Nitric Oxide-releasing Compound Research Articles

Enhancing postharvest quality and extending storage life of Booth 7 avocado (Persea americana Mill.) fruit through sodium nitroprusside (SNP) application during low-temperature storage

Postharvest quality plays a crucial role in marketability, consumer acceptability, and shelf life of avocados. This study investigates the efficacy of nitric oxide (NO) on postharvest physiology and quality of Booth 7 avocados treated with different concentrations of sodium nitroprusside (SNP) - a nitric oxide-releasing compound. Booth 7 avocados were immersed in SNP solutions (0.5 mM, 1.0 mM, and 1.5 mM), while the control fruit was immersed in distilled water for 15 min. After treatment, the fruit was stored at a temperature (T) of 8 ± 1 °C and relative humidity (RH) of 90 ± 5% for 35 days, during which the control fruit exhibited complete deterioration. The results revealed that Booth 7 avocados treated with 1.5 mM SNP concentration exhibited delayed quality changes during storage, reduced ethylene production and respiration rate, reduced alterations in soluble solids and acid content of the fruit flesh, as well as delayed changes in flesh and peel color. Additionally, the activity of total phenolic compounds (TPC), polygalacturonase (PG), and polyphenol oxidase (PPO) enzymes increased significantly, while the activity of the pectin methyl esterase (PME) enzyme was reduced. In conclusion, SNP treatment improved the quality and can be utilized as a potential postharvest preservative for Booth 7 avocados. For producers and distributors, the use of SNP in avocado postharvest management offers the potential to extend shelf life without compromising fruit quality.

بررسی اثر سدیم نیتروپروساید در کاهش سمیت ناشی از کادمیوم در گیاه ریحان (.Ocimum basilicum L)

این تحقیق به منظور بررسی اثر سدیم نیتروپروساید در کاهش سمیت ناشی از کادمیوم در گیاه ریحان به صورت فاکتوریل در قالب طرح کاملا تصادفی در سال 97-1396 اجرا گردید. فاکتورهای اعمال شده در این تحقیق شامل کادمیوم در سطوح صفر (شاهد)، 10، 20 و 30 میلی‌گرم در کیلوگرم خاک و محلول‌پاشی سدیم نیتروپروساید در سطوح صفر (شاهد)، 50 و 100 میکرومولار بود. صفات اندازه‌گیری شامل ارتفاع بوته، خشک بوته، تعداد برگ در بوته، درصد اسانس، مالون دی آلدئید، فعالیت آنزیم آنتی‌اکسیدان سوپراکسید دیسموتاز، پرولین و غلظت کادمیوم بود. نتایج به دست آمده نشان داد که کادمیوم باعث کاهش ارتفاع بوته، وزن خشک بوته، تعداد برگ در بوته، درصد اسانس و فعالیت سوپراکسید دیسموتاز و افزایش پرولین، غلظت کادمیوم و مالون دی آلدئید شد. بیشترین تأثیر کادمیوم در تیمار 30 میلی‌گرم در کیلوگرم خاک به دست آمد. همچنین مشاهده شد که کاربرد سدیم نیتروپروساید توانست موجب افزایش ارتفاع بوته، وزن خشک بوته، تعداد برگ در بوته، درصد اسانس و فعالیت سوپراکسید دیسموتاز و کاهش پرولین، غلظت کادمیوم و مالون دی آلدئید شود، به طور کلی بیشترین تأثیر مثبت در کاربرد 100 میکرومولار سدیم نیتروپروساید حاصل گردید. بر اساس نتایج، محلول‌پاشی سدیم نیتروپروساید توانست باعث بهبود خصوصیات رویشی و کاهش مالون دی آلدئید در شرایط تنش فلز سنگین کادمیوم گردد. می‌توان از نتایج به دست آمده به اثرات مثبت سدیم نیتروپروساید به خصوص در شرایط حضور کادمیوم و کاهش اثرات منفی ناشی از آن تنش در گیاه دارویی ریحان پی برد. بنابراین کاربرد 100 میکرومولار سدیم نیتروپروساید برای تعدیل بخشیدن اثرات منفی ناشی از تنش فلز سنگین کادمیوم پیشنهاد می‌گردد.

Antimicrobial activity and biocompatibility of the mixture of mineral trioxide aggregate and nitric oxide-releasing compound

Background/purposeAntimicrobial activity and biocompatibility of root canal sealer are related to the success of endodontic treatments. This study investigated the efficacy of mixture of mineral trioxide aggregate (MTA) and a NO-releasing compound for the antimicrobial activity, biocompatibility, and physical properties. Materials and methodsMTA was mixed with diethylenetriamine-NO (MTA-NO), and the extracts from MTA and the MTA-NO mixture before and after setting was obtained were investigated the antimicrobial activity against Enterococcus faecalis and Porphyromonas endodontalis. After setting MTA and MTA-NO, pulp cell was incubated in the presence of MTA and MTA-NO disk using Transwell® cell culture insert, and the proliferation assay and mineralization-stimulated factors of the cells were analyzed by MTT assay and real-time RT-PCR, respectively. The physical properties of MTA and the MTA-NO mixture, such as surface hardness and flowability was also analyzed. ResultsThe MTA-NO mixture showed stronger antimicrobial activity against E. faecalis and P. endodontalis than that by MTA. Both MTA and MTA-NO mixture increase the ratio of cell proliferation and induced the expression of alkaline phosphatase, collagen type I, osteocalcin, and osteopontin. Moreover, the induction of gene expression by MTA-NO mixture was higher than that by MTA alone. No significant difference was observed for surface hardness and flowability between MTA and MTA-NO mixture. ConclusionThe addition of a NO-releasing compound to the endodontic treatment using MTA root canal sealer might reduce the risk of bacterial infection and help to regenerate the dental pulp tissue.

NVN1000, a novel nitric oxide-releasing compound, inhibits HPV-18 virus production by interfering with E6 and E7 oncoprotein functions

NVN1000, a novel nitric oxide-releasing compound, inhibits HPV-18 virus production by interfering with E6 and E7 oncoprotein functions

Role of Nitric Oxide and Flavohemoglobin Homolog Genes in Aspergillus nidulans Sexual Development and Mycotoxin Production

Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the Aspergillus nidulans flavohemoglobin gene fhbA induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased nsdD and steA expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi.

Ketamine and thiopental sodium: individual and combined neuroprotective effects on cortical cultures exposed to NMDA or nitric oxide

An N-methyl-D-aspartate (NMDA) blocker, ketamine, has been shown to be neuroprotective both in vivo and in vitro. However, ketamine is not commonly recommended for use in patients suffering from cerebral ischaemia because of its adverse neurological effects. We hypothesized that combined administration of ketamine and thiopental sodium (TPS) would be highly effective in protecting cerebral cortical neurones from ischaemia, with possibly reduced dosages. We examined the degree of neuroprotection provided by various concentrations of ketamine and TPS, alone and in combination, in cortical cultures exposed to NMDA or a nitric oxide-releasing compound (NOC-5) for 24 h. The survival rate (SR) of E16 Wistar rat cortical neurones was evaluated using photomicrographs before and after exposure to these compounds. The SRs of cortical neurones exposed to 30 microM NMDA or NOC-5 were 15.0 (3.8)%, 12.8 (3.1)%, respectively. Higher doses (5, 10 and 50 microM) but not lower doses (<1 microM) of ketamine improved SRs [57.9 (2.2)%, 61.1 (5.4)%, 76.7 (3.0)%, respectively] against NMDA but not NOC. Enhanced survival was observed with combined administration of 5 or 10 microM ketamine and 50 microM TPS [SR 71.3 (4.8)%, 74.7 (3.7)%, respectively, P<0.05 if ketamine alone, P<0.01 if TPS alone], against NMDA-induced neurotoxicity in vitro. Only the highest dose of TPS (50 microM) improved survival after NOC exposure. This neuroprotection was not influenced by ketamine. These data indicate that a low, clinically relevant dose of ketamine offer significant neuroprotection during prolonged exposure to NMDA but not to NOC. Combinations of reduced doses of ketamine and TPS exhibited enhanced neuroprotection against NMDA-induced neurotoxicity. Hence, combinations of these two common i.v. anaesthetics agents could be developed to protect the brain from ischaemia.

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide.

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (approximately 98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (approximately 76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.

Yawning responses induced by local hypoxia in the paraventricular nucleus of the rat

Yawing was induced by microinjections of l-glutamate, cyanide and a nitric oxide-releasing compound (NOC12) into the paraventricular nucleus of the hypothalamus (PVN) in anesthetized, spontaneously breathing rats. To evaluate physiological aspects of yawning, we monitored intercostal electromyogram (EMG) as an index of inspiratory activity, digastric EMG, blood pressure and electrocorticogram (ECoG). Microinjection of l-glutamate in the medial parvocellular subdivision ( mp) elicited a stereotyped yawning response, i.e. an initial depressor response and an arousal shift in ECoG followed by a single large inspiration with mouth opening. The same sequential events were observed during spontaneous yawning, indicating that the mp is responsible for triggering yawning. Microinjection of cyanide into the mp caused the same yawning responses as the ones elicited by microinjection of l-glutamate, suggesting that the mp is sensitive to chemical hypoxia or ischemia within the PVN. Microinjection of NOC12 into the mp elicited a single large inspiration with a variable onset delay, suggesting that diffusible nitric oxide (NO) within the mp may act as a paracrine agent to cause a yawning response. We hypothesize that the mp of the PVN contains an oxygen sensor that causes a yawning response.

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.

Regulation of prostaglandin H2 synthase activity by nitrogen oxides.

Nitric oxide and its derivatives have been shown to both activate and inhibit prostaglandin H(2) synthase 1 (PGHS-1). We set out to determine the mechanisms by which different nitrogen oxide derivatives modulate PGHS-1 activity. To this end, we show that 3-morpholinosydnonimine hydrochloride (SIN-1), a compound capable of generating peroxynitrite, activates purified PGHS-1 and also stimulates PGE(2) production in arterial smooth muscle cells in the presence of exogenous arachidonic acid. The effect of SIN-1 in smooth muscle cells was abrogated by superoxide and peroxynitrite inhibitors, which supports the hypothesis that peroxynitrite is an activating species of PGHS-1. Indeed, authentic peroxynitrite also induced PGE(2) production in arachidonic acid-stimulated cells. In contrast, when cells were exposed to the nitric oxide-releasing compound 1-hydroxy-2-oxo-3-[(methylamino)propyl]-3-methyl-1-triazene (NOC-7), PGHS-1 enzyme activity was inhibited in the presence of exogenous arachidonic acid. Finally, in lipid-loaded smooth muscle cells, we demonstrate that SIN-1 stimulates arachidonic acid-induced PGE(2) production; albeit, the extent of activation is reduced compared to that under normal conditions. These results indicate that formation of peroxynitrite is a key intermediary step in PGHS-1 activation. However, other forms of NO(x)() inhibit PGHS-1. These results may have implications in the regulation of vascular function and tone in normal and atherosclerotic arteries.

Hemoglobin and iron-evoked oxidative stress in the brain: Protection by bile pigments, managanese and S-nitrosoglutathione

In the present in vitro and in vivo study we investigated the pro-oxidant effects of hemoglobin, as well as the antioxidant effects of its metabolites, in the brain. Incubation of rat brain homogenates with hemoglobin (0–10 μM) but not hemin induced lipid peroxidation up to 24 h (EC50 = 1.2 μM). Hemoglobin's effects were similar to ferrous ion (EC50 = 1.7 μM) and were blocked by the chelating agent deferoxamine (IC50 = 0.5 μM) and a nitric oxide-releasing compound S-nitrosoglutathione (IC50 = 40 μM). However, metabolites of hemoglobin — biliverdin and bilirubin — inhibited brain lipid peroxidation induced by cell disruption and hemoglobin (biliverdin IC50 = 12–30 and bilirubin IC50 = 75–170 μM). Biliverdin's antioxidative effects in spontaneous and iron-evoked lipid peroxidation were further augmented by maganese (2 μM) since manganese is an antioxidative transition metal and conjugates with bile pigments. Intrastriatal infusion of hemoglobin (0–24 nmol) produced slight, but significant 20–22% decreases in striatal dopamine levels. Whereas, intrastriatal infusion of ferrous citrate (0–24 nmol) dose-dependently induced a greater 66% depletion of striatal dopamine which was preceded by an acute increase of lipid peroxidation. In conclusion, contrary to the in vitro results hemoglobin is far less neurotoxic than ferrous ions in the brain. It is speculated that hemoglobin may be partially detoxified by heme oxygenase and biliverdin reductase to its antioxidative metabolites in the brain. However, in head trauma and stroke, massive bleeding could significantly produce iron-mediated oxidative stress and neurodegeneration which could be minimized by endogenous antioxidants such as biliverdin, bilirubin, manganese and S-nitrosoglutathione.

The Critical Role of Hepes in SIN-1 Cytotoxicity, Peroxynitrite Versus Hydrogen Peroxide

The cytotoxicity of the superoxide anion radical- and nitric oxide-releasing compound SIN-1 to L929 cells was studied in Krebs-Henseleit buffer, pH 7.4, in the presence and absence of Hepes. SIN-1 cytotoxicity was significantly higher in the presence of Hepes than in the absence of Hepes. The available amount of peroxynitrite formed from SIN-1, however, was significantly decreased by Hepes as indicated by decreased oxidation of dihydrorhodamine 123. On the other hand, Hepes largely increased the formation of H2O2 from SIN-1. Catalase protected the L929 cells from SIN-1 cytotoxicity in the buffer with Hepes. In the buffer without Hepes catalase did not have any protective effect. In contrast, tyrosine and tryptophan provided significant protection against SIN-1 cytotoxicity independent of the presence of Hepes. These results demonstrate that the immediate toxic agent formed from SIN-1 decisively depends on the presence of Hepes. In its absence cytotoxicity is most likely mediated by peroxynitrite while in the presence of Hepes, cytotoxicity is conveyed by co-operative action of hydrogen peroxide and reactive nitrogen species.

Anoxic injury of endothelial cells causes divergent changes in protein kinase C and protein kinase A signaling pathways.

Alterations in protein kinase C (PKC) and cAMP-dependent kinase have been documented in anoxic brain injury. However, the regulation of these signaling enzymes in the cerebrovasculature has not been explored. In this study, cultured brain endothelial cells exposed to anoxic injury (anoxia--20 min/reoxygenation--40 min) showed both a significant increase (p < 0.001) in PKC and decrease (p < 0.01) in cAMP-dependent kinase activity. Analysis of PKC by Western blot indicated an increase in kinase level in response to anoxic injury, whereas there was no change in the level of cAMP-dependent protein kinase, as measured by labeled cAMP binding. Inhibition of nitric oxide synthase did not affect these changes. Addition of the nitric oxide-releasing compound sodium nitroprusside caused a dose-dependent increase in the activity of both signaling systems in endothelial cells. These data demonstrate that anoxic injury of brain endothelial cells in culture causes significant and divergent changes in signaling kinase activity. Abnormalities in brain endothelial PKC and cAMP-dependent kinase could have important consequences for the blood-brain barrier in anoxic brain injury.

Nitric oxide primes pancreatic beta cells for Fas-mediated destruction in insulin-dependent diabetes mellitus.

Fas is an apoptosis-inducing surface receptor involved in controlling tissue homeostasis and function at multiple sites. Here we show that beta cells from the pancreata of newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients express Fas and show extensive apoptosis among those cells located in proximity to Fas ligand-expressing T lymphocytes infiltrating the IDDM islets. Normal human pancreatic beta cells that do not constitutively express Fas, become strongly Fas positive after interleuken (IL)-1beta exposure, and are then susceptible to Fas-mediated apoptosis. NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthase, prevents IL-1beta-induced Fas expression, whereas the NO donors sodium nitroprusside and nitric oxide releasing compound (NOC)-18, induce functional Fas expression in normal pancreatic beta cells. These findings suggest that NO-mediated upregulation of Fas contributes to pancreatic beta cell damage in IDDM.

Molecular Mechanisms of Apoptosis in HL-60 Cells Induced by a Nitric Oxide-Releasing Compound

Nitric oxide (NO) generated from 1-hydroxy-2–0×0–3, 3-bis(2-aminoethyl)-l-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-lβ converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.

Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa

The induction of acrosomal exocytosis in capacitated bull spermatozoa by atrial natriuretic peptide (ANP) was studied in vitro. ANP markedly stimulated acrosomal exocytosis in a calcium-dependent manner. Typically, ANP exerts its action via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that the ANP-induced acrosome reaction was inhibited by the competitive ANPR-A receptor antagonist-anantin, indicating a receptor-mediated effect. We could mimic the effect of ANP on the acrosome reaction by using 8-bromo-cGMP, suggesting that cGMP may serve as a signal transducer mediating the acrosome reaction. Indeed, the ANP-induced acrosome reaction was associated with elevation of cGMP levels. cGMP can also be formed by activation of the soluble form of guanylyl cyclase. Sodium nitroprusside (SNP) stimulated cGMP accumulation and acrosome reaction of capacitated spermatozoa. Thus ANP and the nitric oxide-releasing compound SNP, via activation of guanylyl cyclase (the former activating the particulate and the latter activating the soluble form of the enzyme), may play a significant role in the induction of the acrosome reaction.

Intracerebroventricular administration of a nitric oxide-releasing compound, NOC-18, produces thermal hyperalgesia in rats

This study was undertaken to define the role of nitric oxide (NO) in central nociceptive mechanisms by intracerebroventricular injection of an NO-releasing compound, NOC-18, in rats. The nociceptive threshold was evaluated by the radiant heat tail-flick test. Sixty-nine rats were divided into the seven groups, and the following drugs were injected intracerebroventricularly in 5 μl of saline: no drug (control) ( n = 13), 15μg of NOC-18 ( n = 15); 150 μg of NOC-18 ( n = 9); 100 μg of N-nitro- l-arginine methyl ester (L-NAME) ( n = 8); 15 μg of NOC-18 + 100 μg of L-NAME ( n = 8); 10 μg of methylene blue (MB) ( n = 8); 15 μg of NOC-18 + 10 μg of MB ( n = 8). NOC-18 caused a dose-dependent curtailment (7% and 23% decreases for 15 μg and 150 μg of NOC-18, respectively) of the tail-flick latency during the period from 15 to 120 min. L-NAME caused prolongation (15% maximum) of the tail-flick latency during the period from 15 to 150 min. However, NOC-18-induced hyperalgesia was not influenced by L-NAME. MB also caused prolongation (9% maximum) of the tail-flick latency during the period from 15 to 150 min, and completely blocked the hyperalgesia induced by 15 μg of NOC-18. These findings indicate that the NO-cGMP pathway is directly involved in thermal hyperalgesia in the brain.

Inhibition of Cytochromes P450 by Nitric Oxide and a Nitric Oxide-Releasing Agent

The effect of nitric oxide (NO) on cytochrome P450-mediated benzyloxyresorufin and ethoxyresorufin O-dealkylase activity of rat hepatic postmitochondrial (S-9) or microsomal subfractions, or purified rat liver CYP2B1, was examined. Two distinct inhibitory phases were observed regardless of whether the NO was added prior to initiation of the reactions with NADPH or during the course of substrate turnover. The first was a reversible inhibition characterized by complete cessation of catalytic activity, the duration of which was NO concentration-dependent with an IC50 in the range of 8-60 μM. This phase was followed by a second, irreversible, inhibitory phase characterized by a varying extent of recovery of activity, with inhibition ranging from <1 to 100%. The extent of this diminution in substrate conversion rate was also NO concentration-dependent, with an IC50 in the range of 13-72 μM, and could be partially abrogated by the inclusion of bovine serum albumin in the reaction mixture. Lower IC50 values for both inhibitory phases were obtained in the case of benzyloxyresorufin O-dealkylase activity than in the case of ethoxyresorufin O-dealkylase activity, suggesting a differential susceptibility to inhibition by NO for the two O-dealkylase activities. A nitric oxide-releasing compound ([Et2NN(O)NO]Na, DEA/NO) caused qualitatively similar inhibitory effects on benzyloxyresorufin O-dealkylase activity when added prior to the initiation of the reactions with NADPH, or during the course of substrate turnover. Based upon these results, it is possible that NO may play a role in the regulation of P450 activity in vivo.