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Clonidine, clinically used in the treatment of hypertension, is a central alpha(2)-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca(2+)-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca(2+)-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca(2+)-dependent NOS (NOS-III) activity. However, the value of K(i) for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH(4)) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of K(i) for clonidine binding to NOS-I increased (from [7. 9 +/- 0.4] x 10(-5) M to [8.0 +/- 0.4] x 10(-3) M) as BH(4) concentration was increased (between 3.0 x 10(-7) M and 1.0 x 10(-3) M), at pH 7.5 and 37.0 degrees. In addition, clonidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Finally, clonidine (1.0 x 10(-4) M to 1.0 x 10(-3) M) dose dependently increased the levels of LPS/IFN-gamma-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

Nitric oxide (NO) regulates key aspects of cell metabolism through reversible inhibition of cytochrome c oxidase (CcOX), the terminal electron acceptor (complex IV) of the mitochondrial respiratory chain, in competition with oxygen. Recently, a constitutive mitochondrial NOS corresponding to a neuronal NOS-I isoform (mtNOS-I) has been identified in several tissues. The role of this enzyme might be to generate NO close enough to its target without a significant overall increase in cellular NO concentrations. An effective, selective, and specific NO action might be guaranteed further by a physical interaction between mtNOS-I and CcOX. This possibility has never been investigated. Here we demonstrate that mtNOS-I is associated with CcOX, as proven by electron microscopic immunolocalization and co-immunoprecipitation studies. By affinity chromatography, we found that association is due to physical interaction of mtNOS-I with the C-terminal peptide of the Va subunit of CcOX, which displays a consensus sequence for binding to the PDZ domain of mtNOS-I previously unreported for CcOX. The molecular details of the interaction have been analyzed by means of molecular modeling and molecular dynamics simulations. This is the first evidence of a protein-protein interaction mediated by PDZ domains involving CcOX.

Nitric oxide (NO), produced in different cell types through the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NOreactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acidbase catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2 position has been observed in some NOreactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691 696]. Here, we analyse the threedimensional structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.

Nitric oxide (NO) is a pluripotent regulatory molecule possessing, among others, an antiparasitic activity. In the present study, the inhibitory effect of NO on the catalytic activity of falcipain, the papain-like cysteine protease involved in Plasmodium falciparum trophozoite hemoglobin degradation, is reported. In particular, NO donors S -nitrosoglutathione (GSNO), (±)-( E )-4-ethyl-2-[( E )-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) inhibit dose-dependently the falcipain activity present in the P. falciparum trophozoite extract, this effect likely attributable to S-nitrosylation of the Cys25 catalytic residue. The results represent a new insight into the modulation mechanism of falcipain activity, thereby being relevant in developing new strategies for inhibition of the P. falciparum life cycle.

Amiloride, a diuretic and antihypertensive drug, inhibits Na+ transporting systems, diamine oxidases and the human urinary plasminogen activator. Present results indicate that amiloride is a competitive inhibitor of mouse brain nitric oxide synthase (NOS; Ki = 4.5 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not a nitric oxide (NO) precursor, representing a case for enzyme cross-inhibition. The decreased levels of NO, as a consequence of NOS inhibition, might induce some clinically-adverse amiloride reactions, such as an unexpected reduced antihypertensive effect.

The presence of nitric oxide (NO) pathway has been well demonstrated in the main invertebrate groups, showing parallel findings on the role of NO in vertebrates and invertebrates. Noteworthy is the example of the role played by the nitrergic pathway in the sensorial functions, mainly in olfactory-like systems. On the other hand, the emerging molecular information about NOSs from lower metazoans (Porifera, cnidarians up to higher invertebrates) suggests that NO pathways might represent examples of a parallel evolution of the NOS prototypes in different animal lineages. Nevertheless, increasing evidence suggests that NO is one of the earliest and most widespread signaling molecules in living organisms. Here, we attempt to provide a survey of current knowledge of the synthesis and possible roles of NO and the related signaling pathway in lower metazoans (i.e., Porifera and Cnidaria), two phyla forming a crucial bridge spanning the evolutionary gap between the protozoans and higher metazoans. From the literature data here reported, it emerges that future research on the biological roles of NO in basal metazoans is likely to be very important for understanding the evolution of signaling systems.

The present study demonstrates that a NADPH/Ca2+-dependent nitric oxide synthase (NOS) activity is present in the soluble and in the particulate fractions of fish caudal spinal cord homogenates, both activities being inhibited by calmodulin inhibitors (W7 and/or TFP) and by the NOS inhibitor l -NAME. Moreover, Western blot analysis using either anti-NOS I or anti-NOS III antibodies shows that the soluble enzyme corresponds to the brain NOS (NOS-I) of mammals, whereas the particulate one is likely attributable to NOS I and/or NOS III (ecNOS) enzymes. To confirm the nitric oxide (NO) production by the caudal spinal cord homogenates, the NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored spectroscopically. The present results are consistent with a constitutive, Ca2+-calmodulin-dependent, NO production by the caudal neurosecretory system.

We present here a brief description of the relationships among metals, nitric oxide metabolism, and ageing. In particular, we will discuss the interactions occurring between redox (copper, iron) and non-redox (zinc) metals and nitric oxide, the metal- and nitric oxide-catalyzed formation of thiol adducts (nitrosothiols, mixed disulphides) and the possible involvement of these species in the ageing process.

The activity of NCX 972, a new molecule obtained by adding a nitric oxide (NO) moiety to metronidazole, was tested against six isolates ofEntamoeba histolyticain xenic cultures. NCX 972 released NO into trophozoite cells and enhanced killing of amoebas in a dose- and time-dependent manner compared to metronidazole.

Nitric oxide (NO) is an important cytotoxic and cytostatic mediator for several parasites, including intracellular (e.g. Trypanosoma, Leishmania, Plasmodium, Toxoplasma) and extracellular (e.g. Entamoeba) protozoa and the helminth Schistosoma 1xNitric oxide and parasitic disease. Clark, I.A. and Rockett, K.A. Adv. Parasitol. 1996; 37: 1–56Crossref | PubMedSee all References1. Increasing evidence suggests that parasitic cysteine proteases could represent NO targets, providing molecular bases for the parasiticidal effect of NO (2xNitric oxide inhibits falcipain, the Plasmodium falciparum trophozoite cysteine protease. Venturini, G. et al. Biochem. Biophys. Res. Commun. 2000; 267: 190–193Crossref | PubMed | Scopus (34)See all References, 3xNitric oxide inhibits cruzipain, the major papain-like cysteine protease from Trypanosoma cruzi. Venturini, G. et al. Biochem. Biophys. Res. Commun. 2000; 270: 437–441Crossref | PubMed | Scopus (47)See all References, 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References, 5xNO donors inhibit Leishmania infantum cysteine proteinase activity. Salvati, L. et al. Biochim. Biophys. Acta. 2001; 1545: 357–366Crossref | PubMed | Scopus (49)See all References). NO-donors (e.g. S-nitroso-acetyl-penicillamine, SNAP) inhibit the catalytic activity of cruzipain, falcipain and Leishmania infantum cysteine protease in vitro. L. infantum cysteine protease is inhibited following incubation of promastigotes with SNAP, which leads to parasite killing. Predictably, NO-deprived NO-donors (e.g. N-acetyl-penicillamine) affect neither the enzyme action nor the parasite viability. Moreover, reducing agents (e.g. dithiothreitol) prevent inhibition of parasite cysteine protease and restore enzyme activity 5xNO donors inhibit Leishmania infantum cysteine proteinase activity. Salvati, L. et al. Biochim. Biophys. Acta. 2001; 1545: 357–366Crossref | PubMed | Scopus (49)See all References5.The prevalence and higher reactivity of thiols over other nucleophiles account for the propensity of S-nitrosylation and mixed disulfide bridge formation. The NO-mediated chemical modification(s) of the Cys catalytic residue in cysteine proteases is assisted by neighboring basic and acid amino-acid residues (e.g. the invariant His residue forming the Cys–His catalytic dyad). In particular, S-nitrosylation of the Cys25 catalytic residue of cruzipain is facilitated by His159, which stabilizes the reactive deprotonated form of the Cys25 S atom 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References4 (Fig. 1Fig. 1).Fig. 1The three-dimensional structure of cruzipain. The cysteine-25 catalytic residue, undergoing the nitric-oxide-mediated S-nitrosylation, in addition to the histidine-159 residue, facilitating the Cys25 chemical modification, are shown in ball-and-stick.View Large Image | Download PowerPoint SlideThe NO-mediated chemical modification(s) of cysteine proteases depends on the chemical properties of the NO-donor, in addition to the presence of NO scavengers (e.g. albumin and hemoglobin), reducing agents (e.g. reduced glutathione, GSH), oxidants (e.g. oxygen and related reactive species) and metals. Hemoglobin could also act as a NO carrier because NO release is apparently facilitated by oxygen release in the venous capillaries 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References4. Plasmodium falciparum in blood cultures with low-oxygen tensions similar to those found in the venous capillaries is extremely susceptible to NO (Ref. 6xSensitivity of malaria parasites to nitric oxide at low oxygen tensions. Taylor-Robinson, A.W. and Looker, M. Lancet. 1998; 351: 1630Abstract | Full Text | Full Text PDF | PubMed | Scopus (31)See all ReferencesRef. 6). Flavo-hemoglobin, truncated-hemoglobin and myoglobin could detoxify NO pseudo-enzymatically. The NO-mediated chemical modification(s) of endogenous reducing agents could be induced by exogenous and endogenous NO(-donors). At the physiological GSH concentration, S-nitroso-glutathione (GSNO) formation represents the significant metabolic fate of N2O3. Most of N2O3, obtained from the reaction between O2 with NO is consumed by GSH-nitrosylation. Then, GSNO oxidizes protein thiol(s) by NO-transfer reaction. Alternatively, a direct nucleophilic attack of protein thiol(s) on GSNO, which does not require nitrosothiol cleavage, has been suggested as a potential mechanism for GSNO-mediated protein thiolation. However, reducing agents might revert in part the NO-induced oxidation of Cys residues 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References4. Interestingly, trypanosomes containing low levels of endogenous reducing agents (e.g. trypanothione and GSH) display an increased sensitivity to oxidative stress 7xTrypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Krieger, S. et al. Mol. Microbiol. 2000; 35: 542–552Crossref | PubMed | Scopus (227)See all References7, indicating that GSH is essential for protection against NO cytotoxicity in macrophages and parasites (e.g. Leishmania) 8xGlutathione protects macrophages and Leishmania major against nitric oxide-mediated cytotoxicity. Romao, P.R. et al. Parasitology. 1999; 118: 559–566Crossref | PubMed | Scopus (26)See all References8.Although NO-mediated chemical modification(s) of cysteine proteases accounts for the loss of enzyme activity and the parasiticidal effect of NO, the binding of NO to other parasite molecular targets should be taken into account. In particular, ribonucleotide reductase inhibition has been suggested to explain the cytostatic effect of NO on Trypanosoma brucei gambiense and Trypanosoma brucei brucei. Moreover, the NO-mediated chemical modification(s) of host cysteine-containing proteins could also influence parasite survival 1xNitric oxide and parasitic disease. Clark, I.A. and Rockett, K.A. Adv. Parasitol. 1996; 37: 1–56Crossref | PubMedSee all References, 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References.Considering that cysteine proteases appear as promising targets for anti-parasite chemotherapy 9xDevelopment of cysteine protease inhibitors as chemotherapy for parasitic diseases: insights on safety, target validation, and mechanism of action. McKerrow, J.H. Int. J. Parasitol. 1999; 29: 833–837Crossref | PubMed | Scopus (115)See all References9, NO-releasing drugs could have an enhancing role in the therapeutic treatment of parasitic diseases 10xA topical nitric oxide-generating therapy for cutaneous leishmaniasis. Davidson, R.N. et al. Trans. Roy. Soc. Trop. Med. Hyg. 2000; 94: 319–322PubMedSee all References10.

Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs.

Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed.

The action of copper on the nitric oxide (NO) pathway was investigated in rat C6 glioma cells expressing both inducible and constitutive NO synthase (NOS) isoforms. The inducible NOS-II-mediated NO synthesis (i.e., nitrite production induced by LPS plus IFNgamma) was found to be increased upon copper uptake by cells, this effect being attributable to NOS-II mRNA transcriptional over-expression. On the other hand, the constitutive neuronal isoform (NOS-I) was inhibited after copper uptake, as revealed by the decrease of basal intracellular cGMP levels in C6 cells. Consistently, in vitro experiments showed that copper selectively blocked the catalytic activity of NOS-I, but not of NOS-II. The observed modulation of NOS isoforms by copper in C6 cells is in line with the previous hypothesis that selective inhibition of NOS-I leads to enhanced NO production through transcriptional activation of NOS-II.

Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methyl coumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.

Analogs of L-arginine represent the largest and potentially most useful class of NOS inhibitors. However, no competitive protein inhibitors of NOS activity are known so far. The effect of aprotinin (Kunitz inhibitor) on NOS activity is reported here, aprotinin being one of the most extensively studied globular proteins. Present data indicate that aprotinin, clinically used as a trypsin-like serine proteinase inhibitor, inhibits NOS-I and NOS-II with Ki values of 5.0 x 10(-5) M and 7.8 x 10(-5) M, respectively, at pH 7.5 and 37.0 degrees C, thereby representing the first competitive protein inhibitor of NOS activity. Therefore, the clinical use of aprotinin, as a drug, should be under careful control. In addition, aprotinin and aprotinin-like domains are present in a variety of organs, as well as in the Alzheimer's amyloid beta-protein precursor. Thus, the present findings open the way to novel mechanisms likely to be involved in the modulation of NOS activity, under physiological and pathological conditions.

Considering the structural similarity between gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, and L-arginine, the effect of gabexate mesylate on the nitric oxide (NO) pathway has been investigated. Gabexate mesylate inhibits competitively constitutive and inducible NO synthase (cNOS and iNOS, respectively), with Ki values of 1.0 x 10(-4) M and 5.0 x 10(-3) M, respectively, at pH 7.4 and 37.0 degrees C. However, gabexate mesylate is not an NO precursor. Moreover, like other NOS inhibitors, gabexate mesylate increases iNOS mRNA expression in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Finally, gabexate mesylate inhibits dose-dependently nitrite production (i.e. NO release) in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Thus, this drug should be administered under careful control, since enzyme inhibition may occur also in vivo.

The amyloid peptides Abeta1-42 and Abeta25-35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS-I and NOS-III, respectively) in cell-free assays. The molecular mechanism of NOS inhibition by Ab fragments was studied in detail with Abeta25-35. The inhibitory ability was mostly NADPH-dependent and specific for the soluble form of Abeta25-35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Abeta25-35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal-like PC12 and glioma C6 cell lines were used as cellular models. After Abeta25-35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF-2DA detection system and found to be severely impaired upon Abeta25-35 uptake. Consistent with previous results on the molecular cross-talk between NOS isoforms, repression of constitutive NOS by Abeta25-35 resulted in enhanced expression of inducible NOS (NOS-II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell-free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimer's disease.