2-thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin-dependent enzyme activation and dimerisation

2-thiouracil (TU), an established antithyroid drug and melanoma-seeker, was found to selectively inhibit neuronal nitric oxide synthase (nNOS) in a competitive manner (K(i)=20 microM), being inactive on the other NOS isoforms. The drug apparently interfered with the substrate- and tetrahydrobiopterin (BH(4))-binding to the enzyme. It caused a 60% inhibition of H(2)O(2) production in the absence of L-arginine and BH(4), and antagonised BH(4)-induced dimerisation of nNOS, but did not affect cytochrome c reduction. These results open new perspectives in the understanding of the antithyroid action of TU and provide a new lead structure for the development of selective nNOS inhibitors to elucidate the interdependence of the substrate and pteridine sites and to modulate pathologically aberrant NO formation.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Vascular endothelial dysfunction: does tetrahydrobiopterin play a role?

Tetrahydrobiopterin is one of the most potent naturally occurring reducing agents and an essential cofactor required for enzymatic activity of nitric oxide synthase (NOS). The exact role of tetrahydrobiopterin in the control of NOS catalytic activity is not completely understood. Existing evidence suggests that it can act as allosteric and redox cofactors. Suboptimal concentration of tetrahydrobiopterin reduces formation of nitric oxide and favors "uncoupling" of NOS leading to NOS-mediated reduction of oxygen and formation of superoxide anions and hydrogen peroxide. Recent findings suggest that accelerated catabolism of tetrahydrobiopterin in arteries exposed to oxidative stress may contribute to pathogenesis of endothelial dysfunction present in arteries exposed to hypertension, hypercholesterolemia, diabetes, smoking, and ischemia-reperfusion. Beneficial effects of acute and chronic tetrahydrobiopterin supplementation on endothelial function have been reported in experimental animals and humans. Furthermore, it appears that beneficial effects of some antioxidants (e.g., vitamin C) on vascular function could be mediated via increased intracellular concentration of tetrahydrobiopterin. In this review, the potential role of tetrahydrobiopterin in the pathogenesis of vascular endothelial dysfunction and mechanisms underlying beneficial vascular effects of tetrahydrobiopterin will be discussed.     

GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH4), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a Km for GTP of 6.5 micromoles. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56 degrees C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH4 inhibited enzyme activity by 25% at a concentration of 100 micromoles. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a Ki of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO2- plus NO3-. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH4.     

Nitric oxide synthase is a cytochrome P-450 type hemoprotein.

Nitric oxide has emerged as an important mammalian metabolic intermediate involved in critical physiological functions such as vasodilation, neuronal transmission, and cytostasis. Nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to citrulline and nitric oxide. Cosubstrates for the reaction include molecular oxygen and NADPH. In addition, there is a requirement for tetrahydrobiopterin. NOS also contains the coenzymes FAD and FMN and demonstrates significant amino acid sequence homology to NADPH-cytochrome P-450 reductase. Herein we report the identification of the inducible macrophage NOS as a cytochrome P-450 type hemoprotein. The pyridine hemochrome assay showed that the NOS contained a bound protoporphyrin IX heme. The reduced carbon monoxide binding spectrum shows an absorption maximum at 447 nm indicative of a cytochrome P-450 hemoprotein. A mixture of carbon monoxide and oxygen (80%/20%) potently inhibited the reaction (73-79%), showing that the heme functions directly in the oxidative conversion of L-arginine to nitric oxide and citrulline. Additionally, partially purified NOS from rat cerebellum was inhibited by CO, suggesting that this isoform may also contain a P-450-type heme. NOS is the first example of a soluble cytochrome P-450 in eukaryotes. In addition, the presence of FAD and FMN indicates that this is the first catalytically self-sufficient mammalian P-450 enzyme, containing both a reductase and a heme domain on the same polypeptide.     

Kinetics of NO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry

Nitric-oxide synthase (NOS) catalyzes conversion of L-arginine to nitric oxide, which subsequently stimulates a host of physiological processes. Prior work suggests that NOS is inhibited by NO, providing opportunities for autoregulation. This contribution reports that NO reacts rapidly (ka congruent with 2 x 10(7) M-1 s-1) with neuronal NOS in both its ferric and ferrous oxidation states. Association kinetics are almost unaffected by L-arginine or the cofactor tetrahydrobiopterin. There is no evidence for the distinct two phases previously reported for association kinetics of CO. Small amounts of geminate recombination of NO trapped in a protein pocket can be observed over nanoseconds, and a much larger amount is inferred to take place at picosecond time scales. Dissociation rates are also very fast from the ferric form, in the neighborhood of 50 s-1, when measured by extrapolating association rates to the zero NO concentration limit. Scavenging experiments give dissociation rate constants more than an order of magnitude slower: still quite fast. For the ferrous species, extrapolation is not distinguishable from zero, while scavenging experiments give a dissociation rate constant near 10(-4) s-1. Implications of these results for interactions near the heme binding site are discussed.     

Oxygen metabolism by endothelial nitric-oxide synthase

Nitric-oxide synthase (NOS) catalyzes both coupled and uncoupled reactions that generate nitric oxide and reactive oxygen species. Oxygen is often the overlooked substrate, and the oxygen metabolism catalyzed by NOS has been poorly defined. In this paper we focus on the oxygen stoichiometry and effects of substrate/cofactor binding on the endothelial NOS isoform (eNOS). In the presence of both L-arginine and tetrahydrobiopterin, eNOS is highly coupled (>90%), and the measured stoichiometry of O(2)/NADPH is very close to the theoretical value. We report for the first time that the presence of L-arginine stimulates oxygen uptake by eNOS. The fact that nonhydrolyzable L-arginine analogs are not stimulatory indicates that the occurrence of the coupled reaction, rather than the accelerated uncoupled reaction, is responsible for the L-arginine-dependent stimulation. The presence of 5,6,7,8-tetrahydrobiopterin quenched the uncoupled reactions and resulted in much less reactive oxygen species formation, whereas the presence of redox-incompetent 7,8-dihydrobiopterin demonstrates little quenching effect. These results reveal different mechanisms for oxygen metabolism for eNOS as opposed to nNOS and, perhaps, partially explain their functional differences.     

Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity.

Nitric oxide synthase (NOS) (EC 1.14.23) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. The complex reaction carried out by NOS, which involves NADPH, O2, and enzyme-bound FAD, FMN, and tetrahydrobiopterin (BH4), has only recently begun to be elucidated. Herein we report the characterization of the pterin requirement of murine macrophage NOS. Although purified NOS activity was not dependent on BH4, activity was significantly enhanced by BH4 in a concentration-dependent fashion. NOS purified in the absence of added BH4 was found to contain substoichiometric concentrations of enzyme-bound pterin, where increased concentrations of bound pterin correlated with an increase in activity when assayed in the absence of exogenous BH4. However, NOS purified in the presence of BH4 followed by gel filtration exhibited a 1 mol of pterin:1 mol of NOS 130-kDa subunit stoichiometry and activity that was essentially independent of exogenous BH4. Experiments to probe a redox role for the pterin were carried out using pterin analogues. 6(R,S)-Methyltetrahydropterin was found to increase NOS activity in enzyme purified in the absence of BH4. However, the deaza analogue, 6(R,S)-methyl-5-deazatetrahydropterin, was not only incapable of supporting enzymatic turnover but also inhibited citrulline formation in a concentration-dependent manner. Overall, these results support a role for BH4 in the NOS reaction that involves stabilization of the enzyme and redox chemistry wherein a 1:1 stoichiometry between bound pterin and NOS subunit results in maximum activity.     

Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy

Nitric oxide synthase (EC 1.14.13.39; NOS) converts L-arginine into NO and L-citrulline in a two-step reaction with Nomega-hydroxy-L-arginine (NOHLA) as an intermediate. The active site iron in NOS has thiolate axial heme-iron ligation as found in the related monooxygenase cytochrome P450. In NOS, tetrahydrobiopterin (BH4) is an essential cofactor for both steps, but its function is controversial. Previous optical studies of the reaction between reduced NOS with O2 at -30 degrees C suggested that BH4 may serve as an one-electron donor in the first cycle, implying formation of a trihydrobiopterin radical. We investigated the same reaction under identical conditions with electron paramagnetic resonance spectroscopy. With BH4-containing full-length neuronal NOS we obtained an organic free radical (g-value 2.0042) in the presence of Arg, and a similar radical was observed with the endothelial NOS oxygenase domain in the presence of Arg and BH4. Without substrate the radical yield was greatly (10x) diminished. Without BH4, or with NOHLA instead of Arg, no radical was observed. With 6-methyltetrahydropterin or 5-methyl-BH4 instead of BH4, radicals with somewhat different spectra were formed. On the basis of simulations we assign the signals to trihydropterin radical cations protonated at N5. This is the first study that demonstrates the formation of a protonated trihydrobiopterin radical with the constitutive isoforms of NOS, and the first time the radical was obtained without exogenous BH4. These results offer strong support for redox cycling of BH4 in the first reaction cycle of NOS catalysis (BH4 <--> BH3.H+).     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Catalase activity of oxygenase domain of rat neuronal nitric oxide synthase. Evidence for product formation from L-arginine

Nitric oxide synthases (NOSs) catalyze the formation of nitric oxide from L-arginine. We purified the heme containing, tetrahydrobiopterin-free, oxygenase domain of rat neuronal nitric oxide synthase (nNOSox) overexpressed in Escherichia coli. We found catalase activity in nNOSox. This is significant because H(2)O(2) may also be a product of nitric oxide synthases. We found H(2)O(2) assisted product formation from N-hydroxy-L-arginine and even from L-arginine both in the presence and in absence of tetrahydrobiopterin. We propose how heme moiety of the oxygenase domain alone is sufficient to carry out both steps of the NOS catalysis.     

Involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)* during enzymatic cycling, and the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance spectroscopy, we detected alpha-hydroxyethyl radical (CH(3)*CHOH), produced during the NOS I metabolism of ethanol (EtOH). The generation of CH(3)*CHOH by NOS I was found to be Ca(2+)/calmodulin dependent. Superoxide dismutase prevented CH(3)*CHOH formation in the absence of L-arginine. However, in the presence of L-arginine, the production of CH(3)*CHOH was independent of O(2)* but dependent upon the concentration of L-arginine. Formation of CH(3)*CHOH was inhibited by substituting D-arginine for L-arginine, or inclusion of the NOS inhibitors N(G)-nitro-L-arginine methyl ester, N(G)-monomethyl-L-arginine and the heme blocker, sodium cyanide. The addition of potassium hydrogen persulfate to NOS I, generating the perferryl complex (NOS-[Fe(5+)=O](3+)) in the absence of oxygen and Ca(2+)/calmodulin, and EtOH resulted in the formation of CH(3)*CHOH. NOS I was found to produce the corresponding alpha-hydroxyalkyl radical from 1-propanol and 2-propanol, but not from 2-methyl-2-propanol. Data demonstrated that the perferryl complex of NOS I in the presence of L-arginine was responsible for catalyses of these secondary reactions.     

Oxygen activation in neuronal NO synthase: resolving the consecutive mono-oxygenation steps

The vital signalling molecule NO is produced by mammalian NOS (nitric oxide synthase) enzymes in two steps. L-arginine is converted into NOHA (Nω-hydroxy-L-arginine), which is converted into NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor BH4 (tetrahydrobiopterin) activates dioxygen at the haem site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochrome P450, a haem-iron oxo species may be formed, or direct reaction between a haem-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. In the present paper we analyse the two reaction steps using the G586S mutant of nNOS (neuronal NOS), which introduces an additional hydrogen bond in the active site and provides an additional proton source. In the mutant enzyme, BH4 activates dioxygen as in the wild-type enzyme, but an interesting intermediate haem species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-arginine) indicating that the extra hydrogen bond enables it to discriminate between the two mono-oxygenation steps. This implies that the two steps follow different chemical mechanisms.     

Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities.

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.     

Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) are homodimeric proteins and can form an intersubunit Zn(4S) cluster. We have measured zinc bound to NOS purified from pig brain (0.6 mol/mol of NOS) and baculovirus-expressed rat neuronal NOS (nNOS) (0.49 +/- 0.13 mol/mol of NOS), by on-line gel-filtration/inductively coupled plasma mass spectrometry. Cobalt, manganese, molybdenum, nickel, and vanadium were all undetectable. Baculovirus-expressed nNOS also bound up to 2. 00 +/- 0.58 mol of copper/mol of NOS. Diethylenetriaminepentaacetic acid (DTPA) reduced the bound zinc to 0.28 +/- 0.07 and the copper to 0.97 +/- 0.24 mol/mol of NOS. Desalting of samples into thiol-free buffer did not affect the zinc content but completely eliminated the bound copper ( or =75%) of the bound zinc was released from baculovirus-expressed rat nNOS by p-chloromercuriphenylsulfonic acid (PMPS). PMPS-treated nNOS was strongly (90 +/- 5%) inactivated. To isolate functional effects of zinc release from other effects of PMPS, PMPS-substituted thiols were unblocked by excess reduced thiol in the presence of DTPA, which hindered reincorporation of zinc. The resulting enzyme contained 0.12 +/- 0.05 mol of zinc but had a specific activity of 426 +/- 46 nmol of citrulline.mg(-1).min(-1), corresponding to 93 +/- 10% of non-PMPS-treated controls. PMPS also caused dissociation of nNOS dimers under native conditions, an effect that was blocked by the pteridine cofactor tetrahydrobiopterin (H(4)biopterin). H(4)biopterin did not affect zinc release. Even in the presence of H(4)biopterin, PMPS prevented conversion of NOS dimers to an SDS-resistant form. We conclude that zinc binding is a prerequisite for formation of SDS-resistant NOS dimers but is not essential for catalysis.     

Deuterium isotope effects and product studies for the oxidation of N(omega)-allyl-L-arginine and N(omega)-allyl-N(omega)-hydroxy-L-arginine by neuronal nitric oxide synthase

The nitric oxide synthases (NOS), which require heme, tetrahydrobiopterin, FMN, FAD, and NADPH, catalyze the O2-dependent conversion of L-arginine to L-citrulline and nitric oxide. N(omega)-Allyl-L-arginine, a mechanism-based inactivator of neuronal NOS, also is a substrate, producing L-arginine, acrolein, and H2O (Zhang, H. Q.; Dixon, R. P., Marletta, M. A.; Nikolic, D.; Van Breemen, R.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119, 10888). Two possible mechanisms for this turnover are proposed, one initiated by allyl C-H bond cleavage and the other by guanidino N H cleavage, and these mechanisms are investigated with the use of N(omega)-allyl-L-arginine (1), N(omega)-[1,1-(2)H2]allyl-L-arginine (7), N(omega)-allyl-N(omega)-hydroxy-L-arginine (2) and N(omega)-[1,1-(2)H2]allyl-N(omega)-hydroxy-L-arginine (8) as substrates. Significant isotope effects on the two kinetic parameters, kcat and kcat/Km, are observed in case of 1 and 7 during turnover, but not with 2 and 8. No kinetic isotope effects are observed for either compound in their role as inactivators. These results support a mechanism involving initial C-H bond cleavage of N(omega)-allyl-L-arginine followed by hydroxylation and breakdown to products.     

X-ray absorption spectroscopic analysis of the high-spin ferriheme site in substrate-bound neuronal nitric-oxide synthase

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide through two stepwise oxygenation reactions involving N(omega)-hydroxy-L-arginine, an enzyme-bound intermediate. The N(omega)-hydroxy-L-arginine- and arginine-bound NOS ferriheme centers show distinct, high-spin electron paramagnetic resonance signals. Iron X-ray absorption spectroscopy (XAS) has been used to examine the structure of the ferriheme site in the N(omega)-hydroxy-L-arginine-bound full-length neuronal NOS in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin. Iron XAS shows that the high-spin ferriheme sites in the N(omega)-hydroxy-L-arginine- and arginine-bound forms are strikingly similar, both being coordinated by the heme and an axial thiolate ligand, with an Fe-S distance of ca. 2.29 A. Cu(2+) inhibition slightly affects the spin-state equilibrium, but causes no XAS-detectable changes in the immediate ferriheme coordination environment of neuronal NOS. The structure and ligand geometry of the high-spin ferriheme in arginine-bound neuronal NOS are essentially identical to those of the N(omega)-hydroxy-L-arginine-bound form and only slightly affected by the divalent cation inhibitor of constitutive NOS.     

Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin

Constitutive rat cerebellar nitric oxide synthase (NOS) activity is shown to be inhibited by physiological concentrations of the pineal hormone melatonin. The inhibition was dose-dependent and was coupled to an inhibition of the cyclic GMP production activated by L-arginine. Results also show that calmodulin appears to be involved in this process because its presence in the incubation medium was able to prevent the effect of melatonin on both NOS activity and cyclic GMP production. Moreover, polyacrylamide gel electrophoresis studies suggest that melatonin can interact with calmodulin modifying the binding of the peptide to the synthetic NOS peptide encompassing the calmodulin-binding domain of constitutive NOS from rat cerebellum, the natural mechanism by which calmodulin activates cerebellar NOS. J. Cell. Biochem. 65:430–442. © 1997 Wiley-Liss, Inc.