Treatment with tetrahydrobiopterin overcomes brain death-associated injury in a murine model of pancreas transplantation.

Brain death (BD) has been associated with an immunological priming of donor organs and is thought to exacerbate ischemia reperfusion injury (IRI). Recently, we showed that the essential nitric oxide synthase co-factor tetrahydrobiopterin (BH4) abrogates IRI following experimental pancreas transplantation. We therefore studied the effects of BD in a murine model of syngeneic pancreas transplantation and tested the therapeutic potential of BH4 treatment. Compared with sham-operated controls, donor BD resulted in intragraft inflammation reflected by induced IL-1ß, IL-6, VCAM-1, and P-selectin mRNA expression levels and impaired microcirculation after reperfusion (p < 0.05), whereas pretreatment of the BD donor with BH4 significantly improved microcirculation after reperfusion (p < 0.05). Moreover, BD had a devastating impact on cell viability, whereas BH4-treated grafts showed a significantly higher percentage of viable cells (p < 0.001). Early parenchymal damage in pancreatic grafts was significantly more pronounced in organs from BD donors than from sham or non-BD donors (p < 0.05), but BH4 pretreatment significantly ameliorated necrotic lesions in BD organs (p < 0.05). Pretreatment of the BD donor with BH4 resulted in significant recipient survival (p < 0.05). Our data provide novel insights into the impact of BD on pancreatic isografts, further demonstrating the potential of donor pretreatment strategies including BH4 for preventing BD-associated injury after transplantation.

Iron regulates nitric oxide synthase activity by controlling nuclear transcription.

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts.

The present study demonstrates that murine dermal fibroblasts produce nitrite (NO2-) and nitrate (NO3-) upon treatment with interferon gamma (IFN-gamma). This formation is dependent on L-arginine and can be inhibited by the L-arginine analogue NG-monomethyl-L-arginine. The effect of IFN-gamma is drastically increased by cotreatment with tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), or lipopolysaccharide (LPS). The tested cytokines also induce formation of tetrahydrobiopterin in murine fibroblasts. Inhibition of guanosine triphosphate-cyclohydrolase I, the key enzyme of tetrahydrobiopterin de novo synthesis with 2,4-diamino-6-hydroxy-pyrimidine, leads to decreased formation of NO2- and NO3-. This effect can be reversed by addition of sepiapterin, which provides tetrahydrobiopterin via a salvage pathway. Methotrexate, which inhibits the salvage pathway, blocks the restoration of NO2- and NO3- production by sepiapterin. The cytotoxic effect of combinations of IFN-alpha with TNF-gamma, IL-1, or LPS is attenuated by inhibition of tetrahydrobiopterin synthesis. These results show that intracellular concentrations of tetrahydrobiopterin control the amount of NO2- and NO3- produced in situ and suggest that the role of cytokine-induced tetrahydrobiopterin synthesis is to provide cells with the active cofactor for production of nitrogen oxides.

Donor pretreatment with tetrahydrobiopterin saves pancreatic isografts from ischemia reperfusion injury in a mouse model.

Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B-substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 µM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI-related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia-induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.

The effect of secretory leukocyte protease inhibitor (SLPI) on ischemia/reperfusion injury in cardiac transplantation.

We investigated the role of secretory leukocyte protease inhibitor (SLPI) in ischemia/reperfusion injury in cardiac transplantation. SLPI-/- mouse hearts and wild-type (WT) controls were transplanted immediately or after 10 h of cold ischemia (CI). Recombinant SLPI (rSLPI) was added to the preservation solution or given systemically. After evaluation of myocardial performance, grafts were investigated for histology, SLPI, TNF-alpha, TGF-beta, NF-kappaB and protease expression at indicated time points. Early myocardial contraction was profoundly impaired in SLPI-/- hearts exposed to CI and associated with high intra-graft protease expression. Systemic administration of rSLPI had no effect, however, when SLPI was added to the preservation solution, myocardial contraction was restored to normal. At 10 days, inflammation, myocyte vacuolization and necrosis were significantly more severe in SLPI-/- hearts. SLPI gene expression was detected in WT mice at 12 and 24 h and was significantly higher after CI. SLPI protein was observed at 24 h and 10 days. High intra-graft concentrations of SLPI after administration of rSLPI were inversely correlated with protease levels early and TGF-beta expression late after reperfusion. SLPI plays a crucial role in early myocardial performance and postischemic inflammation after cardiac transplantation. A dual inhibitory effect on protease and TGF-beta expression might be the underlying mechanism.

Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats.

Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.

Clinical significance of neopterin for prognosis and follow-up in ovarian cancer.

Neopterin, a pyrazinopyrimidine compound, is a marker of activation of cell-mediated immunity. The prognostic value of pretherapeutically and of serially measured urinary neopterin levels in patients with ovarian cancer was assessed, in a blinded manner, by analysis of 658 urine specimens from 74 women. The specimens were collected during a 5-year period (January 1981 to January 1986). Thirty-one deaths due to cancer were observed during the study period. By statistical analysis, a significant predictive value of pretherapeutic neopterin levels was found and compared with that of other possible prognostic clinical and laboratory findings. Multivariate analysis, using stratification by tumor stage, demonstrated that this predictive information was independent of other variables. A significant association was found between serial neopterin measurements and the current risk of death during follow-up (P less than 0.0001). In addition, current death risk was correlated with neopterin levels measured 6 months previously (P = 0.026). The histological outcome at surgical reexamination was correlated with the current neopterin levels (P = 0.016). Further, normal neopterin levels in women with evidence of tumor at surgical reexamination were shown to be a sign of better prognosis than elevated levels. Measurement of urinary neopterin levels during follow-up of women with ovarian cancer appears to be a valuable adjunct to conventional techniques, particularly in patients refusing explorative surgery.

Neopterin formation and tryptophan degradation by a human myelomonocytic cell line (THP-1) upon cytokine treatment.

Determination of neopterin [D-erythro-6-(1',2',3'-trihydroxypropyl)pterin] in body fluids is a powerful diagnostic tool in a variety of diseases in which activation of cellular immune mechanisms is involved, such as certain malignancies, allograft rejection, and autoimmune and infectious diseases. In vitro, neopterin is released into the supernatant by peripheral blood-derived monocytes/macrophages upon stimulation with gamma-interferon. In parallel, cleavage of tryptophan by indoleamine 2,3-dioxygenase is induced. We report here that the human myelomonocytic cell line THP-1 forms neopterin and degrades tryptophan upon treatment with gamma-interferon. Like in macrophages alpha-interferon and beta-interferon induce these pathways only to a much smaller degree. The action of interferons is enhanced by cotreatment with tumor necrosis factor alpha, lipopolysaccharide, or dexamethasone. gamma-Interferon-induced neopterin formation and indoleamine 2,3-dioxygenase activity are increased by raising extracellular tryptophan concentrations. The pattern of intracellularly formed pteridines upon stimulation with gamma-interferon shows the unique characteristics of human monocytes/macrophages. Neopterin, monapterin, and biopterin are produced in a 50:2:1 ratio. Thus, the THP-1 cell line provides a permanent, easily accessible in vitro system for studying the induction and mechanism of neopterin formation.

Neopterin as a prognostic indicator in patients with carcinoma of the uterine cervix.

In vitro, neopterin, a pyrazinopyrimidine compound, is excreted by human monocytes-macrophages after induction by supernatants from activated T-lymphocytes or by recombinant gamma-interferon. In vivo, it represents a noninvasive test for activation of cellular immune reactions. To evaluate the prognostic value of pretherapeutic urinary neopterin levels and of serial neopterin measurements during follow-up in women with cervical cancer, 1088 urine specimens from 186 consecutive patients were analyzed. Clinical assessments were made without knowledge of the results of neopterin assays (a "blinded" assessment). During the observation period (June 1980 to March 1984), 27 relapses, 18 metastases, and 26 deaths were seen. The prognostic significance of pretherapeutic neopterin and other possible prognostic clinical and laboratory parameters was tested by the univariate and multivariate Cox proportional hazards model using a stratification according to stage and surgical treatment. The combination of age at diagnosis, pretherapeutical hemoglobin, leukocyte count, and neopterin was found to predict survival best. On the basis of this result, risk groups were identified exhibiting markedly different survival behavior. A highly significant association was found between serial neopterin measurements and the risk for a relapse, metastasis, or death. The data suggest that urinary neopterin levels might be a useful adjuvant parameter in monitoring women with cervical cancer.

Characteristics of interferon induced tryptophan metabolism in human cells in vitro.

Interferon-gamma-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-gamma. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilic acid and 3-hydroxyanthranilic acid). Interferon-gamma regulated only the activity of indoleamine 2,3-dioxygenase. All other enzyme activities detected were independent of interferon-gamma, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyl-L-kynurenine. Increasing the extracellular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK-HEP-1. The activity was unaffected by extracellular L-tryptophan with dermal fibroblasts and A 498. Though interferon-gamma was the most potent inducer of tryptophan metabolism, interferon-alpha and/or -beta showed small but distinct action on some of the cells. In all cells which reacted to interferon-gamma by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-gamma action, that the extent of this induction is influenced by extracellular L-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation of 3-hydroxyanthranilic acid from tryptophan which is regulated by interferon-gamma.

6-Tetrahydrobiopterin functions as a UVB-light switch for de novo melanogenesis.

(6R)-L-erythro 5,6,7,8-tetrahydrobiopterin (6-BH4) and its 7-isomer (7-BH4) function as uncompetitive inhibitors of human and mushroom tyrosinases. Stoichiometry for the binding of [3H]-labeled 6-BH4 to both tyrosinases has been established as 1:1. Stable complexation of 6-BH4 to tyrosinase appears to involve a hydrophilic conserved glutamic acid (Glu131) with a pKa = 4.7. Photo-oxidation by UVB-light and O2 reverses the inhibition of tyrosinase by 6-BH4 and 7-BH4 with the 6-BH4/tyrosinase complex being four-fold more photolabile than 7-BH4/tyrosinase. The photo-oxidation of 6-BH4 by UVB-light can be assessed spectrophotometrically with this reaction yielding 7,8-dihydroxanthopterin as the final product, 7,8-Dihydroxanthopterin neither binds to nor inhibits tyrosinase. By contrast, UVA light does not catalyze the photodegradation of 6-BH4. Taken together, our results indicate that the photo-oxidation of the tetrahydrobiopterins by UVB may represent a photo-switch in the regulation of tyrosinase activity to promote de novo melanogenesis.

Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites.

Recombinant rat liver GTP cyclohydrolase I has been prepared by heterologous gene expression in Escherichia coli and characterized by biochemical and biophysical methods. Correlation averaged electron micrograph images of preferentially oriented enzyme particles revealed a fivefold rotational symmetry of the doughnut-shaped views with an average particle diameter of 10 nm. Analytical ultracentrifugation and quantitative scanning transmission electron microscopy yielded average molecular masses of 270 kDa and 275 kDa, respectively. Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homodecameric protein complex consisting of two fivefold symmetric pentameric rings associated face-to-face. Examination of the amino acid sequence combined with calcium-binding experiments and mutational analysis revealed a high-affinity, EF-hand-like calcium-binding loop motif in eukaryotic enzyme species, which is absent in bacteria. Intrinsic fluorescence measurements yielded an approximate dissociation constant of 10 nM for calcium and no significant binding of magnesium. Interestingly, a loss of calcium-binding capacity observed for two rationally designed mutations within the presumed calcium-binding loop of the rat GTP cyclohydrolase I yielded a 45% decrease in enzyme activity. This finding suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I gene of patients suffering from dopa responsive dystonia.

Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice.

(6R)-5,6,7,8-Tetrahydro-biopterin (H(4)B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H(4)B synthesis. A relative shortage of H(4)B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H(4)B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BLxCBA) were compared. H(4)B levels were determined by high-performance liquid chromatography and NO synthase activity by [(3)H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H(4)B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with N(G)-nitro-L-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H(4)B, the differences between the 2 groups disappeared. Our findings demonstrate that H(4)B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H(4)B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.

Iron modulates interferon-gamma effects in the human myelomonocytic cell line THP-1.

This investigation demonstrates that low concentrations (25 microM) of free and transferrin-bound iron reduce the efficiency of the interferon-gamma (IFN-gamma) signal in the human myelomonocytic cell line THP-1, as seen by decreased production of neopterin, reduced degradation of tryptophan, and impaired expression of major histocompatibility complex (MHC) class II antigens. This inhibitory effect of iron, which is not due to an enhanced cytotoxicity towards THP-1 cells, is increased by enhancement of iron concentrations in a dose-dependent relationship and can be partially reversed by increasing amounts of the cytokine. The iron-mediated inhibition of the effects of IFN-gamma is fully reversed when iron is administered concomitantly with equimolar concentrations of the iron chelator deferoxamine. Furthermore, deferoxamine alone is even able to enhance the efficiency of the IFN-gamma signal. Our data provide evidence that there is an inverse correlation between the intracellular amount of iron, which is not bound to ferritin, and the activity of the IFN-gamma signal. This suggests that iron withholding by the immune cells in the course of inflammatory disorders may also contribute to the enhancement of the cytopathic effect of IFN-gamma. This speculation is confirmed by the observation of high concentrations of immune activation markers such as IFN-gamma and neopterin and low serum iron levels in patients with hypoferric anemia in the course of chronic inflammation.

Neopterin concentrations in cerebrospinal fluid and serum of individuals infected with HIV-1.

Neopterin, a biochemical marker for the activation of cell-mediated immune reactions, was determined in serum and cerebrospinal fluid (CSF) from patients infected with HIV-1. A significant correlation was found between serum and CSF neopterin concentrations, although concentrations of neopterin in serum were more closely correlated with the clinical severity of HIV-1 infection than those in CSF. However, higher CSF levels were observed in patients with neurologic/psychiatric symptoms than in unaffected patients. Also, quotients of CSF neopterin versus serum neopterin concentrations were increased, indicating intrathecal production of neopterin. Positive HIV-1 isolation from peripheral blood mononuclear cells (PBMC) was associated with higher neopterin concentrations in serum, when compared with negative HIV-1 isolation. Neopterin in CSF appears to be a suitable biochemical marker in patients with HIV-1 infection for detecting overt neurologic/psychiatric disturbances. The data suggest that in HIV-1 infected patients, cell-mediated immune reactions might be activated intrathecally and might contribute to neuropsychiatric disease.

Negative correlation between blood cell counts and serum neopterin concentration in patients with HIV-1 infection.

Hematopoietic disturbances are common in patients with HIV-1 infection. Recent studies on immune activation markers such as neopterin demonstrate that HIV-1 infection is associated with chronic immune activation. We investigated a possible association between serum neopterin concentrations and blood cell counts (CD4+ T cells, white blood cells, platelets, red blood cells) and hemoglobin and hematocrit in 94 HIV-1-seropositive individuals [52 Walter Reed (WR) stage 1, 31 WR2, one WR5, and 10 WR6]. There were significant negative correlations between neopterin concentrations and CD4+ T cells, hemoglobin, hematocrit and platelets. These correlations were also significant if either only WR1 and WR2 patients or the entire set of data were considered for calculations. Thus, hematological abnormalities are associated with chronic immune activation in patients with HIV-1 infection. Large amounts of neopterin are released by human macrophages on stimulation with interferon-gamma (IFN gamma), and tumor necrosis factor alpha (TNF alpha) further enhances the effect of IFN gamma. Therefore, our data suggest that activated immune cells and specific cytokines such as IFN gamma and TNF alpha are involved inhibiting hematopoiesis.

Urinary excretion of porphyrins is increased in patients with HIV-1 infection.

Urine concentrations of total porphyrins and of porphyrin precursors, delta-aminolaevulinic acid and porphobilinogen, were analysed in the first-morning urine samples of 36 people with established HIV-1 infection. For comparison, we also analysed the urine samples of 26 healthy HIV-seronegative people. In patients with HIV-1 infection concentrations of total porphyrins were found to be significantly higher than in healthy controls. Furthermore, there was a close correlation between concentrations of total porphyrins and porphobilinogen and urine neopterin concentrations in patients. Our data indicate an association between chronic immune activation and altered porphyrin metabolism. The data may provide a rationale for the observation of acute porphyria in patients with HIV-1 infection.

Coordinated induction of inducible nitric oxide synthase and GTP-cyclohydrolase I is dependent on inflammatory cytokines and interferon-gamma in HaCaT keratinocytes: implications for the model of cutaneous wound repair.

Recently we demonstrated a strong expression of inducible nitric oxide synthase (iNOS) and GTP-cyclohydrolase I (GTP-CH I) in the basal keratinocytes of the epidermis adjacent to the wound and of the hyperproliferative epithelium during wound healing. To identify possible mediators of iNOS and GTP-CH I expression during this process, we analyzed the regulation of iNOS and GTP-CH I expression in cultured human keratinocytes. We found a large and long lasting coinduction of iNOS and GTP-CH I expression upon simultaneous treatment of quiescent cells with inflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, but not with serum growth factors. The stimulatory effect of interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma is strongly synergistic on iNOS and GTP-CH I expression, because these factors alone stimulated GTP-CH I expression, although to a much lesser extent. Furthermore, iNOS mRNA levels are not influenced at all by stimulation with IL-1beta and revealed only a weak induction after treatment with tumor necrosis factor-alpha and interferon-gamma. Induction of iNOS and GTP-CH I gene expression upon cytokine and interferon-gamma exposure is independent of de novo protein synthesis. Because these cytokines are present at the wound site, they might be responsible for iNOS and GTP-CH I induction during cutaneous repair. Serum, which is released upon hemorrhage, is likely to play no stimulatory role in iNOS and GTP-CH I induction during wound healing.

Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects.

Tetrahydrobiopterin (H4-biopterin) is an essential cofactor of a set of enzymes that are of central metabolic importance, i.e. the hydroxylases of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan, of ether lipid oxidase, and of the three nitric oxide synthase (NOS) isoenzymes. As a consequence, H4-biopterin plays a key role in a vast number of biological processes and pathological states associated with neurotransmitter formation, vasorelaxation, and immune response. In mammals, its biosynthesis is controlled by hormones, cytokines and certain immune stimuli. This review aims to summarize recent developments concerning regulation of H4-biopterin biosynthetic and regulatory enzymes and pharmacological effects of H4-biopterin in various conditions, e.g. endothelial dysfunction or apoptosis of neuronal cells. Also, approaches towards gene therapy of diseases like the different forms of phenylketonuria or of Parkinson's disease are reviewed. Additional emphasis is given to H4-biopterin biosynthesis and function in non-mammalian species such as fruit fly, zebra fish, fungi, slime molds, the bacterium Nocardia as well as to the parasitic protozoan genus of Leishmania that is not capable of pteridine biosynthesis but has evolved a sophisticated salvage network for scavenging various pteridine compounds, notably folate and biopterin.

Identification of imidazole as L-arginine-competitive inhibitor of porcine brain nitric oxide synthase.

Imidazole acts as a heme-site inhibitor of nitric oxide synthase (NOS). We used this compound to investigate whether the substrate L-arginine binds directly to the heme or to a separate domain of brain NOS. Enzyme kinetic experiments showed that imidazole enhanced the apparent Km for L-arginine without affecting maximal enzyme activity, and binding studies revealed that the inhibitor displaced the radioligand NG-nitro-L-[3H]arginine in a concentration-dependent fashion. These results demonstrate that imidazole exerts its effects on NOS in an L-arginine-competitive manner and that the substrate site of the enzyme may be identical with the prosthetic heme group.