Role of PheE15 gate in ligand entry and nitric oxide detoxification function of Mycobacterium tuberculosis truncated hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

The Regulation of Skeletal and Cardiac Muscle Blood Flow in Humans. Studies by positron emission tomography with special reference to exercise, adenosine and nitric oxide

Virtually every cell and organ in the human body is dependent on a proper oxygen supply. This is taken care of by the cardiovascular system that supplies tissues with oxygen precisely according to their metabolic needs. Physical exercise is one of the most demanding challenges the human circulatory system can face. During exercise skeletal muscle blood flow can easily increase some 20-fold and its proper distribution to and within muscles is of importance for optimal oxygen delivery. The local regulation of skeletal muscle blood flow during exercise remains little understood, but adenosine and nitric oxide may take part in this process. In addition to acute exercise, long-term vigorous physical conditioning also induces changes in the cardiovasculature, which leads to improved maximal physical performance. The changes are largely central, such as structural and functional changes in the heart. The function and reserve of the heart’s own vasculature can be studied by adenosine infusion, which according to animal studies evokes vasodilation via it’s a_2A receptors. This has, however, never been addressed in humans in vivo and also studies in endurance athletes have shown inconsistent results regarding the effects of sport training on myocardial blood flow. This study was performed on healthy young adults and endurance athletes and local skeletal and cardiac muscle blod flow was measured by positron emission tomography. In the heart, myocardial blood flow reserve and adenosine A_2A receptor density, and in skeletal muscle, oxygen extraction and consumption was also measured. The role of adenosine in the control of skeletal muscle blood flow during exercise, and its vasodilator effects, were addressed by infusing competitive inhibitors and adenosine into the femoral artery. The formation of skeletal muscle nitric oxide was also inhibited by a drug, with and without prostanoid blockade. As a result and conclusion, it can be said that skeletal muscle blood flow heterogeneity decreases with increasing exercise intensity most likely due to increased vascular unit recruitment, but exercise hyperemia is a very complex phenomenon that cannot be mimicked by pharmacological infusions, and no single regulator factor (e.g. adenosine or nitric oxide) accounts for a significant part of exercise-induced muscle hyperemia. However, in the present study it was observed for the first time in humans that nitric oxide is not only important regulator of the basal level of muscle blood flow, but also oxygen consumption, and together with prostanoids affects muscle blood flow and oxygen consumption during exercise. Finally, even vigorous endurance training does not seem to lead to supranormal myocardial blood flow reserve, and also other receptors than A_2A mediate the vasodilator effects of adenosine. In respect to cardiac work, atheletes heart seems to be luxuriously perfused at rest, which may result from reduced oxygen extraction or impaired efficiency due to pronouncedly enhanced myocardial mass developed to excel in strenuous exercise.

Morphological and physiological alterations induced by lactofen in soybean leaves are reduced with nitric oxide

Lactofen is a diphenylether herbicide recommended to control broad-leaved weeds in soybean (Glycine max) fields and its mechanism of action is the inhibition of protoporphyrinogen-IX oxidase (Protox), which acts in the chlorophyll biosynthesis. This inhibition results in an accumulation of protoporphyrin-IX, which leads to the production of reactive oxygen species (ROS) that cause oxidative stress. Consequently, spots, wrinkling and leaf burn may occur, resulting in a transitory crop growth interruption. However, nitric oxide (NO) acts as an antioxidant in direct ROS scavenging. Thus, the aim of this work was to verify, through phytometric and biochemical evaluations, the protective effect of NO in soybean plants treated with the herbicide lactofen. Soybean plants were pre-treated with different levels of sodium nitroprusside (SNP), a NO-donor substance, and then sprayed with 168 g a.i. ha-1 lactofen. Pre-treatment with SNP was beneficial because NO decreased the injury symptoms caused by lactofen in young leaflets and kept low the soluble sugar levels. Nevertheless, NO caused slower plant growth, which indicates that further studies are needed in order to elucidate the action mechanisms of NO in signaling the stress caused by lactofen in soybean crop.

Role of nitric oxide and superoxide in Giardia lamblia killing

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (~60% dead trophozoites). This effect was inhibited (>90%) by an NO synthase inhibitor (200 µM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 µM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (~35% dead trophozoites at 1 mM). The mixture of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoite killing and this effect is not mediated by peroxynitrite

The myth of nitric oxide in central cardiovascular control by the nucleus tractus solitarii

Considerable evidence suggests that nitroxidergic mechanisms in the nucleus tractus solitarii (NTS) participate in cardiovascular reflex control. Much of that evidence, being based on responses to nitric oxide precursors or inhibitors of nitric oxide synthesis, has been indirect and circumstantial. We sought to directly determine cardiovascular responses to nitric oxide donors microinjected into the NTS and to determine if traditional receptor mechanisms might account for responses to certain of these donors in the central nervous system. Anesthetized adult Sprague Dawley rats that were instrumented for recording arterial pressure and heart rate were used in the physiological studies. Microinjection of nitric oxide itself into the NTS did not produce any cardiovascular responses and injection of sodium nitroprusside elicited minimal depressor responses. The S-nitrosothiols, S-nitrosoglutathione (GSNO), S-nitrosoacetylpenicillamine (SNAP), and S-nitroso-D-cysteine (D-SNC) produced no significant cardiovascular responses while injection of S-nitroso-L-cysteine (L-SNC) elicited brisk, dose-dependent depressor and bradycardic responses. In contrast, injection of glyceryl trinitrate elicited minimal pressor responses without associated changes in heart rate. It is unlikely that the responses to L-SNC were dependent on release of nitric oxide in that 1) the responses were not affected by injection of oxyhemoglobin or an inhibitor of nitric oxide synthesis prior to injection of L-SNC and 2) L- and D-SNC released identical amounts of nitric oxide when exposed to brain tissue homogenates. Although GSNO did not independently affect blood pressure, its injection attenuated responses to subsequent injection of L-SNC. Furthermore, radioligand binding studies suggested that in rat brain synaptosomes there is a saturable binding site for GSNO that is displaced from that site by L-SNC. The studies suggest that S-nitrosocysteine, not nitric oxide, may be an interneuronal messenger for cardiovascular neurons in the NTS

Anticonvulsant and proconvulsant roles of nitric oxide in experimental epilepsy models

The effect of acute (120 mg/kg) and chronic (25 mg/kg, twice a day, for 4 days) intraperitonial injection of the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine (L-NOARG) was evaluated on seizure induction by drugs such as pilocarpine and pentylenetetrazole (PTZ) and by sound stimulation of audiogenic seizure-resistant (R) and audiogenic seizure-susceptible (S) rats. Seizures were elicited by a subconvulsant dose of pilocarpine (100 mg/kg) only after NOS inhibition. NOS inhibition also simultaneously potentiated the severity of PTZ-induced limbic seizures (60 mg/kg) and protected against PTZ-induced tonic seizures (80 mg/kg). The audiogenic seizure susceptibility of S or R rats did not change after similar treatments. In conclusion, proconvulsant effects of NOS inhibition are suggested to occur in the pilocarpine model and in the limbic components of PTZ-induced seizures, while an anticonvulsant role is suggested for the tonic seizures induced by higher doses of PTZ, revealing inhibitor-specific interactions with convulsant dose and also confirming the hypothesis that the effects of NOS inhibitors vary with the model of seizure

Reversal by methylene blue of tetanic fade induced in cats by nitric oxide

Previous data from our laboratory have indicated that nitric oxide (NO) acting at the presynaptic level increases the amplitude of muscular contraction (AMC) of the phrenic-diaphragm preparations isolated from indirectly stimulated rats, but, by acting at the postsynaptic level, it reduces the AMC when the preparations are directly stimulated. In the present study we investigated the effects induced by NO when tetanic frequencies of stimulation were applied to in vivo preparations (sciatic nerve-anterior tibial muscle of the cat). Intra-arterial injection of NO (0.75-1.5 mg/kg) induced a dose-dependent increase in the Wedensky inhibition produced by high frequencies of stimulation applied to the motor nerve. Intra-arterial administration of 7.2 µg/kg methylene blue did not produce any change in AMC at low frequencies of nerve stimulation (0.2 Hz), but antagonized the NO-induced Wedensky inhibition. The experimental data suggest that NO-induced Wedensky inhibition may be mediated by the guanylate cyclase-cGMP pathway

Constitutive nitric oxide synthase (cNOS) activity in Langerhans islets from streptozotocin diabetic rats

Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

Cytotoxic activity of BCG-activated macrophages against L929 tumor cells is nitric oxide-dependent

The tumoricidal activity of activated macrophages has been attributed largely to the release of tumor necrosis factor (TNF), or to the production of reactive oxygen or nitrogen intermediates. The L929 tumor cell line (a murine fibroblast-like cell) when treated with actinomycin D (ActD) has been used to measure TNFa cytotoxicity. In the present study, we determined the cytotoxic activity of BCG-activated peritoneal macrophages against ActD-untreated L929 tumor cells. Furthermore, we measured the production of hydrogen peroxide (H2O2), nitric oxide (NO) and TNF by macrophages cultured in the presence or absence of L929 cells. As expected, BCG-activated macrophages produced significant amounts of H2O2 (16.0 ± 3.0 µM), TNF (512 U/ml) and NO (71.5 ± 3.2 µM). TNF (256 U/ml) and NO (78.9 ± 9.7 µM) production was unchanged in co-cultures of L929 cells with BCG-activated macrophages but H2O2 production was totally inhibited. The cytotoxic activity was dependent on NO release since L-NAME (2.5, 5.0 and 10 mM), which blocks NO synthase, inhibited the killing of L929 cells. Addition of anti-TNF (20 µg/ml) antibodies to the cultures did not affect the tumoricidal activity of macrophages. Our results indicate that macrophage-mediated killing of L929 cells is largely dependent on NO production but independent of H2O2 or TNF release.

Cellular signaling with nitric oxide and cyclic GMP

During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

The role of nitric oxide in reproduction

Nitric oxide (NO) plays a crucial role in reproduction at every level in the organism. In the brain, it activates the release of luteinizing hormone-releasing hormone (LHRH). The axons of the LHRH neurons project to the mating centers in the brain stem and by afferent pathways evoke the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine monophosphate (cGMP). NO also activates the release of LHRH which reaches the pituitary and activates the release of gonadotropins by activating neural NO synthase (nNOS) in the pituitary gland. In the gonad, NO plays an important role in inducing ovulation and in causing luteolysis, whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it via prostaglandins (PG).

Effects of a neuronal nitric oxide synthase inhibitor on lipopolysaccharide-induced fever

It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg/kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02). The coinjection of LPS and 7-NI was followed by a significant (P<0.02) hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.