Tissue‐type plasminogen activator induces plasmin‐dependent proteolysis of intracellular neuronal nitric oxide synthase

Background information. Despite its pro‐fibrinolytic activity, tPA (tissue plasminogen activator) is a serine protease known to influence a number of physiological and pathological functions in the central nervous system. Accordingly, tPA was reported to mediate some of its functions in the central nervous system through NMDA (N‐methyl‐d ‐aspartate) receptors, LRP (low‐density lipoprotein receptor‐related protein) or annexin II. Results. We provide here both in vitro and in vivo evidence that tPA could mediate proteolysis and subsequent delocalization of neuronal nitric oxide synthase, thereby reducing endogenous neuronal nitric oxide release. We also demonstrate that although this effect is independent of NMDA receptors, LRP signalling and calpain‐mediated proteolysis, it is dependent on the ability of tPA to promote the conversion of plasminogen into plasmin. Conclusion. Altogether, these results demonstrate a new function for tPA in the central nervous system, which most likely contributes to its pleiotropic functions.     

Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures

Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro . The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Inhibition of neuronal nitric oxide synthase activity by N1-acetyl-5-methoxykynuramine, a brain metabolite of melatonin

We assessed the effects of melatonin, N(1)-acetyl-N (2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10(-11)-10(-3) m), but not AFMK, inhibited nNOS activity in vitro in a dose-response manner. The IC(50) value for AMK (70 microm) was significantly lower than for melatonin (>1 mm). A 20% nNOS inhibition was reached with either 10(-9) m melatonin or 10(-11) m AMK. AMK inhibits nNOS by a non-competitive mechanism through its binding to Ca(2+)-calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mm norharmane, an indoleamine-2,3-dioxygenase inhibitor. In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhibition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase

Hypoxia/ischaemia is known to trigger neuronal death, but the role of neuronal nitric oxide synthase (nNOS) in this process is controversial. Nitric oxide (NO) inhibits cytochrome oxidase in competition with oxygen. We tested whether NO derived from nNOS synergises with hypoxia to induce neuronal death by inhibiting mitochondrial cytochrome oxidase. Sixteen hours of hypoxia (2% oxygen) plus deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added glutamate or by added azide (an uncompetitive inhibitor of cytochrome oxidase), indicating that nNOS does not act downstream of glutamate or cytochrome oxidase. Hypoxia plus deoxyglucose-induced glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this. Hypoxia inhibited cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting cytochrome oxidase in competition with oxygen. These data indicate that hypoxia synergises with NO from nNOS to induce neuronal death via cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/stroke-induced neuronal death in vivo.     

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

A novel pathway for receptor‐mediated post‐translational activation of inducible nitric oxide synthase

Inducible nitric oxide synthase (iNOS) is a major source of nitric oxide during inflammation whose activity is thought to be controlled primarily at the expression level. The B1 kinin receptor (B1R) post‐translationally activates iNOS beyond its basal activity via extracellular signal regulated kinase (ERK)‐mediated phosphorylation of Ser⁷⁴⁵. Here we identified the signalling pathway causing iNOS activation in cytokine‐treated endothelial cells or HEK293 cells transfected with iNOS and B1R. To allow kinetic measurements of nitric oxide release, we used a sensitive porphyrinic microsensor (response time = 10 msec.; 1 nM detection limit). B1Rs signalled through Gαi coupling as ERK and iNOS activation were inhibited by pertussis toxin. Furthermore, transfection of constitutively active mutant Gαi Q204L but not Gαq Q209L resulted in high basal iNOS‐derived nitric oxide. G‐βγ subunits were also necessary as transfection with the β‐adrenergic receptor kinase C‐terminus inhibited the response. B1R‐dependent iNOS activation was also inhibited by Src family kinase inhibitor PP2 and trans‐fection with dominant negative Src. Other ERK‐MAP kinase members were involved as the response was inhibited by dominant negative H‐Ras, Raf kinase inhibitor, ERK activation inhibitor and MEK inhibitor PD98059. In contrast, PI3 kinase inhibitor LY94002, calcium chelator 1,2‐bis‐(o‐Aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid, tetraacetoxymethyl ester (BAPTA‐AM), protein kinase C inhibitor calphostin C and protein kinase C activator PMA had no effect. Angiotensin converting enzyme inhibitor enalaprilat also directly activated B1Rs to generate high output nitric oxide via the same pathway. These studies reveal a new mechanism for generating receptor‐regulated high output nitric oxide in inflamed endothelium that may play an important role in the development of vascular inflammation.     

Transient characteristics of universal cells on human‐induced pluripotent stem cells and their differentiated cells derived from foetal stem cells with mixed donor sources

IntroductionIt is important to prepare ‘hypoimmunogenic’ or ‘universal’ human pluripotent stem cells (hPSCs) with gene‐editing technology by knocking out or in immune‐related genes, because only a few hypoimmunogenic or universal hPSC lines would be sufficient to store for their off‐the‐shelf use. However, these hypoimmunogenic or universal hPSCs prepared previously were all genetically edited, which makes laborious processes to check and evaluate no abnormal gene editing of hPSCs.MethodsUniversal human‐induced pluripotent stem cells (hiPSCs) were generated without gene editing, which were reprogrammed from foetal stem cells (human amniotic fluid stem cells) with mixing 2‐5 allogenic donors but not with single donor. We evaluated human leucocyte antigen (HLA)‐expressing class Ia and class II of our hiPSCs and their differentiated cells into embryoid bodies, cardiomyocytes and mesenchymal stem cells. We further evaluated immunogenic response of transient universal hiPSCs with allogenic mononuclear cells from survival rate and cytokine production, which were generated by the cells due to immunogenic reactions.ResultsOur universal hiPSCs during passages 10‐25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21‐22 survived and continued beating even after treatment with allogenic mononuclear cells.     

Enhancement of nitric oxide production by association of nitric oxide synthase with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive dye

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.     

Chronic hyperammonemia reduces the activity of neuronal nitric oxide synthase in cerebellum by altering its localization and increasing its phosphorylation by calcium-calmodulin kinase II

Impaired function of the glutamate-nitric oxide-cGMP pathway contributes to cognitive impairment in hyperammonemia and hepatic encephalopathy. The mechanisms by which hyperammonemia impairs this pathway remain unclear. Understanding these mechanisms would allow designing clinical treatments for cognitive deficits in hepatic encephalopathy. The aims of this work were: (i) to assess whether chronic hyperammonemia in vivo alters basal activity of neuronal nitric oxide synthase (nNOS) in cerebellum and/or its activation in response to NMDA receptor activation and (ii) to analyse the molecular mechanisms by which hyperammonemia induces these alterations. It is shown that hyperammonemia reduces both basal activity of nNOS and its activation following NMDA receptor activation. Reduced basal activity is because of increased phosphorylation in Ser847 (by 69%) which reduces basal activity of nNOS by about 40%. Increased phosphorylation of nNOS in Ser847 is because of increased activity of calcium-calmodulin-dependent protein kinases (CaMKII) which in turn is because of increased phosphorylation at Thr286. Inhibiting CaMKII with KN-62 normalizes phosphorylation of Ser847 and basal NOS activity in hyperammonemic rats, returning to values similar to controls. Reduced activation of nNOS in response to NMDA receptor activation in hyperammonemia is because of altered subcellular localization of nNOS, with reduced amount in post-synaptic membranes and increased amount in the cytosol.     

Inhibition of neuronal nitric oxide synthase (n-cNOS) reverses the corticotrophin-induced behavioral effects in rats

The nitric oxide (NO) synthase inhibitor N^G-monomethyl-L-arginine (N-NMMA) and the competitive substrate for NO synthase L-arginine were used to determine the role of endogenous NO on the behavioral and neuroendocrine responsiveness following systemic corticotrophin in dexamethasone-suppressed rats. Corticotrophin (50-200 mU/kg, s.c.) dose-dependently decreased behavioral activity in the actimeter and produced significant anxiolytic and anti-risk activity in the plus-maze behavior test, without affecting systolic blood pressure. Rats given corticotrophin showed significant increased plasma corticosterone and reduced adrenal ascorbic acid level. These behavioral and adrenal responses of corticotrophin were dose dependently blocked by metyrapone (20 and 50 mg/kg, i.p.), an inhibitor of steroid 11-hydroxylase in adrenal and neural tissues that block steroidogenesis. Intracerebroventricular administration of L-NMMA (20 g/rat in 10 l) significantly prevented the behavioral hypoactivity and anxiolytic-like responses of corticotrophin without influencing the adrenal responsiveness. The effect of L-NMMA was completely reversed by preadministration of L-arginine (300 mg/kg, i.p.). These results suggest that neuronal nitric oxide pathway plays an important modulating role in the behavioral effects of corticotrophin by mechanisms other than those involving cardiovascular effects.     

Hypothermic storage of coronary endothelial cells reduces nitric oxide synthase activity and expression

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.     

一氧化氮促进体外培养大鼠脑室下区神经干细胞的分化

目的：探讨一氧化氮（NO）对新生大鼠体外培养的神经干细胞（NSCs）分化的作用。方法：采用常规方法分离新生大鼠脑室下区（SVZ）组织,进行NSCs体外培养。用DETA／NO作为NO供体,用L-NAME作为一氧化氮合酶（NOS）抑制剂。免疫荧光法检测NSCs标志物-巢蛋白（nestin）、神经元标志物-8Ⅲ型微管蛋白（Tuj-1）和星型胶质细胞标志物-胶质原纤维酸性蛋白（GFAP）的表达,还检测了神经元型NOS的表达。用Greiss还原法检测培养液中总NO的浓度。结果：培养的神经球均为nestin阳性、BIdu阳性和nNOS阳性。NSCs和40μmol／L、50μmol／L、60μmol／LDEFA／N0共培养5d,实验组培养液中N0浓度较对照组显著增高（P〈0．01）,相应实验组分化的神经元数和星型胶质细胞数较对照组明显增加（P〈0．01和P〈0．05）。NSCs和100μmol／L、150μmol／L、200μmol／LL-NAME共培养5d,实验组培养液中NO浓度较对照组降低（P〈0．05）,相应实验组分化的神经元数和星型胶质细胞数也较对照组减少（P〈0．05）。结论：NO能直接促进大鼠SVZ体外培养的NSCs分化。     

PIN-bodies: a new class of antibody-like proteins with CD4 specificity derived from the protein inhibitor of neuronal nitric oxide synthase

By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.