共查询到20条相似文献,搜索用时 15 毫秒
1.
P A Bush N E Gonzalez J M Griscavage L J Ignarro 《Biochemical and biophysical research communications》1992,185(3):960-966
This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from L-arginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NOx (NO+nitrite [NO2-] + nitrate [NO3-]) by chemiluminescence after reduction of NOx to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37 degrees C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 microM; 18 microM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum. 相似文献
2.
Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate 总被引:117,自引:0,他引:117
Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666]. 相似文献
3.
Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands 总被引:23,自引:0,他引:23
M Palacios R G Knowles R M Palmer S Moncada 《Biochemical and biophysical research communications》1989,165(2):802-809
The formation of nitric oxide (NO) by an L-arginine:NO synthase and its stimulation of the soluble guanylate cyclase was studied in rat whole adrenal and bovine cortex and medulla cytosol. In the presence of L-arginine, the stimulation of soluble guanylate cyclase was accompanied by the formation of citrulline and NO2-, formed from NO. The NO synthase was NADPH- and Ca(2+)-dependent and was inhibited by several L-arginine analogues. These results indicate that rat and bovine adrenal cytosol contains an L-arginine:NO synthase. 相似文献
4.
Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum. 总被引:5,自引:0,他引:5
P A Bush N E Gonzalez L J Ignarro 《Biochemical and biophysical research communications》1992,186(1):308-314
The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum. 相似文献
5.
M Takema K Inaba K Uno K Kakihara K Tawara S Muramatsu 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(6):1928-1933
It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells. 相似文献
6.
Müllner N Lázár A Hrabák A 《The international journal of biochemistry & cell biology》2002,34(9):1080-1090
Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells).We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important. 相似文献
7.
G W Cox G Melillo U Chattopadhyay D Mullet R H Fertel L Varesio 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(10):3290-3296
We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity. 相似文献
8.
The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO2; 30 microM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 microM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 microM) and N-methyl-N'-nitro-N-nitrosoguanidine (200 microM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis. 相似文献
9.
Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells 总被引:5,自引:0,他引:5
The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor. 相似文献
10.
Besides NO, neuronal NO synthase (nNOS) also produces superoxide (O(2)(-.) at low levels of L-arginine. Recently, heat shock protein 90 (hsp90) was shown to facilitate NO synthesis from eNOS and nNOS. However, the effect of hsp90 on the O(2)(-.) generation from NOS has not been determined yet. The interrelationship between its effects on O(2)(-.) and NO generation from NOS is also unclear. Therefore, we performed electron paramagnetic resonance measurements of O(2)(-.) generation from nNOS to study the effect of hsp90. Purified rat nNOS generated strong O(2)(-.) signals in the absence of L-arginine. In contrast to its effect on NO synthesis, hsp90 dose-dependently inhibited O(2)(-.) generation from nNOS with an IC(50) of 658 nM. This inhibition was not due to O(2)(-.) scavenging because hsp90 did not affect the O(2)(-.) generated by xanthine oxidase. At lower levels of L-arginine where marked O(2)(-.) generation occurred, hsp90 caused a more dramatic enhancement of NO synthesis from nNOS as compared to that under normal L-arginine. Significant O(2)(-.) production was detected from nNOS even at intracellular levels of L-arginine. Adding hsp90 prevented this O(2)(-.) production, leading to enhanced nNOS activity. Thus, these results demonstrated that hsp90 directly inhibited O(2)(-.) generation from nNOS. Inhibition of O(2)(-.) generation may be an important mechanism by which hsp90 enhances NO synthesis from NOS. 相似文献
11.
Constitutive and inducible nitric oxide synthases incorporate molecular oxygen into both nitric oxide and citrulline. 总被引:15,自引:0,他引:15
A M Leone R M Palmer R G Knowles P L Francis D S Ashton S Moncada 《The Journal of biological chemistry》1991,266(35):23790-23795
Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO. 相似文献
12.
Vicetti Miguel RD Cherpes TL Watson LJ McKenna KC 《Journal of immunology (Baltimore, Md. : 1950)》2010,185(11):6706-6718
To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment, tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors, whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms), suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However, CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically, the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer, tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly, in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion, the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress. 相似文献
13.
The macrophage response to bacteria: flow of L-arginine through the nitric oxide and urea pathways and induction of tumoricidal activity 总被引:3,自引:0,他引:3
R Keller R Keist S Klauser A Schweiger 《Biochemical and biophysical research communications》1991,177(2):821-827
The consequences of the interaction of heat-killed bacteria and lipopolysaccharide (LPS) with a pure population of bone marrow-derived mononuclear phagocytes (BMM?) were investigated, utilizing changes in the flow of L-arginine and expression of tumoricidal activity as parameters of macrophage (m?) function. Gram negative bacteria and LPS proved potent in inducing the flow of L-arginine through the nitric oxide and the urea pathways but were mostly poor in eliciting tumoricidal activity. Gram positive bacteria affected the metabolism of L-arginine only little but were often efficient in triggering tumoricidal activity. The findings show that the m? response to bacteria, which may determine the outcome of their interaction with the host, may differ considerably depending on the type of bacteria. 相似文献
14.
Stewart AG Dusting GJ Giarracca RG Harris T Lim Y Sobey CG 《Mediators of inflammation》1993,2(5):349-356
The generation of nitrite (NO(2) (-)) was used as an index of the production of nitric oxide by human and rat polymorphonuclear leukocytes (PMN) and rat peritoneal macrophages. Human peripheral blood PMN did not produce significant levels of NO(2) (-). Attempts to induce NO(2) (-) generation in human PMN by incubation with GM-CSF (1 nM), TNFalpha (0.3 nM), endotoxin (1 mug/ml) or formyl-Met-Leu-Phe (100 nM) for up to 16 h were not successful. Addition of human PMN primed by GM-CSF (1 nM) to rabbit aortic ring preparations precontracted with phenylephrine had no effect on tone. In contrast to these observations, PMN, isolated from the peritoneum of oyster glycogen treated rats, generated NO(2) (-) via a pathway sensitive to inhibition by the nitric oxide synthase inhibitor, N(G)-monomethyl L-arginine. However, peripheral blood rat PMN obtained from the same animals did not produce NO(2) (-), even during prolonged incubation for periods of up to 16 h. It is suggested that detectable NO production by PMN requires NO synthase activity to be induced either by the process of PMN migration or by exposure to certain cytokines produced locally at the site of inflammation. 相似文献
15.
Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells 总被引:12,自引:0,他引:12
M Lepoivre B Chenais A Yapo G Lemaire L Thelander J P Tenu 《The Journal of biological chemistry》1990,265(24):14143-14149
The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway. 相似文献
16.
17.
Ichiro Kikkawa Shinji Saito Kaoru Tominaga Yuichi Hoshino Yoshio Ooi Masayasu Nakano 《Microbiology and immunology》1998,42(9):591-598
Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), NG-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them. 相似文献
18.
Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia. 总被引:4,自引:0,他引:4
Dawn R S Steiner Norberto C Gonzalez John G Wood 《Journal of applied physiology》2002,93(4):1411-1418
Systemic hypoxia results in oxidative stress due to a change in the reactive oxygen species (ROS)-nitric oxide (NO) balance. These experiments explored two mechanisms for the altered ROS-NO balance: 1) decreased NO synthesis by NO synthase due to limited O(2) substrate availability and 2) increased superoxide generation. ROS levels and leukocyte adherence in mesenteric venules of rats during hypoxia were studied in the absence and presence of an NO donor [spermine NONOate (SNO)] and of the NO precursor L-arginine. We hypothesized that if the lower NO levels during hypoxia were due to O(2) substrate limitation, L-arginine would not prevent hypoxia-induced microvascular responses. Graded hypoxia (produced by breathing 15, 10, and 7.5% O(2)) increased both ROS (123 +/- 6, 148 +/- 11, and 167 +/- 3% of control) and leukocyte adherence. ROS levels during breathing of 10 and 7.5% O(2) were significantly attenuated by SNO (105 +/- 6 and 108 +/- 3%, respectively) and L-arginine (117 +/- 5 and 115 +/- 2%, respectively). Both interventions reduced leukocyte adherence by similar degrees. The fact that the effects of L-arginine were similar to those of SNO does not support the idea that NO generation is impaired in hypoxia and suggests that tissue NO levels are depleted by the increased ROS during hypoxia. 相似文献
19.
Cossenza M Cadilhe DV Coutinho RN Paes-de-Carvalho R 《Journal of neurochemistry》2006,97(5):1481-1493
The synthesis of nitric oxide (NO) is limited by the intracellular availability of L-arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular L-arginine which supports an increase in the production of NO. Although L-[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin-layer chromatography analysis showed that free intracellular L-[3H]arginine was six times higher in CHX-treated than in control cultures. Extracellular L-[3H]citrulline levels increased threefold in CHX-treated groups, an effect blocked by NG-nitro-L-arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular L-[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2-amino 5-phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40-50% in the incorporation of 35[S]methionine or L-[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular L-arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin-dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin-dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity. 相似文献
20.
Sonia Eligini Benedetta Porro Alessandro Lualdi Isabella Squellerio Fabrizio Veglia Elisa Chiorino Mauro Crisci Anna Garlaschè Marta Giovannardi Josè-Pablo Werba Elena Tremoli Viviana Cavalca 《PloS one》2013,8(8)