Biomedical subjects
Publications and source records attributed to M A Moro.
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Nitric oxide (NO) has been proposed to exert neuroprotective actions against oxidative damage acting directly as an antioxidant; in addition, it has also been suggested that NO might be cytoprotective by increasing cyclic GMP concentrations via activation of soluble guanylate cyclase. In this context, we have previously shown that cyclic GMP elevations confer cytoprotection against the neurotoxicity induced by SIN-1 in the presence of superoxide dismutase, conditions in which cell death seems to be a consequence of hydrogen peroxide (H2O2) formation. We have now found that H2O2 (20-100 microM) causes neurotoxicity in 1-week-old rat cortical neurones and that this effect is inhibited by the NO donor DETA-NONOate (1-10 microM). We have also found that 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylate cyclase, reverses the effect induced by DETA-NONOate, and that this action of ODQ is mimicked by 8-(4-chlorophenylthio)guanosine-3',5'-monophosphorothioate (Rp-8-pCPT-cGMPS), an inhibitor of cyclic GMP-dependent protein kinase, suggesting that the pathway affording protection involves activation of this kinase by cyclic GMP elevations. Simultaneously, ODQ inhibits the elevation of cyclic GMP concentrations induced by DETA-NONOate (1-3 microM) in cortical cells. Finally, we have also shown that the cyclic GMP mimetic, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cyclic GMP) inhibits the neurotoxicity induced by H2O2 (30-40 microM). Taken together, these data demonstrate that NO-induced cyclic GMP elevations confer cytoprotection against H2O2-induced neuronal cell death.
We have recently demonstrated that inducible nitric oxide synthase (iNOS) is expressed in rat forebrain slices exposed to oxygen and glucose deprivation (OGD). Now, we have found that the expression of iNOS after OGD is time-dependent since 20 min of OGD produces the appearance of iNOS at earlier times than 10 min of OGD. OGD also causes neurotoxicity in this model, as revealed by the increase in excitatory amino acid, neuron specific enolase and lactate dehydrogenase (LDH) efflux to the incubation solution. Finally, the administration of the NMDA receptor antagonist MK-801 (100 nM) inhibits both the expression of iNOS and the release of LDH. Our findings demonstrate that this method may be considered an useful in vitro model of ischemia-reperfusion to determine the therapeutic role of neuroprotective tools.
The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.
OBJECTIVES: To test whether nitric oxide synthase (NOS) is expressed in primary otolaryngologic tumors and whether this expression is associated with the degree of malignancy. STUDY DESIGN: Twenty-six samples from the primary localization of human pharyngolaryngeal squamous cell carcinoma. MATERIALS AND METHODS: the activity of calcium-dependent and calcium-independent NOS was analyzed by the conversion of L-[14C]-arginine into L-[14C]-citrulline. RESULTS: NOS activity is below detectable levels in pharyngolaryngeal mucosa from noncancer patients. In the primary localization of the tumor, calcium-independent NOS activity is maximal at early stages of tumor growth, whereas calcium-dependent activity increases from early to advanced stages. CONCLUSIONS: These data suggest that tumor growth and malignancy is associated with a change in the enzymatic source of NO from calcium-independent NOS to calcium-dependent NOS isoform in primary localization. These data suggest that the inhibition of calcium-independent NOS activity in early stages and/or inhibition of calcium-dependent NOS activity in later stages could delay growth of solid tumors.
Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS-immunoreactive multipolar neurons found in layers II-VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS-positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II-VI of aged rat cortex. While no iNOS-immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine-positive cells outnumbered total NOS-positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine-positive cells. Western blots of brain extracts showed a several-fold increase in both nNOS- and iNOS-immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine-containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation.
It has been suggested that large amounts of nitric oxide (NO) produced by inducible NO synthase are involved in the mechanisms of neurotoxicity after cerebral ischaemia. We have recently demonstrated that inducible NO synthase was expressed within hours after rat forebrain slices were exposed to oxygen-glucose deprivation. Therefore, we sought to determine whether NO produced by inducible NO synthase contributes to tissue damage in this model, by using a new, highly selective, inhibitor of inducible NO synthase, N-(3-(aminomethyl)benzyl)acetamidine (1400W). We found that incubation with 1400W from the start of the oxygen-glucose deprivation period until the end of the experiment decreases tissue damage determined as lactate dehydrogenase (LDH) efflux 4 h after the oxygen-glucose deprivation period, the time at which inducible NO synthase expression is maximal in this model. This effect may be a result of direct inhibition of inducible NO synthase activity, raising the possibility of a clinical use of selective inhibitors of this NO synthase isoform in the management of cerebral ischaemia.
OBJECTIVE: Peroxynitrite (ONOO-) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO- in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO- on human platelets, a system in which we have previously shown that ONOO- has both pro- and anti-aggregatory effects. METHODS: Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO- was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. RESULTS: Peroxynitrite (50-400 microM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO- acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma, ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50-100 microM) of ONOO- inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. CONCLUSIONS: Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO- to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO- in biological systems. We hypothesize that the conversion of ONOO- to NO is the critical factor determining the outcome of ONOO- exposure in vivo.
The nitrovasodilator 3-morpholinosydnonimine (SIN-1) slowly decomposes to release both nitric oxide (NO) and superoxide (O2-) and thereby produces peroxynitrite (ONOO-), a powerful oxidant which has been proposed to mediate the toxic actions caused by NO. Indeed, ONOO has been shown to cause neuronal death and it has been proposed to occur in different disorders of the CNS such as brain ischaemia, AIDS-associated dementia, amyothrophic lateral sclerosis, etc. We have found that SIN-1 was only slightly toxic to 1-week-old rat cortical neurones in primary culture (LC50=2.5+/-0.5 mM). Superoxide dismutase (SOD; 100 U/ml) significantly increased SIN-1-induced toxicity, an effect that was enhanced in the presence of HbO2, abolished by catalase and accompanied by the formation of hydrogen peroxide (H2O2). We have also found that 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylate cyclase, enhances cell death induced by SIN-1 (0.2-0.5 mM) + SOD (100 U/ml) in a concentration-dependent way (EC50=0.073+/-0.004 microM). Simultaneously, ODQ inhibits the elevation of cyclic GMP concentrations induced by SIN-1 + SOD in cortical cells (IC50=0.022+/-0.014 microM). Finally, we have also shown that the cyclic GMP mimetic, 8-bromo-cyclic GMP reverses the potentiating effect induced by ODQ on SIN-1 + SOD-induced neuronal death and inhibits the neurotoxicity induced by H2O2 (100 microM). Taken together, these data suggest that H2O2 is the species responsible for the potentiation by SOD of SIN-1-induced cell death and that cyclic GMP elevations confer selective cytoprotection against this H2O2-mediated component of cell death.
Nitric oxide (NO) overproduction has been postulated to contribute significantly to ischaemia-reperfusion neurotoxicity. Inducible or type II NO synthase (iNOS) synthesizes NO in large quantities for long periods of time. Therefore we investigated the expression and localization of iNOS after oxygen and glucose deprivation in rat forebrain slices. In this experimental model, calcium-independent NOS activity reached a maximum 180 min after the end of a 20 min oxygen-glucose deprivation period. During the same period of time, the calcium-independent activity was absent in control forebrain slices. To test whether this calcium-independent NOS activity was due to the expression of iNOS, the effects of the addition of dexamethasone, cycloheximide and pyrrolidine dithiocarbamate were determined. All of them inhibited the induction of the calcium-independent NOS activity measured in the rat forebrain slices after oxygen and glucose deprivation. Furthermore, oxygen and glucose deprivation caused the expression of the gene encoding iNOS in rat forebrain slices, as assessed by the detection of iNOS message and protein in these samples. A sixfold increase in the iNOS mRNA levels was observed at 180 min and the time-course of the expression of iNOS mRNA was in agreement with the temporal profile of iNOS enzymatic activity. Immunohistochemistry analysis revealed that iNOS was highly expressed in neurones, astrocytes and microglial cells. These results demonstrate for the first time that iNOS is expressed in neurones after oxygen and glucose deprivation, and that this expression occurs in short periods of time. These findings suggest that NO can play an important pathogenic role in the tissue damage that occurs after cerebral ischaemia.
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Peroxynitrite (ONOO-) is a powerful oxidant which is formed from the reaction between nitric oxide (NO) and superoxide anion. It has therefore been proposed to mediate the toxic actions caused by NO. Since ONOO- may be formed in the central nervous system (CNS) in pathological conditions such as brain ischaemia, we decided to investigate whether this molecule induces the release of the endogenous excitatory amino acids glutamate and aspartate from neurones. We selected as biological model acutely dissociated rat cerebellar granule neurones in suspension to allow a direct interaction between ONOO- and target cells. Peroxynitrite caused a concentration-dependent release of aspartate but not of glutamate from dissociated cerebellar granule neurones. Peroxynitrite-induced aspartate release was inhibited by dithiothreitol, tetrodotoxin, and in Na+-deprived solutions and not affected by EGTA or pre-incubation with the cytosolic Ca2+ chelator BAPTA/AM. Peroxynitrite also induced an increase in intracellular Ca2+ concentration which was not affected in the presence of EGTA. These data show that ONOO- causes release of aspartate from cerebellar granule neurones and that this effect might arise from an alteration of Na+ membrane permeability leading subsequently to reversal of a Na+-dependent plasma membrane transporter of this excitatory amino acid. In addition, ONOO- alters Ca2+ homeostasis likely due to Na+ overload. Taken together, these findings may help and elucidate some of the intimate mechanisms of NO-induced neuronal damage in pathological circumstances.
We studied the effects of lamotrigine [3,5-diamino-6-(2,3-dichlorophenyl)- 1,2,4-triazine], a new antiepileptic compound, on naloxone-precipitated morphine withdrawal in mice. Pretreatment with lamotrigine (5-100 mg/kg, s.c.) reversed in a dose-dependent way the withdrawal-induced increase in cerebellar Ca(2+)-dependent nitric oxide (NO) synthase activity and reduced the number of escape jumps and other motor symptoms of abstinence, at doses that did not modify locomotor activity (25-50 mg/kg). Pretreatment with the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydroxy-5H- dibenzo[a,d]cyclohepten-5,10-imine; dizocilpine] (0.1-0.3 mg/kg, s.c.) also reversed the increase in cerebellar Ca(2+)-dependent NO synthase activity. However, although MK-801 reduced the number of escape jumps and other motor symptoms of abstinence, its effects were not clearly dose-dependent. Furthermore, the highest dose of MK-801 tested (0.3 mg/kg) caused an impairment of the locomotor behaviour in naive mice. Thus, lamotrigine may represent a new and useful agent for the treatment of opiate abstinence.
Nitric oxide (NO) and peroxynitrite both inhibit respiration by brain submitochondrial particles, the former reversibly at cytochrome c oxidase, the latter irreversibly at complexes I-III. Both GSH (IC50 =10 microM) and glucose (IC50 = 8 mM) prevented inhibition of respiration by peroxynitrite (ONOO-), but neither glucose (100 mM) nor GSH (100 microM) affected that by NO. Thus, unless ONOO- is formed within mitochondria it is unlikely to inhibit respiration in cells directly, because of reactions with cellular thiols and carbohydrates. However, the reversible inhibition of respiration cytochrome c oxidase by NO is likely to occur (e.g. in the brain during ischaemia) and could be responsible for cytotoxicity.
The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.
The opiate withdrawal induced by administration of naloxone to morphine-dependent mice correlates with an increment of calcium- dependent nitric oxide synthase (NOS) activity in the cerebellum. L-NAME, an irreversible competitive inhibitor of NOS (0.5, 5, 25, 50 mg/kg) injected sc. 45 min. prior to naloxone significantly reduced the number of escape jumps and other motor symptoms of abstinence. In addition, L-NAME also decreased NOS activity in cerebellum. L-arginine, but not D-arginine, when coadministered with L-NAME, prevented both the inhibition of NOS activity and the reduction of withdrawal symptoms induced by L-NAME in morphine-withdrawn animals. These results demonstrate a hyperactivity of the L-arginine: NO pathway in opiate withdrawal and suggests the possibility of a therapeutic use of NOS inhibitors in this state.
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1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.