thiourea and 7-nitroindazole

Nitric oxide (NO) has been recently shown to enhance µ-opioid receptor (MOR) desensitisation in locus coeruleus (LC) neurons. The aim of this study was to evaluate by single-unit extracellular recordings in rat brain slices whether the neuronal NO synthase is involved in MOR desensitisation in LC neurons. As expected, a high concentration of the opioid agonist Met(5)-enkephalin (ME; 10 µM, 10 min) strongly desensitised the inhibition induced by a test application of ME (0.8 µM, 1 min), whereas lower ME concentrations (1 and 3 µM) only weakly desensitised it. The neuronal NO synthase inhibitors 7-nitroindazole (10-100 µM), S-methyl-L-thiocitrulline (0.01-10 µM) and N(ω)-propyl-L-arginine (1-10 µM) attenuated ME (10 µM)-induced opioid desensitisation, although the endothelial NO synthase inhibitor N(5)-(1-iminoethyl)-L-ornithine (3-30 µM) failed to change it. The NO donor sodium nitroprusside (1 mM), but not its inactive analog potassium ferricyanide (1 mM), enhanced the ME (3 µM)-induced desensitisation and prevented the effect of S-methyl-L-thiocitrulline (10 µM). Sodium nitroprusside (1 mM) failed to change the desensitisation of α2-adrenoceptors by noradrenaline (100 µM, 10 min). These results suggest the contribution of NO and a neuronal type of NO synthase in homologous MOR desensitisation in rat LC neurons.

Topics: Action Potentials; Animals; Arginine; Citrulline; Dose-Response Relationship, Drug; Enkephalin, Methionine; Ferricyanides; Indazoles; Locus Coeruleus; Male; Neural Inhibition; Nitric Oxide Synthase Type I; Nitroprusside; Norepinephrine; Ornithine; Rats; Receptors, Opioid, mu; Thiourea

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO-producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)-expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single-cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long-term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I(NaP) ), voltage-gated Ca currents (I(Ca) ) and small-conductance calcium-activated potassium (SK) channels. We show that NOS inhibitors 7-nitroindazole and S-methyl-l-thiocitrulline resulted in a decrease in I(NaP) , and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I(NaP) , riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I(Ca) , and blocked spontaneous and depolarization-induced spiking, suggesting that intrinsic NO controlled I(Ca) via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self-produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.

Topics: Action Potentials; Animals; Apamin; Calcium Channels; Citrulline; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Guanylate Cyclase; Indazoles; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Potassium Channel Blockers; Receptors, Cytoplasmic and Nuclear; Riluzole; Small-Conductance Calcium-Activated Potassium Channels; Snails; Sodium Channel Blockers; Sodium Channels; Soluble Guanylyl Cyclase; Thiourea

Direct renal nitric oxide (NO) measurements were infrequent and no simultaneous measurements of renal cortical and medullary NO and local perfusion. Large-surface NO electrodes were placed in renal cortex and medulla of anaesthetised rats; simultaneously, renal blood flow (RBF, index of cortical perfusion) and medullary laser-Doppler flux (MBF) were determined. NO synthase inhibitors: nonselective (L-NAME) or selective for neuronal NOS (nNOS) (S-methyl-thiocitrulline, SMTC), and NO donor (SNAP), were used to manipulate tissue NO. Baseline tissue NO was significantly higher in medulla (703+/-49 NM) than in cortex (231+/-17 nM). Minimal cortical and medullary NO current measured after maximal L-NAME dose (2.4 mg kg(-1) i.v.) was taken as tissue NO zero kevel. This dose decreased RBF and MBF significantly (-43%). SMTC, 1.2 mg kg(-1) h(-1) i.v., significantly decreased tissue NO by 105+/-32 nM in cortex and 546+/-64 nM in medulla, RBF and MBF decreased 30% and 20%, respectively. Renal artery infusion of SNAP, 0.24 mg kg(-1) min(-1) significantly increased tissue NO by 139+/-18 nM in cortex and 948+/-110 nM in medulla. Since inhibition of nNOS decreased medullary NO by 80% and MBF by 20% only, this isoform has probably minor role in the maintenance of medullary perfusion.

Topics: Anesthesia; Animals; Blood Flow Velocity; Citrulline; Enzyme Inhibitors; Indazoles; Ion-Selective Electrodes; Kidney; Kidney Cortex; Kidney Medulla; Laser-Doppler Flowmetry; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Polarography; Rats; Rats, Wistar; Renal Circulation; Reproducibility of Results; S-Nitroso-N-Acetylpenicillamine; Thiourea; Time Factors

In order to understand the heme distal structure of neuronal nitric oxide synthase (nNOS), we studied cyanide binding to the ferric wild-type and substrate binding site mutants, Glu592Ala and Tyr588His, of the isolated oxygenase domain in the absence and presence of substrates and inhibitors. Cyanide bound to isolated heme-bound oxygenase domains (nNOSox) in the absence of the substrates with the dissociation constant (K(d)) of 3.1 mM. The presence of the substrates, L-Arg and NHA, did not change the K(d) value. However, cyanide binding was almost abolished in the presence of inhibitors such as NAME, thiocitrulline and 7-NI. The effect of the inhibitors were not observed for the Glu592Ala mutant, while similar strong inhibiting effects were observed for the Tyr588His mutant. We discuss the binding fashion of those inhibitors to the heme substrate binding site of nNOS.

Topics: Arginine; Binding Sites; Citrulline; Cyanides; Enzyme Inhibitors; Indazoles; Mutagenesis, Site-Directed; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Spectrophotometry, Ultraviolet; Thiourea

Cyclooxygenase-2 (COX-2) expression in rat kidney is localized to the macula densa and the immediately proximal cTALH and increases after salt restriction. Either ACE inhibitors or AT1 receptor blockers increase COX-2 expression in both control and salt-restricted animals, suggesting that the RAS activation feedback inhibits renal cortical COX-2 expression. To determine whether increased COX-2 expression in response to ACE inhibition mediated increases in renin production, rats were treated with Captopril for 1 week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the Captopril group. This increase was partially reversed by simultaneous treatment with SC58236. Kidney renin activity also increased in the Captopril group compared with control, which was also significantly inhibited by SC58236 treatment. Because of the localization of bNOS to MD and surrounding cTALH, the current study investigated the role of NO in the regulation of COX-2 expression. Rats were fed a normal diet, low salt diet or low salt diet combined with captopril and half of them were treated with the neuronal NOS inhibitor, 7-NI, and half with vehicle. After 7 days, mRNA was extracted and the microsome proteins purified from renal cortex. COX-2 mRNA expression was measured by Northern-blot and normalized with GAPDH. 7-NI treatment decreased COX-2 mRNA and immunoreactive COX-2 expression in each group. In summary, these studies indicate that COX-2 from macula densa/cTALH is a regulator of renin production and release. Angiotensin II may be a negative regulator of cTALH/macula densa COX-2 expression, and NO may mediate increased renal cortical COX-2 expression seen in volume depletion. These studies suggest important interactions between the NO and COX-2 systems in the regulation of arteriolar tone and the renin-angiotensin system by the macula densa.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Citrulline; Cyclooxygenase 2; Diet, Sodium-Restricted; Enzyme Inhibitors; Indazoles; Isoenzymes; Kidney; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Prostaglandin-Endoperoxide Synthases; Rats; Renin-Angiotensin System; RNA, Messenger; Thiourea

We have previously shown that cyclooxygenase-2 (COX-2) is localized to the cortical thick ascending limb of the loop of Henle (cTALH)/macula densa of the rat kidney, and expression increases in response to low-salt diet and/or angiotensin-converting enzyme (ACE) inhibition. Because of the localization of neuronal nitric oxide synthase (nNOS) to macula densa and surrounding cTALH, the present study investigated the role of nitric oxide (NO) in the regulation of COX-2 expression. For in vivo studies, rats were fed a normal diet, low-salt diet or low-salt diet combined with the ACE inhibitor captopril. In each group, one-half of them were treated with the nNOS inhibitors 7-nitroinidazole (7-NI) or S-methyl-thiocitrulline. Both of these NOS inhibitors inhibited increases in COX-2 mRNA and immunoreactive protein in response to low salt and low salt+captopril. For in vitro studies, COX-2 expression was studied in primary cultures of rabbit cTALH cells immunodisssected with Tamm-Horsfall antibody. Basal COX-2 immunoreactivity expression was stimulated by S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, and intracellular cGMP concentration. The cultured cells expressed immunoreactive nNOS, and 7-NI inhibited basal COX-2 immunoreactivity expression, which could be partially overcome by cGMP. In summary, these studies indicate that NO is a mediator of increased renal cortical COX-2 expression seen in volume depletion and suggest important interactions between the NO and COX-2 systems in the regulation of arteriolar tone and the renin-angiotensin system by the macula densa.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Cells, Cultured; Citrulline; Cyclooxygenase 2; Dibutyryl Cyclic GMP; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Indazoles; Isoenzymes; Kidney Cortex; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Penicillamine; Prostaglandin-Endoperoxide Synthases; Rabbits; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Chloride, Dietary; Thiourea

Methamphetamine (METH)-induced dopaminergic neurotoxicity is associated with hyperthermia. We investigated the effect of several neuronal nitric oxide synthase (nNOS) inhibitors on METH-induced hyperthermia and striatal dopaminergic neurotoxicity. Administration of METH (5 mg/kg; q. 3 h x 3) to Swiss Webster mice produced marked hyperthermia and 50-60% depletion of striatal dopaminergic markers 72 h after METH administration. Pretreatment with the nNOS inhibitors S-methylthiocitrulline (SMTC; 10 mg/kg) or 3-bromo-7-nitroindazole (3-Br-7-NI; 20 mg/kg) before each METH injection did not affect the persistent hyperthermia produced by METH, but afforded protection against the depletion of dopaminergic markers. A low dose (25 mg/kg) of the nNOS inhibitor 7-nitroindazole (7-NI) did not affect METH-induced hyperthermia, but a high dose (50 mg/kg) produced significant hypothermia. These findings indicate that low dose of selective nNOS inhibitors protect against METH-induced neurotoxicity with no effect on body temperature and support the hypothesis that nitric oxide (NO) and peroxynitrite have a major role in METH-induced dopaminergic neurotoxicity.

Topics: Animals; Citrulline; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Fever; Indazoles; Male; Methamphetamine; Mice; Neostriatum; Nerve Degeneration; Neurons; Neuroprotective Agents; Neurotoxins; Nitric Oxide; Nitric Oxide Synthase; Thiourea

Mechanisms of the modulation of sympathetic activity by neuronal NO were studied in vagotomized anesthetized pigs. Inhibition of neuronal NO synthase (nNOS) within the brain stem by intracerebroventricular (ICV) administration of 7-nitroindazole (7-NI, 1 mmol/L) or S-methyl-L-thiocitrulline (MeTC, 0.1 mmol/L) caused slight increases in renal sympathetic nerve activity (RSNA) but did not affect arterial blood pressure (BP) or cardiac output (CO). However, the sympathoexcitatory effects of glutamate (0.5 mL, 0.1 mol/L ICV) that were associated with marked increases in BP, CO, and heart rate were potentiated by both nNOS inhibitors. Furthermore, 7-NI and MeTC significantly enhanced the responses of RSNA, BP, and CO to activation of somatosympathetic reflexes via stimulation of the left greater sciatic nerve (nervus ischiadicus, 10 to 20 V, 30 Hz, 1-millisecond pulses). Subsequent systemic inhibition of either the neuronal (by 7-NI) or all isoforms of NOS by NG-nitro-L-arginine-methyl ester (20 mg/kg) had no significant additional effects on these responses. The effects of NOS inhibition were effectively counteracted by the endogenous NOS substrate L-arginine and by S-nitroso-N-acetyl-penicillamine (SNAP), a stable analogue of endogenous S-nitroso factors. Disruption of sympathoinhibitory baroreflex mechanisms by bilateral cutting of the carotid sinus nerves caused increases in RSNA and slightly increased responses to all excitatory stimuli but had no effects on the actions of the NOS inhibitors or SNAP. These results suggest that modulation of glutamate effects by nNOS-derived NO may be an important mechanism by which NO affects sympathetic activity in pigs.

Topics: Animals; Arginine; Brain Stem; Citrulline; Enzyme Inhibitors; Glutamic Acid; Heart Rate; Hemodynamics; Indazoles; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Penicillamine; S-Nitroso-N-Acetylpenicillamine; Swine; Sympathetic Nervous System; Thiourea; Vagotomy

The N-methyl-D-aspartate (NMDA) receptor-nitric oxide (NO) pathway has been linked to opiate withdrawal. Pretreatments with four inhibitors of NO synthase, 7-nitro indazole, 3-bromo-7-nitro indazole, S-methyl-L-thiocitrulline and aminoguanidine, which exhibit different isoform selectivity in vitro, were evaluated for their ability to attenuate signs of naloxone-precipitated morphine withdrawal. In separate experiments, effects of NO synthase inhibitors on blood pressure were measured in naive and morphine-dependent rats. 7-Nitro indazole, 3-bromo-7-nitro indazole and S-methyl-L-thiocitrulline, which are specific inhibitors of the constitutive isoforms, produced dose-dependent reductions of several signs of withdrawal. Blood pressure was unaffected by the indazoles, whereas S-methyl-L-thiocitrulline produced a strong vasoconstrictor response. Aminoguanidine, which selectively inhibits inducible NO synthase, reduced fewer signs of opioid withdrawal, had a lower relative potency and exhibited no vasopressor activity. These data suggest that constitutive isoforms, but not the inducible isoform of NO synthase, have a primary role in NO-mediated processes that modulate the opioid withdrawal syndrome in the rat.

Topics: Analysis of Variance; Animals; Blood Pressure; Citrulline; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction; Enzyme Inhibitors; Guanidines; Heart Rate; Indazoles; Isoenzymes; Male; Morphine; Naloxone; Narcotic Antagonists; Nitric Oxide Synthase; Rats; Rats, Inbred F344; Substance Withdrawal Syndrome; Thiourea

We examined the effects of potent neuronal nitric oxide synthase inhibitors, 3-bromo-7-nitro indazole (3-Br-7-NI) and S-methyl-L-thiocitrulline (S-Me-TC) on general behaviour, vigilance stages and electroencephalographic (EEG) power spectra in rats. In addition, we studied the effect of 7-nitro indazole (7-NI) on EEG power spectra in rats during dark and light periods. 3-Br-7-NI induced ptosis and decrease of slow wave sleep and rapid eye movement sleep in the rat. 7-NI and 3-Br-7-NI reduced the EEG power density in all frequency bands in the rat, suggesting a depression of central neuronal activity. This effect of 7-NI was more prominent during the day than during the night, indicating a circadian variation in the nitric oxide synthase (NOS) response to NOS inhibitor. EEG power was the most reduced in the 7-9 Hz range of the rhythmic slow activity (theta rhythm), which is in accordance with decreased locomotion observed following administration of NOS inhibitors. Although S-Me-TC is the most potent NOS inhibitor in vitro experiments, it had less effect on vigilance and EEG power in the rat than other NOS inhibitors used in this study, probably due to its short lasting and blood pressure raising effect. The present results indicate that nitric oxide exerts an excitatory and circadian dependent effect in the central neuronal structures involved in the regulation of vigilance.

Topics: Animals; Arousal; Child; Citrulline; Dimethyl Sulfoxide; Electroencephalography; Electromyography; Humans; Indazoles; Motor Activity; Neurons; Nitric Oxide Synthase; Rats; Rats, Wistar; Sleep; Thiourea

Rat striata were exposed to 15 mM quinolinic acid (QUIN), or QUIN plus the nitric oxide synthase inhibitors S-methyl-L-thiocitrulline dihydrochloride (L-MIN) or 7-nitroindazole monosodium salt (7-NINA) for 21 days. Co-administration of 100 microM or 1 mM L-MIN with QUIN significantly reduced lesion volume compared to QUIN alone. Co-administration of 1 microM or 10 microM L-MIN with QUIN had no significant effect. There was no significant effect of 7-NINA co-administered with QUIN compared to QUIN alone. L-MIN reduction of lesion volume supports the contention that neuronal nitric oxide synthase is a mediator of excitotoxic injury.

Topics: Animals; Citrulline; Enzyme Inhibitors; Indazoles; Male; Microdialysis; Neostriatum; Neurotoxins; Nitric Oxide Synthase; Quinolinic Acid; Rats; Rats, Sprague-Dawley; Thiourea