Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.     

PDT诱导的凋亡细胞对巨噬细胞NO合成的影响

NO作为细胞间信息传递的重要调节因子,在肿瘤的发生、发展以及转移过程中被广泛研究。一氧化氮合酶是合成NO的关键酶,诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)通常在应激、荷瘤等病理状态下被激活,产生大量NO。NO具有细胞毒性,与机体免疫反应及细胞凋亡有关,在许多致癌和抑癌机制中扮演着重要角色。实验探讨了光动力学疗法(photodynamic therapy,PDT)处理产生的小鼠乳腺癌凋亡细胞对巨噬细胞产生NO的影响,从而确定活化的巨噬细胞在肿瘤生长中的作用。     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin.

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.     

Effect of N-acetylaspartylglutamate (NAAG) on non-quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non-quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-beta-aspartylglutamate (betaNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect. Inhibition of NMDA receptors by dl-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N(G)-nitro-l-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.     

Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.     

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-l-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca²⁺-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

Involvement of nitric oxide and potassium channels in the bradykinin-induced vasodilatation in the rat kidney perfused ex situ

The role of nitric oxide (NO), K(+) channels, and arachidonic acid metabolism, via cytochrome P450 and cyclooxygenase pathways, in the renal vasodilatory effect of bradykinin was examined in the isolated rat kidney perfused ex situ with a blood-free solution. Bradykinin (BK, 0.25-1.0 microM) induced a dose-dependent reduction of 10-35% in the relative renal vascular resistance (rRVR) of isolated kidneys preconstricted with phenylephrine (PHE, 0.17-0.35 microM). The vasodilating effect of 0.5 microM bradykinin was significantly inhibited by the nitric oxide synthase inhibitors, N(G)-nitro-L-arginine (95% inhibition) and N(G)-nitro-L-arginine methyl ester (45-75% inhibition). Clotrimazole, an inhibitor of cytochrome P450 pathway but not indomethacin, a cyclooxygenase inhibitor, reduced the renal vasodilator response to bradykinin by 84%. The nonspecific K(+) channel inhibitor, tetraethylammonium ion (TEA) and the selective inhibitor of Ca(2+)-activated K(+) channels, charybdotoxin (ChTX) greatly attenuated the vasodilator response to bradykinin by approximately 84% and 79%, respectively. These two K(+) channel inhibitors showed similar effects on vasodilatation induced by S-nitroso-acetyl-D,L-penicillamine (1 microM), a nitric oxide donor. The results suggest that bradykinin releases nitric oxide which, by opening potassium channels specifically the Ca(+)-dependent type, mediates the renal vasodilator response to bradykinin in the isolated kidney perfused ex situ.     

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

The distribution of putative nitric oxide releasing neurones in the locust abdominal nervous system: a comparison of NADPHd histochemistry and NOS-immunocytochemistry

Nitric oxide is well established as a signalling molecule in the nervous system of vertebrates and invertebrates. In this study we evaluate the usefulness of NADPHdiaphorase histochemistry and immunocytochemistry for detecting the presence of nitric oxide synthase in locusts. We describe the distribution of putative nitric oxide releasing neurones and stained neuropiles in the locust ventral nerve cord, in particular the abdominal ganglia and abdominal neuromeres. NADPHdiaphorase histochemistry revealed prominent staining in all neuropilar regions and a specific distribution pattern of stained cell bodies in all examined ganglia. Nitric oxide synthase immunocytochemistry, using a commercially available universal antibody, labelled cells in corresponding positions within the ganglia. This was confirmed by double labelling of alternate sections. Western blot analysis demonstrated that in locusts this universal NOS-antibody binds to a protein of similar size to nitric oxide synthase identified in other insect species. The antibody also labelled axons in most peripheral nerves of all examined ganglia, whereas NADPHdiaphorase histochemistry only revealed such stained fibres within peripheral nerves in some preparations, because they may have been masked by intense background staining. We therefore conclude that nitric oxide synthase-immunocytochemistry and NADPHd histochemistry are both good markers for the presence of nitric oxide synthase in the locust ventral nerve cord, and that nitric oxide may be used as a signalling molecule by efferent neurones in locusts.     

Nitric oxide underlies the differentiation of PC12 cells induced by depolarization with high KCl

Nitric oxide (NO) acts as a cytostatic agent to induce neuronal differentiation of PC12 cells after nerve growth factor (NGF) treatment. We newly subcloned PC12K cells that extended neurites after depolarization with high KCl. Here we present evidence that the neuronal differentiation of PC12K cells caused by depolarization with high KCl is mediated by endogenous NO. The outgrowth of neurites was significantly inhibited by 2 mM N-nitro-L-arginine methyl ester (L-NMAE), and 10 mM L-NAME was necessary for complete inhibition. The inhibition of NGF-dependent neurite outgrowth by L-NAME was abolished by depolarization of cells with KCl. The expression of neuronal- and endothelial-NO-synthase in PC12K cells was confirmed by immuno-cytochemical and immuno-blotting analyses with the respective monoclonal antibodies. However, the expression of inducible-NO synthase was not observed in PC12K cells cultured with high KCl under the depolarization conditions with 45 mM KCl. We observed the increase of NO in the differentiated PC12K cells using diaminofluorescein, a novel fluorescent indicator for NO.     

Apoptotic Pathways Mobilized in Microglia and Neurones as a Consequence of Chromogranin A-Induced Microglial Activation

Senile plaques of Alzheimer's brain are characterized by activated microglia and immunoreactivity for the peptide chromogranin A. We have investigated the mechanisms by which chromogranin A activates microglia, producing modulators of neuronal survival. Primary cultures of rat brain-derived microglia display a reactive phenotype within 24 h of exposure to 10 nM chromogranin A, culminating in microglial death via apoptotic mechanisms mediated by interleukin-1beta converting enzyme. The signalling cascade initiated by chromogranin A triggers nitric oxide production followed by enhanced microglial glutamate release, inhibition of which prevents microglial death. The plasma membrane carrier inhibitor aminoadipate and the type II/III metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin-sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase-3-dependent neuronal apoptosis. Brain-derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved; nitric oxide synthase inhibitors are ineffective.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.     

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2ú-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2ú- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.     

Influence of Nitric Oxide Synthase Inhibitors on Embryo Destruction and Morphological Features in Pre- and Postimplantation Mouse Embryos

Nitric oxide is an important intraovarian regulatory factor. The periimplantation period is a critical phase in mouse development. Although it was shown that nitric oxide plays an essential role during gestation, its role in the preimplantation period is not yet fully clear. We studied the involvement of nitric oxide in developmental competence (embryonic defects and morphology of pre- and postimplantation embryos) using nitric oxide synthase inhibitors, which suppress all forms of nitric oxide synthase, and female mice, to which the inhibitors had been administered before their mating with intact males. The level of mortality of pre- and postimplantation embryos in females mated to intact males increased soon after the administration of inhibitors. Studies of the morphology of embryos have shown that there was a delay in embryogenesis at the stages of cleavage and gastrulation. The results obtained suggest that nitric oxide is a potent regulator of embryonic differentiation, specifically in pre- and postimplantation mouse embryos.     

Influence of nitric oxide synthase inhibitors on embryonic destructions and morphological features in pre- and postimplantation mouse embryos

Nitric oxide is an important intraovarian regulatory factor. The periimplantation period is a critical phase in mouse development. Although it was shown that nitric oxide plays an essential role during gestation, its role in the preimplantation period is not yet fully clear. We studied the involvement of nitric oxide in developmental competence (embryonic defects and morphology of pre- and postimplantation embryos) using nitric oxide synthase inhibitors, which suppress all forms of nitric oxide synthase, and female mice, to which the inhibitors had been administered before their mating with intact males. The level of mortality of pre- and postimplantation embryos in females mated to intact males increased soon after the administration of inhibitors. Studies of the morphology of embryos have shown that there was a delay in embryogenesis at the stages of cleavage and gastrulation. The results obtained suggest that nitric oxide is a potent regulator of embryonic differentiation, specifically in pre- and postimplantation mouse embryos.     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.