suggest that increasing BH 4 availability with sepiapterin could also have stimulatory effects on the transcription rate of iNOS mRNA in nuclear runoff assays (results not shown). However, the intrinsic biochemical mechanisms of these phenomena remain to be elucidated. It should be noted that an increment in BH 4 levels per se, like that brought about by sepiapterin supplementation, was not able to induce iNOS activity or mRNA expression in HMC. In addition, the existence of BH 4 -dependent proteins involved in mRNA stabilization and/or transcription factors has not been documented. Thus, a direct effect of BH 4 on iNOS mRNA expression seems unlikely. Clearly, other possibilities of interaction between BH 4 availability and iNOS induction need to be considered, including the potential contribution of the NO generated as a consequence of iNOS activity. NO has been reported to exert both negative and positive feedback on iNOS activity and expression (41,42). In astroglial cells, the expression of iNOS mRNA elicited by a combination of IL-1␤ and IFN-␥ has been found to be amplified in the presence of NOS inhibitors or NO-trapping agents and to be reduced by NO donors (41). Interestingly, BH 4 has been reported to inactivate NO by inducing its oxidation in a 1:1 stoichiometry (43). Therefore, BH 4 could be acting as a NO scavenging agent in HMC, thus attenuating NO negative feedback on iNOS expression. In contrast, in rat mesangial cells, NO has been described to potentiate IL-1␤-induced iNOS expression (42). In this context, BH 4 availability would influence iNOS activity and consequently the amount of NO generated, which in turn would be responsible for the potentiation of iNOS induction (42). However, in neither of these experimental systems did NO appear to influence iNOS mRNA stability (41,42). Under our experimental conditions, the manipulation of the NO generated by means of the NO donor sodium nitroprusside and the NOS inhibitor L-NAME did not result in substantial changes in cytokine-induced iNOS expression or in its potentiation by sepiapterin. Although detailed studies would be needed to clarify completely the effects of NO on iNOS expression, our results do not support a major role for NO in the amplification of iNOS induction observed in HMC.
The results herein reported suggest that the induction of iNOS in HMC results from the integration of multiple signals. On one hand, cytokines would directly stimulate responsive elements on the iNOS gene. At the same time, they would indirectly potentiate the activity and expression of iNOS by stimulating the synthesis of BH 4 , which would contribute to the catalytic activity of iNOS and to iNOS mRNA expression. In vivo, the supply of BH 4 to perform these functions could come from several sources: from the activation of the BH 4 biosynthetic pathway in the mesangial cells and/or in the endothelial cells that line the capillaries of the glomerulus. In human endothelial cells, interferon-␥, bacterial lipopolysaccharide, IL-1, and TNF-␣ have been reported to stimulate the synthesis of BH 4 (15,35,44,45). Interestingly, up to 90% of the newly synthesized BH 4 is released into the culture medium (45). This release is not random, but vectorially directed into the basal compartment, thereby providing underlining smooth muscle cells with the cofactor needed for NO production (46). Our results suggest that the secreted BH 4 can also potentiate iNOS mRNA expression in HMC. Importantly, some of the so-called deactivating cytokines, including IL-4 and IL-10, have been reported to suppress profoundly the BH 4 synthesis elicited by inflammatory stimuli in endothelial cells, leading to the complete disappearance of extracellular BH 4 (45). These deactivating cytokines are also known to inhibit iNOS activity and expression in various cell types (47,48). It would be interesting to determine whether the inhibitory effect of these cytokines on iNOS expression can be mediated, at least in part, by the down-regulation of BH 4 biosynthesis, and if so, whether GTP cyclohydrolase I can be a target for their action.
In conclusion, our observation that the availability of BH 4 can modulate the expression of iNOS in HMC strengthens the hypothesis that this cofactor can play an important role in the regulation of NO generation and consequently of vascular tone. Therefore, the pharmacological manipulation of BH 4 levels emerges as a valuable approach for the management of NOrelated pathophysiological processes.