cyclic-gmp and Nerve-Degeneration

It has been reported that N-methyl-D-aspartate receptor (NMDAR)-triggered neurotoxicity is related to excessive Ca(2+) loading and an increase in nitric oxide (NO) concentration. However, the molecular mechanisms that underlie these events are not completely understood. NMDARs and neuronal NO synthase each binds to the scaffolding protein postsynaptic density (PSD)-93 through its PDZ domains. In this study, we determined whether PSD-93 plays a critical role in NMDAR/Ca(2+)/NO-mediated neurotoxicity. We found that the targeted disruption of the PSD-93 gene attenuated the neurotoxicity triggered by NMDAR activation, but not by non-NMDAR activation, in cultured mouse cortical neurons. PSD-93 deficiency reduced the amount of NMDAR subunits NR2A and NR2B in synaptosomal fractions from the cortical neurons and significantly prevented NMDA-stimulated increases in cyclic guanosine 3',5'-monophosphate and Ca(2+) loading in the cortical neurons. These findings indicate that PSD-93 deficiency could block NMDAR-triggered neurotoxicity by disrupting the NMDAR-Ca(2+)-NO signaling pathway and reducing expression of synaptic NR2A and NR2B. Since NMDARs, Ca(2+), and NO play a critical role during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders.

Topics: Animals; Brain Diseases, Metabolic; Calcium Signaling; Cells, Cultured; Cyclic GMP; Excitatory Amino Acid Agonists; Gene Targeting; Guanylate Kinases; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neurotoxins; Nitric Oxide; Presynaptic Terminals; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Synaptic Transmission

Excessive production of nitric oxide (NO) by microglia is at least in part responsible for the pathogenesis of various neurodegenerative disorders including Parkinson disease, but at the same time NO may also play a distinct role as a signaling molecule such as an activator of soluble guanylyl cyclase. Here we investigated potential roles of the NO-soluble guanylyl cyclase-cyclic GMP signaling pathway in the regulation of dopaminergic neurodegeneration. Activation of microglia by interferon-gamma (IFN-gamma) followed by lipopolysaccharide (LPS) caused dopaminergic cell death in rat midbrain slice cultures, which was dependent on NO production. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, as well as KT5823, an inhibitor of cyclic GMP-dependent protein kinase, exacerbated dopaminergic cell death induced by IFN-gamma/LPS. Conversely, 8-bromo-cyclic GMP attenuated IFN-gamma/LPS cytotoxicity on dopaminergic neurons. Notably, although heme oxygenase-1 (HO-1) was expressed prominently in cells other than dopaminergic neurons in control cultures, robust expression of HO-1 was induced in surviving dopaminergic neurons challenged with IFN-gamma/LPS. ODQ and KT5823 decreased, whereas 8-bromo-cyclic GMP increased, the number of dopaminergic neurons expressing HO-1 after IFN-gamma/LPS challenge, without parallel changes in HO-1 expression in other cell populations. An NO donor 3-(4-morpholinyl)sydnonimine hydrochloride also induced HO-1 expression in dopaminergic neurons, which was abolished by ODQ and augmented by 8-bromo-cyclic GMP. Moreover, IFN-gamma/LPS-induced dopaminergic cell death was augmented by zinc protoporphyrin IX, an HO-1 inhibitor. The NO donor cytotoxicity on dopaminergic neurons was also augmented by ODQ and zinc protoporphyrin IX. These results indicate that the NO-cyclic GMP signaling pathway promotes the induction of HO-1 specifically in dopaminergic neurons, which acts as an endogenous protective system to limit inflammatory degeneration of this cell population.

Topics: Animals; Animals, Newborn; Cyclic GMP; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Gene Expression Regulation; Heme Oxygenase-1; In Vitro Techniques; Inflammation; Interferon-gamma; Mesencephalon; Nerve Degeneration; Nitric Oxide; Polysaccharides; Rats; Rats, Wistar; Signal Transduction; Tyrosine 3-Monooxygenase

Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5' end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study, we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function.

Topics: Animals; Animals, Genetically Modified; Cyclic GMP; Female; Genetic Vectors; Guanylate Cyclase; Hybridization, Genetic; Male; Models, Neurological; Moloney murine leukemia virus; Mutagenesis, Insertional; Nerve Degeneration; Transcriptional Activation; Zebrafish

The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP(+)-induced cytosolic phospholipase A(2) (cPLA(2)) activation of dopaminergic neurons. MPP(+) activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca(2+)](i) level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA(2), and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF(3)), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA(2) on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA(2) phosphorylation at Ser505. Inhibitors of cPLA(2) and PKG increased viability and reduced MPP(+)-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA(2) phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA(2).

Topics: Animals; Arachidonic Acid; Calcium; Catalytic Domain; Cell Death; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; In Vitro Techniques; Mice; Mice, Inbred C57BL; Models, Biological; Nerve Degeneration; Nitric Oxide Synthase Type I; Parkinsonian Disorders; PC12 Cells; Phospholipases A2, Cytosolic; Protein Kinase C; Rats; Serine; Signal Transduction; Substantia Nigra

We have characterized lipopolysaccharide (LPS) preconditioning-induced neuroprotective mechanisms against nitric oxide (NO) toxicity. Pretreatment of rat cortical cultures with LPS attenuated neurotoxicity of NO donors, including sodium nitroprusside (SNP) and diethylamine NONOate (NONOate). A transiently increased expression of endothelial nitric oxide synthase (eNOS) accompanied by an increase in NO production was observed during LPS preconditioning. Application of NOS inhibitors including L-N(5)-(1-iminoethyl)-ornithine (L-NIO) and L-nitroarginine methylester (L-NAME) abolished LPS-dependent protection against SNP toxicity. The LPS effect was also blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). Consistently, application of 8-bromo-cyclic GMP (8-Br-cGMP), a slowly degradable cGMP analogue capable of PKG activation, was neuroprotective. LPS preconditioning resulted in a heightened neuronal expression of Bcl-2 protein that was abolished by L-NAME and KT5823, the respective inhibitors of NOS and PKG. Together, our results reveal the signaling cascade of "LPS --> eNOS --> NO --> cGMP/PKG --> Bcl-2" that might have contributed to the LPS protective effects in cortical neurons.

Topics: Animals; Blotting, Western; Brain; Cells, Cultured; Cyclic GMP; Immunohistochemistry; Ischemic Preconditioning; Lipopolysaccharides; Microscopy, Confocal; Microscopy, Fluorescence; Nerve Degeneration; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction

We have investigated the effects of low (10 mg/kg) and high (100 mg/kg) doses of L-DOPA on the expression and activity of neuronal nitric oxide synthase (nNOS) and guanylyl cyclase (GC) in the striatum and midbrain of mice. L-DOPA was administered subchronically for 11 days (beginning 3 days after last MPTP/NaCl injection) or for 14 days (with dosing started immediately following the last MPTP/NaCl injection). Adult mice received three intraperitoneal (i.p.) injections of physiological saline or MPTP at 2h intervals (total dose of 40 mg/kg). Normal and MPTP-injected mice were treated twice a day for 11 or 14 days with low (10/2.5 mg/kg bw) or high (100/25mg/kg bw) doses of L-DOPA/benserazide. The present study indicates that several days of treatment with L-DOPA does not affect MPTP-activation of the nNOS/sGC/cGMP pathway or the neurodegenerative processes that occur in the striatum and midbrain of mice. In normal mice, L-DOPA upregulates the expression and activity of nNOS and GC to levels found in MPTP-injected mice. Due to upregulation of nNOS and GC, cGMP levels in the mouse striatum and midbrain are also elevated, however, significantly lower in mice administrated with low dose of L-DOPA. In both investigated brain regions of normal mice cGMP-dependent PDEs activities were elevated after low dose administration of L-DOPA, but no change in PDEs activities has been detected in MPTP and high L-DOPA-injected mice as compared to control values. The enhancement of nNOS mRNA and GCbeta1 mRNA levels were generated by both doses of L-DOPA, given in a time-dependent fashion. L-DOPA-injected for 11 or 14 days caused a decrease in TH protein levels in the striatum and midbrain, respectively; this result was noted irrespective of dose. L-DOPA therapy did not prevent the MPTP-induced decrease in TH protein levels in either investigated brain region.

Topics: Animals; Blotting, Western; Cyclic GMP; Dopamine; Dopamine Agents; Dopamine Plasma Membrane Transport Proteins; Electrophoresis, Polyacrylamide Gel; Guanylate Cyclase; Immunohistochemistry; Levodopa; Mesencephalon; Mice; Mice, Inbred C57BL; Microdialysis; MPTP Poisoning; Neostriatum; Nerve Degeneration; Nerve Tissue Proteins; Nitric Oxide Synthase Type I; Phosphoric Diester Hydrolases; RNA, Messenger; Synaptosomes

Polychlorinated biphenyls (PCB) are persistent environmental contaminants whose chronic exposure can affect nervous system development and function. The cellular and molecular mechanisms underlying neuronal damage are not yet clear. In the present study, we investigated whether nitric oxide (NO) could be involved in aroclor 1254 (A1254; a PCB mixture)-induced cytotoxicity in SH-SY5Y human neuroblastoma cells. Prolonged exposure (24 hr) to A1254 (10-100 microg/ml) caused a dose-dependent reduction of cell viability that was attenuated in the presence of a calcium entry blocker, gadolinum (Gd(3+)) at 10 microM, a concentration able to block voltage-sensitive calcium channels. In addition, A1254 caused an increase of cytosolic calcium that was dependent on extracellular calcium, as measured by fura-2 videomicroscopy. A1254-induced calcium rise may stimulate NO production through an activation of neuronal NOS (nNOS). Indeed, the concomitant addition of the selective nNOS inhibitor N(omega)-propyl-L-arginine (NPLA) and A1254 prevented cell injury, suggesting that NO production plays a major role in A1254-evoked cell injury. Furthermore, the exposure (14 hr) to A1254 (30 microg/ml) produced an up-regulation of the expression of beta isoform of nNOS. This up-regulation was calcium dependent and was accompanied by an enhancement of NO production as demonstrated by an increase of nitrite formation. Moreover, A1254-induced cell injury was prevented when KT 5823, a selective cGMP/PKG inhibitor, was added concomitantly to 30 microg/ml A1254. These results suggest that PCB-induced cell death in neuroblastoma cells is mediated by an activation of the cGMP/PKG pathway triggered by NO production.

Topics: Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cell Death; Cell Line, Tumor; Cell Survival; Chlorodiphenyl (54% Chlorine); Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Environmental Exposure; Enzyme Inhibitors; Humans; Nerve Degeneration; Neurons; Neurotoxins; Nitric Oxide; Nitric Oxide Synthase Type I; Oxidative Stress; Polychlorinated Biphenyls; Signal Transduction; Up-Regulation

The NO-cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)-deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co-culture of neonatal DRG neurons with either Schwann cells pre-treated with an NO donor or a membrane-permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO-sGC-cGMP-mediated NGF and NT3 synthesis by Schwann cells protect neurons.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Communication; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Cyclic GMP; Cytoprotection; Ganglia, Spinal; Guanylate Cyclase; Nerve Degeneration; Neuroglia; Neurons; Neurons, Afferent; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Peripheral Nervous System; Rats; Rats, Wistar; Receptor, Nerve Growth Factor; Receptor, trkC; Receptors, Cytoplasmic and Nuclear; Schwann Cells; Signal Transduction; Soluble Guanylyl Cyclase; Transfection

In axotomised adult rat dorsal root ganglion (DRG), many neurons show a marked increase in expression of neuronal nitric oxide synthase (nNOS). It has been established that NO functions as a neuron-glial signalling molecule by generating cGMP in glia cells that surround the neuron in DRG. Furthermore, in cultures of dissociated DRG deprived of nerve growth factor, many neurons expressed nNOS and cGMP and subsequently died if either enzyme's activity was inhibited suggesting that NO-cGMP pathway could be neuroprotective in stressed DRG neurons. This has now been tested in vivo. It was found, 10 days after sciatic axotomy that nNOS was expressed in 36% of DRG neurons in the L5 and L6 ganglia giving rise to the damaged nerve, compared with 6% in contralateral ganglia. Almost all nNOS neurons and 24% of non-nNOS neurons expressed c-Jun in their nuclei. Ten days following axotomy, treatment with the relatively selective nNOS-blocker, 1-(2-trifluoromethylphenyl) imidazole (TRIM), caused morphology changes in approximately 50% of neurons that consisted of vacuolations, blebbing and highly irregular cell boundaries. Sham operated, TRIM treated, nerve-sectioned, vehicle treated, and controls did not show these changes. These observations further support the view that NO could be neuroprotective in some injured/stressed primary sensory neurons.

Topics: Animals; Apoptosis; Axotomy; Cell Survival; Cyclic GMP; Enzyme Inhibitors; Female; Ganglia, Spinal; Imidazoles; Immunohistochemistry; Male; Nerve Degeneration; Neurons, Afferent; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Sciatic Nerve; Signal Transduction; Stress, Physiological

Subunit c of ATP synthase functions as a high conductance ion channel, tightly regulated by calcium. We have suggested that the pathogenesis of Batten syndromes involving overaccumulation of subunit c are linked to the protein's ion channel function. In normal electrically excitable tissue the channel could act as a pacer setting nodal voltage via control of cation entry. The channel conductance is controlled by voltage, calcium, cyclic nucleotides and polyamines. We discuss the pathogenic role that subunit c could play in the electrically excitable tissues of retina, brain and heart where Batten neurodegeneration is seen. Focus is given to potential links between subunit c and the known mutant gene products in the Batten diseases, the process of apoptosis, and the requirement of the growing brain for gradients of cGMP, a ligand of the subunit c channel.

Topics: Animals; Apoptosis; Calcium; Cattle; Cyclic GMP; Ion Channel Gating; Microscopy, Electron; Mitochondrial Proton-Translocating ATPases; Nerve Degeneration; Neuronal Ceroid-Lipofuscinoses; Proton-Translocating ATPases; Rats; Sheep

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.

Topics: Animals; Animals, Newborn; Apoptosis; Arginine; Autocrine Communication; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Female; Ganglia, Spinal; Immunohistochemistry; Male; Nerve Degeneration; Neurons, Afferent; Nitric Oxide; Nitric Oxide Synthase; Peripheral Nervous System Diseases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Stress, Physiological

Nitric oxide (NO) synthase (NOS)-containing cerebrocortical neurons degenerate in patients with amyotrophic lateral sclerosis (ALS) and dementia, and in transgenic mice expressing a mutated superoxide dismutase gene (G93A) associated with familial ALS. The cerebral cortex of transgenic mice displayed decreased NOS activity (p<0.001) and cGMP levels (p<0.01), but no changes in NOS content indicating that less NO is produced. Therefore, NOSN degeneration is not caused by elevated NO.

Topics: Amino Acid Substitution; Animals; Blotting, Western; Cerebellum; Cerebral Cortex; Cyclic GMP; Disease Models, Animal; Genotype; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Degeneration; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Polymerase Chain Reaction; Superoxide Dismutase

We studied the mechanism of nitric oxide (NO) toxicity in cultured rat spinal motoneurons. Treatment with the NO donor NOC-18 (NOC) resulted in slow motoneuron death, ending in apoptosis. The observed motoneuron death was completely prevented by hemoglobin. Treatment with inhibitors of the known intracellular targets of NO, soluble guanylate cyclase, polyADP-ribose polymerase (PARP) and superoxide, did not result in any significant protection against NOC-induced motoneuron death. ATP levels were reduced as soon as 3 h after the start of NOC treatment, suggesting a direct inhibition of cellular energy production. NOC toxicity could be blocked by the general voltage-gated calcium channel blocker cobalt, but not by specific blockers of various subtypes of calcium channels.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Calcium; Cells, Cultured; Cobalt; Cyclic GMP; Electrophysiology; Motor Neurons; Nerve Degeneration; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Rats

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K+-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K+-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K+-induced taurine release. We conclude that a substantial part of K+-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K+-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.

Topics: Animals; Corpus Striatum; Cyclic GMP; Male; Microdialysis; Nerve Degeneration; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Potassium Chloride; Quinolinic Acid; Rats; Rats, Wistar; Taurine; Time Factors

The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief (15 min) exposure of the neurones to glutamate caused a dose-dependent (0.01-1 mM) increase in cyclic GMP levels together with delayed (24 h) neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide (.NO) synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (NAME; 1 mM) or by the N-methyl-D-aspartate (NMDA) glutamate-subtype receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV; 0.1 mM). Glutamate exposure (0.1 mM and 1 mM) followed by 24 h of incubation caused the inhibition of succinate-cytochrome c reductase (20-25%) and cytochrome c oxidase (31%) activities in the surviving neurones, without affecting NADH-coenzyme-Q1 reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose (by 26%) or succinate (by 39%) as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion (20%) by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving .NO biosynthesis.

Topics: 2-Amino-5-phosphonovalerate; Animals; Antimycin A; Cell Respiration; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Glutathione; Mitochondria; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Radiation-Protective Agents; Rats; Rats, Wistar; Rotenone; Succinic Acid; Uncoupling Agents

Cyclic guanosine 3',5'-monophosphate (cGMP) levels were determined in retinas from a strain of Labrador Retrievers with inherited retinal dystrophy manifesting at early stages of retinal differentiation. The cGMP contents of dystrophic retinas of dogs from 1 to 4 months of age (n = 7) were significantly higher (p = 0.001) than in age-matched controls of the same breed (n = 11). Ultrastructure along the vertical retinal meridian was studied in developing retinas and findings were related to those of age-matched wild-type controls of the same breed. Slow central to peripheral progression of degeneration was observed in affected dogs. No differences were found in total cGMP-phosphodiesterase (PDE) activity, in PDE subunit composition as determined by Western blotting of 2-month-old homozygote affected retinas, or in the amino acid sequence deduced from the nucleotide sequence of the PDE beta-subunit as compared to controls. This model of photoreceptor degeneration thus is the first case of an apparent abnormality of cGMP metabolism that is not associated with a defect in the PDE catalytic subunits, and it is also the first reported model not associated with severe developmental abnormalities and rapid degeneration.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blotting, Western; Cyclic GMP; Disease Models, Animal; Dog Diseases; Dogs; Electroretinography; Enzyme Activation; Microscopy, Electron; Nerve Degeneration; Photoreceptor Cells; Reference Values; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Trypsin

The NMDA antagonist and neuroprotective effects of RPR 104632 (2H-1,2,4-benzothiadiazine-1-dioxide-3-carboxylic acid), a new benzothiadiazine derivative, with affinity for the glycine site of the NMDA receptor-channel complex are described. RPR 104632 antagonized the binding of [3H]5,7-dichlorokynurenic acid to the rat cerebral cortex, with a Ki of 4.9 nM. This effect was stereospecific, since the (-)-isomer was 500-fold more potent than the (+)-isomer. The potent affinity of RPR 104632 for the glycine site was confirmed by the observation that RPR 104632 inhibited [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in the presence of N-methyl-D-aspartate (NMDA) (IC50 = 55 nM), whereas it had no effect on the competitive NMDA site or on the dissociative anaesthetic site. RPR 104632 inhibited the NMDA-evoked increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC50 = 890 nM) in a non-competitive manner and markedly reduced NMDA-induced neurotoxicity in rat hippocampal slices and in cortical primary cell cultures. These results suggest that RPR 104632 is a high-affinity specific antagonist of the glycine site coupled to the NMDA receptor channel with potent neuroprotective properties in vitro.

Topics: Aminoquinolines; Animals; Benzothiadiazines; Binding, Competitive; Cerebral Cortex; Cyclic GMP; Excitatory Amino Acid Antagonists; Hippocampus; In Vitro Techniques; Kynurenic Acid; Nerve Degeneration; Phencyclidine; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

To investigate whether nitric oxide (NO) plays a role in degenerative neurologic disease (DND), we measured nitrite, nitrate and cyclic GMP in cerebrospinal fluid (CSF) samples from patients with Parkinson's disease (PD), spinocerebellar ataxia (SCA) and amyotrophic lateral sclerosis (ALS). We found no significant change in CSF nitrite, nitrate or cyclic GMP in patients with any DND compared with control values. These results suggest that NO production is preserved in PD, SCA and ALS.

Topics: Aged; Amyotrophic Lateral Sclerosis; Biomarkers; Cyclic GMP; Female; Humans; Male; Middle Aged; Nerve Degeneration; Nervous System Diseases; Nitrates; Nitric Oxide; Nitrites; Parkinson Disease; Spinocerebellar Degenerations

In the presence of physiological concentrations of Mg2+ and in glycine-free buffer, the relationship between KA-mediated generation of NO and neurotoxicity in cultures of cerebellar granule cells of the rat was examined. The neuronal damage elicited by KA was not dependent on the presence of L-arginine, a precursor of NO, since neither the potency nor magnitude of KA-mediated cell death was altered in either the absence or presence of exogenously applied L-arginine. Similarly, with the exception of 4-hydroxy-azobenzene-4'-sulfonic acid, disodium salt dihydrate (HBS), the salt associated with NG-monomethyl-L-arginine (di-(p-hydroxyazobenzene-p'-sulfonate) (MA(HBS)), treatment with several different competitive NO synthetase inhibitors did not provide protection against the toxicity of KA. However, the ability of KA to induce neuronal damage was significantly decreased in cerebellar granule cells treated with either HBS or alpha-tocopherol (VE). On the basis of these results, it is concluded that the generation of free radicals may be involved in the process of KA-elicited neuronal death in cultures of cerebellar granule cells but that this is unrelated to the synthesis of NO. This conclusion agrees with both in vivo and in vitro studies, implicating the involvement of free radicals in non-NMDA mediated neuronal damage.

Topics: Animals; Arginine; Azo Compounds; Cell Death; Cells, Cultured; Cerebellum; Cyclic GMP; Kainic Acid; Kinetics; Magnesium; Nerve Degeneration; Neurons; Nitric Oxide; Nitroarginine; Rats; Rats, Wistar

Incubation of a primary culture of rat striatal neurons with sodium nitroprusside (SNP), a known source of nitric oxide (NO), showed a concentration-dependent increase in cyclic GMP levels with an EC50 value of 13.7 microM. Twenty-four hours following incubation with 10 microM SNP, striatal neurons underwent degeneration as assessed immunohistochemically and biochemically. In contrast, potassium ferricyanate at concentrations up to 1 mM had no effect on striatal neuron viability. These results indicate that SNP has neurotoxic actions in-vitro, an effect that may involve NO as a second messenger.

Topics: Animals; Cell Survival; Cells, Cultured; Corpus Striatum; Culture Media; Cyclic GMP; Dose-Response Relationship, Drug; L-Lactate Dehydrogenase; Nerve Degeneration; Neurons; Nitroprusside; Osmolar Concentration; Rats

Topics: Acetyltransferases; Adenylyl Cyclases; Animals; Bacterial Toxins; Cyclic AMP; Cyclic GMP; Cycloheximide; Drug Interactions; Enzyme Induction; Male; Nerve Degeneration; Norepinephrine; Nucleotides, Cyclic; Organ Culture Techniques; Ouabain; Phosphoric Diester Hydrolases; Pineal Gland; Potassium; Rats; Serotonin; Synapses; Time Factors; Vibrio cholerae; Xanthines

The activities of guanylate cyclase, guanosine 3', 5'-monophosphate (cyclic GMP) phosphodiesterase and 5'-nucleotidase were measured during postnatal development in retinas of control and C3H/HeJ mice. In control retina, each of these enzyme activities increases in conjunction with photoreceptor cell differentiation and maturation. In C3H retina, guanylate cyclase and 5-nucleotidase activities increase with photoreceptor cell development and decrease with photoreceptor cell death. However, the activity of a class of cyclic GMP phosphodiesterase which distinguishes the photoreceptor cells of control mice and those of several other species is not demonstrable in retina of C3H mice at any age. It is suggested that the deficiency in cyclic GMP phosphodiesterase activity may account for the accumulation of cyclic GMP which has been shown to occur in the C3H photoreceptor cells before they degenerate.

Topics: Aging; Animals; Animals, Newborn; Cyclic GMP; Guanylate Cyclase; Mice; Mice, Inbred Strains; Nerve Degeneration; Nucleotidases; Phosphoric Diester Hydrolases; Photoreceptor Cells; Retina; Species Specificity

As a result of an early deficiency in cyclic nucleotide phosphodiesterase activity, guanosine 3',5'-monophosphate accumulates in retinal photoreceptor cells before they begin to degenerate. It is suggested that degeneration of the photoreceptor cells is related to an imbalance in their metabolism or function which is caused by the elevated levels of cyclic guanosine monophosphate.

Topics: Adenylyl Cyclases; Animals; Animals, Newborn; Chromatography, Ion Exchange; Chromosome Aberrations; Chromosome Disorders; Cyclic AMP; Cyclic GMP; Mice; Mice, Inbred C3H; Mice, Inbred DBA; Nerve Degeneration; Phosphoric Diester Hydrolases; Photoreceptor Cells; Protein Binding; Retina; Time Factors