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• W2048091395 abstract "Dopamine-β-hydroxylase (DβH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the β-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase-dependent mechanism. In this report, we investigated the relationship between NO and DβH enzymatic activity. In the initial in vitroexperiments, the activity of purified DβH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC50 of 1 mm. The inclusion of either azide or GSH partially restored DβH activity, suggesting the involvement of the reactive nitrogen oxide species, N2O3. Treatment of human neuroblastoma cells (SK-N-MC) with diethylamine/NO decreased cellular DβH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Co-culture of the SK-N-MC cells with interferon-γ and lipopolysaccharide-activated macrophages, which release NO, also reduced the DβH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N2O3, can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation. Dopamine-β-hydroxylase (DβH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the β-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase-dependent mechanism. In this report, we investigated the relationship between NO and DβH enzymatic activity. In the initial in vitroexperiments, the activity of purified DβH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC50 of 1 mm. The inclusion of either azide or GSH partially restored DβH activity, suggesting the involvement of the reactive nitrogen oxide species, N2O3. Treatment of human neuroblastoma cells (SK-N-MC) with diethylamine/NO decreased cellular DβH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Co-culture of the SK-N-MC cells with interferon-γ and lipopolysaccharide-activated macrophages, which release NO, also reduced the DβH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N2O3, can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation. dopamine-β-hydroxylase nitric oxide nitric-oxide synthase inducible nitric-oxide synthase reactive nitrogen oxide species diethylamine buthionine suloximine lipopolysaccharide interferon-γ phosphate-buffered saline l-N-methylarginine Norepinephrine and epinephrine are critical determinants of transient neuronal regulation of local vascular tone and arterial pressure. DβH,1 the third enzyme in the biosynthetic pathway of norepinephrine, can hydroxylate the β-carbon of a variety of phenylethylamine derivatives. DβH is a tetrameric copper-containing oxidoreductase with conserved homology among mammalian species (1.Nagatsu T. Neurosci. Res. 1991; 12: 315-345Crossref PubMed Scopus (105) Google Scholar) that is localized in both noradrenergic and adrenergic neurons of the central nervous system, sympathetic ganglia, and adrenal medulla (1.Nagatsu T. Neurosci. Res. 1991; 12: 315-345Crossref PubMed Scopus (105) Google Scholar, 2.Axelrod J. Pharmacol. Rev. 1972; 24: 233-243PubMed Google Scholar). The physiological regulation of DβH activity includes axonal transport rate, transcriptional factors such as nerve growth factor, and cyclic AMP (3.Kobayashi K. Kurosawa Y. Fujita K. Nagatsu T. Nucleic Acids Res. 1989; 17: 1089-1102Crossref PubMed Scopus (138) Google Scholar, 4.Thoenen H. Angeletti P.U. Levi-Montalcini R. Kettler R. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1598-1602Crossref PubMed Scopus (240) Google Scholar). Germ line mutations in DβH result in systemic hypotension mediated by increased dopamine levels in cerebrospinal fluid and blood (5.Robertson D. Haile V. Perry S.E. Robertson R.M. Phillips III, J.A. Biaggioni I. Hypertension. 1991; 18: 1-8Crossref PubMed Scopus (123) Google Scholar, 6.Rush R.A. Geffen L.B. Crit. Rev. Clin. Lab. Sci. 1980; 12: 241-277Crossref PubMed Scopus (79) Google Scholar). Nitric oxide (nitrogen monoxide, NO) is a prominent cellular messenger involved in several important biological processes including the regulation of neurotransmission and cardiovascular function (7.Wink D.A. Mitchell J.B. Free Radical Biol. Med. 1998; 25: 434-456Crossref PubMed Scopus (1321) Google Scholar). The physiological effects of NO occur with relatively low concentrations and primarily involve direct reactions with metal-containing proteins and radicals (7.Wink D.A. Mitchell J.B. Free Radical Biol. Med. 1998; 25: 434-456Crossref PubMed Scopus (1321) Google Scholar). Nitric oxide fluxes, generated from nitric-oxide synthase (NOS) isoenzymes, are present in endothelia, and selected neurons mediate these direct effects (8.Moncada S. Palmer R.M. Higgs E.A. Biochem. Pharmacol. 1989; 38: 1709-1715Crossref PubMed Scopus (1102) Google Scholar). In contrast, indirect effects of NO are likely to occur during pathological states. Indirect effects require relatively higher levels of NO and involve the formation of reactive nitrogen oxide species (RNOS) prior to target modification. In general, this chemistry is a consequence of the NO fluxes generated by the isoenzyme, inducible nitric-oxide synthase (iNOS) (7.Wink D.A. Mitchell J.B. Free Radical Biol. Med. 1998; 25: 434-456Crossref PubMed Scopus (1321) Google Scholar). Nitric oxide regulates the release and uptake of dopamine and the activity of a number of catecholamines in the striatum and other dopaminergic cells (9.Palacios M. Knowles R.G. Palmer R.M. Moncada S. Biochem. Biophys. Res. Commun. 1989; 165: 802-809Crossref PubMed Scopus (206) Google Scholar, 10.Hanbauer I. Wink D. Osawa Y. Edelman G.M. Gally J.A. Neuroreport. 1992; 3: 409-412Crossref PubMed Scopus (255) Google Scholar, 11.Lonart G. Cassels K.L. Johnson K.M. J Neurosci. Res. 1993; 35: 192-198Crossref PubMed Scopus (144) Google Scholar, 12.Lorrain D.S. Hull E.M. Neuroreport. 1993; 5: 87-89Crossref PubMed Scopus (196) Google Scholar, 13.Seilicovich A. Lasaga M. Befumo M. Duvilanski B.H. del Carmen Diaz M. Rettori V. McCann S.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11299-11302Crossref PubMed Scopus (85) Google Scholar). l-N-monomethylarginine, a competitive inhibitor of NOS, enhances nerve-induced contraction in the guinea pig ileum and pulmonary artery and in the dog mesenteric artery segments (14.Cederqvist B. Wiklund N.P. Persson M.G. Gustafsson L.E. Neurosci. Lett. 1991; 127: 67-69Crossref PubMed Scopus (39) Google Scholar, 15.Toda N. Okamura T. Jpn. J. Pharmacol. 1990; 52: 170-173Crossref PubMed Scopus (99) Google Scholar). In anococcygeus muscle preparations, the contractile response to noradrenergic stimulation is enhanced in the presence ofl-N-monomethylarginine as well (16.Li C.G. Rand M.J. Clin. Exp. Pharmacol. Physiol. 1989; 16: 933-938Crossref PubMed Scopus (167) Google Scholar, 17.Gibson A. Mirzazadeh S. Hobbs A.J. Moore P.K. Br. J. Pharmacol. 1990; 99: 602-606Crossref PubMed Scopus (172) Google Scholar). These studies show that an interrelationship exists between catecholamines and NO in the neuronal-endothelial control of blood flow. In this study, we sought to determine if there was an effect of NO on DβH and under what conditions these might exist in vivo. A modified spectrophotometric assay (18.Kato T. Kuzuya H. Nagatsu T. Biochem. Med. 1974; 10: 320-328Crossref PubMed Scopus (110) Google Scholar) was used to detect DβH activityin vitro. This assay was based on the conversion of substrate tyramine to octopamine, and the octopamine was converted top-hydroxybenzaldehyde by sodium periodate treatment. Thep-hydroxybenzaldehyde is measured spectrophotometrically. Bovine adrenal DβH (40 units/liter; Sigma) in PBS was mixed with DEA/NO (a generous gift from Dr. Joseph Saavedra, SCAI, Frederick, MD) and incubated at 37 °C for 30 min. Then the DβH with or without exposure to DEA/NO was added to a substrate mixture (600 μl). The substrate mixture contained N-ethylmaleimide (40 mm), sodium acetate buffer (200 mm, pH 5.0), tyramine (20 mm), sodium fumarate (10 mm), pargyline (1 mm), catalase (0.1 mg), fresh ascorbic acid (10 mm), and PBS (400 μl). The reaction mixtures were incubated at 37 °C for 30 min with shaking. The reaction was terminated by adding 0.2 ml of 3 m cold trichloroacetic acid. The mixture was immediately centrifuged at 2500 rpm for 10 min (MX/TX-160, TOMY Technology), and the supernatant was transferred to a microbio-spin column (400-μl bed volume) of GA-50W-X4 (H+, 200–400 mesh; Bio-Rad). After washing with 2 ml of distilled water, the absorbed reaction product octopamine was eluted with 1 ml of 4m ammonia. Octopamine in the eluate was converted top-hydroxybenzaldehyde by adding 0.2 ml of 2% sodium periodate, and the excess sodium periodate was reduced by adding 0.2 ml of 10% sodium metabisulfite. The absorbance was measured by a dual wavelength spectrophotometer at 330 nm. The blank value was obtained by substituting water for the enzyme. One hundred mm DEA/NO in NaOH and NaNO2 in water were used as stock to detect the NO inhibition effect. Preventing the inhibition of DβH activity by DEA/NO was preformed by directly adding GSH (Sigma) and sodium azide (Sigma) into the reaction before incubation. To eliminate the possibility that DEA/NO might interact with octopamine directly, the absorbance was measured after the co-incubation of 5 mmoctopamine (Sigma) with 5 mm DEA/NO. The neuroblastoma cell line SK-N-MC (ATCC, Rockville, MD) was grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. When the culture was 85–90% confluent, cells were harvested, suspended at a density of 107 cells/ml in PBS, and stored at −70 °C. To investigate the dose response and time course of inhibition of DβH activity, various amounts of DEA/NO were added to the cell cultures with different incubation times according to the experimental design. The cells were trypsinized, harvested, counted, and lysed by freezing and thawing three times and then centrifuged for 30 min at 14,000 rpm at 4 °C. The supernatant was transferred to a new tube, and the DβH activity was measured as described above. To eliminate the possibility that the reduction of DβH activity was caused by a decreased number of viable cells, the cell growth rate was examined by a nonradioactive cell proliferation assay (MTS assay; Promega). The SK-N-MC cells, with and without DEA/NO treatment, were washed with the culture medium and subcultured into a 96-well plate. The number of viable cells was measured by the MTS assay in the first, second, third, and fourth day after 24 h of subculture. Purified bovine adrenal DβH (40 units/liter) was exposed to 5 mm fresh and decomposed DEA/NO in PBS for 2 h at room temperature before the enzyme reaction. The decomposed DEA/NO was made by incubating DEA/NO at 37 °C for 1 h in PBS with shaking. The enzyme activities were measured at 15-min intervals throughout the total 2-h incubation time. The activity of the purified DβH without DEA/NO was measured as a positive control. The cultured neuroblastoma cells (SK-N-MC) were incubated with 0 and 5 mm DEA/NO for 1 h. The cells were washed with PBS four times and continually cultured with fresh medium. The cells were collected for DβH activity measurement at 0, 1, 2, 3, 4, and 5 h after DEA/NO exposure. Immortalized C3 murine microglial cells (kindly provided by Dr. C. A. Colton, Georgetown University, Washington, D. C.) were seeded (2 × 106 cells) into 100-mm2 Petri dishes and were stimulated for 12 h with both IFN-γ (20 ng/ml) and LPS (20 ng/ml). The nitrosative capacity of C3 cells under these conditions was monitored by the release of nitrite and the ability to form triazole from the target compound, 2,3-diaminonaphthalene, present in the medium. Nitric oxide was measured using fluorescence spectroscopy (19.Miles A.M. Wink D.A. Cook J.C. Grisham M.B. Methods Enzymol. 1996; 268: 105-120Crossref PubMed Google Scholar). SK-N-MC cells were trypsinized, washed with the culture medium, and seeded into the C3 cultures at an effector-to-target ratio of 1:3. The iNOS inhibitorl-N-methylarginine (NMA) was added into the co-culture either at the beginning of co-culture or 4 h before harvest to investigate whether the inhibition can be prevented or restored. Following 12 h of co-incubation, the cells were suspended, washed, and lysed as described previously. The activity of DβH was detected with a dual wavelength spectrophotometer. To rule out the possibility that the overnight co-culture may affect the SK-N-MC cells' growth and viability, the number of viable SK-N-MC cells after co-culture was counted by bright line hemocytometer (Sigma) with trypan blue staining. The concentration of GSH was determined in both DEA/NO-treated (5 mm, 1 h) and nontreated SK-N-MC cells. Intracellular GSH was determined as described previously (20.Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5554) Google Scholar). The cells were washed with PBS four times, harvested, and then suspended in 2 ml of 0.69% sulfosalicylic acid and stored at −70 °C. Spectra were performed on a UV-visible spectrophotometer, model U-3000/U-3300 (Hitachi). To investigate whether a selective inhibitor of GSH synthesis, buthionine suloximine (BSO; Sigma), affected DβH inhibition by NO, SK-N-MC cells were treated with 5 mm fresh BSO for 12 h before DEA/NO was added into the culture. After DEA/NO treatment, the cells were washed with PBS, harvested, and lysed as described above. In each experiment, experimental and control cells were matched for seeding density, number of passages, and the percentage of confluence. The experiments were repeated at least three times. Student's pairedt tests were used to determine the significance of differences between the means of experiments and controls. To verify the reliability of the spectrophotometric assay for the detection of DβH activity, purified bovine adrenal DβH activity was measured. In this assay, the reaction containing both enzyme and substrate was incubated, and the absorbance was measured. The enzyme activities (absorbance) showed a linear relation with the amount of DβH added from a range of 0–80 units/liter (Fig.1 A). To determine the effect of NO on DβH activity, the protein was exposed to the reaction buffer with different amounts of the NO donor DEA/NO. Exposure of DβH to increasing concentrations of DEA/NO in vitro resulted in a marked decrease in the conversion of tyramine to octopamine (Fig.1 B). The absolute absorbance of octopamine remained the same with and without DEA/NO co-incubation (data not shown). The concentration of DEA/NO required to reduce DβH activity by 50% was 1 mm. Nitrite is a by-product of the decomposition of DEA/NO, which can produce both NO and RNOS under acidic conditions. Because the assay for DβH was performed under acidic conditions (21.Williams D.L. Nitric Oxide. 1997; 1: 522-527Crossref PubMed Scopus (36) Google Scholar), we determined the effect of nitrite on DβH. The effect of increasing nitrite on DβH caused a decrease in DβH activity (Fig. 1 B). This result suggests that NO/RNOS, produced in the presence of nitrite under acidic conditions, could be responsible for this inhibition (Fig.1 B). Despite the effect of nitrite, complete inhibition required 50 mm nitrite, a dose 10 times higher than DEA/NO to inhibit the enzyme activity to the same extent. These results suggest that the vast majority of the inhibition by DEA/NO resulted from the liberation of nitric oxide and not nitrite; thus, increasing nitric oxide concentrations inhibit DβH. The inhibition of DβH activity by DEA/NO could be mediated by either NO or an RNOS, such as N2O3. A significant amount of N2O3 is formed at 5 mmconcentrations of DEA/NO, which has been shown to inhibit other enzymes (22.Wink D.A. Nims R.W. Darbyshire J.F. Chistodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. Chem. Res. Toxicol. 1994; 7: 519-525Crossref PubMed Scopus (359) Google Scholar). To determine the nature of the chemical intermediate responsible for the decomposition, the same experiments were performed in the presence of azide, an efficient scavenger of N2O3 (21.Williams D.L. Nitric Oxide. 1997; 1: 522-527Crossref PubMed Scopus (36) Google Scholar, 23.Wink D.A. Grisham M.B. Miles A.M. Nims R.W. Krishna M.C. Pacelli R. Teague D. Poore C.M. Cook J.A. Ford P.C. Methods Enzymol. 1996; 268: 120-130Crossref PubMed Google Scholar). Exposure of DEA/NO to DβH in the presence of sodium azide showed abrogation of the inhibitory effect of DEA/NO (Fig. 2 A). The same concentration of azide had no effect on the activity of the enzyme. Furthermore, when GSH was substituted with the azide as a scavenger of N2O3, abrogation of DEA/NO-mediated inhibition also was observed (Fig. 2 B). It should be noted that 10 mm GSH inhibited the assay, which is not surprising, because previous reports have shown that thiol-containing substances such as cystamine inhibited DβH (24.Terry L.C. Craig R. Neuroendocrinology. 1985; 41: 467-475Crossref PubMed Scopus (36) Google Scholar). However, the pretreatment of the isolated protein in the presence of GSH with excess DEA/NO results in nearly a 100% conversion to S-nitrosoglutathione (22.Wink D.A. Nims R.W. Darbyshire J.F. Chistodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. Chem. Res. Toxicol. 1994; 7: 519-525Crossref PubMed Scopus (359) Google Scholar). These results suggest that the S-nitrosothiol,S-nitrosoglutathione, does not inhibit DβH. Hence, scavenging of N2O3 by GSH resulted in the protection of the enzyme. Because NO does not directly react with either azide or GSH, these data clearly indicate that N2O3, not NO, was responsible for the inhibition of DβH activity in this in vitro experimental model. As shown above, the purified enzyme can readily be inhibited by N2O3. To extend these findings, we selected a human neuroblastoma cell line as a model to detect and measure the possible alteration of DβH activity by NO. Human neuroblastoma (SK-N-MC) cells have been shown to contain endogenous DβH (25.Brewster M.A. Berry D.H. Ann. Clin. Lab. Sci. 1979; 9: 343-346PubMed Google Scholar). Although we did not detect any DβH activity in the cell culture medium (data not shown), we were able to measure the activity of DβH in the SK-N-MC cell lysate. The activity of DβH was positively correlated with the number of cells used for lysis (Fig.3 A). The different negative controls including boiled cell lysate, buffer only, 100 μm specific DβH inhibitor, fusaric acid (Sigma) (26.Kato T. Wakui Y. Nagatsu T. Ohnishi T. Biochem. Pharmacol. 1978; 27: 829-831Crossref PubMed Scopus (49) Google Scholar), and zero incubation time showed either very low or no DβH activity (Fig. 3 B). The DβH activity in the cell lysates was partially decreased when DEA/NO was added to the culture before cellular lysing of the cells (Fig. 3 C). Unlike the results with the isolated protein (Fig. 1 B), 5 mm DEA/NO reduced cellular DβH activity to a lesser extent, i.e.50%. An increase of DEA/NO concentrations had no further effect on the reduction of DβH activity. A major difference between the experiments, exposing DEA/NO directly to the isolated protein or to the DβH-containing cell lysate, is the presence of cellular defenses against NO/RNOS such as GSH. It has been shown that GSH protects cells against N2O3 toxicity (22.Wink D.A. Nims R.W. Darbyshire J.F. Chistodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. Chem. Res. Toxicol. 1994; 7: 519-525Crossref PubMed Scopus (359) Google Scholar); therefore, GSH may be expected to attenuate the inhibitory effect of DEA/NO. SK-N-MC cells contain 20 μg of GSH/mg of protein, which is considerably more than most other mammalian cells. Exposure of 10 mm DEA/NO only reduced GSH levels modestly (data not shown). To determine if GSH affected the DEA/NO-mediated inhibition of DβH, cells were pretreated with BSO to deplete intracellular GSH. In controlled experiments, the BSO treatment had no effect on the DβH activity of the cells. However, BSO increased the DEA/NO-mediated inhibition from 50 to 90% (Fig. 3 D). These results indicate that the presence of intracellular GSH plays a role in partially protecting DβH from NO-mediated inhibition. The cells treated with DEA/NO did not show any significant alteration of cell growth rate in comparison with the non-DEA/NO-treated control. The doubling times of the cells, with and without DEA/NO, were 64 and 60 h, respectively. We investigated whether the inhibition of DβH activity by NO is reversible or irreversible in our experimental models. The activity of the purified DβH was inhibited completely by 5 mm of the fresh DEA/NO, and even when the incubation time was prolonged to 2 h, there was no recovery of the enzyme activity (Fig.4 A). Because the half-life of DEA/NO is about 4 min at 37 °C (27.Keefer L.K. Nims R.W. Davies K.M. Wink D.A. Methods Enzymol. 1996; 268: 281-293Crossref PubMed Google Scholar), DEA/NO cannot generate any NO in this system when the preincubation time is extended to 2 h at room temperature. The composite spectra for the decomposition of DEA/NO in our experimental conditions show that the concentration of DEA/NO is almost down to zero after a 2-h period (data not shown). The activity of the DβH treated with decomposed DEA/NO showed a slight reduction because of the DEA/NO by-product nitrite (Fig. 4 A), which is consistent with the data showing DβH activity after exposure to NaNO2 only (Fig. 1 B). After removing 5 mm DEA/NO exposed for 1 h, no recovery of DβH activity was detected in the continuing culture of SK-N-MC cells, even up to 5 h (Fig. 4 B). Although 5 mm of DEA/NO inhibited both the purified and the cellular DβH, it could be argued that these conditions are not relevant in biological systems. Therefore, we sought to determine if NO, derived from activated microglial cells, could inhibit DβH activity in co-cultured SK-N-MC cells. SK-N-MC cells were plated in Petri dishes containing murine macrophage C3 cells untreated or activated by IFN-γ and LPS. The C3 cells showed no detectable DβH activity (data not shown). A marked inhibition of DβH was observed in the SK-N-MC cells co-cultured with activated C3 cells (Fig.5). This inhibition was abated in the presence of the NOS inhibitor NMA during the co-culture (Fig. 5). However, the activity of DβH was not restored by the addition of NMA for 4 h after 8 h of co-culture (Fig. 5). The number and the viability of the SK-N-MC cells after co-culture were found to be similar to those of the companion cultures without C3 cells (>95%). These data indicate that NO, derived from iNOS, could produce an environment to inhibit DβH similar to 5 mm DEA/NOin vivo. Because either DEA/NO or cytokine-activated C3 cells inhibited DβH activity in the SK-N-MC cells, we determined the relative NO concentration that was produced by these two methods. A solution of 10 mm DEA/NO was dissolved in an appropriate buffer at 37 °C. Aliquots of samples were taken every 30 s at different time points and evaluated by a chemiluminescent method. The flux of NO rose to 50–100 μm and then decreased as described previously (23.Wink D.A. Grisham M.B. Miles A.M. Nims R.W. Krishna M.C. Pacelli R. Teague D. Poore C.M. Cook J.A. Ford P.C. Methods Enzymol. 1996; 268: 120-130Crossref PubMed Google Scholar). We then examined NO production by the cytokine-activated C3 cells in which aliquots were obtained hourly. As seen in Table I, the activated C3 cells produced 10–15 μm of nitrite during the course of the experiment where DEA/NO generated 10 mm (data not shown).Table INitrosative capacity of activated microgliaStimulantsNitriteTriazolenm/hNone30 ± 0.10IFN-γ + LPS910 ± 3272 ± 9Data are derived from triplicate values ± S.E. of the nitrite andN-nitrosated product of 2,3-diaminonaphthalene, triazole that accumulated over a period of 3 h. Blank values have been subtracted. Open table in a new tab Data are derived from triplicate values ± S.E. of the nitrite andN-nitrosated product of 2,3-diaminonaphthalene, triazole that accumulated over a period of 3 h. Blank values have been subtracted. We have investigated whether nitrosative stress generated by activated macrophages can alter the noradrenergic function by regulating the DβH enzymatic activity. Our results show that the activity of DβH, the enzyme that catalyzes the conversion of dopamine to norepinephrine, is inhibited by NO, derived from either a synthetic donor or macrophage/microglial iNOS. DEA/NO decomposes to release a high flux of NO at a physiological pH (27.Keefer L.K. Nims R.W. Davies K.M. Wink D.A. Methods Enzymol. 1996; 268: 281-293Crossref PubMed Google Scholar). In an aerobic environment, NO can combine with molecular oxygen to form RNOS such as N2O3 (28.Ford P.C. Wink D.A. Stanbury D.M. FEBS Lett. 1993; 326: 1-3Crossref PubMed Scopus (401) Google Scholar). The RNOS scavenger compounds, GSH and azide (21.Williams D.L. Nitric Oxide. 1997; 1: 522-527Crossref PubMed Scopus (36) Google Scholar), attenuated DEA/NO-mediated inhibition of DβH (Fig. 2, A and B), indicating that N2O3, rather than NO alone, was the effector molecule. N2O3 can transfer an NO+ equivalent to nucleophilic sites in a reaction known as nitrosation (22.Wink D.A. Nims R.W. Darbyshire J.F. Chistodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. Chem. Res. Toxicol. 1994; 7: 519-525Crossref PubMed Scopus (359) Google Scholar, 29.Wink D.A. Vodovotz Y. Laval J. Laval F. Dewhirst M.W. Mitchell J.B. Carcinogenesis. 1998; 19: 711-721Crossref PubMed Scopus (625) Google Scholar). Enzymes containing cysteine-zinc finger motifs, unprotonated lysyl, or reduced cystyl residues at key sites are susceptible to nitrosation-mediated inhibition (29.Wink D.A. Vodovotz Y. Laval J. Laval F. Dewhirst M.W. Mitchell J.B. Carcinogenesis. 1998; 19: 711-721Crossref PubMed Scopus (625) Google Scholar). In addition, N2O3 can nitrosate tyrosine residues in proteins (30.Simon D.I. Mullins M.E. Jia L. Gaston B. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4736-4741Crossref PubMed Scopus (184) Google Scholar). Previous studies indicated that multiple residues are critical for DβH activity (31.Sams C.F. Matthews K.S. Biochim. Biophys. Acta. 1984; 787: 61-70Crossref PubMed Scopus (4) Google Scholar). Incubation of DβH with diazonium tetrazole, iodoacetamide, or diethylpyrocarbonate caused complete inhibition of the enzyme activity, indicating that lysine, tyrosine, and histidine residues are critical for catalytic function. Interestingly, the only thiol sites in DβH are oxidized on cystine bridges that maintain the tetrameric structure of the protein complex. This precludes their accessibility to N2O3. The reaction of the DβH complex with dithiothreitol reduces it into a dimeric form without affecting DβH activity (31.Sams C.F. Matthews K.S. Biochim. Biophys. Acta. 1984; 787: 61-70Crossref PubMed Scopus (4) Google Scholar). Similar to other metalloenzymes, such as P450 hydroxylase and cytochrome coxidase in the mitochondrial respiration chain (7.Wink D.A. Mitchell J.B. Free Radical Biol. Med. 1998; 25: 434-456Crossref PubMed Scopus (1321) Google Scholar), our data showed that DβH activity was irreversibly inhibited by 5 mm of DEA/NO and microglia-derived NO. The higher local concentration of NO could form N2O3 and cause indirect effects on DβH. The formation of a benzylic radical intermediate in the hydroxylation of DβH has been proposed (32.Fitzpatrick P.F. Villafranca J.J. Arch. Biochem. Biophys. 1987; 257: 231-250Crossref PubMed Scopus (42) Google Scholar). Our data show that the unpaired electron of NO does not contribute to DβH inhibition. However, the benzylic radical mechanism could play a role under turnover conditions (data not shown). Taken together, these data suggest that nitrosation-mediated inhibition of DβH activity may occur at either lysine and/or tyrosine residues. Additional studies are needed to further test this hypothesis. Neuroblastoma is composed of primitive cells derived from the neural crest and contains a high level of DβH activity roughly equivalent to adrenal medulla tissue (25.Brewster M.A. Berry D.H. Ann. Clin. Lab. Sci. 1979; 9: 343-346PubMed Google Scholar). While 5 mm DEA/NO inhibited the purified enzyme completely, this concentration of DEA/NO reduced the activity of DβH within intact SK-N-MC neurons by only 50% (Fig.3 C). The inclusion of BSO resulted in full (>90%) inhibition, showing that the thiol-containing tripeptide GSH was a protective agent against nitrosative DβH inhibition for only 50% of the intracellular enzyme population. GSH concentrations of 5 mm were sufficient to protect purified DβH (Fig.2 B). SK-N-MC cells contain a reduced GSH pool in the range of 12–30 mm. These data demonstrated that GSH was unable to protect approximately half of the intracellular DβH against nitrosative inhibition despite relatively high levels. DβH exists in both cytoplasmic and membrane-bound forms in approximately equal amounts (5.Robertson D. Haile V. Perry S.E. Robertson R.M. Phillips III, J.A. Biaggioni I. Hypertension. 1991; 18: 1-8Crossref PubMed Scopus (123) Google Scholar, 33.Sokoloff R.L. Frigon R.P. O'Connor D.T. J. Neurochem. 1985; 44: 411-420Crossref PubMed Scopus (12) Google Scholar). The reaction between NO and molecular oxygen to form N2O3 is accelerated greatly in the hydrophobic phase of lipid bilayers (34.Liu X. Miller M.J.S. Joshi M.S. Thomas D.D. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2175-2179Crossref PubMed Scopus (532) Google Scholar). The insertion of DβH into membranes may augment nitrosative inhibition by 1) placing it in an environment of relatively higher NO+equivalents and 2) concealing lysine and/or tyrosine sites from the GSH protection. The hydroxylation reaction catalyzed by DβH requires the reduction of ascorbate as the electron donor. Sequestration of the active site of DβH into the hydrophobic phase of the membrane may be necessary to avoid interference from the high cytoplasmic pool of reduced GSH. By this mechanism, GSH may maintain DβH in an inactive state as it is transported down axons restricting norepinephrine synthesis to nerve terminals. The term nitrosative stress has been used to describe biological components that are nitrosated by species derived from N2O3 (7.Wink D.A. Mitchell J.B. Free Radical Biol. Med. 1998; 25: 434-456Crossref PubMed Scopus (1321) Google Scholar). A nitrosative stress microenvironment can occur under very distinct conditions including high concentrations of NO for prolonged periods of time. Constitutive NOS are unlikely to produce these conditions, because they produce NO for only short periods of time generally, and they also are inhibited by high fluxes of NO (35.Griscavage J.M. Hobbs A.J. Ignarro L.J. Adv. Pharmacol. 1995; 34: 215-234Crossref PubMed Scopus (146) Google Scholar). Therefore, the iNOS would be the most likely source. Nitrosative stress can occur following the expression of iNOS in activated leukocytes (36.Espey M.G. Miranda K.M. Pluta R.M. Wink D.A. J. Biol. Chem. 2000; 275: 11341-11347Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Co-culture of SK-N-MC cells with macrophages/microglia stimulated to produce N2O3, resulted in DβH inhibition equivalent to that observed with 5 mm DEA/NO (Fig. 5). The inhibition of iNOS with l-N-monomethylarginine fully restored DβH activity, ruling out the potential influence of other inflammatory mediators. These findings suggest that a condition of nitrosative stress is required in vivo to inhibit DβH. The possibility of DβH inhibition in cortex, cerebellum, hypothalamus, and adrenal medulla may be a significant feature of nitrosative stress, particularly during infection that localizes the leukocytes to express iNOS in the proximity of catecholaminergic systems. Under these circumstances, the resultant increase in both dopamine and NO with a decrease in norepinephrine would increase vasodilation, regional blood flow, and tissue permeability. Nitrosated DβH may then be replaced by the GSH-protected pool mobilized from the cellular perikarya. Chronic nitrosation, however, may lead to a more severe dysregulation of the catecholamine balance. NO, as an important intercellular messenger, may regulate catecholamine and blood flow by the modulation of DβH activity. We thank D. Dudek for editorial assistance." @default.
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