cyclic-gmp and 2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline

Phosphodiesterase (PDE) inhibitors are currently considered promising therapeutic targets for treatment of cognitive impairment in diseases such as Schizophrenia and Alzheimer's disease. Inhibitors of PDE2A and PDE9A have emerged as potential candidates shown to improve synaptic plasticity and memory function in animals. However, the functional relevance of their putative different localization in the neuron is not understood. Thus, this study aims at elucidating potential presynaptic effects of PDE2A inhibition in comparison to the inhibition of PDE9A. For this purpose, we used paired-pulse facilitation (PPF), a model of short-term synaptic plasticity related to presynaptic function. First, we performed a series of experiments to validate the model in acute rat hippocampal slices using several reference substances including calcium channel blockers, glutamatergic receptor antagonists, and GPCR agonists. Second, we analysed the effect of PDE2A and PDE9A inhibition and their role regulating the influence that the second messengers cAMP and cGMP exert on basal transmission. Our results show that the interplay between the adenylyl cyclase activator forskolin, the soluble guanylyl cyclase activator BAY 41-8543 and the PDE2A inhibitor PF-999 reveals a primarily presynaptic mechanism of action of PDE2A inhibition. On the contrary, inhibition of PDE9A did not alter PPF under similar conditions. In conclusion, these data provide new evidence supporting a role of PDE2A modulating short-term synaptic plasticity. Moreover, this function of PDE2A is suggested to rely on an active modulation of the cAMP hydrolysis as a response to changes in cGMP levels at the presynaptic level.

Topics: Adenosine; Animals; CA1 Region, Hippocampal; Cadmium Chloride; Colforsin; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Long-Term Potentiation; Male; Morpholines; Neurons; Oxadiazoles; Presynaptic Terminals; Pyrimidines; Quinoxalines; Rats; Rats, Wistar

We investigated the role of nitric oxide (NO) on mitochondrial complexes activity, following short-term non-desensitizing activation of AMPA receptors with kainate (KA) plus cyclothiazide (CTZ), in cultured rat hippocampal neurons. In these conditions, we observed a decrease in the activity of mitochondrial complexes I, II/III, and IV. A selective neuronal nitric oxide synthase inhibitor, 7-Nitroindazole, prevented the decrease in the activity of mitochondrial complex I, but not for the other complexes. Exposure to KA plus CTZ also increased cyclic GMP levels significantly, and led to increased levels of 3-nitrotyrosine, a biomarker for peroxynitrite production. Taken together, our results suggest that non-desensitizing activation of AMPA receptors causes inhibition of mitochondrial complex I via peroxynitrite.

Topics: Analysis of Variance; Animals; Antihypertensive Agents; Benzothiadiazines; Cells, Cultured; Cyclic GMP; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Immunohistochemistry; Kainic Acid; Microtubule-Associated Proteins; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Proton Pumps; Quinoxalines; Rats; Receptors, AMPA; Tyrosine

The mechanisms regulating the cerebellar microcirculation during neural activity are poorly understood. One of the major neural inputs to the cerebellar cortex is the climbing fiber (CF), a pathway that uses excitatory amino acids, including glutamate, as a transmitter. We studied whether CF activation increases cerebellar blood flow (BFcrb) and, if so, we investigated the role of glutamate receptors, nitric oxide (NO) and cGMP, in the response.. The CF were activated by harmaline administration (40 mg/kg, i.p.) in halothane-anesthetized rats with a cranial window placed over the cerebellar vermis. BFcrb was monitored by a laser-Doppler probe, and arterial pressure and blood gases were controlled.. With Ringer superfusion, harmaline produced sustained increases in BFcrb that peaked 20 minutes after administration (+115 +/- 13%; n=6; P<.05). The increases in BFcrb were substantially reduced by superfusion with tetrodotoxin (10 micromol/L; -91 +/- 5%; n=5; P<.05 from Ringer). The response was also attenuated by the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxaline (100 micromol/L; -70 +/- 6%; P<.05; n=5), but not by the N-methyl-D-aspartate receptor blocker 2-amino-5-phosphonopentanoic acid (500 micromol/L; P>.05; n=5). The response was attenuated by the nonselective NO synthase (NOS) inhibitor nitro-L-arginine (1 mmol/L; -73 +/- 5%; n=6) or by 7-NI (50 mg, i.p.; -71 +/- 5%; n=5), a relatively selective neuronal NOS inhibitor. The soluble guanylyl cyclase inhibitor 1H-1,2,4oxadiazolo[4,3-a]quinoxalin-1-one (100 micromol/L) attenuated the response to harmaline (-73 +/- 5; P<.05; n=6) but not to superfusion with adenosine (P>.05; n=5) or 8-bromo-cGMP (P>.05; n=5).. Activation of the CF system increases BFcrb. The response depends on activation of glutamate receptors and is in large part mediated by NO via stimulation of soluble guanylyl cyclase. Glutamate receptors NO and cGMP are important factors in the mechanisms of functional hyperemia in cerebellar cortex.

Topics: 2-Amino-5-phosphonovalerate; Animals; Blood Pressure; Carbon Dioxide; Cerebellum; Cyclic GMP; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glutamic Acid; Harmaline; Male; Nerve Fibers; Nitric Oxide; Nitroarginine; Oxygen; Partial Pressure; Penicillamine; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; S-Nitroso-N-Acetylpenicillamine; Tetrodotoxin; Ultrasonography, Doppler, Transcranial

We investigated whether the neuropeptide galanin affects the nitric oxide synthase/cyclic GMP pathway in rat hippocampus by measuring in vivo the extracellular cyclic GMP levels during microdialysis. Galanin (2.5 and 3.5 nmol; i.c.v.) dose-dependently raised the extracellular levels of cyclic GMP in the ventral but not the dorsal hippocampus. The effect of 3.5 nmol galanin was blocked by local application of tetrodotoxin and inhibited by the high-affinity galanin antagonist M40 (galanin-[1-12]-Pro3-[Ala-Leu]2-Ala amide). The non-competitive N-methyl-D-aspartate receptor antagonist dizocilpine maleate (30 microM infused into the ventral hippocampus or 0.2 mg/kg, i.p.) and the competitive one, 3-([R]-carboxypiperazin-4-yl)-propyl-phosphonic acid (50 microM infused), but not local perfusion of the AMPA antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (15 microM) abolished the galanin-evoked cyclic GMP response in the hippocampus. Inhibitors of nitric oxide synthase, L-Arg(NO2)-OMe.HCl and 7-nitroindazole monosodium salt, applied locally, blocked the galanin-induced increase in hippocampal extracellular cyclic GMP. This increase was also prevented by local application of 1H-(1,2,4)oxadiazolo(4,3a) quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase. The galanin receptors mediating the rise in cyclic GMP reside outside the hippocampus, as galanin (0.35-3 nmol) locally applied had no effect. The results provide in vivo evidence that galanin stimulates the N-methyl-D-aspartate receptor/nitric oxide synthase/cyclic GMP pathway in the ventral hippocampus, which may be of importance in memory processes.

Topics: Animals; Cyclic GMP; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Galanin; Hippocampus; Locomotion; Male; Microdialysis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Peptide Fragments; Piperazines; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

Monitoring of extracellular cGMP during intracerebral microdialysis in freely moving rats permits the study of the functional changes occurring in the glutamate receptor/nitric oxide (NO) synthase/guanylyl cyclase pathway and the relationship of these changes to animal behaviour. When infused into the rat hippocampus in Mg2+-free medium, cyclothiazide, a blocker of desensitization of the AMPA-preferring receptor, increased cGMP levels. The effect of cyclothiazide (300 microM) was abolished by the NO synthase inhibitor L-NARG (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (100 microM). During cyclothiazide infusion the animals displayed a pre-convulsive behaviour characterized by frequent "wet dog shakes" (WDS). Neither L-NARG nor ODQ decreased the WDS episodes. Both cGMP and WDS responses elicited by cyclothiazide were prevented by blocking NMDA receptor function with the glutamate site antagonist CGS 19755 (100 microM), the channel antagonist MK-801 (30 microM) or Mg2+ ions (1 mM). The AMPA/kainate receptor antagonists DNQX (100 microM) and NBQX (100 microM) abolished the WDS episodes but could not inhibit the cyclothiazide-evoked cGMP response. DNQX or NBQX (but not MK-801) elevated, on their own, extracellular cGMP levels. The cGMP response elicited by the antagonists appears to be due to prevention of a glutamate-dependent inhibitory GABAergic tone, since infusion of bicuculline (50 microM) caused a strong cGMP response. The results suggest that (a) AMPA/kainate receptors linked to the NO/cGMP pathway in the hippocampus (but not NMDA receptors) are tonically activated and kept in a desensitized state by endogenous glutamate; (b) blockade of AMPA/kainate receptor desensitization by cyclothiazide leads to endogenous activation of NMDA receptors; (c) the hippocampal NO/cGMP system is under a GABAergic inhibitory tone driven by non-NMDA ionotropic receptors; (d) the pre-convulsive episodes observed depend on hippocampal NMDA receptor activation but not on NO and cGMP production.

Topics: Animals; Behavior, Animal; Benzothiadiazines; Cyclic GMP; Diuretics; Drug Interactions; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Hippocampus; Male; Nitric Oxide; Nitric Oxide Synthase; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors

Intrahippocampal perfusion of bicuculline (50 microM) in Mg2+-free medium caused elevation of extracellular cGMP and epileptic-like behaviour. Both effects were partially prevented by blocking NMDA receptors with MK-801 or Mg2+ ions. Similarly, the GABA(B) receptor antagonists CGP52432 (0.1-30 microM) and CGP35348 (0.3-1 mM) evoked increases of extracellular cGMP. CGP52432 also elicited behavioural responses ranging from wet dog shakes to convulsions. MK-801 or Mg2+ ions reduced the effects of CGP52432. Local application of muscimol (100-300 microM) or (-)baclofen (300 microM) caused inhibition of extracellular cGMP. Administration of the AMPA/kainate receptor antagonist NBQX (100 microM) caused cGMP elevation which was almost abolished by co-perfusion of muscimol and (-)baclofen. In the presence of physiological Mg2+, perfusion of AMPA (30 microM) failed to affect cGMP levels, although rats displayed wet dog shakes episodes. When AMPA was co-perfused with low concentrations of bicuculline or CGP52432, cGMP elevations were observed in 60% of the rats. Addition of both antagonists to AMPA resulted in 85% of rats displaying a cGMP response. To conclude: (a) extracellular hippocampal cGMP is controlled by inhibitory GABA(A) and GABA(B) receptors tonically activated through GABAergic interneurons receiving AMPA/kainate-mediated glutamatergic inputs; (b) the GABAergic receptors are not endogenously saturated and can be further stimulated by exogenous agonists; (c) blockade of the GABA-mediated inhibition causes increase of cGMP and epileptic-like behaviour, due largely to endogenous activation of NMDA receptors; (d) reproducible cGMP responses to AMPA can be observed when the inhibitory GABAergic inputs to the NO/guanylyl cyclase system are blocked, confirming the previously proposed existence of AMPA/kainate receptors able to increase the nucleotide synthesis.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Behavior, Animal; Cyclic GMP; Epilepsy; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; Hippocampus; Microdialysis; Nitric Oxide; Quinoxalines; Rats; Receptors, AMPA; Receptors, GABA-A; Receptors, GABA-B; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate