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

This study characterizes the receptor subtypes and investigates some of the mechanisms by which glutamate, injected intraplantarly (i.pl.) into the mouse paw, produces nociception and paw oedema. I.pl. injection of glutamate induced a rapid-onset, dose-related pain response associated with oedema formation, with mean ED(50) values of 2.6 (1.6-4.3) and 0.5 (0.4-0.7) micromol/kg, respectively. Pretreatment with Chicago sky blue 6B (100 microg/kg), an inhibitor of glutamate uptake, caused a significant (about sixfold) reduction of the mean ED(50) value for glutamate-induced nociception, but not paw oedema. NMDA receptor antagonist MK 801, given by systemic (i.p.), intracerebroventricular (i.c.v.), i.pl. or intrathecal (i.t.) routes, produced graded inhibition of glutamate-induced nociception. Non-NMDA receptor antagonists NBQX or GAMS, metabotropic antagonist E4CPG, and also the antagonist that acts at the NMDA receptor-associated glycine binding site felbamate, significantly inhibited the nociception induced by glutamate. L(omega)-N-nitro-arginine (given i.p., i.t., i.pl. or i.c.v.) prevented the nociception and paw oedema caused by glutamate, an effect that was reversed by L-arginine but not by D-arginine. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), given i.pl., greatly potentiated glutamate-induced nociception and oedema formation. Finally, the i.pl. injection of glutamate was accompanied by a graded increase in the nitrite levels of the hindpaw exudate. It is concluded that the nociception caused by i.pl. injection of glutamate probably involves the activation of NMDA and non-NMDA receptors by a mechanism which largely depends on the activation of L-arginine-nitric oxide pathway. Glutamate-induced paw oedema seems to be primarily mediated by non-NMDA ionotropic glutamate receptors and release of nitric oxide.

Topics: Animals; Azo Compounds; Coloring Agents; Dizocilpine Maleate; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Extremities; Glutamic Acid; Glutamine; Male; Mice; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitroarginine; Nociceptors; Penicillamine; Quinoxalines; Trypan Blue

Crus II is an area of the cerebellar cortex that receives trigeminal afferents from the perioral region. We investigated the mechanisms of functional hyperemia in cerebellum using activation of crus II by somatosensory stimuli as a model. In particular, we sought to determine whether stimulation of the perioral region increases cerebellar blood flow (BFcrb) in crus II and, if so, whether the response depends on activation of 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-kainate receptors and nitric oxide (NO) production. Crus II was exposed in anesthetized rats, and the site was superfused with Ringer. Field potentials were recorded, and BFcrb was measured by laser-Doppler flowmetry. Crus II was activated by electrical stimulation of the perioral region (upper lip). Perioral stimulation evoked the characteristic field potentials in crus II and increased BFcrb (34 +/- 6%; 10 Hz-25 V; n = 6) without changing arterial pressure. The BFcrb increases were associated with a local increase in glucose utilization (74 +/- 8%; P < 0.05; n = 5) and were attenuated by the AMPA-kainate receptor antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline (-71 +/- 3%; 100 microM; P < 0.01; n = 5). The neuronal NO synthase inhibitor 7-nitroindazole (7-NI, 50 mg/kg; n = 5) virtually abolished the increases in BFcrb (-90 +/- 2%; P < 0.01) but did not affect the amplitude of the field potentials. In contrast, 7-NI attenuated the increase in neocortical cerebral blood flow produced by perioral stimulation by 52 +/- 6% (P < 0.05; n = 5). We conclude that crus II activation by somatosensory stimuli produces localized increases in local neural activity and BFcrb that are mediated by activation of glutamate receptors and NO. Unlike in neocortex, in cerebellum the vasodilation depends almost exclusively on NO. The findings underscore the unique role of NO in the mechanisms of synaptic function and blood flow regulation in cerebellum.

Topics: Animals; Blood Pressure; Brain Stem; Carbon Dioxide; Cerebellar Cortex; Cerebellum; Cerebrovascular Circulation; Electric Stimulation; Enzyme Inhibitors; Glucose; Hyperemia; Indazoles; Laser-Doppler Flowmetry; Male; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Somatosensory Cortex; Tetrodotoxin

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 used transgenic mice with Purkinje cell dysfunction (PO3 line) to study the role of these neurons in the increase in cerebellar blood flow (BFcrb) produced by stimulation of the cerebellar parallel fibers (PF). Mice (age 8-10 wk) were anesthetized (halothane) and artificially ventilated. Arterial pressure and end-tidal CO2 were monitored continuously. Arterial blood gases were measured. The PF were stimulated electrically (100 microA, 30 Hz; 40 s), and the increases in BFcrb were monitored by a laser-Doppler flow probe. First, we characterized the increases in BFcrb and the field potentials produced by PF stimulation in normal mice. PF stimulation evoked the typical field potentials and increased BFcrb by 60 +/- 4% (100 microA, 30 Hz; n = 10). The increases in BFcrb were attenuated by the broad-spectrum glutamate receptor antagonist kynurenate (-84 +/- 3%; P < 0.05 analysis of variance; n = 5), by the DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (-62 +/- 6%; P < 0.05; n = 5), and by the nitric oxide synthase inhibitor N omega-nitro-L-arginine (-46 +/- 7%; P < 0.05; n = 5). In PO3 transgenic mice, the increases in BFcrb produced by PF stimulation were reduced (P < 0.001) at every stimulus intensity and frequency tested (residual increase at 100 microA, 30 Hz: 19 +/- 2%; n = 6). The field potentials evoked by PF stimulation also were abnormal in that they lacked the late negative wave (n = 6), a finding consistent with lack of depolarization of Purkinje cells. The residual flow response in the transgenics was abolished by N omega-nitro-L-arginine (n = 5; P > 0.05). Ultrastructural studies showed that the density of PF-Purkinje cell synapses is reduced in PO3 mice, whereas the morphology of molecular layer interneurons (stellate cells) is normal. The findings suggest that Purkinje cells are responsible for a sizable component of the flow response whereas molecular layer interneurons mediate the remainder of the response. The study provides evidence that mouse mutants with spontaneous or genetically engineered cerebellar abnormalities could be useful to study the cellular and molecular correlates of functional hyperemia in the central nervous system.

Topics: Action Potentials; Animals; Blood Flow Velocity; Cerebellum; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Kynurenic Acid; Laser-Doppler Flowmetry; Mice; Mice, Transgenic; Microscopy, Electron; Nitric Oxide Synthase; Nitroarginine; Purkinje Cells; Quinoxalines; Receptors, AMPA; Synapses

Hyperbaric O2 exposure causes seizures by an unknown mechanism. Cerebral blood flow (CBF) may affect seizure latency, although no studies have demonstrated a direct relationship. Awake rats (male, Sprague-Dawley, 350-450 g), instrumented for measuring electroencephalographic activity (EEG) and CBF (laser-Doppler flowmetry), were exposed to 100% O2 at 4 or 5 atm (gauge pressure) until EEG seizures. Compression with O2 caused vasoconstriction to about 70% of control flow that was maintained for various times. CBF then suddenly, but transiently, increased at a time that was reliably related to seizure latency (r=0.8, p<0.01). Additional animals were treated with agents that have diverse pharmacology and their effects on CBF and latency were measured. Glutamate receptor antagonists MK-801 (1 or 4 mg/kg) and ketamine (20-100 mg/kg) significantly increased CBF by 60-80% and decreased seizure latency from about 17+/-8 min (+/-S.D.) in controls to 5+/-1 and 6+/-2 min, respectively. In opposite, a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNA)(25 mg/kg) decreased CBF by about 25% and increased time to seizure to 60+/-16 min. If these effects occur in humans, non-invasive measurement of CBF could potentially improve the safety and reliability of hyperbaric O2 usage in clinical and diving applications. It also appears that the effect of drugs on seizure latency can be explained, at least in part, by their effect on CBF.

Topics: Analysis of Variance; Animals; Cerebrovascular Circulation; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hyperbaric Oxygenation; Ketamine; Male; Nitric Oxide Synthase; Nitroarginine; Quinoxalines; Rats; Rats, Sprague-Dawley; Reaction Time; Seizures

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[3H]-arginine to L-[3H]-citrulline. 2. Elevation of extracellular K+ to 20, 35 or 60 mM concentration-dependently augmented L-[3H]-citrulline production. The response to 35 mM KCl was abolished by N(G)-nitro-L-arginine (100 microM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl2 concentration up to 10 mM also prevented the K+ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity. 3. However, the effect of high K+ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)-quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine. 4. The 35 mM K+ response was insensitive to omega-conotoxin GVIA (1 microM) and nifedipine (100 microM), but could be prevented in part by omega-agatoxin IVA (0.1 and 1 microM). The inhibition caused by 0.1 microM omega-agatoxin IVA (approximately 30%) was enhanced by adding omega-conotoxin GVIA (1 microM) or nifedipine (100 microM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 microM) the K+-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 microM omega-conotoxin GVIA plus 100 microM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX. 5. It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca2+ entering cells mainly through channels of the P/Q-type. Other Ca2+ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

Topics: Alanine; Arginine; Calcium Channel Blockers; Calcium Channels; Citrulline; Dizocilpine Maleate; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Humans; In Vitro Techniques; Mecamylamine; Nicotinic Antagonists; Nitric Oxide Synthase; Nitroarginine; Potassium; Potassium Chloride; Quinoxalines; Temporal Lobe

We have studied in a well-characterized in vitro neuronal system, cultures of cerebellar granule cells, the toxicity of polyamines endogenously present in the brain: spermine, spermidine, and putrescine. Twenty-four-hour exposure of mature (8 days in vitro) cultures to 1-500 microM spermine resulted in a dose-dependent death of granule cells, with the half-maximal effect being reached below 50 microM concentration. Putrescine was moderately toxic but only at 500 microM concentration. Spermidine was tested at 50 and 100 microM concentration and its toxicity was evaluated to be about 50% that of spermine. Neuronal death caused by spermine occurred, at least in part, by apoptosis. Spermine toxicity was completely prevented by competitive (CGP 39551) and noncompetitive (MK-801) antagonists of the NMDA receptor, but was unaffected by a non-NMDA antagonist (NBQX) or by antagonists of the polyamine site present on the NMDA receptor complex, such as ifenprodil. A partial protection from spermine toxicity was obtained through the simultaneous presence of free radical scavengers or through inhibition of the free radical-generating enzyme nitric oxide synthase, known to be partially effective against direct glutamate toxicity. The link between spermine toxicity and glutamate was further strengthened by the fact that, under culture conditions in which glutamate toxicity was ineffective or much reduced, spermine toxicity was absent or very much decreased. Exposure to spermine was accompanied by a progressive accumulation of glutamate in the medium of granule cell cultures. This was attributed to glutamate leaking out from dying or dead cells and was substantially prevented by the simultaneous presence of MK-801 or CGP 39551. The present results demonstrate that polyamines are toxic to granule cells in culture and that this toxicity is mediated through the NMDA receptor by interaction of exogenously added polyamines with endogenous glutamate released by neurons in the medium. The involvement of brain polyamines, in particular spermine and spermidine, in excitotoxic neuronal death is strongly supported by our present results.

Topics: 2-Amino-5-phosphonovalerate; Animals; Apoptosis; Aspartic Acid; Butylated Hydroxytoluene; Cells, Cultured; Cerebellar Cortex; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Free Radical Scavengers; Glutamic Acid; L-Lactate Dehydrogenase; Male; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Nitric Oxide Synthase; Nitroarginine; Piperidines; Putrescine; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermidine; Spermine; Vitamin E

The endothelium-derived relaxing factor, probably nitric oxide (NO), is a potent vasodilator that regulates the vascular tone in several vascular beds, including the brain. We explored the possibility that NO might be of importance for the increase of cerebral blood flow (CBF) associated with activity of the well-defined neuronal circuits of the rat cerebellar cortex. Laser-Doppler flowmetry was used to measure increases of cerebellar blood flow evoked by trains of electrical stimulations of the dorsal surface. The evoked increases of CBF were frequency-dependent, being larger on than off the parallel fiber tracts, suggesting that conduction along parallel fibers and synaptic activation of target cells were important for the increase of CBF. This was verified experimentally since the evoked CBF increases were abolished by tetrodotoxin and reduced by 10 mM Mg2+ and selective antagonists for non-N-methyl-D-aspartate receptors. The cerebellar cortex contains high levels of NO synthase. This raised the possibility that NO was involved in the increase of CBF associated with neuronal activation. NO synthase inhibition by topical application of NG-nitro-L-arginine attenuated the evoked CBF increase by about 50%. This effect was partially reversed by pretreatment with L-arginine, the natural substrate for the enzyme, while NG-nitro-D-arginine, the inactive enantiomer, had no effect on the evoked CBF increases. Simultaneous blockade of non-N-methyl-D-aspartate receptors and NO synthase had no further suppressing effect on the blood flow increase than either substance alone, suggesting that the NO-dependent flow rise was dependent on postsynaptic mechanisms. These findings are consistent with the idea that local synthesis of NO is involved in the transduction mechanism between neuronal activity and increased CBF.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Amino Acid Oxidoreductases; Amino Acids; Animals; Anticonvulsants; Arginine; Carbon Dioxide; Cerebellar Cortex; Cerebrovascular Circulation; Drug Interactions; Electric Stimulation; Isomerism; Magnesium; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Regional Blood Flow; Tetrodotoxin