Correlation between brain nitric oxide synthase activity and opiate withdrawal

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prevented both the inhibition of NOS activity and the reduction of withdrawal symptoms induced by L-NAME in morphine-withdrawn animals.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.Abbreviations EAA Excitatory amino acid - LC Locus coeruleus - L-NAME N-nitro L-arginine methyl ester - NMDA N-methyl-D-aspartate - NO Nitric oxide - NOS Nitric oxide synthase     

Geissoschizine methyl ether,an indole alkaloid extracted from Uncariae Ramulus et Uncus,is a potent vasorelaxant of isolated rat aorta

Effects of geissoschizine methyl ether, an indole alkaloid isolated from the hook of Uncariae Ramulus et Uncus, on vascular responses were examined using isolated strips of rat aorta. Geissoschizine methyl ether (10(-7)-10(-4) M) relaxed norepinephrine (5x10(-8) M)-induced contraction in a dose-dependent manner. The potency (50% efficacy concentration, EC(50)=0.744 microM) was approximately 14 times greater than that (EC(50)=10.6 microM) of hirsutine, one of the indole alkaloids isolated from Uncariae Ramulus et Uncus that demonstrates a vasorelaxant effect by Ca(2+)-channel blocking. The vasorelaxant effect of geissoschizine methyl ether found at the lower concentrations (10(-7)-3x10(-6) M) on the norepinephrine-induced contraction was abolished by pretreatment with N(G)-nitro-L-arginine (10(-4) M), an inhibitor of nitric oxide synthesis, or by denuding aortas of endothelium, while the effects at the higher concentrations (10(-5)-10(-4) M) were not completely prevented by either N(G)-nitro-L-arginine and deendothelialization. Furthermore, geissoschizine methyl ether did not relax high K(+)-, Ca(2+)- and a Ca(2+)-channel agonist Bay K8644-induced contractions at the lower concentrations that markedly relaxed the norepinephrine-induced contractions, while the higher concentrations of geissoschizine methyl ether relaxed the high K(+)-, Ca(2+)- and Bay K8644-induced contractions. These results suggest that the vasorelaxant effect of geissoschizine methyl ether is composed of two different mechanisms: endothelial dependency with nitric oxide and endothelial independency with voltage-dependent Ca(2+)-channel blocking.     

Mildronate: an antiischemic drug for neurological indications

Mildronate (3-(2,2,2-trimethylhydrazinium)propionate; MET-88; meldonium, quaterine) is an antiischemic drug developed at the Latvian Institute of Organic Synthesis. Mildronate was designed to inhibit carnitine biosynthesis in order to prevent accumulation of cytotoxic intermediate products of fatty acid beta-oxidation in ischemic tissues and to block this highly oxygen-consuming process. Mildronate is efficient in the treatment of heart ischemia and its consequences. Extensive evaluation of pharmacological activities of mildronate revealed its beneficial effect on cerebral circulation disorders and central nervous system (CNS) functions. The drug is used in neurological clinics for the treatment of brain circulation disorders. It appears to improve patients' mood; they become more active, their motor dysfunction decreases, and asthenia, dizziness and nausea become less pronounced. Since the brain does not utilize fatty acids as fuel other mechanisms of action of mildronate in CNS should be considered. Several reports indicate the possible existence of an alternative, non-carnitine dependent mechanism of action of mildronate. Our recent findings suggest that CNS effects of mildronate could be mediated by stimulation of the nitric oxide production in the vascular endothelium by modification of the gamma-butyrobetaine and its esters pools. It is hypothesized that mildronate may increase the formation of the gamma-butyrobetaine esters. The latter are potent cholinomimetics and may activate eNOS via acetylcholine receptors or specific gamma-butyrobetaine ester receptors. This article summarizes known pharmacological effects of mildronate, its pharmacokinetics, toxicology, as well as the proposed mechanisms of action.     

Nitric oxide effect on 5-hydroxytryptamine-induced vasoconstrictions of isolated smooth muscle

The aim of the present study was to investigate a nitric oxide (NO) involvement in the mediation of a 5-HT-induced vasoconstriction response in the rat portal vein in vitro. Isolated rat portal vein segments were placed in organ baths for isometric force measurement. 5-HT (3 x 10(-8) M-3 x 10(-4) contracted portal vein preparations (EC50 = 7 x 10(-7) M) in a concentration-dependent manner. The vasoconstriction induced by 5-HT was significantly increased in endothelium-denuded vessels. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME 100 microM) enhanced the contractive response to 5-HT either in intact or endothelium-denuded vessels. However, ketanserin (0.1 microM) abolished 5-HT-induced vasoconstrictions (EC50 = 4.6 x 10(-8) M). Furthermore, a non-selective 5-HT(1B/D) receptor agonist, sumatriptan (1 x 10(-10) M-1 x 10(-5) M), induced a reduction of spontaneous rhythmic contractions also in endothelium-intact vessels. However, 5-HT- induced vasoconstriction was unaffected by propranolol (10 microM). The data support the hypothesis of the existence of serotonergic innervations modulating the contractility of vascular smooth muscle. These findings are consistent with the hypothesis that the vasoconstrictor activity of 5-HT in smooth muscle was mediated by activation of 5-HT(1B/D) and 5-HT(2B) receptor subtypes involving the endothelium-dependent mechanism.     

Modulation of adrenergic contraction of dog pulmonary arteries by nitric oxide and prostacyclin.

The aim of this work was to investigate the influence of endothelium-derived nitric oxide and prostaglandins on the contractile responses of isolated dog pulmonary arteries to electrical field stimulation and noradrenaline. Electrical field stimulation (1-8 Hz, 20 v, 0.25 ms duration, for 30 s) produced frequency-dependent contractions that were abolished by tetrodotoxin, guanethidine and, prazosin (all at 10(-6) M). Noradrenaline induced concentration-dependent contractions with an EC50, of 1.85 x 10(-6) M. The increases in tension induced by electrical stimulation and noradrenaline were of greater magnitude in arteries denuded of endothelium. In segments with endothelium, N(G)-nitro-L-arginine methyl ester (10(-4) M) or indomethacin (10(-5) M) had no effects on the basal tone, but significantly enhanced the neurogenic and noradrenaline-induced contractions. The potentiation by N(G)-nitro-L-arginine methyl ester of electrical stimulation-induced contractile responses was partially reversed by L-arginine (10(-4) M). In the presence of N(G)-nitro-L-arginine methyl ester together with indomethacin the electrical stimulation-induced contractile responses were higher than those obtained when only N(G)-nitro-L-arginine methyl ester or indomethacin was used. N(G)-nitro-L-arginine methyl ester and indomethacin did not influence neurogenic-induced contractile responses of endothelium-denuded arteries. The results suggest that endothelial cells of isolated dog pulmonary arteries depress the contractile response to electrical field stimulation of intramural nerves and that endothelium-derived dilator prostaglandins and nitric oxide may interact to inhibit contractile effects of adrenergic stimulation.     

Spontaneous nitric oxide in hepatocyte monolayers and inhibition of compound-induced apoptosis

Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Following isolation, hepatocytes produce large amounts of nitric oxide (NO), which is known to have both pro- and anti-apoptotic effects in hepatocytes in vivo and in vitro. Previous work has not determined the effect of these increased levels of NO on the response of hepatocytes to apoptotic stimuli. Here we report that levels of nitrites are elevated in hepatocyte monolayers from 24 h onwards. Addition of the inducible nitric oxide synthase (iNOS) inhibitor, Nω-nitro- -arginine methyl ester (L-NAME), to the medium inhibited this increase in nitrites. These results indicate that the increase in nitrite is most likely due to the formation of NO. Elevated nitrite levels had no effect either on basal levels of apoptosis or on ATP and GSH. Apoptosis was induced by transforming growth factor β-1 (TGFβ-1) or glycochenodeoxycholate (GCDC). Both compounds caused moderate hepatocyte apoptosis; however, addition of L-NAME prior to exposure significantly increased the level of apoptosis observed with the two compounds. Both TGFβ-1 and GCDC had no effect on hepatocyte ATP or GSH levels; however, as a consequence of secondary necrosis, TGFβ-1 exposure significantly increased levels of lactate dehydrogenase (LDH) leakage. These findings indicate that the increased levels of NO associated with the culture of hepatocytes have an inhibitory effect on compound-induced apoptosis in the cells.     

Putative role of nitric oxide synthase isoforms in the changes of nitric oxide concentration in rat brain cortex and cerebellum following sevoflurane and isoflurane anaesthesia

We have previously observed an increase in nitric oxide (NO) content in rat brain cortex following halothane, sevoflurane or isoflurane anaesthesia. This study was undertaken in order to determine whether isoform-specific nitric oxide synthase (NOS) inhibitors and inducers could modify these increases in NO contents. Rats were subjected to isoflurane and sevoflurane anaesthesia with concomitant administration of neuronal nitric oxide synthase (nNOS) inhibitor 7-Nitro-indazole (7-NI), inducible nitric oxide synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) or lipopolysaccharide. NO concentration in different organs was measured by electron paramagnetic resonance (EPR) spectroscopy. 7-NI significantly decreased NO concentration in cerebellum but not in brain cortex, whereas AMT decreased NO in all the organs studied. Anaesthesia significantly increased NO concentration in brain cortex and decreased that in cerebellum. AMT abolished the NO increase in brain cortex. Anaesthesia enhanced the drastic increase in NO concentration in brain cortex after intraventricular lipopolysaccharide administration. Isoflurane was found to inhibit recombinant nNOS and iNOS activities at high concentrations (EC50=20 mM). Our data suggest a putative role for iNOS in the increase in NO levels produced by isoflurane and sevoflurane, whereas nNOS activity is probably inhibited during anaesthesia.     

Synthesis and biological evaluation of novel 5-benzylidenethiazolidine-2,4-dione derivatives for the treatment of inflammatory diseases

Twenty-two compounds based on thiazolidine-2,4-dione moiety were synthesized and evaluated for the inhibitory potency on the production of nitric oxide (NO), inducible nitric oxide synthase (iNOS) activity, and the generation of prostaglandin E(2) (PEG(2)). (Z)-N-(3-chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acetamide (3I), superior to the commercial anti-inflammatory drug indomethacin, significantly inhibited iNOS activity (IC(50) = 8.66 μM), iNOS-mediated NO, and cyclooxygenase (COX)-2-derived PGE(2) production (IC(50) = 4.16 and 23.55 μM, respectively) on lipopolysaccharide (LPS)-induced RAW 264.7 cells. Docking study revealed that 3I was perfectly docking into the active site of murine iNOS and suppressed the expression of iNOS protein as evidenced by Western blot analysis. At the dose of 50 mg/kg, oral administration of 3I possessed protective properties in both carrageenan-induced paw edema and adjuvant-induced arthritis rat models.     

Possible role of nitric oxide in 5-hydroxytryptamine-induced increase in vascular permeability in mouse skin

In order to test the hypothesis that a 5-hydroxytryptamine (5-HT)-induced increase in vascular permeability results from a cascade triggered by activation of the synthesis of nitric oxide (NO), the vascular permeability was investigated using the Pontamine sky blue leakage method in male mice. Subcutaneous injection of 5-HT induced a dose-related increase of vascular permeability at the injection site. The vascular permeability induced by 5-HT was inhibited by pretreatment with intraperitoneal injection of ketanserin (5-HT_2A antagonist) and methysergide (5-HT_1/2A antagonist), less efficiently by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine (NAN-190) (5-HT_1A antagonist), but not by granisetron (5-HT₃ antagonist). Increase in vascular permeability induced by 5-HT was inhibited by concurrent intravenous administration of NO synthase inhibitors N^G-nitro-Lrarginine methyl ester (L-NAME) and methylene blue but not by the inactive enantiomer N^G-nitro-D-arginine methyl ester (D-NAME). These results suggest that 5-HT increases vascular permeability by activating the 5-HT receptors and that endogenous NO is involved in this effect of 5-HT. Correspondence to: E. Fujii at the above address     

Antileishmanial activity of hydrolyzable tannins and their modulatory effects on nitric oxide and tumour necrosis factor-alpha release in macrophages in vitro.

A series of 27 hydrolyzable tannins and related compounds was tested for antiparasitic effects against both extracellular promastigote and intracellular amastigote Leishmania donovani organisms. In parallel, the compounds were evaluated for their immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and interferon (IFN)-like properties using several functional assays. Of the series of polyphenols tested, only gallic acid (54 microM NO) and its methyl ester (32 microM NO) induced murine macrophage-like RAW 264.7 cells to release NO in appreciable amounts (IFN-gamma/LPS 119 microM NO). The in vitro TNF-inducing potential of the polyphenols examined increased in the order of oligomeric ellagitannins (EC(50) > 25 microg/ml) < monomeric ellagitannins, gallotannins (EC(50) 8.5 to > 25 microg/ml) < C-glucosidic ellagitannins, dehydroellagitannins (EC(50) 0.6 - 2.8 microg/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, promastigotes of Leishmania donovani were assayed in the presence of these polyphenols and the results showed that none of the compounds was significantly toxic (EC(50) > 25 microg/ml) to the extracellular forms. In contrast, all polyphenols showed pronounced antileishmanial activities (EC(50) < 0.4 - 12.5 versus 7.9 microg/ml for Pentostam) against intracellular amastigotes of L. donovani residing within RAW cells. Noteworthy, most compounds exhibited low cytotoxicity against the murine host cells (EC(50) >25 microg/ml). Furthermore, some ellagitannins and the majority of dehydroellagitannins induced potent interferon-like activities as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L929 cells. This is the first report on hydrolyzable tannins as a new class of natural products with leishmanicidal activity including their potential for inducing the release of NO, TNF and IFN-like activity in macrophage-like RAW cells.     

Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects

Selected nucleophile/nitric oxide adducts [compounds which contain the anionic moiety, XN(O-)N = O] were studied for their ability to release nitric oxide spontaneously in aqueous solution and for possible vasoactivity. The diversity of structures chosen included those in which the nucleophile residue, X, was that of a secondary amine [Et2N, as in [Et2NN(N = O)O]Na, 1], a primary amine [iPrHN, as in [iPrHNN(N = O)O]Na, 2], a polyamine, spermine [as in the zwitterion H2N(CH2)3NH2+(CH2)4N[N(N = O)O-](CH2)3NH2, 3], oxide [as in Na[ON(N = O)O]Na, 4], and sulfite [as in NH4[O3SN(N = O)O]NH4, 5]. The rate constants (k) for decomposition in pH 7.4 phosphate buffer at 37 degrees C, as measured by following loss of chromophore at 230-260 nm, were as follows: 1, 5.4 x 10(-3) s-1; 2, 5.1 x 10(-3) s-1; 3, 0.30 x 10(-3) s-1; 4, 5.0 x 10(-3) s-1; and 5, 1.7 x 10(-3) s-1. The corresponding extents of nitric oxide release (ENO) were 1.5, 0.73, 1.9, 0.54, and 0.001 mol/mol of starting material consumed, respectively, as determined from the integrated chemiluminescence response. Vasodilatory activities expressed as the concentrations required to induce 50% relaxation in norepinephrine-constricted aortic rings bathed in pH 7.4 buffer at 37 degrees C (EC50) were as follows: 1, 0.19 microM; 2, 0.45 microM; 3, 6.2 microM; 4, 0.59 microM; and 5, 62 microM. Vasorelaxant potency (expressed as 1/EC50) was strongly correlated with the quantity of .NO calculated from the physicochemical data to be released in the interval required to achieve maximum relaxation at the EC50 doses (r = 0.995). This suggests that such nucleophile/.NO adducts might generally be useful as vehicles for the nonenzymatic generation of nitric oxide, in predictable amounts and at predictable rates, for biological purposes. The particular significance for possible drug design is underscored in the very favorable potency comparison between several of these agents and the established nitrovasodilators sodium nitroprusside and glyceryl trinitrate (EC50 values of 2.0 and greater than 10 microM, respectively) in parallel aortic ring tests.     

Effect of a selective inducible nitric oxide synthase inhibitor on intraocular nitric oxide production in endotoxin-induced uveitis rabbits: in vivo intraocular microdialysis study

Inducible nitric oxide (NO) synthase (iNOS) is believed to contribute to the pathogenesis of endotoxin-induced uveitis (EIU). In the present study, we investigated the inhibitory effects of N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, and S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBITU), a potent and selective iNOS inhibitor, on intraocular NO production in EIU rabbits using an in vivo intraocular microdialysis technique. The flare level in the anterior chamber increased from 1h after the injection of 100 micro g/kg lipopolysaccharide (LPS), and continued to increase for 24h. Aqueous humor protein concentrations were significantly increased at 24h after LPS-injection. These changes were significantly reduced by L-NAME (10mg/kg) and PBITU (1mg/kg), but not by D-NAME (10mg/kg). The increase in NO(2)(-) and NO(3)(-) levels in the dialysate induced by LPS was significantly inhibited by L-NAME (10mg/kg) and PBITU (1mg/kg), but not by D-NAME (10mg/kg). These results suggest that activation of iNOS may play a key role in the development of EIU, and selective inhibitors of iNOS may have therapeutic applications in the treatment of EIU.     

The effect of nitric oxide synthase blockade on responses to morphine in rat aortic rings

1 It has been suggested that opioids may play an indirect role in the regulation of the peripheral circulation through the control of nitric oxide (NO) release in vascular tissue. The current study was undertaken to investigate the effect of nitric oxide synthase (NOS) blockade on responses to morphine in phenylephrine (PE)- or KCl-precontracted rat aortic rings. 2 Morphine (3 x 10(-8) - 3 x 10(-5) M) administration did not cause any significant effect on basal tonus of endothelium-intact or endothelium-denuded preparations. Morphine produced concentration-dependent relaxation responses in endothelium-intact as well as in endothelium-denuded rat aortic rings precontracted by PE or KCl. Removal of endothelium did not significantly alter the relaxation responses to morphine. 3 The relaxant responses to morphine were significantly and partially inhibited by pretreatment of tissues with naloxone (NAL, 3 x 10(-5) M) for 5 min. The inhibitory effect of NAL on relaxant responses to morphine in PE- or KCl-precontracted rings did not differ significantly between endothelium-intact and endothelium-denuded preparations. 4 Incubation of endothelium-intact or endothelium-denuded rat aortic rings with NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) for 20 min did not cause a significant inhibition on relaxation responses to morphine. 5 These findings confirmed the presence of opiate receptors in rat thoracic aorta, but suggested that mechanisms other than NO release play a role in the relaxant effect of morphine on rat aortic rings.     

Attenuation of morphine tolerance and dependence by aminoguanidine in mice

The effect of aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, on morphine-induced tolerance and dependence in mice was investigated in this study. Acute administration of aminoguanidine (20 mg/kg, p.o.) did not affect the antinociceptive effect of morphine (10 mg/kg, s.c.) as measured by the hot plate test. Repeated administration of aminoguanidine along with morphine attenuated the development of tolerance to the antinociceptive effect of morphine. Also, the development of morphine dependence as assessed by naloxone-precipitated withdrawal manifestations was reduced by co-administration of aminoguanidine. The effect of aminoguanidine on naloxone-precipitated withdrawal was enhanced by concurrent administration of the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, dizocilpine (0.25 mg/kg, i.p.) or the non-specific nitric oxide synthase (NOS) inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME; 5 mg/kg, i.p.) and antagonized by concurrent administration of the nitric oxide (NO) precursor, l-arginine (50 mg/kg, p.o.). Concomitantly, the progressive increase in NO production, but not in brain glutamate level, induced by morphine was inhibited by repeated administration of aminoguanidine along with morphine. Similarly, co-administration of aminoguanidine inhibited naloxone-induced NO overproduction, but it did not inhibit naloxone-induced elevation of brain glutamate level in morphine-dependent mice. The effect of aminoguanidine on naloxone-induced NO overproduction was potentiated by concurrent administration of dizocilpine or l-NAME and antagonized by concurrent administration of l-arginine. These results provide evidence that blockade of NO overproduction, the consequence of NMDA receptor activation, by aminoguanidine, via inhibition of iNOS, can attenuate the development of morphine tolerance and dependence.     

Design and synthesis of novel NO‐donor‐ferulic acid hybrids as potential antiatherosclerotic drug candidatesa

Novel NO‐donor‐ferulic acid hybrids were designed and synthesized through a symbiotic approach using ferulic acid and three different NO‐donating groups, such as nitric ester, 4‐hydroxyl‐3‐phenylfuroxan, and 4‐hydroxymethyl‐3‐phenylsulfonylfuroxan. Antioxidant, nitric oxide release, and vasodilator properties studies showed that the target phenylsulfonylfuroxan 14, especially 14c, while keeping the antioxidant activity, showed more NO release activity and vasodilating activity than isosorbide dinitrate (ISDN). Thus, 14c may be considered a novel potent anti‐atherosclerosis drug candidate. Drug Dev Res 72: 405–415, 2011. © 2011 Wiley‐Liss, Inc.     

Perinatal lead exposure affects nitric oxide and cyclooxygenase pathways in aorta of weaned rats.

Perinatal Pb exposure may modulate arterial tone through nitric oxide (NO) and cyclooxygenase products. To investigate this, Wistar dams received 1000 ppm of Pb or sodium acetate (control) in drinking water during pregnancy and lactation. Curves were constructed in phenylephrine-precontracted intact and/or denuded rings of thoracic aortas of weaned (23-day-old) male pups from their responses to N(omega)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and ACh in the absence or presence of indomethacin (10(-5)M, cyclooxygenase inhibitor) or L-NAME (3 x 10(-7)M and 3 x 10(-4)M). Blood lead concentration and systolic blood pressure (SBP) were higher in intoxicated than control pups (blood lead microg/dl: control < 3.0, Pb 58.7 +/- 6.5*; SBP mmHg: control 111.4 +/- 2.3, Pb 135.5 +/- 2.4*). In L-NAME-treated rings maximal responses increased in Pb-exposed rats, and were higher in intact than in denuded aortas (contraction [% of phenylephrine] intact: control 184.3 +/- 23.7, Pb 289.1 +/- 18.3*; denuded: control 125.1 +/- 4.5, Pb 154.8 +/- 13.3*). ACh-induced relaxation in intact aortas from Pb-exposed rats presented rightward shift in L-NAME presence (EC50 x 10(-7)M: control 1.32 [0.33-5.18], Pb 4.88 [3.56-6.69]*) but moved left under indomethacin (EC50 x 10(-7)M: control 8.95 [3.47-23.07], Pb 0.97 [0.38-2.43]*). *p < 0.05 significant relative to the respective control; N = 7-9. Endothelium removal abolished ACh-induced relaxation. Perinatal Pb exposure caused hypertension associated with alterations in the production and/or release of basal and stimulated endothelium-derived relaxing factors-NO and constricting cyclooxygenase products. These findings may help explain the contribution of NO and cyclooxygenase products to the etiology and/or maintenance of Pb-induced hypertension and could ultimately lead to therapeutic advantages in plumbism.     

P物质对血管内皮细胞一氧化氮分泌及表达的影响

目的探讨P物质（SP）对血管内皮细胞一氧化氮（NO）分泌及表达的影响.方法采用不同浓度P物质（10-9mol/L、10-8mol/L、10-7mol/L和10-6mol/L）作用于人脐静脉血管内皮细胞（HUVEC）,分别于15 min、30 min、1 h、3 h、6 h和12 h检测培养上清液中NO含量,并应用免疫组化方法检测HUVEC内诱生型一氧化氮合成酶（iNOS）和内皮型一氧化氮合成酶（eNOS）的表达.结查10-9mol/L～10-6mol/L浓度范围的SP作用15 min,均可使HUVEC内eNOS表达增强,分泌NO水平上升,并于1 h后达峰值,与对照组比较具有显著性差异（P＜0.05）,其中10-8mol/LSP浓度组作用最为明显;而不同浓度的SP对HUVEC内iNOS的表达与对照组比较无显著性差异.结论SP可通过增强HUVEC内eNOS的表达来促进NO的分泌;SP对HUVEC内iNOS的表达则无明显影响.     

The role of the NO/NMDA pathways in the development of morphine withdrawal induced by naloxone in vitro.

The effect of nitric oxide (NO)/N-methyl-d-aspartate (NMDA) pathways on naloxone-induced withdrawal contracture was studied in vitro in a model of acute morphine dependence in the isolated guinea pig ileum. Exposure of the isolated guinea pig ileum to morphine (10(-5) M) for 5 min resulted in acute dependence, characterized by a strong withdrawal contracture induced by naloxone (5x10(-5) M). The NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 5x10(-4) M) as well as the soluble guanylate cyclase inhibitor methylene blue (MB; 10 microM) were found to significantly attenuate the naloxone-induced withdrawal contracture. In addition, the NO precursor L-arginine (5x10(-4) M) as well as the NO donors sodium nitroprusside (SNP; 1 microM) and sodium azide (NaZ; 10 microM) were able to revert the effect of L-NAME returning the amplitude of naloxone-induced contracture to the same level in control morphine-dependent ilea. We also demonstrated that the competitive NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP-5; 50 microM) potently reduced the amplitude of naloxone-induced contracture in the same model, an effect that was reversed by co-administration of the excitatory amino acid L-glutamate (40 microM). This in vitro study confirms the implication of the NO/NMDA pathways in morphine dependence.     

Evidence that A(2a) and not A(2b) purinoceptors are coupled to production of nitric oxide in the central regulation of blood pressure

In a previous study we reported that nitric oxide (NO) partially mediates centrally the decrease of arterial blood pressure induced by adenosine A₂ subtype receptor stimulation. The present study confirms the earlier suggestion and shows that in adult male normotensive anaesthetized rats 5′-N-ethylcarboxamidoadenosine (NECA), a non-selective adenosine receptor agonist, centrally injected induced a significant decrease of arterial blood pressure. Moreover, the observation that intracerebroventricular (i.c.v.) administration of N^ω-nitro- -arginine methyl ester ( -NAME), a NO synthase inhibitor, +8-(3-chlorostyryl)-caffeine (CSC), antagonist of A_2a receptors, did not reduce furthermore the hypotensive effect induced by NECA injection, demonstrated that NO is involved only via A_2a and not via A_2b adenosine subtype receptors in the central regulation of blood pressure.     

Vanadate-induced nitric oxide production: role in osteoblast growth and differentiation

Nitric oxide (NO) has been shown to act as a mediator of cytokines in bone tissue. We have previously demonstrated that vanadium compounds are insulin- and growth factor-mimetic compounds in osteoblasts in culture, although high doses are toxic to these cells. In this study, we measured NO production in two osteoblast-like cells (UMR106 and MC3T3E1) incubated with different concentrations (2.5–100 μM) of vanadate. Vanadate induced NO release in a biphasic manner, with levels being significantly increased at concentrations over 50 μM. The NO donor, sodium nitroprusside, mimicked the vanadate effect: it inhibited cell growth and alkaline phosphatase activity in a dose-dependent manner. Vanadate enhanced the NO synthases, the endothelial and inducible (eNOS and iNOS) isoforms, in a dose-dependent manner. Experiments performed with the ionophore A23187 and EGTA suggested that vanadate-induced NO production involves Ca²⁺-dependent and -independent mechanisms. Altogether, our results suggest that NO may play a critical role in the bioactivity of vanadium in osteoblast-like cells.