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AIMS/HYPOTHESIS: Hypertension, endothelial dysfunction and insulin resistance are associated conditions that share oxidative stress and vascular inflammation as common features. Adiponectin is an abundant plasma adipokine that plays a physiological role in modulating lipid metabolism and exerts a potent anti-inflammatory activity. We hypothesised that adiponectin levels decrease in response to oxidative stress and that this may promote the development of hypertension, endothelial dysfunction and insulin resistance. METHODS: Rats were infused with angiotensin II (AngII) or its vehicle, either alone or in combination with tempo1 (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl), a membrane-permeable metal-independent superoxide dismutase mimetic, or tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor for nitric oxide synthase activity. Heart rate, systolic blood pressure, body weight and serum levels of adiponectin were measured on day 7 of treatment, and then the animals were killed. Vessel tone and superoxide production were measured ex vivo in thoracic vascular rings. The expression of adiponectin mRNA in adipose tissue was assessed by Northern blotting, and in 3T3-L1 adipocytes exposed to H2O2 by real-time PCR. The expression of NAD(P)H oxidase subunit mRNAs in the rats was assessed by RT-PCR and real-time PCR. RESULTS: Hypertension and endothelial dysfunction were induced in rats by infusion of AngII and reversed by administration of tempol. Plasma concentrations of adiponectin and adipose tissue levels of adiponectin mRNA were decreased in AngII-infused rats, and this effect was prevented by cotreatment with tempol or BH4. The production of superoxide anions (O2-) was significantly increased in the aortae of AngII-treated rats, and this increase was prevented by the administration of tempol or BH4. Levels of mRNAs that encode NAD(P)H oxidase components, including p22phox, gp91phox, p47phox and Rac1, were similarly increased in adipose tissue, aortae and hearts of AngII-infused rats. Cotreatment of rats with tempol or BH4 reversed AngII-induced increases in NAD(P)H oxidase subunit mRNAs. Fully differentiated 3T3-L1 adipocytes, also exhibited diminished adiponectin mRNA levels when exposed to low concentrations of H2O2. CONCLUSIONS/INTERPRETATION: Our results demonstrate that AngII-induced oxidative stress and endothelial dysfunction are accompanied by a decrease in adiponectin gene expression. Since antioxidants were observed to prevent the actions of AngII, and H2O2 on its own suppressed adiponectin expression, we conclude that adiponectin gene expression is negatively modulated by oxidative stress. Plasma adiponectin levels may provide a useful indicator of oxidative stress in vivo, and suppressed levels may contribute to the proinflammatory and metabolic derangements associated with type 2 diabetes, coronary artery disease and the metabolic syndrome.

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Nitric oxide (NO) is a free-radical product of mammalian cell metabolism that plays diverse and important roles in the regulation of cell function. Biological actions of NO arise as a direct consequence of chemical reactions between NO or NO-derived species and protein targets. Reactions of NO with transition metals in target proteins have garnered the most attention to date as the principal mechanism of NO signaling; nonetheless, S-nitrosylation of protein Cys residues is rapidly moving to center stage in importance. In general, however, there has been a delay in adequate appreciation of the role of S-nitrosylation in biological signaling by NO. This lag is attributed to a poor understanding of the basis for selective targeting of NO to particular thiols, and methodological limitations in accurately quantifying this modification--recent breakthroughs in concepts and methods diminish these barriers. Here, we consider the wheres and whys of protein S-nitrosylation and its basis for specificity. Protein S-nitrosylation potentially represents a ubiquitous and fundamental mechanism for posttranslational control of protein activity on a par with that of O-phosphorylation.

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Endothelial dysfunction accompanies suboptimal glucose control in patients with diabetes mellitus. A hallmark of endothelial dysfunction is a deficiency in production or bioavailability of vascular nitric oxide (NO). Here we demonstrate that acute exposure of human endothelial cells to glucose, at levels found in plasma of diabetic patients, results in a significant blunting of NO responses to the endothelial nitric oxide synthase (eNOS) agonists bradykinin and A-23187. Monitoring of NO generation by purified recombinant bovine eNOS in vitro, using amperometric electrochemical detection and an NO-selective porphyrinic microelectrode, showed that glucose causes a progressive and concentration-dependent attenuation of detectable NO. Addition of glucose to pure NO solutions similarly elicited a sharp decrease in NO concentration, indicating that glucose promotes NO loss. Electrospray ionization-tandem mass spectrometry, using negative ion monitoring, directly demonstrated the occurrence of a covalent reaction involving unitary addition of NO (or a derived species) to glucose. Collectively, our findings reveal that hyperglycemia promotes the chemical inactivation of NO; this glucose-mediated NO loss may directly contribute to hypertension and endothelial dysfunction in diabetic patients.

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This article presents data linking eNOS function and nitric oxide (NO) generation or availability to vascular remodeling and signaling, hyperlipidemias, advanced glycated end products and hyperglycemia. These data should be viewed within the broader framework of endothelial cell dysfunction (ECD). It is possible that vascular dysfunctions are capable of triggering early preclinical forms of generalized ECD. Accordingly, it is important to learn more about simple noninvasive ways to assess the functional state of eNOS and the bioavailability of NO. Here we discuss the growing body of evidence--which suggests that disturbances of NO production or availability are major determinants of ECD--and the need for therapeutic efforts toward correction of eNOS activity and NO levels in blood vessels.

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Nitric oxide (NO) is a key biosignaling molecule produced in both peripheral tissues and the central nervous system by a family of enzymes known as nitric oxide synthases (NOSs). NOSs convert L-arginine to stoichiometric quantities of NO and L-citrulline using molecular oxygen and NADPH as cofactors. Techniques for measurement of NO and NOS activity are essential to demonstrate the role of NO and NO-derived species in biological systems. This unit describes two methods for detection of NO: a direct method employing chemiluminescent detection and one based on quantification of the stable oxidation products with detection using the Griess reagent. Additionally, NOS activity can be quantified by measuring the conversion of radiolabeled L-arginine to radiolabeled L-citrulline.

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L-arginine is the substrate for nitric oxide (NO) production by each of the 3 NO synthase (NOS) isoforms encoded by the mammalian genome. Despite the pivotal roles of NO in mammalian physiology and pathophysiology, the source of arginine for NO synthesis is not clearly defined. In this context, it is notable that cell types that do not have a complete urea cycle often possess the urea cycle enzymes argininosuccinate synthase and argininosuccinate lyase; together, these enzymes confer the ability to regenerate arginine from the NOS product, L-citrulline. Herein, the authors summarize evidence to support the view that argininosuccinate synthase and argininosuccinate lyase function in an arginine-citrulline cycle, providing a ready source of arginine for high-output NO synthesis. The arginine-citrulline cycle is induced in vascular cells by the same cytokines that trigger iNOS expression and provides the preferred source of substrate for NO production. Evidence suggests that argininosuccinate synthase activity is rate-limiting to high-output NO synthesis and, hence, represents a novel target for the treatment of pathophysiological conditions arising from NO overproduction.

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NO production by the endothelial and neuronal isoforms of nitric oxide synthase (cNOS) is regulated on a moment-to-moment basis by calmodulin binding, triggered by transient elevations in intracellular-free calcium levels. Nonetheless, additional modes of cNOS regulation are implicit in the discoveries of stimuli that elicit a sustained increase in cNOS activity despite undetectable or transient increases in intracellular Ca2+ in endothelial cells; such stimuli include shear-stress, oestrogen, insulin or insulin-like growth factor treatment of endothelial cells. Recently, we identified a peptide insertion within the FMN-binding domain of mammalian NOSs that is unique to calcium-dependent isoforms, and not shared with inducible NOS or ancestral flavoproteins. Evidence suggests that this insertion serves as a fundamental control element, analogous to intrinsic autoinhibitory peptides that have been demonstrated to regulate activity of other calmodulin-dependent enzymes. Thus, the peptide insertion of cNOSs appears to function as structural element that is displaced upon calmodulin binding, resulting in dysinhibition of NO synthesis. Once displaced, the peptide may also be subject to transient chemical modifications and protein-protein interactions that modulate autoinhibitory function. Herein we summarize our present knowledge and speculate on mechanisms by which calmodulin and the autoinhibitory peptide conspire to regulate cNOS activity.

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The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 microM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on L-arginine but not D-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 microM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

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Facial pain of patients with craniomandibular disorders might be caused by muscle overload. However, the activity of masticatory muscles of healthy individuals is still unknown. The aim of this study was therefore a first attempt to clarify this question by recording the masseter muscle activity of healthy subjects during sleep by means of portable recorders. The study was performed on 21 healthy subjects selected after telephone and questionnaire screenings and clinical examination from among randomly selected inhabitants of Zürich. The masseter EMG was recorded during seven nights in each subject's natural environment with the electrodes in reproducible position. The signal was analyzed for number, amplitude, and duration of contraction periods defined as signal portions above a threshold which could contain sub-threshold signal portions shorter than the standby time of 5 sec. The signal amplitude was expressed in percent of the amplitude recorded during maximum voluntary clenches (%MVC). An average of 71.9 +/- 28.7 contraction episodes per night (men, 74.7 +/- 30.1; women, 65.0 +/- 23.8; p = 0.043), i.e., of 10.5 +/- 3.8 per hour (men, 11.0 +/- 4.0; women, 9.3 +/- 3.0; p = 0.005), was found. The average mean amplitude was 26.2 +/- 6.4% MVC (men, 27.0 +/- 6.8; women, 24.4 +/- 4.5; p = 0.009). The duration of the episodes had a mode of 0.5 sec, and the group mean of the integral of the amplitude over time was 123.7 +/- 157.9% MVC (men, 138.9 +/- 184.0; women, 85.9 +/- 28.2; p = 0.005). Healthy subjects showed intermittent periods of masseter activity during sleep which, on average, were of rather low intensity and short duration.

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Immunostimulants trigger vascular smooth muscle cells (VSMC) to express the inducible isoform of NO synthase (iNOS) and increased arginine transport activity. Although arginine transport in VSMC is considered to be mediated via the y+ system, we show here that rat VSMC in culture express the cat-1 gene transcript as well as an alternatively spliced transcript of the cat-2 gene. An RT-PCR cloning sequence strategy was used to identify a 141-base nucleotide sequence encoding the low-affinity domain of alternatively spliced CAT-2A and a 138-base nucleotide sequence encoding the high-affinity domain of CAT-2B in VSMC activated with lipopolysaccharide (LPS) in combination with interferon-gamma (IFN). With this sequence as a probe, Northern analyses showed that CAT-1 mRNA and CAT-2B mRNA are constitutively present in VSMC, and the expression of both mRNAs was rapidly stimulated by treatment with LPS-IFN, peaked within 4 h, and decayed to basal levels within 6 h after LPS-IFN. CAT-2A mRNA was not detectable in unstimulated or stimulated VSMC. Arginine transporter activity significantly increased 4-10 h after LPS-IFN. iNOS activity was reduced to almost zero in the absence of extracellular arginine uptake via system y+. Induction of arginine transport seems to be a prerequisite to the enhanced synthesis of NO in VSMC. Moreover, this work demonstrates tissue expression of CAT mRNAs with use of a model of LPS injection in rats. RT-PCR shows that the expression of CAT-1 and CAT-2B mRNA in the lung, heart, and kidney is increased by LPS administration to rats, whereas CAT-2A mRNA is abundantly expressed in the liver independent of LPS treatment. These findings suggest that together CAT-1 and CAT-2B play an important role in providing substrate for high-output NO synthesis in vitro as well as in vivo and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane arginine transport.

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Glycated proteins, including serum albumin, may be involved in the pathogenesis of diabetic vasculopathy. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMC) may, in part, promote atherosclerosis by increasing local oxidative stress. We therefore investigated whether VSMC exposed to glycated human serum albumin (GHSA) produce nitric oxide (NO) by increasing iNOS expression through transcriptional activation of the iNOS gene and whether this process is dependent on nuclear factor kappaB (NF-kappaB) activation. Treatment of VSMC with GHSA causes activation of NF-kappaB and the iNOS promoter. Induction of NF-kappaB and the iNOS promoter by GHSA exhibited dose-dependent kinetics at concentrations ranging from 3 to 1000 microgram/ml. GHSA alone was a weak inducer of NO production in VSMC as measured by determining nitrite levels, and interferon-gamma alone was totally ineffective, whereas the combination of GHSA and interferon-gamma was a strong stimulus. This synergy for NO production corresponded to Northern blot analyses of iNOS mRNA expression. Thus, GHSA may promote atherosclerosis in part by activation of NF-kappaB and upregulation of iNOS, thereby fostering local inflammation and oxidative stress.

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Appreciation of the role of nitric oxide (NO) in mammalian cell biology has toppled the paradigm that biological signaling is initiated exclusively by noncovalent, lock-and-key-type interactions with receptor proteins. Remarkably, nitric oxide is a free radical that signals by chemical reaction with its protein targets, resulting in covalent modifications and a stable alteration in protein structure and function. Although most proteins may be coerced to react with NO in vitro, the specific proteins that are functionally modified by NO within cells will depend on the concentration of NO and the composition of the intracellular milieu. A further level of complexity is introduced into NO signaling by the fact that reactions can occur with NO directly, or secondarily with NO-derived species. Much to the surprise of those who thought that reactive molecules are generated and act only under pathophysiological conditions (e.g., ischemia-reperfusion injury), NO has emerged as a prototype molecule that signals by chemistry in normal physiology. The unique attributes and importance of NO were recently recognized by the Nobel Prize Committee, with their decision to award the 1998 Prize in Medicine to Drs Furchgott, Ignarro, and Murad, pioneers in NO biology. This review surveys what we believe to be the most important mechanisms and targets of signaling by NO.

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OBJECTIVE: Immunostimulants increase nitric oxide (NO) and tetrahydrobiopterin (BH4) synthesis in vascular smooth muscle cells (VSMC) by coinducing expression of an isoform of NO synthase (iNOS) and GTP cyclohydrolase I (GTPCH). GTPCH is the first and rate-limiting enzyme in the synthesis of BH4, a cofactor of NO synthases. Given the adrenomedullin (AM) increases NO production, this effect of AM may involve modulation of BH4 synthesis in cytokine-stimulated VSMC. METHODS: We investigated the effects of AM on the synthesis of NO and BH4, the expression of iNOS and GTPCH mRNA, and the promoter activity of iNOS and GTPCH genes in rat VSMC stimulated with interleukin-1 (IL-1). RESULTS: IL-1 increased both NO and BH4 synthesis as well as the abundance of iNOS and GTPCH mRNA. AM significantly increased both NO and BH4 synthesis caused by IL-1 stimulation. AM also augmented the IL-1-induced increase in the abundance of iNOS and GTPCH mRNA. IL-1 activated the iNOS promoter activity as well as the GTPCH promoter activity in VSMC. AM alone had no effect on the activity of either the iNOS or the GTPCH promoter, nor did it potentiate the activation by IL-1 of either of these promoters. CONCLUSION: These results suggest that AM increases IL-1-induced NO and BH4 synthesis by enhancing the expression of iNOS and GTPCH genes at the post-transcriptional level. Thus, the potentiating effect of AM on NO synthesis appears to be associated with an increased expression of both genes necessary for cellular NO synthesis in VSMC.

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It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [3H]Nomega-nitro-L-arginine or [3H]BH4, but [3H]BH4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity.

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2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered to be a selective and direct-acting inhibitor of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the pathway for synthesis of tetrahydrobiopterin (BH4). Accordingly, DAHP has been widely employed to distinguish whether de novo BH4 synthesis is required in a given biological system. Although it has been assumed that DAHP inhibits GTPCH by direct competition with substrate GTP, this has never been formally demonstrated. In view of apparent structural homology between DAHP and BH4, we questioned whether DAHP may mimic BH4 in its inhibition of GTPCH by an indirect mechanism, involving interaction with a recently cloned 9.5-kDa protein termed GTPCH Feedback Regulatory Protein (GFRP). We show by reverse transcription-polymerase chain reaction that GFRP mRNA is constitutively expressed in rat aortic smooth muscle cells and further induced by treatment with immunostimulants. Moreover, functional GFRP is expressed and immunostimulant-induced BH4 accumulates in sufficient quantity to trigger feedback inhibition of GTPCH. Studies with DAHP reveal that GFRP is also essential to achieve potent inhibition of GTPCH. Indeed, DAHP inhibits GTPCH by dual mechanisms. At a relatively low concentration, DAHP emulates BH4 and engages the GFRP-dependent feedback inhibitory system; at higher concentrations, DAHP competes directly for binding with GTP substrate. This knowledge predicts that DAHP would preferably target GTPCH in tissues with abundant GFRP.

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Lipoteichoic acid (LTA), a component of the membrane of gram-positive bacteria, induces an isoform of nitric oxide (NO) synthase (iNOS) in vascular smooth muscle; this process may be associated with the vascular failure observed in gram-positive septic shock. The aim of the present work was to study the cellular mechanisms involved in the induction of NO synthesis by LTA from Staphylococcus aureus in cultured rat vascular smooth muscle cells. LTA induces the gene expression of iNOS and GTP cyclohydrolase I (GTPCH) as well as the formation of NO and tetrahydrobiopterin (BH4), effects which are synergistic with interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTPCH, inhibits both the increase in cellular levels of BH4 as well as the concomitant formation of NO caused by LTA in combination with interferon-gamma. This inhibition by DAHP is reversed by co-addition of sepiapterin which is a substrate for BH4 synthesis. Thus, BH4 synthesis is an absolute requirement for the induction of NO synthesis by LTA in vascular smooth muscle. Our findings also suggest that interrupting pterin synthesis may be an effective target for pharmacologic interventions aimed at limiting NO overproduction in gram-positive shock.

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Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

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Because tetra-hydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) formation, we investigated whether BH4 synthesis is required for cytokine-induced NO production in cultured rat cardiac myocytes. The total biopterin content of untreated cardiac myocytes was below our limit of detection. However, treatment with interleukin-1 alpha (IL-1 alpha) + interferon-gamma (IFN-gamma) caused a significant rise in biopterin levels and induced NO synthesis. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I (the rate-limiting enzyme for de novo BH4 synthesis), completely abolished the elevation in biopterin levels induced by IL-1 alpha + IFN-gamma. DAHP also caused a concentration-dependent inhibition of (IL-1 alpha + IFN-gamma)-induced NO synthesis. Similarly, N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, blocked increases in biopterin levels as well as NO synthesis induced by IL-1 alpha + IFN-gamma. Sepiapterin, substrate for BH4 synthesis via the pterin salvage pathway, prevented this inhibition by DAHP or N-acetylserotonin, and this effect was blocked by methotrexate. Sepiapterin and, to a lesser extent, BH4 dose dependently enhanced (IL-1 alpha + IFN-gamma)-induced NO synthesis, suggesting that the concentration of BH4 limits the rate of NO production. Inducible NO synthase mRNA and GTP cyclohydrolase I mRNA were induced by IL-1 alpha + IFN-gamma in parallel. We thus demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes.

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