Selective Neuronal Nitric Oxide Synthase Inhibitor Research Articles

Nitric oxide modulates elicitation of reflex swallowing from the pharynx in rats

The pharynx is very important for elicitation of reflex swallowing. The region of the pharynx is innervated by the pharyngeal branch of the glossopharyngeal nerve (GPN-ph). Nitric oxide (NO) plays an important role in various physiological functions. The purpose of this study is to investigate the contribution of NO to reflex swallowing evoked by electrical stimulation of the GPN-ph. Swallowing was evoked in urethane-anesthetized rats by application of repetitive electrical stimulation (10- to 20-microA amplitude, 10- to 20-Hz frequency, 1.0-ms duration) to the central cut end of the GPN-ph or superior laryngeal nerve. Swallowing was identified by electromyographic activity of the mylohyoid muscle. Latency to the first swallow and the interval between swallows were measured. Intravenous administration of N(G)-nitro-L-arginine (L-NNA, 0.6 mg/kg), a nonselective inhibitor of NO synthase (NOS), extremely prolonged latency to the first swallow and the interval between swallows evoked by the GPN-ph. Intraperitoneal administration of 7-nitroindazole (5.0 mg/kg), a selective inhibitor of neuronal NOS, significantly prolonged latency to the first swallow and the interval between swallows evoked by the GPN-ph. Administration of L-arginine (an NO donor, 500 mg/kg) and sodium nitroprusside (an NO releaser, 0.6 mg/kg) restored the suppression of swallowing induced by the NOS inhibitor. Superior laryngeal nerve-evoked swallowing was suppressed by administration of a higher dose of L-NNA (6.0 mg/kg). Swallowing evoked by water stimulation of the pharynx was also suppressed by L-NNA (0.6 mg/kg). These results suggest that NO plays an important role in signal processing for initiation of reflex swallowing from the pharynx.

The effects of tempol, 3-aminobenzamide and nitric oxide synthase inhibitors on acoustic injury of the mouse cochlea

Oxygen free radicals have been implicated in the pathogenesis of acoustic injury of the cochlea. The purpose of this study was to evaluate the effects of tempol (a superoxide anion scavenger), 3-aminobenzamide (a poly (ADP-ribose) synthetase (PARS) inhibitor), N-nitro- l-arginine (a non-selective nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (a selective neuronal NOS inhibitor) and aminoguanidine (a selective inducible NOS inhibitor) on acoustic injury. Mice were exposed to a 4 kHz pure tone of 110–128 dB SPL for 4 h. Tempol, 3-aminobenzamide or N-nitro- l-arginine was intraperitoneally administered immediately before the onset of acoustic overexposure, while 7-nitroindazole or aminoguanidine was intraperitoneally administered every 12 h starting immediately before the onset of acoustic overexposure. The threshold shift of the auditory brainstem response (ABR) and hair cell loss were then evaluated one and two weeks after acoustic overexposure. Tempol and 3-aminobenzamide significantly protected the cochlea against acoustic injury, whereas the NOS inhibitors did not exert any protective effect. These findings suggest that reactive oxygen species and PARS are involved in acoustic injury of the cochlea. However, further study is necessary to elucidate the roles of nitric oxide and nitric oxide synthase in acoustic injury.

Nitric oxide in obstructive uropathy: role of endothelial nitric oxide synthase.

Nitric oxide (NO) ameliorates fibrosis in experimental obstructive uropathy. Previously, we demonstrated that renal fibrosis was decreased after 2 weeks of unilateral ureteral obstruction in inducible nitric oxide synthase (iNOS) knock-out mice given L-arginine supplemented drinking water. We proposed that the 2 constitutive isoforms of nitric oxide synthase (NOS) mediated down-regulation of renal fibrosis in response to prolonged ureteral obstruction. To determine the specific role of endothelial NOS (eNOS) versus neuronal NOS in modulating renal fibrosis due to obstructive uropathy, we evaluated renal injury following unilateral ureteral obstruction in C57BL/6J mice subjected to biochemical inhibition of the constitutive isoforms of NOS and in eNOS knockout mice. Four groups of C57BL/6J mice were studied. Complete unilateral ureteral obstruction was created by ligating the right ureter at age 8 weeks. A single daily intraperitoneal injection of 30 mg./kg. S-methyl-L-thiocitrulline (SMLT), a selective neuronal and endothelial NOS inhibitor was started 24 hours before ureteral obstruction and administered to half of the study animals. SMLT treated mice and control animals were further subdivided to receive either regular tap water or 1% L-arginine (weight/volume) supplemented water after unilateral ureteral obstruction. Animals were sacrificed on postoperative day 3, 7 or 14. In addition, eNOS knockout mice with unilateral ureteral obstruction were given tap water or L-arginine supplemented water to drink and sacrificed after 14 days. Urine specimens from the bladder and the obstructed renal pelvis along with serum were collected. Nitrite level in each fluid was determined. Renal morphology and cortical thickness were assessed in the normal and obstructed kidneys. Interstitial fibrosis was evaluated using trichrome stain. SMLT was well tolerated by C57BL/6J mice. Serum nitrite levels and nitrite excretion in bladder urine were similar in all SMLT treated groups throughout the duration of unilateral ureteral obstruction. A reduction of pelvic urine nitrite levels by 89%, 68% and 48% versus bladder urine nitrite levels was observed after 3, 7 and 14 days of unilateral ureteral obstruction (p <0.05). Administration of SMLT resulted in a significant increase in bladder urine nitrite level at 7 days and in pelvic urine nitrite levels at 14 days. No significant histological differences in the obstructed kidney were seen after 3, 7 or 14 days of unilateral ureteral obstruction in SMLT treated versus control mice regardless of whether they received tap water or L-arginine supplemented drinking water. In eNOS knockout mice with unilateral ureteral obstruction for 14 days L-arginine supplementation had no effect on pelvic urine nitrite levels and did not alter renal histopathology or cortical thickness. NO production is decreased in the obstructed kidney in mice with unilateral ureteral obstruction. Biochemical inhibition of constitutive NOS did not modulate renal injury after 14 days of unilateral ureteral obstruction. In contrast to previous findings with iNOS knockout mice, dietary supplementation with L-arginine had no effect on the degree of fibrosis in the obstructed kidney in SMLT treated C57BL/6J or eNOS knockout mice. We conclude that NO derived from eNOS within the kidney has a pivotal role in protecting against renal fibrosis in response to unilateral ureteral obstruction.

The cannabinoid receptor antagonist SR-141716A induces penile erection in male rats: Involvement of paraventricular glutamic acid and nitric oxide

The cannabinoid CB1 receptor antagonist SR141716A (0.5, 1 and 2 μg) induces penile erection when injected into the paraventricular nucleus of male rats. The pro-erectile effect of SR 141716A occurs concomitantly with an increase in the concentration of NO 2 − and NO 3 − in the paraventricular dialysate obtained by means of intracerebral microdialysis. Both penile erection and NO 2 − increase induced by SR 141716A were reduced by the prior injection into the PVN of the cannabinoid CB1 agonists WIN 55,212-2 (5 μg) or HU 210 (5 μg), given into the paraventricular nucleus at doses unable to induce penile erection or to modify NO 2 − concentration. SR 141716A responses were also reduced by nitro- l-arginine methylester (20 μg), a non-selective NO synthase inhibitor, S-methyl- l-thiocitrulline (20 μg), a selective neuronal NO synthase inhibitor, the excitatory amino acid NMDA receptor antagonist dizocilpine ((+)MK 801) (1 μg), or the GABA A receptor agonist muscimol (0.2 μg) injected into the PVN 15 min before SR 141716A. In contrast, the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (20 μg), the GABA B receptor agonist baclofen (0.2 μg), the mixed dopamine receptor antagonist cis-flupenthixol (10 μg), and the oxytocin receptor antagonist d(CH 2) 5Tyr(Me)-Orn 8-vasotocin (1 μg), were ineffective. Despite its inability to reduce penile erection and NO 2 − increase induced by SR 141716A when injected into the PVN, d(CH 2) 5Tyr(Me)-Orn 8-vasotocin (1 μg) reduced almost completely penile erection without reducing paraventricular NO 2 − increase when injected into the lateral ventricles 15 min before SR 141716A. The present results show that SR 141716 induces penile erection by a mechanism (possibly activation of excitatory amino acid neurotransmission), which causes the activation of neuronal NO synthase in paraventricular oxytocinergic neurons mediating penile erection.

Influence of the selective neuronal NO synthase inhibitor ARL 17477 on nitrergic neurotransmission in porcine stomach

Selective neuronal NOS (nNOS) inhibitors have been developed for possible application in cerebral ischemia and neurodegenerative disorders. To investigate the degree of interference with peripheral nNOS, the influence of the selective nNOS inhibitor ARL 17477 was studied on electrically induced nitrergic relaxations in pig gastric fundus strips and on gastric fundic compliance in conscious pig. Circular muscle strips of porcine gastric fundus were electrically stimulated (10 s trains at 4 Hz, 0.1 ms and 40 V). ARL 17477 inhibited the electrically induced relaxations in a concentration-dependent way (3 × 10 − 6 M–10 − 4 M). The inhibitory effect of ARL 17477 developed more progressively than that of N G-nitro- l-arginine methyl ester ( l-NAME; 3 × 10 − 4 M). In conscious pigs, instrumented with a fundic cannula, l-NAME (20 mg/kg i.v.) significantly increased mean arterial blood pressure and decreased fundic compliance in the fasted state (71 ± 13 ml/mm Hg versus 185 ± 37 ml/mm Hg after saline; P < 0.05). ARL 17477 (3 mg/kg, i.v.) did not influence blood pressure but influenced gastric fundic volume–pressure curves in a similar way as l-NAME. Plasma concentration analysis of ARL 17477 indicated a half-life of less than 30 min in pig. ARL 17477 thus inhibits the effect of nitrergic neurons in the pig gastric fundus in vitro, leading to inhibited gastric compliance in the conscious pig. The study indicates that selective nNOS inhibitors, applied for cerebral disorders, might also interfere with neuronal nitrergic regulation of gastrointestinal motility.

Role of nitric oxide and prostanoids in attenuation of rapid baroreceptor resetting

Because the regulation of vascular function involves complex mutual interactions between nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) products, we examined the contribution of NO and prostanoids derived from the COX pathway in modulating aortic baroreceptor resetting during an acute (30 min) increase in arterial pressure in anesthetized rats. Increase in pressure was induced either by administration of the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or aortic coarctation (COA) with or without treatment with the COX inhibitor indomethacin (INDO) or the selective neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)imidazole (TRIM). The activity of the aortic depressor nerve and arterial pressure were simultaneously recorded, and the degree of resetting was determined by the shift of the pressure-nerve activity curve using the ratio [delta systolic pressure at 50% of maximum baroreceptor activity/delta systolic pressure] x 100. The magnitude of pressure rise was similar in the different groups (59 +/- 6, 53 +/- 5, 53 +/- 5, 45 +/- 5, 49 +/- 3, and 41 +/- 3 mmHg for COA, L-NAME, INDO+COA, INDO+L-NAME, TRIM+COA, and TRIM+INDO+COA, respectively, P = 0.27). The degree of resetting that occurred with L-NAME or COA combined with treatment with TRIM was attenuated compared with COA alone (7 +/- 4, 5 +/- 2, and 31 +/- 6%, respectively, P = 0.04). INDO failed to influence baroreceptor resetting to higher pressure but prevented L-NAME- and TRIM-induced effects (20 +/- 7, 21 +/- 8, and 32 +/- 6% for INDO+COA, INDO+L-NAME, and INDO+TRIM+COA, respectively; P = 0.38). Baroreceptor gain was affected only by l-NAME. These findings indicate that NO, probably from neuronal origin, may exert stimulatory influence on the degree of rapid baroreceptor resetting to hypertension that involves COX-derived prostanoids.

Effects of 7-nitroindazole and N-nitro- l-arginine methyl ester on changes in cerebral blood flow and nitric oxide production preceding development of hyperbaric oxygen-induced seizures in rats

Hyperbaric oxygen (HBO 2) exposure induces increases in cerebral blood flow (CBF) and extracellular concentrations of nitric oxide (NO) that precede the appearance of central nervous system toxicity, which may manifest as convulsions. To elucidate the origins of NO production during HBO 2 exposure, we examined the effects of the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole (7-NI), and the non-selective NOS inhibitor, N-nitro- l-arginine methyl ester ( l-NAME), on changes in CBF and NO metabolites (NO x , nitrite and nitrate) using a laser Doppler flow probe and in vivo microdialysis techniques, respectively. Rats were anesthetized, artificially ventilated, and pressurized to 5 atmosphere absolute (ATA) with pure oxygen for 60 min. In rats treated with vehicle, CBF and NO x levels in the cortex increased to 201% and 239% of basal levels, respectively, before the onset of electrical discharges, measured by electroencephalogram. The increase in CBF and NO x was completely inhibited by 7-NI and l-NAME. Both drugs also inhibited the appearance of electrical discharges for 60 min. Dynamic changes in CBF and NO x were not significantly different between 7-NI and l-NAME. These findings suggest that neuronal NOS is the main mediator of NO production associated with increase in CBF leading to the appearance of electrical discharge during HBO 2 exposure.

Neuronal nitric oxide synthase inhibition differentially affects oxytocin and vasopressin secretion in salt loaded rats

Nitric oxide, an endogenous gas produced by nitric oxide synthase (NOS), has been described as a neuromodulator of hormone secretion, including the neurohypophysial peptides oxytocin (OT) and vasopressin (AVP), hormones involved in the sodium and water homeostasis. The presence of NOS in the hypothalamic nuclei as well as in the circumventricular organs suggests a nitrergic regulation of OT and AVP secretion. Thus, the aim of this study was to evaluate the effect of 7-nitroindazole (7-NI), a selective inhibitor of neuronal NOS, in the plasma OT and AVP levels in rats submitted to a short and long-term salt loading. We also evaluated the NOS activity in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Our data showed an increase of plasma OT and AVP levels in both short and long-term salt loading. The augment of plasma OT and AVP levels was accompanied by an increase of NOS activity in the SON and PVN. The injection of 7-NI potentiated the increase of plasma OT induced by salt loading, but inhibited the increase of plasma AVP in the same experimental conditions. These results indicate that, under short and prolonged osmotic stimulation, nitric oxide may differentially control the neurohypophysial secretion.

Roles of nitric oxide and prostaglandins in pathogenesis of delayed colonic transit after burn injury in rats

Burn injury has been shown to impair gut transit, but the exact mechanism remains unknown. The present study investigated whether nitric oxide synthase (NOS) and cyclooxygenase (COX) mediated changes in burn-induced colonic transit. After rats underwent 30% total body surface area burn injury, they were injected with S-methylisothiourea (SMT, selective inducible NOS inhibitor), 7-nitronidazole (7-NI, selective neuronal NOS inhibitor), and nimesulide (NIM, selective COX-2 inhibitor), respectively. The protein and mRNA of NOS and COX-2 were measured by Western blot analysis and real-time RT-RCR, and localization of NOS and COX-2 protein was determined by immunohistochemistry. Our results showed that colonic transit assessed by the geometric center was delayed from 3.47+/-0.28 in controls to 2.21+/-0.18 after burn (P<0.009). SMT and NIM significantly improved colonic transit in burned rats but had no effect in sham-operated rats. 7-NI failed to modify delayed transit in burned rats but significantly delayed colonic transit in sham-operated rats. Both protein and mRNA of inducible NOS and COX-2 increased significantly but not neuronal NOS in burned rats. Inducible NOS protein expression was noted not only in epithelial cells but also in neurons of the myenteric ganglia in burned rats. These findings suggest that nitric oxide (NO) produced by neuronal NOS plays an important role in mediating colonic transit under the physiological condition. NO produced by inducible NOS and prostaglandins synthesized by COX-2 are both involved in the pathogenesis of delayed colonic transit after burn injury. Inducible NOS expression in neurons of the myenteric ganglia may contribute to dysmotility with burn injury.

P2-095 The effect of ovariectomy on pathology and phenotype in Tau (P301L) transgenic mice

In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether l-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,β-methylene ATP) or NO precursor (l-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (l-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with d-NAME (an isomer of l-NAME) or N5-(1-iminoethyl)-l-ornithine (l-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with l-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of l-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and/or developing therapeutic strategies for the diseases associated with CCA blood flow that supplies intra- and extra-cranial tissues.

Adrenomedullin in the rostral ventrolateral medulla increases arterial pressure and heart rate: roles of glutamate and nitric oxide.

This study was done to investigate the effects of microinjections of adrenomedullin (ADM), a vasoactive neuropeptide, in the rostral ventrolateral medulla (RVLM) on mean arterial pressure (MAP) and heart rate (HR) in urethane-anesthetized rats, and to assess the potential roles of glutamate and nitric oxide (NO) in these effects. Unilateral injections of ADM (0.01 or 0.1 pmol) into the RVLM significantly increased MAP and HR in a dose-dependent manner, whereas ADM at 0.001 pmol was ineffective. Microinjections of ADM (0.01 pmol) outside the RVLM had no effects on MAP or HR. Coinjections of a putative ADM receptor antagonist, ADM(22-52) (0.01 pmol), abolished the increases in MAP and HR evoked by ADM (0.01 pmol). The vasopressor effects of ADM (0.01 pmol) in the RVLM were abolished by coinjections of either dizocilpine hydrogen maleate (a selective NMDA glutamate receptor antagonist, 500 pmol) or 6-cyano-7-nitroquinoxaline-2,3-dione (a selective non-NMDA glutamate receptor antagonist, 50 pmol). The ADM-induced vasopressor effects were also abolished by coadministration of either 7-nitroindazole sodium salt (a selective neuronal NO synthase inhibitor, 0.05 pmol) or methylene blue (a soluble guanylyl cyclase inhibitor, 100 pmol). These results suggest that ADM in the RVLM stimulates increases in MAP and HR through ADM receptor-mediated mechanisms. These effects are mediated by glutamate via both NMDA and non-NMDA receptors. NO, derived from neuronal NO synthase, also contributes to the ADM-induced vasopressor effects via a soluble guanylyl cyclase-associated signaling pathway.

Interaction of spinal nitric oxide and prostaglandins after L5-L6 spinal nerve ligation in the rat: an isobolographic analysis.

Interaction of spinal nitric oxide and prostaglandins after L5-L6 spinal nerve ligation in the rat: an isobolographic analysis.

Positive N-methyl- d-aspartate receptor modulation by selective glycine transporter-1 inhibition in the rat dorsal spinal cord in vivo

In this study we have employed the selective glycine transporter-1 (GlyT-1) and GlyT-2 transporter inhibitors R-(−)- N-methyl- N-[3-[(4-trifluoromethyl)phenoxy]-3-phenyl-propyl]glycine (1:1) lithium salt (Org 24598) and 4-benzyloxy-3,5-dimethoxy- N-[1-(dimethylaminocyclopently)methyl]benzamide (Org 25543), respectively, and microdialysis perfusion to determine the effect of GlyT transporter inhibition on extracellular amino acid concentrations in the lumbar dorsal spinal cord of the halothane-anaesthetised rat. Reverse dialysis of Org 24598 (0.1–10 μM) induced a concentration-related increase in extracellular glycine accompanied by a progressive increase in citrulline, but not aspartate, glutamate or GABA, efflux. Org 25543 (10 μM) by the same route induced a similar increase in glycine levels without affecting the efflux of other amino acids quantified. To test the hypothesis that the increase in citrulline efflux resulted from activation of the N-methyl- d-aspartate receptor (NMDA-R)/nitric oxide synthase (NOS) signalling cascade, the sensitivity was determined of GlyT-1 inhibition-induced effects to NMDA-R antagonism or NOS inhibition. Co-administration by reverse dialysis of the selective NMDA-R channel blocker MK-801 (0.5 mM) or the selective antagonist of the strychnine-insensitive glycine site, 7-chlorokynurenic acid (1 mM), with Org 24598 (10 μM) did not affect the uptake inhibition-induced increase in glycine efflux, but did significantly attenuate the increase in extracellular citrulline. Similarly, co-administration with Org 24598 of the isoform non-selective and selective neuronal NOS inhibitors N ω-nitro- l-arginine methyl ester (1 mM) or 1-(2-trifluoromethylphenyl)imidazole (0.2 mM), respectively, prevented Org 24598-induced citrulline efflux with no effect on increased glycine efflux. These data provide evidence that the observed increased in extracellular citrulline is a consequence of positive modulation of NMDA-R, secondary to increased extracellular glycine and support a protective role for GlyT-1 against fluctuations in extracellular glycine uptake at glutamatergic synapses in the dorsal spinal cord. Such a mechanism could be important to NMDA-R-mediated synaptic plasticity in the spinal cord and be of relevance to the clinical usage of GlyT-1 inhibitors.

Potent and selective conformationally restricted neuronal nitric oxide synthase inhibitors.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) in pathologically elevated synthesis of nitric oxide has great therapeutic potential. We previously reported nitroarginine-containing dipeptide amides and some peptidomimetic analogues as potent and selective inhibitors of neuronal NOS (nNOS). Here we report conformationally restricted dipeptides derived from the dipeptide L-Arg(NO2)-L-Dbu-NH2 (8). The selectivities for nNOS over endothelial NOS and inducible NOS of the most potent nNOS inhibitor (10a) among these compounds are comparable to that of the parent compound. An unsubstituted amide bond is necessary for potency against nNOS. The stereochemistry of compound 10a was optimum for potency and selectivity and thus provides the binding conformation of the parent compound with nNOS.

Intact carrageenan-induced thermal hyperalgesia in mice lacking inducible nitric oxide synthase

To date, the exact role of inducible nitric oxide synthase (iNOS) in inflammatory pain remains controversial. In the present study, we combined a pharmacological strategy (using a selective iNOS inhibitor) with a genomic strategy (using mice lacking the iNOS gene) to address the function of iNOS in the central mechanism of carrageenan-induced persistent inflammatory pain. In the wild type mice, intrathecal administration of L- N 6-(1-iminoethyl)-lysine, a selective iNOS inhibitor, significantly inhibited thermal hyperalgesia in the late phase but not in the early phase of carrageenan inflammation. Moreover, iNOS mRNA expression in the lumbar enlargement segments of the spinal cord was dramatically induced at 24 h (late phase) after injection of carrageenan into a hind paw. Interestingly, targeted disruption of iNOS gene did not affect carrageenan-induced thermal hyperalgesia in either the early (2–6 h) or late phase. In the lumbar enlargement segments of iNOS knockout mice, nitric oxide synthase (NOS) enzyme activity remained at a similar level to that of the wild type mice at 24 h after carrageenan injection. We found that intrathecal administration of 7-nitroindazole (a selective neuronal NOS inhibitor), but not L- N 5-(1-iminoethyl)-ornithine (a selective endothelial NOS inhibitor), significantly reduced carrageenan-induced thermal hyperalgesia in both the early phase and the late phase in iNOS knockout mice. We also found that expression of neuronal NOS but not endothelial NOS in the lumbar enlargement segments was significantly increased in iNOS knockout mice compared with wild type mice at 24 h after carrageenan injection. Our results indicate that neuronal NOS might compensate for the function of iNOS in the late phase of carrageenan-induced inflammatory pain in iNOS knockout mice. This suggests that iNOS may be sufficient, but not essential, for the late phase of the carrageenan-induced thermal hyperalgesia.

Homocysteine-induced brain lipid peroxidation: effects of NMDA receptor blockade, antioxidant treatment, and nitric oxide synthase inhibition.

The effect of homocysteine (HCY) on lipid peroxidation (LP), a current mechanism of oxidative neurotoxicity, was investigated in rat brain synaptosomes. LP was assessed by measuring the amount of thiobarbituric acid-reactive substances (TBARS) formed from synaptosomal fractions following HCY treatment. Increasing HCY concentrations (5-1000 micro M) enhanced the TBARS formation in brain synaptosomes in a concentration-dependent manner. When compared at equimolar concentrations (100 micro M), the oxidative potency of HCY was lower than that of the oxidant ferrous sulfate, similar to that produced by glutamate (Glu) and the mitochondrial toxin 3-nitropropionic acid, and higher than that of the Glu agonists, kainate and quinolinate. The N-methyl-D-aspartate receptor (NMDAr) antagonist dizocilpine (MK-801) completely blocked the HCY-induced LP at concentrations between 5 to 1000 micro M, whereas the well-known antioxidant N-acetylcysteine (NAC) was less effective, but still protective against the HCY oxidative toxicity at higher concentrations (400 and 1000 micro M). Three nitric oxide synthase (NOS) inhibitors, 7-nitroindazole (7-NI), Nomega-nitro-L-arginine (L-NARG) and Nomega-nitro-L-arginine methyl ester (L-NAME), were also tested on HCY-induced LP at increasing concentrations. Both nonspecific NOS inhibitors (L-NARG and L-NAME) decreased more effectively the HCY-induced LP than did the selective neuronal NOS inhibitor, 7-NI. These results show that submillimolar concentrations of HCY can induce oxidative injury to nerve terminals, and this effect involves NMDAr stimulation, NOS activation, and associated free radicals formation.

Use of an electrochemical nitric oxide sensor to detect neuronal nitric oxide production in conscious, unrestrained rats

Introduction: Amperometric sensors that directly measure nitric oxide (NO) are readily employed in pharmacologic research. While several of these sensors have been developed, none has been investigated for use in conscious, freely moving animals. An approach was developed and validated for real-time quantitation of neuronal NO production in rats without restricting locomotor activity or other potentially useful behavioral endpoints. Methods: Male Sprague–Dawley rats were equipped with a femoral vein or intraperitoneal cannula. A guide cannula and an amperometric NO sensor were placed in the left and right hippocampus, respectively. Following recovery, rats received a 6-h intravenous infusion of saline, l-arginine (an NO precursor; 250 or 500 mg/kg/h), or incremental intraperitoneal 7-nitroindazole (an NO synthase inhibitor; 200-mg/kg loading dose and 100 mg/kg every 2 h). The sensor recorded NO production continuously and microdialysis samples were collected incrementally throughout the experiment. Griess assay analysis of microdialysate samples was compared to sensor readings in vivo. In vitro degradation of an NO donor also was used to validate sensor performance. Results: Exogenous administration of l-arginine resulted in incremental increases in the neuronal NO signal. A reduction in NO production was observed during administration of 7-nitroindazole, a selective neuronal NO synthase inhibitor. A significant correlation was observed in vitro between the Griess assay analysis, an indirect analytical approach, and the NO sensor readings. The lack of a strong correlation between these measures in vivo is consistent with the indirect nature of the Griess assay. Discussion: The current approach allows real-time determination of neuronal NO production in unrestrained rats. This model will be invaluable in evaluating pharmacologic issues regarding brain tissue NO synthesis, assessing brain NO synthase as a molecular target, and establishing the effects of pharmacologic agents on neuronal NO production.

Endothelial nitric oxide modulates perivascular sensory neurotransmission in the rat isolated mesenteric arterial bed.

1. A possible role of nitric oxide (NO) as a modulator of capsaicin-sensitive sensory neurotransmission in blood vessels was investigated in the rat isolated mesenteric arterial bed. 2. Electrical field stimulation (EFS) of methoxamine-preconstricted mesenteric beds elicited frequency-dependent vasorelaxation mediated by capsaicin-sensitive sensory nerves. N(G)-nitro-L-arginine methyl ester (L-NAME, 10 and 300 microM) and 7-nitroindazole (7-NI, 100 microM), inhibitors of nitric oxide synthase (NOS), augmented sensory neurogenic vasorelaxation. D-NAME (300 microM), 6-aminoindazole (100 microM) and N(omega)-propyl-L-arginine (50 nM), a selective inhibitor of neuronal NOS, were without effect. The effect of 10 microM L-NAME was reversed by L-arginine (1 mM), the substrate for NOS. 3. L-NAME (300 microM) and 7-NI (100 microM) had no significant effect on vasorelaxations to calcitonin gene-related peptide (CGRP), the principal motor neurotransmitter of capsaicin-sensitive sensory nerves in rat mesenteric arteries, or to capsaicin, indicating a prejunctional action. The inhibitors of NOS had no effect on vasorelaxation to forskolin, but augmented vasorelaxation to sodium nitroprusside (SNP). 4. Removal of the endothelium augmented sensory neurogenic vasorelaxation, but did not affect vasorelaxation to CGRP, indicating a prejunctional action of endothelial NO. 5. In the absence of endothelium, L-NAME (300 microM) inhibited, and 7-NI (100 microM) caused no further augmentation of sensory neurotransmission. 6. SNP (100 nM), a nitric oxide donor, attenuated sensory neurogenic relaxations to EFS. 7. In rat isolated thoracic aortic rings, L-NAME (100 microM) and 7-NI (100 microM) attenuated concentration-dependent relaxations to acetylcholine. 8. These data show that NO modulates sensory neurotransmission evoked by EFS of the rat isolated mesenteric arterial bed, and that when NO synthesis is blocked sensory neurogenic relaxation is augmented. The source of NO is the vascular endothelium.

Behavioral effects of 7-nitroindazole on hyperbaric oxygen toxicity

The aim of this study was to evaluate the role of nitric oxide (NO) upon hyperbaric oxygen (HBO) toxicity in male Sprague–Dawley rats during exposure to 0.5 MPa >99% O 2. In the first experiment, the selective neuronal NO synthase inhibitor 7-nitroindazole (7-NI) was injected intraperitoneally (ip) in 15 rats. Another 15 rats received vehicle injections of peanut oil intraperitoneally. Latency to observable tonic-clonic convulsions and motor activity during the HBO exposure were scored and compared between the control group and the 7-NI group. The results showed that injection of 7-NI (30 mg/kg) significantly prolonged the latency to observable tonic-clonic convulsions. The 7-NI group also showed a significant decrease in motor activity compared with the control group. A second experiment was performed to measure the effect of 7-NI injections upon open-field activity during normobaric conditions. Twenty-four male Sprague–Dawley rats were randomly divided into three groups, each consisting of eight rats receiving 30 mg/kg 7-NI injections, 10 mg/kg 7-NI injections or vehicle injections of peanut oil intraperitoneally, respectively. The results showed that injection of 7-NI led to a significant dose-dependent reduction in horizontal and vertical activities. This study shows that 7-NI prolongs the latency to hyperoxia-induced seizures. However, it also demonstrates that 7-NI in doses ranging from 30 to 10 mg/kg has a secondary effect upon motor behavior in general. It can therefore not be ruled out that the protective effect of 7-NI upon HBO intoxication is partly due to reduced motor activity.

Facilitatory role of NO in neural norepinephrine release in the rat kidney

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [N(omega)-nitro-L-arginine methyl ester (L-NAME) or N(omega)-nitro-L-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with L-arginine prevented the effects of L-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO(2), suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO(2) rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.