Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP).
2. Using 10 μM of each donor compound, mean±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min⁻¹ (for SNP) to 3.15±0.29 nmol min⁻¹ (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 10⁸ platelets (for SNP) to 2.67±0.31 pmol per 10⁸ platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC).
3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34).
4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8-bromo cyclic GMP).
5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu⁺) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO.
6. Cyclic GMP accumulation in response to GSNO (10⁻⁹–10⁻⁵M) was undetectable following treatment of platelets with ODQ (100 μM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu⁺ ions were required for the cyclic GMP-independent pathway to operate.
7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP.
8. The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (CMPS) produced a concentration-dependent inhibition of aggregation of ODQ-treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP-independent inhibition of platelet aggregation.
9. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP-dependent and -independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP-independent pathway may depend upon a cellular system which involves both copper (I) (Cu⁺) ions and surface membrane thiol groups. The potent anti-platelet action of GSNO results from its ability to exploit this cyclic GMP-independent mechanism.

     

Nicotine administration stimulates the in vivo N-methyl-D-aspartate receptor/nitric oxide/cyclic GMP pathway in rat hippocampus through glutamate release

1. The in vivo effects of nicotine on the nitric oxide (NO) synthase/cyclic GMP pathway of the adult rat hippocampus have been investigated by monitoring the levels of extracellular cyclic GMP during microdialysis in conscious unrestrained animals.
2. Intraperitoneal (i.p.) administration of nicotine caused elevation of cyclic GMP levels which was prevented by mecamylamine. The effect of nicotine was abolished by local infusion of the NO synthase inhibitor N^G-nitro-L-arginine (L-NOARG) or by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ).
3. Local administration of the NMDA receptor antagonists cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid ({"type":"entrez-protein","attrs":{"text":"CGS19755","term_id":"875773122","term_text":"CGS19755"}}CGS19755) and dizocilpine (MK-801) inhibited by about 60% the nicotine-induced elevation of cyclic GMP. Nicotine was able to stimulate cyclic GMP outflow also when administered directly into the hippocampus; the effect was sensitive to mecamylamine, L-NOARG, ODQ or MK-801.
4. Nicotine, either administered i.p. or infused locally, produced augmentation of glutamate and aspartate extracellular levels, whereas the outflows of γ-aminobutyric acid (GABA) and glycine remained unaffected. Following local administration of high concentrations of nicotine, animals displayed symptoms of mild excitation (sniffing, increased motor and exploratory activity) during the first 20–40 min of infusion, followed by wet dog shake episodes; these behavioural effects were prevented by mecamylamine or MK-801, but not by L-NOARG or by ODQ.
5. It is concluded that (a) nicotine stimulates the production of NO and cyclic GMP in the hippocampus; (b) this occurs, at least in part, through release of glutamate/aspartate and activation of NMDA receptors. Modulation of the NMDA receptor/NO synthase/cyclic GMP pathway may be involved in the cognitive activities of nicotine.

     

Effect of simvastatin on endothelium-dependent vaso-relaxation and endogenous nitric oxide synthase inhibitor

AIM: To investigate the effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthesis inhibitor asymmetric dimethylarginine (ADMA) in rats and cultured ECV304 cells. METHODS: Endothe-lial injury was induced by a single injection of low density lipoprotein (LDL) (4 mg/kg, 48 h) in rats or incubation with LDL (300 mg/L) or oxidative-modified LDL (100 mg/L) in cultured ECV304 cells, and vasodilator responses to acetylcholine (ACh) in the aortic rings and the level of ADMA, nitrite/nitrate (NO) and tumor necrosis factor-alpha (TNF-α) in the serum or cultured medium were determined. And the adhesion of the monocytes to endothe-lial cells and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the cultured ECV304 cells were measured. RESULTS: A single injection of LDL decreased endothelium-dependent relaxation to ACh, markedly increased the serum level of endogenous ADMA and TNF-α, and reduced serum level of NO. Pretreatment with simvastatin (30 or 60 mg/kg) markedly attenuated inhibition of vasodilator responses to ACh, the increased level of TNF-α and the decreased level of NO by LDL, but no effect on serum concentration of endogenous ADMA. In cultured ECV304 cells, LDL or ox-LDL markedly increased the level of ADMA and TNF-α and potentiated the adhesion of monocytes to endothelial cells, concomitantly with a significantly decrease in the activity of DDAH and serum level of NO. Pretreatment with simvastatin (0.1, 0.5, or 2.5 μmol/L) markedly decreased the level of TNF-α and the adhesion of monocytes to endothelial cells, but did not affect the concentration of endogenous ADMA and the activity of DDAH. CONCLUSION: Simvastatin protect the vascular endothelium against the damages induced by LDL or ox-LDL in rats or cultured ECV304 cells, and the beneficial effects of simvastatin may be related to the reduction of inflammatory cytokine TNF-α level.     

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca²⁺]_i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10⁻⁷ M), NO (10⁻¹⁰–10⁻⁶ M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca²⁺]_i and cation influx as indicated by fura-2 fluorimetry.
2. Zaprinast (10⁻⁴ M), increased NO-stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca²⁺]_i reached after administration of NO, the initial decreases in [Ca²⁺]_i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast.
3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10⁻⁵ M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10⁻¹⁰–10⁻⁸ M, but the decrease in [Ca²⁺]_i or cation influx caused by higher concentrations of NO (10⁻⁷–10⁻⁶ M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10⁻⁷–10⁻⁵ M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO.
4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca²⁺]_i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca²⁺]_i and smooth muscle relaxation to NO.

     

Regional distribution of cyclic GMP and its metabolizing enzymes within the hippocampal formation of the guinea-pig

Using quantitative histochemical methods the distribution of cyclic GMP, guanylate cyclase and cyclic GMP phosphodiesterase among regions of the guinea-pig hippocampal formation was examined. The four regions examined were the dentate gyrus, hippocampus, subiculum and entorhinal cortex. The guanylate cyclase activity was lowest in the hippocampus and only half that of the other regions which had similar activity. In contrast, cyclic GMP phosphodiesterase activity varied little among the four regions. Basal cyclic GMP content was similar in hippocampus, subiculum and entorhinal cortex; however, the dentate gyrus had nearly twice this basal level. Seizures produced by pentylenetetrazol treatment caused a variable (4–7-fold) increase in levels of cyclic GMP in all the regions. The hippocampus had the lowest level of cyclic GMP during seizure activity. This may be the result of the lower guanylate cyclase in the hippocampus as compared to the other regions studied.     

Endogenous nitric oxide generation linked to ryanodine receptors activates cyclic GMP / protein kinase G pathway for cell proliferation of neural stem/progenitor cells derived from embryonic hippocampus

Nitric oxide (NO) activates the cyclic GMP (cGMP) / protein kinase G (PKG) pathway during physiological processes in numerous types of cells. Here, we evaluated whether this NO/cGMP/PKG pathway is involved in the proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampus of embryonic mice. In culture, the exposure to the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) significantly decreased the number of viable cells and 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cells, as well as the levels of intracellular reactive oxygen species, extracellular NO(2), and intracellular cGMP. Like L-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and PKG inhibitor KT5823 also decreased cell viability and BrdU incorporation. The membrane-permeable cGMP analogue 8-bromo-cGMP partially abolished the L-NAME-induced decrease in the BrdU incorporation. BrdU incorporation was decreased by Ca(2+)-channel blockers, including dantrolene, MK-801, ifenprodil, and nifedipine. Interestingly, the NO(2) level was decreased by dantrolene, but not by the other 3 blockers. L-NAME and ODQ attenuated phosphorylation of Akt, but not that of extracellular signal-regulated kinases or epidermal growth factor receptors. Our data suggest that endogenous NO generation linked to dantrolene-sensitive ryanodine receptors activates the cGMP/PKG signaling pathway for positive regulation of proliferation of hippocampal NPCs derived from embryonic mice.     

戊四唑癫痫大鼠脑组织一氧化氮含量和一氧化氮合酶活性变化

探讨一氧化氮在戊四唑癫病发机制中的作用。方法每天注射戊四唑建立在鼠癫痫模型,测定癫病发作后大鼠大脑皮质,海马一氧化氮和一氧化氮合酶活性变化,结果癫痫发作后海马ＮＯ含量和ＮＯＳ活性显著升高,结 戊四唑诱导的癫痫中具有致痫性。     

神经元型一氧化氮合酶在心脏功能调节中的作用

神经元型一氧化氮合酶(nNOS)不仅在神经细胞中表达,也存在于心肌细胞。生理状况下,nNOS源性的一氧化氮(NO)通过自分泌以负反馈的调节方式维持细胞内Ca2+浓度稳态,防止细胞内Ca2+浓度超载,对于维持正常的心功能具有重要意义。病理状况下,nNOS的活性发生明显变化,nNOS源性的NO可能参与了心脏疾病的发生和发展。     

Modification of cholinergically induced convulsive activity and cyclic GMP levels in the CNS

Soman, an organophosphorus cholinesterase inhibitor, was used to produce cholinergic stimulation and convulsions in rats. Soman (1 or 2 × LD₅₀) elevated cyclic 3′5′ guanosine monophosphate (cGMP) levels in the cerebellum, before the onset of cholinergic symptoms, during the stage prior to convulsions, at doses which produced no convulsive activity (1/2LD₅₀) and greatly elevated cGMP levels at the onset and during convulsions. Pretreatment which reduced the convulsive activity of soman also attenuated the increase in cGMP in the cerebellum.The results indicated that soman-induced convulsions and the resulting increase in cerebellar cGMP concentrations could not be related solely to increased cholinergic activity or to muscarinic effects. Pretreatment of poisoned animals with antimusarinic or antinicotinic compounds or atropine plus an oxime (HI-6) which protected animals from the lethal effects of soman, had little effect on severity of convulsions or cGMP levels. On the other hand, both parameters were sensitive to anticonvulsants (i.e. clonazepam) which, are not thought to produce their effects via anticholinergic actions. In addition, intracerebroventricular injections of dibutyryl cGMP (dbcGMP) caused convulsions which were blocked by small doses of clonazepam but were only slightly affected by atropine or mecamylamine, given alone.Stimulation of climbing fibres to the cerebellum may result in the release of an excitatory amino acid which may subsequently cause elevation of cGMP. However, soman failed to alter cerebellar concentrations of putative excitatory or inhibitory neurotransmitters while destruction of these fibres with 3-acetylpyridine prior to injection of soman failed to block either the convulsions or the increase in cGMP concentrations.These results suggest that cGMP may play a role in the initiation and continuation of soman-induced convulsions which were not related to specific cholinergic receptors but to general neuronal excitation. The results also indicate the involvement of non-cholinergic mechanisms in soman-induced convulsions, and in the rise in cerebellar cGMP levels; however, the identity of these mechanisms was not known.     

Effects of statins on nitric oxide/cGMP signaling in human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVECs) were established as in vitro models for the modulation of endothelial function and cell viability by statins. Emphasis was placed on the biphasic effects of the drugs on nitric oxide (NO) bioavailability and cytotoxicity, as well as drug interference with the interaction of endothelial NO synthase (eNOS) with caveolin-1 (Cav-1). Incubation of HUVECs with fluvastatin, lovastatin or cerivastatin for 24 h caused an approximately 3-fold upregulation of eNOS expression that was associated with increased eNOS activity and accumulation of cGMP. Cerivastatin exhibited the highest potency with an EC50 of 13.8 ± 2nM after 24 h, while having no effect after only 30 min. The effects of statins on eNOS expression were similar in control and Cav-1 knockdown cells, but the increase in eNOS activity was less pronounced in Cav-1-deficient cells. Statin-triggered cyto-toxicity occurred at ~10-fold higher drug concentrations (maximal toxicity at 1–10 µM), was sensitive to mevalonate, and was significantly enhanced in the presence of N^G-nitro-L-arginine. The overexpression of eNOS induced by clinically relevant concentrations of statins may contribute to the beneficial vascular effects of the drugs in patients. Stimulation of NO synthesis and cytotoxicity appear to share a common initial mechanism but involve distinct downstream signaling cascades that exhibit differential sensitivity to HMG-CoA reductase inhibition.     

The potential of stimulating nitric oxide formation in the treatment of hypertension

Introduction: Hypertension is a leading cause of morbidity and mortality worldwide. A major pathophysiological factor contributing to hypertension is reduced nitric oxide (NO) bioavailability. Strategies to address this pathophysiological mechanism could offer significant advantages.

Areas covered: In this review we aimed at examining a variety of drugs (statins, beta-adrenergic receptor blockers, calcium channel blockers, angiotensin converting enzyme inhibitors, angiotensin II type-1 receptor blockers) used to treat hypertension and other cardiovascular diseases, particularly with respect to their potential of increasing NO bioavailability and activity in the cardiovascular system. There is now evidence supporting the notion that many cardiovascular drugs activate NO signaling or enhance NO bioavailability as a contributing mechanism to their beneficial cardiovascular effects. Moreover, other drugs may attenuate NO inactivation by superoxide and other reactive oxygen species by exerting antioxidant effects. More recently, the NO oxidation products nitrite and nitrate have been acknowledged as sources of NO after recycling back to NO. Activation of the nitrate-nitrite-NO pathway is an alternate pathway that may generate NO from both anions and exert antihypertensive effects.

Expert opinion: In this review, we provide an overview of the possible mechanisms by which these drugs enhance NO bioavailability and help in the therapy of hypertension.     

Inhibition of nitric oxide formation and superoxide generation during reduction of LY83583 by neuronal nitric oxide synthase

6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000×g rat cerebellar supernatant preparation in a concentration-dependent manner (IC₅₀ value=12.9 μM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a K_i value of 2.57 μM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl₂/calmodulin, indicating that the presence of CaCl₂/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl₂/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.     

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.     

Effect of simvastatin on nitric oxide synthases (eNOS,iNOS) and arginine and its derivatives (ADMA,SDMA) in ischemia/reperfusion injury in rat liver

Hydroxymethylglutaryl-CoA reductase inhibitors play a role in nitric oxide synthesis. In this study, the impact of simvastatin (SV) on the levels of nitric oxide synthases, and arginine (Arg) and its derivatives was evaluated in rat liver under ischemia-reperfusion (I/R) conditions. Rats received SV (25 mg/kg) (groups S and S-IR) or saline solution (groups C and C-IR) intragastrically for 21 days. The livers of groups C and S were homogenized after treatment while those of groups C-IR and S-IR underwent ischemia and reperfusion before homogenization. Endothelial (eNOS) and inducible (iNOS) nitric oxide synthase concentrations were determined in the homogenates. Alanine and asparagine aminotransferase (ALT, AST, respectively), arginine (Arg), and asymmetric (ADMA) and symmetric (SDMA) methylarginine levels were determined in the blood before I/R and during reperfusion. I/R injury produced significant increases in aminotransferase, ADMA, eNOS, and iNOS, but decreases in Arg and Arg/ADMA levels. Arg concentration increased significantly after warm ischemia in the S-IR group, but decreased significantly during the first 30 minutes of reperfusion in both the S-IR and C-IR groups. eNOS concentration was significantly higher in group S than in group C. Both I/R and SV exerted no influence on SDMA concentration. SV exerted a protective action by increasing eNOS levels under normal conditions and Arg levels after ischemia and by preventing a significant increase in iNOS concentration after I/R. SV had no effect on ADMA concentration under normal and pathological conditions.     

Relationship between myoglobin contents and increases in cyclic GMP produced by glyceryl trinitrate and nitric oxide in rabbit aorta,right atrium and papillary muscle

Summary Effects of glyceryl trinitrate (GTN) and nitric oxide (NO) on the cardiac functions and myocardial cyclic GMP (cGMP) contents were examined in comparison with those in the aorta and correlated with myoglobin (an inhibitor of soluble guanylate cyclase) contents using the preparations isolated from the reserpinized rabbit.GTN (10^–10-10^–4mol/l) produced a dose-dependent relaxation in the aorta. However, this compound exerted no effect on the rate of the spontaneous beat of the right atrium and the contraction of the papillary muscle. A transient and significant increase in cGMP was observed in the aorta with GTN (3 × 10^–6 mol/l). Although the increase was also observed in the right atrium, it was much smaller. No definite change was observed in papillary muscle. Increases in cGMP produced by NO (3 × 10^–6 mol/l) were larger and significant in all tissues; (AUC_cGMP(GTN)/AUC_cGMP(NO)) ratio was 30.1 for the aorta, 65.0 for the right atrium and 16.3% for the papillary muscle. Although higher concentrations of NO were necessary in the right atrium and papillary muscle to induce increases in cGMP, no differences were noted in the three tissues as regards the maximum accumulation of this substance. Furthermore, kinetic analysis of NO-induced increases in tissue cGMP indicated no marked difference in the production rate among the three tissues, while the rate of elimination of cGMP was lower in the aorta than in the atrium or the papillary muscle. The increases in cGMP observed in these three tissues were inversely related to the contents of myoglobin in respective tissues. No effect on myocardial function was observed with NO up to the concentration of 3 × 10^–5 mol/l.These results suggest that myoglobin, an endogenous inhibitor of activation of soluble guanylate cyclase by NO, was responsible for the lower production of cGMP by NO and GTN in the myocardial tissue. Correspondence to T. Ishibashi at the above address     

Dinoprostone potentiates cytokines and lipopolysaccharides inducing nitric oxide production in cultured rat hepatocytes 1

目的：探讨地诺前列酮（Ｄｉｎ）对细胞因子（ＩＬ１β,ＴＮＦα,ＩＦＮγ）及脂多糖（ＬＰＳ）诱导的原代培养大鼠肝细胞一氧化氮（ＮＯ）生成的影响,以及环腺苷一磷酸（ｃＡＭＰ）信号通路在诱导过程中的作用．方法：将吲哚美辛（Ｉｎｄ）,Ｄｉｎ,弗司扣林（Ｆｏｒ）及双丁酰环鸟苷一磷酸（ｄｂｃＧＭＰ）分别加入含细胞因子ＴＮＦα,ＩＬ１β,ＩＦＮγ及ＬＰＳ的培养液中,原代培养肝细胞２４ｈ后,采用Ｇｒｉｅｓ反应法测定细胞培养上清中ＮＯ含量,放射免疫法测定细胞内ｃＡＭＰ水平．结果：Ｉｎｄ明显抑制ＬＰＳ与细胞因子刺激所致ＮＯ生成,反之,Ｄｉｎ及Ｆｏｒ促进ＮＯ生成;在同样刺激条件下,Ｄｉｎ与Ｆｏｒ使细胞内ｃＡＭＰ水平增高,而在对照组细胞未观察到上述反应．ｄｂｃＧＭＰ则无论炎性刺激存在与否均无此作用．结论：Ｄｉｎ协同细胞因子及ＬＰＳ诱导肝细胞ＮＯ生成,细胞内第二信使ｃＡＭＰ通路参与此诱导过程．     

Platelet aggregation responses are critically regulated in vivo by endogenous nitric oxide but not by endothelial nitric oxide synthase

Background and purpose:

Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.

Experimental approach:

We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.

Key results:

Acute NOS inhibition by N_ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3^−/− mice but responses in NOS-3^−/− mice were enhanced by L-NAME.

Conclusions and implications:

Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3.     

Rewarding properties of sildenafil citrate in mice: role of the nitric oxide-cyclic GMP pathway

Rationale Sildenafil citrate is widely prescribed for erectile dysfunction and acts by inhibiting phosphodiesterase type-5, resulting in accumulation of cyclic-guanosine monophosphate (cGMP) via activation of nitric oxide synthase (NOS). The nitric oxide (NO) system is relevant to the rewarding effects of various drugs of abuse. Several epidemiologic studies indicate that sildenafil is abused in a recreational fashion. Objectives In the present study, the rewarding properties of sildenafil and probable involvement of the NO-cGMP pathway were investigated in adult male NMRI mice. Methods The ability of sildenafil citrate (1–40 mg/kg) to produce conditioned place preference (CPP) was studied in an unbiased CPP paradigm. The effects of NO precursor l-arginine, nonselective NOS inhibitor N ^G-nitro-l-arginine methyl ester (l-NAME), and the inhibitor of guanylyl cyclase methylene blue (MB) on sildenafil-induced CPP were assessed. Results Mice that received sildenafil (20 and 40 mg/kg) in one environment during conditioning phase displayed a preference for this environment. Both l-NAME (5 mg/kg) and MB (1 mg/kg) in combination with sildenafil (20 mg/kg) suppressed the acquisition of sildenafil-induced place preference. Lower and per se noneffective dose of sildenafil (10 mg/kg) and l-arginine (60 mg/kg), when coadministered, exerted a significant place conditioning. Conclusions Sildenafil shows rewarding properties that may involve the NO-cGMP pathway.     

The kappa-opioid agonist (+/-)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway

In view of the scarce information about the analgesic mechanism of kappa-opioid receptor agonists, the objective of the present study was to determine whether nitric oxide (NO) is involved in the peripheral antinociception of bremazocine, a kappa-opioid receptor agonist. Three drugs all interfering with the L-arginine/NO/cyclic GMP pathway were tested using the rat paw model of carrageenan-induced (250 microg) hyperalgesia: (a) N(G)-nitro-L-arginine (a nonselective NO-synthase inhibitor), (b) methylene blue (a guanylate cyclase inhibitor), and (c) zaprinast (a cyclic GMP phosphodiesterase inhibitor). Intraplantar administration of bremazocine (20, 40 and 50 microg) caused a dose-dependent peripheral antihyperalgesia against carrageenan-induced hyperalgesia. The possibility of the higher dose of bremazocine (50 microg) having central or systemic effect was excluded since administration of the drug into the left paw did not elicit antinociception in the contralateral paw. However, when the dose of bremazocine was increased to 100 microg, a significant increase in the nociceptive threshold was observed, as measured in the hyperalgesic contralateral paw. Peripheral antihyperalgesia induced by bremazocine (50 microg) was significantly reduced in a dose-dependent manner when N(G)-nitro-L-arginine (6, 9, 12 and 25 microg) or methylene blue (250, 375 and 500 microg) was injected before. Previous treatment with 50 microg of zaprinast (which had no effect when administered alone) potentiated the antihyperalgesic effect of bremazocine (20 microg).Our data suggest that bremazocine elicits peripheral antinociception by activation of the L-arginine/NO/cyclic GMP pathway and that nitric oxide is an intermediary in this mechanism, forming cyclic GMP.     

香菇多糖对巨噬细胞一氧化氮和一氧化氮合酶活性的影响

目的研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性,探讨LTN的免疫调节作用机理.方法采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性.观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响.结果LTN能使小鼠腹腔巨噬细胞NO生成增加,iNOS活性增高,并呈作用剂量依赖关系.3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞N0的生成和iNOS活性.结论LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成.提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关.