Altered Cerebrospinal Fluid Concentrations of Hydrophobic and Hydrophilic Compounds in Early Stages of Multiple Sclerosis-Metabolic Profile Analyses.

The lack of a single predictive or diagnostic test in multiple sclerosis (MS) remains a major obstacle in the patient's care. The aim of this study was to investigate metabolic profiles, especially lipids in cerebrospinal fluid (CSF) using 1H-NMR spectroscopy and metabolomics analysis to discriminate MS patient group from the control ones. In this study, 19 MS patients and 19 controls, without neurological problems, patients were enrolled. To obtain the CSF metabolic profiles, NMR spectroscopy was used. Hydrophilic and hydrophobic compounds were analyzed using univariate and multivariate supervised analysis orthogonal partial least square discriminant analysis (OPLS-DA). Targeted OPLS-DA analysis of 32 hydrophilic and 17 hydrophobic compounds obtained 9 hydrophilic metabolites and 8 lipid functional groups which had the highest contribution to patient's group separation. Lower concentrations of CSF hydrophilic and hydrophobic compounds were observed in MS patients as compared to control group. Acetone, choline, urea, 1,3-dimethylurate, creatinine, isoleucine, myo-inositol, leucine, and 3-OH butyrate; saturated and monounsaturated acyl groups of ω-9, ω-7, ω-6, ω-3, and fatty acid, triglycerides, 1,3-DG, 1-MG, and unassigned component signal at 3.33 ppm were the most important signal compounds in group separation. Analysis of metabolic profile of raw CSF and their lipid extract shows decreased levels of many compounds and led to the conclusion that MS patients could have a disturbance in many metabolic pathways perhaps leading to the decreased level of acetyl-CoA and/or inflammation. CSF metabolic profile analyses could be used as a fingerprint for early MS diagnosis.

The effect of subchronic, intermittent L-DOPA treatment on neuronal nitric oxide synthase and soluble guanylyl cyclase expression and activity in the striatum and midbrain of normal and MPTP-treated mice.

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.

Alterations of the expression and activity of midbrain nitric oxide synthase and soluble guanylyl cyclase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice.

The study was aimed at investigating the expression and the activity of neuronal nitric oxide synthase, and of soluble guanylyl cyclase and phosphodiesterase activities that regulate guanosine 3',5'-cyclic monophosphate level in the midbrain, in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections. Adult male mice of the C57/BL strain were given three i.p. injections of physiological saline or three i.p. injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution in physiological saline at 2 h intervals (summary 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dose: 40 mg/kg), and were killed 3, 7, or 14 days later. mRNA, protein level, and/or activities of neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase and guanosine 3',5'-cyclic monophosphate were determined. Immunohistochemistry showed about 75% decrease in the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed increased midbrain guanylyl cyclase and total nitric oxide synthase activities at 3, 7, and 14 days post-treatment. The specific neuronal nitric oxide synthase inhibitor 7-nitroindazole (10 microM) and the specific inducible nitric oxide synthase inhibitor 1400W (10 microM) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced excess in nitric oxide synthase activity by 63-70 and 13-25%, respectively. The increases in total midbrain nitric oxide synthase activity were accompanied by elevated guanosine 3',5'-cyclic monophosphate, enhanced expression of neuronal nitric oxide synthase and of the beta1 subunit of guanylyl cyclase at both mRNA and protein levels that persisted up to the end of the observation period, and by enhanced neuronal nitric oxide synthase and guanylyl cyclase beta1 immunoreactivities in substantia nigra pars compacta 7 and 14 days after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The increases in guanylyl cyclase activity were found to occur exclusively due to increased maximal enzyme activity. No 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced change in phosphodiesterase activity has been detected in any brain region studied. 7-Nitroindazole prevented a significant increase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced midbrain guanosine 3',5'-cyclic monophosphate level and neurodegeneration of dopaminergic neurons. These results raise the possibility that the nitric oxide/guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling pathway may play a role in maintaining dopaminergic neurons function in substantia nigra pars compacta.

Macrophage migration inhibitory factor induces cell death and decreases neuronal nitric oxide expression in spinal cord neurons.

Macrophage migration inhibitory factor is a potent proinflammatory cytokine; however, its role in spinal cord injury is poorly understood. Therefore, the aim of the present study was to investigate the effects of macrophage migration inhibitory factor on spinal cord neuron survival and viability. Due to the importance of nitric oxide metabolism in these events, part of our study was also focused on the influence of recombinant macrophage migration inhibitory factor on neuronal nitric oxide expression. Exposure of cultured mouse spinal cord neurons to macrophage migration inhibitory factor markedly increased cellular oxidative stress as measured by 2',7'-dichlorofluorescein fluorescence and intracellular calcium levels. In addition, an antagonist of the inositol 1,4,5-triphosphate receptor, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, completely blocked the macrophage migration inhibitory factor-induced increase in intracellular calcium levels. Macrophage migration inhibitory factor treatment also decreased cell viability, increased cellular lactate dehydrogenase release, and induced chromatin condensation and aggregation in cultured spinal cord neurons. Finally, exposure to macrophage migration inhibitory factor markedly decreased expression and activity of neuronal nitric oxide, accompanied by a decrease in cellular guanosine 3'5'-cyclic monophosphate levels. The present results indicate that macrophage migration inhibitory factor can induce dysfunction of spinal cord neurons, leading to cell death through oxidative stress and intracellular calcium-dependent pathways.

Diversity of endurance training effects on antioxidant defenses and oxidative damage in different brain regions of adolescent male rats.

Studies on the effect of physical activity on brain oxidative stress, performed mostly in adult rats, have shown that moderate aerobic activity increases resistance to oxidative stress and reduces cellular damage. These effects can greatly differ between various brain regions. The postnatal period of the highest brain sensitivity to various stimuli is adolescence. We hypothesized that endurance training will modify brain antioxidant barrier differently in various regions, depending on their role in locomotion. Therefore, we studied the effect of moderate intensity endurance training on the activities of selected antioxidant enzymes (superoxide dismutase, gluthathione peroxidase and catalase and the contents of thiobarbituric acid-reactive substances (the key index of lipid peroxidation) and glutathione in several brain regions with dissimilar relationship to locomotion, as well as in circulating blood. Additionally, we investigated the effect of the training on nitric oxide synthase activity that may be a major player in exercise-related oxidative stress in brain regions that are directly involved in the locomotion control and execution (the striatum, midbrain and cerebellum). The training significantly enhanced nitric oxide synthase activity only in the latter three regions. Surprisingly, it elevated the activities of all studied antioxidant enzymes (excepting gluthathione peroxidase) in the neocortex, while no appreciable change in these activities was found in either the cerebellum (except for elevated catalase activity), or the striatum, or the midbrain. The training also elevated total glutathione content (a key protector of brain proteins under the conditions of enhanced nitric oxide production) in the cerebellum and striatum, but not in the other regions. The observed brain changes greatly differed from those in circulating blood and did not prevent the training-related increases in oxidative damage as evidenced by elevations in cerebellar and striatal thiobarbituric acid-reactive substances. These data suggest an increased susceptibility of adolescent brain to enhanced physical activity-related oxidative stress.

Activation of serotonergic 5-HT1A receptor reduces Ca(2+)- and glutamatergic receptor-evoked arachidonic acid and No/cGMP release in adult hippocampus.

Stimulation of glutamatergic NMDA receptor in adult rat hippocampal synaptoneurosomes induces statistically significant Ca(2+)-dependent liberation of arachidonic acid (AA) and nitric oxide (NO)-activated cGMP synthesis. NMDA acting for 5 min at 100 microM markedly increases, by approx. 25%, Ca(2+)-mediated AA release from phospholipids of hippocampal synaptoneurosomes. Prolonged stimulation of NMDA receptor up to 10 min has smaller stimulatory effect and enhances AA release by about 6%. Moreover, NMDA activates NO-dependent cGMP production by approx. 5 times more than the Ca2+ itself. Release of both these second messengers is completely blocked by the competitive NMDA antagonist, APV (100 microM). The NMDA-mediated cGMP elevation completely depends on NO action, and is abolished by the specific inhibitor of NO synthase, NG-nitro-L-arginine. Moreover, serotonin at 10 microM in the presence of 10 microM pargyline, potently decreases both Ca(2+)- and NMDA receptor-mediated AA and cGMP release in hippocampal synaptoneurosomes. The agonist of 5-HT1A receptor, buspirone, in a way similar to serotonin itself, counteracts the Ca(2+)- and also NMDA receptor-evoked AA release and cGMP accumulation. An antagonist of 5-HT1A receptor, NAN-190, eliminates the effect of serotonin and buspirone on AA and NO/cGMP liberation. An antagonist of serotonergic 5-HT2 receptor, ketanserin, has no effect on the Ca2+ and serotonin action. These results indicate that serotonin, through 5-HT1A receptor, potently antagonizes the action of excitatory amino acid for AA release and NO/cGMP synthesis in the adult rat hippocampus. In conclusion, the interaction of serotonin with the glutamatergic system in the hippocampus may play an important role in the modulation of a signal transduction pathway, and by this molecular mechanism serotonin may exert a neuroprotective effect on hippocampal neurons.

Effect of spinal cord compression on cyclic 3',5'-guanosine monophosphate in the white matter columns of rabbit.

Changes in the level of cyclic 3',5'-guanosine monophosphate (cGMP) were studied one day after a surgically induced spinal cord constriction performed at the Th7 segment level in the dorsal, lateral and ventral white matter columns and in the non-compartmentalized white matter of Th5-Th6 segments, i.e., above the site of the spinal cord constriction and in Th8-Th9 segments, located below the spinal cord constriction. The midthoracic spinal cord constriction caused a significant decrease in the level of cGMP in the ventral column of Th5-Th6 segments and a significant increase in the lateral column of Th8-Th9 segments. The level of cGMP in the dorsal column, located either rostrally or caudally to the site of the spinal cord injury, remained unchanged. In addition, no significant changes in the level of cGMP were found in the non-compartmentalized white matter of Th5-Th6 and Th8-Th9 segments in response to constriction of the Th7 segment.

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents.

Upregulation and activation of phospholipases A2 (PLA2) and cyclooxygenases (COX) leading to prostaglandin E2(PGE2) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE2 production in primary rat astrocytes in response to agents that activate PLA2 including pro-inflammatory cytokines (IL-1beta, TNFalpha and IFNgamma), the P2 nucleotide receptor agonist ATP, and oxidants (H2O2 and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE2 production that was marked by increased expression of secretory sPLA2 and COX-2, but not COX-1 and cytosolic cPLA2. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA2 phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE2. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H2O2 or menadione, markedly increased PGE2 production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE2 production and may contribute to chronic inflammation seen in Alzheimer's disease.

Hydroxylamine attenuates the effects of simulated subarachnoid hemorrhage in the rat brain and improves neurological outcome.

Some of the neurological deficits that emerge after aneurysmal subarachnoid hemorrhage (SAH) in humans are presumably caused by ischemic brain damage consequential to SAH-induced delayed cerebral vasospasm. This vasospasm probably results from an imbalance among vasoactive factors released from both the clot formed by extravasated blood and adjacent tissues, and in particular from a decrease in the endothelium-derived relaxing factor nitric oxide (NO). Brain ischemia is also known to elevate brain production and deposition of beta-amyloid, and to induce a delayed increase in total NO synthase (NOS) activity due to induction of expression of so-called induced NOS isoform, phenomena that may secondarily contribute to SAH-related brain damage. The aim of this study was to investigate the effects of treatment with the intracellular NO donor hydroxylamine on: (i) basilar arterial wall that remained in a direct contact with the clot, (ii) formation of the beta-amyloid precursor protein (beta-APP), (iii) total brain NOS activity, and (iv) neurological outcome in a 'two-hemorrhage' rat SAH model. Intraperitoneal (i.p.) administration of 0.18 mmol/kg hydroxylamine hydrochloride (12.5 mg/kg) twice daily for 7 days beginning immediately after the first 'hemorrhage' (intracisternal blood injection) reduced basilar arterial wall damage and attenuated post-SAH neurological deficit. It also reduced the SAH-related increases in hippocampal and cortical beta-APP immunoreactivities and hippocampal NOS activity measured 24 h after commencement of the treatment. These results indicate that intracellular NO donors that yield NO through the action of widely distributed enzymes in brain cells (cytochromes, catalase) can attenuate detrimental effects of SAH.

Arachidonate transport through the blood-retina and blood-brain barrier of the rat after reperfusion of varying duration following complete cerebral ischemia.

The permeability-surface area product (PS) of [1-14C]arachidonate at the blood-retina and blood-brain barrier was determined by short carotid perfusion in young Wistar rats 1 or 6 h after recovery period following complete cerebral ischemia induced by temporary cardiac arrest. For the retina and structures of visual system, hypothalamus and olfactory bulb there was no significant difference over sham-operated rats among mean PSs. For cortex, hippocampus and striatum, significant increases were found at both time intervals of recovery after cardiac arrest. The ischemia-reperfusion model was characterized by a significant increase in tissue conjugated diene in the hippocampus and microsomal lysophosphatidylcholine acyltransferase activity in the cortex. Consistent with these findings, we also show ultrastructural evidence mainly represented by partial opening of interendothelial junctions and mild signs of tissue edema in surrounding neuropil, suggesting barrier leakiness predominantly in the cortex, hippocampus and striatum but almost absent in the retina microvessels. Our results indicate that ischemia-reperfusion does affect influex through blood-brain barrier into regional structures of rat central nervous system of arachidonate, a metabolic substrate and lipid mediator rapidly incorporated into microcapillary and brain lipids. The data also suggested that: (i) reactive oxyradicals were moderately generated during the early phase of ischemic-reperfusion process in the rat; (ii) after reperfusion, in vitro susceptibility of different brain regions to iron-induced peroxidation was highest in the hippocampus and lowest in the cortex and striatum; (iii) membrane phospholipid repair mechanisms were activated at the same time.

Arachidonate transport through the blood-retina and blood-brain barrier of the rat during aging.

The permeability-surface area product (PS) of [I-14C]arachidonate at the blood-retina (BRB) and blood-brain barrier (BBB) was determined after short carotid perfusion in Wistar rats at 4, 12 and 28 months of age. For the visual system structures, parietal and frontal cortex, striatum, hypothalamus, hippocampus and olfactory bulb there was no significant difference among mean PSs in any age group. Our results indicate that: (1) arachidonate is able to cross at relevant rate BRB and BBB; (2) in all brain regions except retina, optic tract and hippocampus, blood barriers have a transport capacity for arachidonate significantly higher than that for docosahexaenoate and palmitate as well; (3) aging does not affect influx into retina and other structures of rat central nervous system of the arachidonate, a metabolic substrate rapidly incorporated into microcapillary and brain lipids, and for which simple diffusion transport across the BRB and BBB may be postulated.

Biphasic enhancement of nitric oxide synthase activity and cGMP level following brain ischemia in gerbils.

This study was aimed to examine properties and changes in nitric oxide synthase (NOS) activity and cGMP level during reperfusion after 5 min of brain ischemia in gerbils. Animals were treated 5 min before ischemia with NOS inhibitors: N-Nitro-L-arginine (NNLA), or 7-Nitroindazole (7-NI), or with the inhibitor of guanylate cyclase, LY 83583, or with hydrocortisone for 7 days before ischemia. Northern blot analysis was performed using specific cDNA for inducible NOS. It was observed that ischemia significantly enhances NOS activity and cGMP level. During reperfusion, biphasic increase in NOS activity and cGMP level took place with two peaks 15 min and 2 h after ischemia. NNLA, 7-NI, and LY 83583 eliminated enhancements of NOS activity and cGMP level, whereas glucocorticoid remained without effect. There was no activation of gene encoding inducible NOS (iNOS). Our results indicate that ischemia-reperfusion activates constitutive NOS. It is suggested that nitric oxide (NO) production during reperfusion is related to neuronal degeneration and that inhibitor of NOS offers a new therapeutical strategies.

NMDA receptor mediated nitric oxide dependent cGMP synthesis in brain cortex and hippocampus. Effect of ischemia on NO related biochemical processes during reperfusion.

The stimulation of NMDA receptor activates NO dependent cGMP biosynthesis with dynamic and extent different for hippocampus and brain cortex. The significantly higher NO mediated cGMP level was observed in hippocampus than in brain cortex. NMDA receptor stimulation increases NO mediated cGMP formation about 8 fold in hippocampus and 2.5 fold in brain cortex as compared to basal value (2 mM CaCl2). The activity of NO synthase and the basal level of cGMP in unstimulated slices were only slightly higher in hippocampus then in brain cortex. The CA2+ calmodulin dependent NO synthase was found in brain membrane and cytosol fraction. The enzyme activity was not affected by glucocorticoids, even after 20 days of hydrocortisone treatment in a dose of 40 mg/kg b.w. Brain ischemia induced by ligation of both common carotid arteries in gerbils increases significantly NOS activities as well as the level of cGMP and putrescine but decreases mono-ADP-ribosylation of brain proteins during reperfusion period. The ischemia evoked changes of NOS/cGMP were eliminated by specific inhibitor of neuronal form of NOS, 7-Nitrodazole (7NI) administered in a dose of 25 mg/kg b.w. 5 min. before ischemia. This inhibitor has no effect on the level of putrescine enhanced during ischemia and also biphasically during reperfusion. The inhibitor of guanylate cyclase, LY 83583 administered in a dose of 6 mg/kg b.w. 5 min before ischemia diminishes not only the enhanced level of cGMP but also NOS activity stimulated by ischemia. These results indicate that activation of NMDA receptor stimulates more significantly NO/cGMP production in hippocampus than in brain cortex suggesting the role of NO in neuronal form of NOS and inhibitor of guanylate cyclase protect the brain against excessive production of nitric oxide and cGMP during ischemia-reperfusion. These compounds may offer a new strategy in the therapy of brain ischemia.

[Influence of NMDA receptor stimulation in brain cortex and hippocampus on NO dependent cGMP synthase. Effect of ischemia on NO related biochemical processes during recirculation]. / Wplyw stymulacji receptora NMDA w hipokampie i korze mózgu na synteze cGMP zaleznego od NO. Procesy biochemiczne tlenku azotu w okresie recyrkulacji po krótkotrwalej ischemii mózgu.

Stimulation of NMDA receptor increases NO-dependent cGMP synthesis. A significantly higher cGMP level was observed in hippocampus (about 8-fold increase) than in cerebral cortex (2.5-fold increase), as compared to basal value. The activity of NO synthase (NOS) and the basal level of cGMP in unstimulated slices were only slightly higher in hippocampus than in the cortex. About 60% of NOS total activity was found in the brain membrane fraction. The enzyme activity was not affected by glucocorticoids, even after 20 days of hydrocortisone treatment in dose of 40 mg/kg b.w. Brain ischemia induced by ligation of the both common carotid arteries in gerbils (Meriones unquiculatus) significantly increased NOS activity as well as cGMP and putrescine concentrations but decreased mono-ADP-ribosolation of proteins. Changes of NOS activity and cGMP concentration evoked by ischemia were decreased by specific inhibitor of the neuronal form of NOS (nNOS), 7-nitrodazole and the inhibitor of guanylate cyclase, LY 83,583 administered respectively in a dose of 25 mg/kg b.w. and 6 mg/kg b.w. 5 min. before ischemia. The inhibitor of nNOS, 7NI, did not change the concentration of putrescine during ischemia and reperfusion. Our results indicated that these inhibitors could protect the brain against excessive production of nitric oxide and biochemical processes dependent on it. In this way they may offer a new strategy in the therapy of brain ischemia.

Diverging oxidative damage and heat shock protein 72 responses to endurance training and chronic testosterone propionate treatment in three striated muscle types of adolescent male rats.

The aim of the study was to assess the effects of a combination of anabolic-androgenic steroid abuse and endurance training during adolescence on selected aspects of oxidative stress and antioxidant defenses in various striated muscle types. The effects were studied of testosterone propionate (TP) treatment (8 and 80 mg/kg/week, for 6 weeks), given alone or in combination with moderate-intensity endurance training, starting at adolescence, on thiobarbituric acid-reactive substances and heat shock protein 72 (Hsp72) contents, and androgen receptorm(AR) mRNA level in the heart left ventricle, soleus and extensor digitorum longus of male Wistar rats. TP treatment alone markedly elevated thiobarbituric acid-reactive substances only in the left ventricle and soleus; this effect was but marginally enhanced by endurance training. The training alone markedly elevated Hsp72 content in all muscles studied. TP treatment alone dose-dependently upregulated Hsp72, while the lower TP dose slightly curtailed the effect of the training. Low-dose TP treatment alone elevated, whereas high-dose TP treatment alone lowered androgen receptor mRNA level in the soleus and extensor digitorum longus. Endurance training alone elevated AR mRNA in all muscles studied, whereas TP treatment dose-dependently counteracted this effect. Exercise-associated rise in body temperature was significantly less in the TP-treated rats. We came to the conclusion that chronic suprapharmacological TP treatment might exert a protective effect on muscle cell proteins in adolescent sedentary rats, but it markedly enhanced lipid peroxidation. These effects were unlikely to result from an androgen receptor-mediated genomic action of testosterone. Exercise-related heat stress, and not oxidative stress, was mainly responsible for Hsp72 upregulation in striated muscles of chronic TP-treated endurance-trained adolescent male rats.

Influence of the therapy with pergolide mesylate plus L-DOPA and with L-DOPA alone on serum cGMP level in PD patients.

The aim of our study was to investigate serum cGMP level in patients treated with L-DOPA alone and L-DOPA with pergolide mesylate (PM) and to compare the obtained values with age-matched healthy subjects. PD patients treated with L-DOPA or with PM + L-DOPA had significantly higher cGMP levels in serum as compared to control. Moreover, cGMP level was significantly higher after treatment with PM + L-DOPA then L-DOPA alone. We did not observed significant differences in UPDRS scores between both PD groups. Our data suggest that changes in serum cGMP level in PD patients are related with PM + L-DOPA or L-DOPA alone therapy.

Level of nitric oxide-dependent cGMP in patients with migraine.

It is believed that nitric oxide (NO) plays a significant role in migraine attacks. This molecule is formed due to the conversion of L-arginine into L-citrulline. The target receptor for NO is ferrum in the heme group of cytoplasmic guanyl cyclase, the enzyme catalyzing cyclic guanosine monophosphate (cGMP) formation. To confirm this hypothesis, cGMP and nitrite level in the blood serum were measured in patients with migraine. The group under study included 37 subjects suffering from migraine with and without aura and 40 normal control subjects. The cGMP was measured during a migraine attack and 60 min following the administration of sumatriptan 6 mg subcutaneously. A statistically significant increase in cGMP level was observed in patients during a migraine attack compared to the controls. This level decreased after the administration of sumatriptan, but it was still higher than in the controls. No correlation was found between the increased cGMP level and pain intensification with clinical symptoms of migraine. The results suggest the participation of biochemical changes in migraine pathogenesis in the L-arginine-NO-cGMP pathway.

NMDA receptor-dependent nitric oxide and cGMP synthesis in brain hemispheres and cerebellum during reperfusion after transient forebrain ischemia in gerbils: effect of 7-Nitroindazole.

In this study, the N-Methyl-D-Aspartate (NMDA) receptor-dependent nitric oxide and cyclic GMP (cGMP) synthesis in the course of reperfusion after 5 min of ischemia in gerbil brain hemispheres and cerebellum were investigated. Moreover, the role of the neuronal isoform of nitric oxide (NO) synthase (nNOS) in liberation of NO in postischemic brain and the involvement of NO in membrane lipoperoxidations activated during reperfusion were evaluated. Enhancement of Ca2+/calmodulin-regulated NOS activity and cGMP level in brain hemispheres and in cerebellum during reperfusion was found to be coupled to the activation of the NMDA receptor. cGMP concentration 40% above the control level was observed to persist up to 7 days after ischemia. The amount of conjugated double bounds in membrane lipids and the level of thiobarbituric acid reactive substances were increased exclusively in brain hemispheres, indicating activation of lipid peroxidation. The NMDA receptor antagonist, MK-801, eliminated, and a rather selective nNOS inhibitor, 7-Nitroindazole (7-NI) attenuated, NMDA receptor-evoked enhancement of NOS activity and cGMP level in brain hemispheres and in cerebellum during reperfusion. Moreover, 7-NI decreased significantly membrane lipid peroxidation during the early time of reperfusion. Histological examination demonstrated that 7-NI protects against death a selected population of neuronal cells in CA1 layer of hippocampus. It is suggested that NMDA receptor dependence of NO release during reperfusion is responsible for the degeneration of some populations of neurons and that the effect is mediated by activation of free radical formation and lipid peroxidation. Moreover, in cerebellum, ischemia-evoked activation of glutamatergic system stimulates NO-dependent signal transmission. Our results indicated that 7-NI has a significant ameliorating effect on biochemical alterations evoked by ischemia, suggesting nNOS inhibitors as a potential therapeutic agents in reperfusion injury.

Aging modulates nitric oxide synthesis and cGMP levels in hippocampus and cerebellum. Effects of amyloid beta peptide.

The biological roles of nitric oxide (NO) and cGMP as inter- and intracellular messengers have been intensively investigated during the last decade. NO and cGMP both mediate physiological effects in the cardiovascular, endocrinological, and immunological systems as well as in central nervous system (CNS). In the CNS, activation of the N-methyl-D-aspartic acid (NMDA) type of glutamatergic receptor induces Ca(2+)-dependent NOS and NO release, which then activates soluble guanylate cyclase for the synthesis of cGMP. Both compounds appear to be important mediators in long-term potentiation and long-term depression, and thus may play important roles in the mechanisms of learning and memory. Aging and the accumulation of amyloid beta (A beta) peptides are important risk factors for the impairment of memory and development of dementia. In these studies, the mechanism of basal- and NMDA receptor-mediated cGMP formation in different parts of adult and aged brains was evaluated. The relative activity of the NO cascade was determined by assay of NOS and guanylate cyclase activities. In addition, the effect of the neurotoxic fragment 25-35 of A beta (A beta) peptide on basal and NMDA receptor-mediated NOS activity was investigated. The studies were carried out using slices of hippocampus, brain cortex, and cerebellum from 3- and 28-mo-old rats. Aging coincided with a decrease in the basal level of cGMP as a consequence of a more active degradation of cGMP by a phosphodiesterase in the aged brain as compared to the adult brain. Moreover, a loss of the NMDA receptor-stimulated enhancement of the cGMP level determined in the presence of cGMP-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was observed in hippocampus and cerebellum of aged rats. However, this NMDA receptor response was preserved in aged brain cerebral cortex. A significant enhancement of the basal activity of NOS by about 175 and 160% in hippocampus and cerebellum, respectively, of aged brain may be involved in the alteration of the NMDA receptor response. The neurotoxic fragment of A beta, peptide 25-35, decreased significantly the NMDA receptor-mediated calcium, and calmodulim-dependent NO synthesis that may then be responsible for disturbances of the NO and cGMP signaling pathway. We concluded that cGMP-dependent signal transduction in hippocampus and cerebellum may become insufficient in senescent brain and may have functional consequences in disturbances of learning and memory processes. A beta peptide accumulated during brain aging and in Alzheimer disease may be an important factor in decreasing the NO-dependent signal transduction mediated by NMDA receptors.