Basolateral amygdala lesions disrupt latent inhibitionin rats

Hypothermia is a well-known phenomenon which accompanies hypoglycemia in mammals. The present study was designed to test the hypothesis that nitric oxide (NO) plays a role in insulin-induced hypothermia. The body temperature (Tb) of awake, unrestrained rats was measured before and after systemic infusion of insulin (2U x kg(-1) x h(-1)), and intracerebroventricular administration of NG-nitro-(L)-arginine methyl ester (L-NAME, a nonselective NO synthase inhibitor, 200 microg/1 microl). We observed a significant reduction in body temperature after insulin infusion. L-NAME alone caused no significant change in body temperature. When the two treatments were combined, no change in Tb was observed. The data indicate that NO plays a key role in insulin-induced hypothermia.     

Reduced ventromedial hypothalamic neuronal nitric oxide synthase and increased sensitivity to NOS inhibition in dietary obese rats: further evidence of a role for nitric oxide in the regulation of energy balance

Inhibition of hypothalamic nitric oxide (NO) decreases energy intake, and changes in hypothalamic NO synthase (NOS) have been observed in genetically obese rodents, but it is not known if NO is involved in the development of diet-induced obesity (DIO). We therefore measured changes in hypothalamic neuronal NOS (nNOS) in DIO and investigated effects of peripheral and central inhibition of NOS in this model. Expression of nNOS in relation to changes in nutritional state was measured by immunohistochemistry, with radiochemical detection. The effect of chronic intraperitoneal (i.p.) administration of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day) on energy intake, bodyweight and hypothalamic nitric oxide content was assessed in both chow-fed and DIO animals. Twenty-four hour energy intake after acute intracerebroventricular (i.c.v.) of L-NAME was also measured. Diet-induced obese animals had a statistically significant 32% reduction in the number of nNOS-immunolabelled cells in the ventromedial hypothalamus compared to chow-fed controls. Intraperitoneal administration of L-NAME decreased hypothalamic NO content in both chow-fed and DIO. Energy intake was reduced by 16% in DIO over 16 days, whereas energy intake was only reduced by 11% in chow-fed animals, although both were statistically significant. L-NAME significantly reduced body weight gain in DIO but not in chow-fed rats. L-NAME administered i.c.v. decreased 24 h energy intake to a greater extent in DIO rats, by 18%, compared with a 10% reduction in chow-fed rats. Ventromedial hypothalamic expression of nNOS is sensitive to changes in nutritional state. Despite having reduced nNOS, dietary obese rats were more sensitive to the effects of NOS inhibition than lean controls, suggesting a role for NO in the development of hyperphagia and obesity in rats fed a palatable diet.     

Nitric Oxide Synthase Inhibition Delays Axonal Degeneration and Promotes the Survival of Axotomized Retinal Ganglion Cells

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.     

Different effects between 7-nitroindazole and L-NAME on cerebral hemodynamics and hippocampal lesions during kainic acid-induced seizures in newborn rabbits

We examined the effects of 7-nitroindazole (7-NI) and N-omega-nitro-L-arginine methyl ester (L-NAME) on the endogenous nitric oxide (NO) production in vivo, cerebral hemodynamics, and hippocampal lesions to investigate the different roles between endothelial NOS (eNOS) and neuronal NOS (nNOS) during kainic acid (KA)-induced seizures in newborn rabbits. After a pre-treatment with 7-NI (50 mg/kg, i.p.), L-NAME (20 mg/kg, i.v.) or saline (1 ml, i.v.), KA (12 mg/kg, i.v.) was administered. NO production in the brain, regional cerebral blood flow (rCBF), cerebral oxygenation (concentrations of oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), and total hemoglobin (tHb) in the brain tissue), and electroencephalography (EEG) were continuously monitored throughout the experiment lasting at least 60 min after the KA administration. There was a significant increase in NO generation in the brain during KA-induced seizures, which was inhibited by a pre-treatment with 7-NI or L-NAME. KA-induced seizures also increased rCBF significantly, which was inhibited not by 7-NI but by L-NAME. L-NAME pre-treatment caused a significant decrease in HbO2 and a significant increase in HbR during KA-induced seizures, compared with 7-NI and saline pre-treatment. EEG abnormalities and Neuronal damages in the hippocampus were significantly lower in 7-NI- and L-NAME-treated animals respectively, than in saline-treated animals. The present data demonstrated that the selective nNOS inhibitor, 7-NI, attenuated neither rCBF nor cerebral oxygenation during the seizures, while the non-selective NOS (nNOS and eNOS) inhibitor, L-NAME, attenuated both. These findings suggest that NO, probably originating from eNOS, may play an important role in the cerebral circulation. Both 7-NI and L-NAME inhibited the NO production and EEG abnormalities during the seizures that led to less damage to the hippocampus.     

Bombesin produces hypothermia in rats pretreated with 2-deoxy-D-glucose

Previous research has shown that microinfusion of bombesin into the preoptic area (POA) decreases core body temperature in rats that are food-deprived or made hypoglycemic with insulin. The present study employed 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to further investigate the importance of a reduction in glucose utilization in the production of bombesin-induced hypothermia. Rats (n = 7) were pretreated with 2-DG (0, 25, 50, 100, 200 mg/kg; IP) followed by bombesin (100 ng/1.0 microliters) microinfusions into the POA. The highest dose of 2-DG (200 mg) was also tested in the absence of bombesin as a control. Pretreatment with 2-DG resulted in a dose-related reduction in Tb following bombesin. Injections of 2-DG alone did not significantly alter Tb. The results provide additional evidence that the production of bombesin-induced hypothermia in fasted rats is linked to a reduction in glucose utilization.     

Role of nitric oxide on ATP-induced Ca2+ signaling in outer hair cells of the guinea pig cochlea

Recently, a negative feedback effect of nitric oxide (NO) on the adenosine 5'-triphosphate (ATP)-induced Ca2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca2+ -sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor, and suramin, a P2 receptor antagonist. ATP failed to induce NO production in the Ca2+ -free solution. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca2+ concentrations ([Ca2+]i), while L-NAME inhibited it. SNAP accelerated ATP-induced Mn2+ quenching in fura-2 fluorescence, while L-NAME suppressed it. 8-Bromoguanosine-cGMP, a membrane permeable analog of cGMP, mimicked the effects of SNAP. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase inhibited the ATP-induced [Ca2+]i increase. Selective neuronal NOS inhibitors, namely either 7-nitro-indazole or 1-(2-trifluoromethylphenyl) imidazole, mimicked the effects of L-NAME regarding both ATP-induced Ca2+ response and NO production. Immunofluorescent staining of neuronal nitric oxide synthase (nNOS) in isolated OHCs showed the localization of nNOS in the apical region of OHCs. These results suggest that the ATP-induced Ca2+ influx via a direct action of P2X receptors may be the principal source for nNOS activity in the apical region of OHCs. Thereafter, NO can be produced while conversely enhancing the Ca2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.     

Blockade of nitric oxide formation enhances thermal and behavioral responses in rats during turpentine abscess

OBJECTIVE: The purpose of this study was to investigate the role of nitric oxide (NO) during the development of fever and other symptoms of sickness behavior (i.e. anorexia, cachexia) in response to localized tissue inflammation caused by injection of turpentine in freely moving biotelemetered rats. METHODS: To determine the role of NO in turpentine-induced fever, we injected the NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) intraperitoneally simultaneously or 5 h after turpentine injection. RESULTS: Rats responded with fever to intramuscular injection of 20 microl of turpentine that commenced 6 h after injection and reached peak values 11 h after injection. Although turpentine did not significantly alter food and water intake, it caused a drop in body weight. Rats injected with turpentine and treated with L-NAME responded with a substantial rise in fever, independently of the time of L-NAME injection. The rise in body temperature (T(b)) due to turpentine injection began slightly sooner and reached the maximal T(b) value faster in rats treated with L-NAME than in the ones treated with saline (control for L-NAME). The enhanced decrease in food and water intake in rats treated with a combination of L-NAME and turpentine was also observed. As a result, L-NAME-injected rats responded with a profound drop in body mass due to turpentine, independently of the time of L-NAME injection. L-NAME alone did not affect food and water intake, but slightly suppressed the gain of body mass. CONCLUSION: These results indirectly indicate that NO is involved in pyrogenic and behavioral responses in rats during turpentine abscess.     

Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.     

Role of nitric oxide synthase against MPTP neurotoxicity in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway injury similar to that observed in Parkinson's disease. Many hypotheses have been proposed to explain the mechanisms underlying MPTP neurotoxicity. Previous work showed that the inhibitor of neuronal nitric oxide synthase (nNOS) might produce protection against MPTP-induced dopaminergic toxicity. To exactly test the role of NO in MPTP neurotoxicity, we examined the effect of nNOS inhibitor 7-nitroindazole, in comparison with that of nonselective NOS inhibitor (L-NAME), immunosuppressant (FK-506), monoamine oxidase (MAO) inhibitors (clorgyline and pargyline), N-methyl-D-aspartate receptor antagonist (MK-801) and Ca2+ antagonist (amlodipine). Among seven compounds, 7-nitroindazole produced dose-dependent protection against MPTP-induced depletion of striatal dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid (DOPAC) in mice. Clorgyline and pargyline also showed a significant effect on MPTP-induced dopamine depletion in the mouse striatum. However, both compounds did not protect against MPTP-induced depletion of striatal DOPAC Our immunohistological study with tyrosine hydroxylase (TH) and microtuble-associated protein 2 (MAP 2) showed that 7-nitroindazole or pargyline can protect against MPTP-induced depletion of TH and MAP 2 immunostained neurons in the substantia nigra. Furthermore, these compounds reduced a marked increase in GFAP-positive astrocytes of the mouse striatum after MPTP treatments. The present study demonstrates that nNOS inhibitor 7-nitroindazole as well as MAO inhibitors clorgyline and pargyline can produce dose-dependent neuroprotection against the dopaminergic neurotoxicity of MPTP. However, nonselective NOS inhibitor L-NAME, immunosuppressant FK-506, NMDA receptor antagonist MK-801 and Ca2+ antagonist amlodipine did not show a beneficial effect on MPTP neurotoxicity.     

Involvement of nitric oxide pathway in the acute anticonvulsant effect of melatonin in mice

Melatonin, the major hormone produced by the pineal gland, is shown to have anticonvulsant effects. Nitric oxide (NO) is a known mediator in seizure susceptibility modulation. In the present study, the involvement of NO pathway in the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced clonic seizures was investigated in mice. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) significantly increased the clonic seizure threshold induced by intravenous administration of PTZ. This effect was observed as soon as 1 min after injection and lasted for 30 min with a peak effect at 3 min after melatonin administration. Combination of per se non-effective doses of melatonin (10 and 20 mg/kg) and nitric oxide synthase (NOS) substrate L-arginine (30, 60 mg/kg) showed a significant anticonvulsant activity. This effect was reversed by NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for melatonin effect. Pretreatment with L-NAME (30 mg/kg) and N(G)-nitro-L-arginine (L-NNA, 10 mg/kg) inhibited the anticonvulsant property of melatonin (40 and 80 mg/kg) and melatonin 40 mg/kg, respectively. Specific inducible NOS (iNOS) inhibitor aminoguanidine (100 and 300 mg/kg) did not affect the anticonvulsant effect of melatonin, excluding the role of iNOS in this phenomenon, while pretreatment of with 7-NI (50 mg/kg), a preferential neuronal NOS inhibitor, reversed this effect. The present data show an anticonvulsant effect for melatonin in i.v. PTZ seizure paradigm, which may be mediated via NO/L-arginine pathway by constitutively expressed NOS.     

Nitric Oxide and Glutamate Interaction in the Control of Cortical and Hippocampal Excitability

PURPOSE: We investigated the role of nitric oxide (NO) as a new neurotransmitter in the control of excitability of the hippocampus and the cerebral cortex, as well as the possible functional interaction between NO and the glutamate systems. METHODS: The experiments were performed on anesthetized rats. The bioelectrical activities of the somatosensory cortex and the CA1 region of the hippocampus of these rats were recorded. Pharmacologic inhibition of NO synthase (NOS) through the nonselective and brain-selective inhibitors, N-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), was performed. RESULTS: The treatments caused the appearance of an interictal discharge activity in both the structures. The latency of induction and the duration of the interictal discharge activity were strictly related to the dose of NOS inhibitor used. In some cases, after L-NAME treatment at high doses, it was possible to note spike and wave afterdischarge activity in the hippocampus. All the NOS inhibitor-mediated excitatory effects were abolished by intraperitoneal (i.p.) pretreatment with the N-methyl-D-aspartic acid (NMDA) receptor antagonists (DL-2-amino-5-phosphonovaleric acid, 2-APV; dizolcipine, MK-801) and partly suppressed after the i.p. injection of the non-NMDA antagonist (6-cyano-7-nitroquinoxaline-2,3-dione; CNQX). CONCLUSIONS: All data showed that the reduction of NO levels in the nervous system causes the functional prevalence of the excitatory neurotransmission, which is probably due to an NMDA overactivity caused by the absence of the NO-mediated modulatory action. Thus, it is possible to hypothesize a neuroprotective role for NO, probably through a selective desensitization of the NMDA receptors.     

Mutual regulation between the intercellular messengers nitric oxide and brain-derived neurotrophic factor in rodent neocortical neurons

The diffusible factors, nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) are both suggested to be intercellular messengers that have similar synaptic activities and developmental influences in the brain. In the present study, we have analysed their mutual regulation with respect to their production in rodent neocortical neurons. Some of the cultured rat neocortical neurons exhibited immunoreactivity for both neuronal NO synthase (NOS) and the BDNF receptor trkB. Neuronal NOS appeared to be activated autonomously and produced NO in culture as monitored by nitrite accumulation. Inhibition of the endogenous NO production in culture by a NOS inhibitor, NG-monomethyl-L-arginine (NMMA), enhanced basal expression of BDNF mRNA and protein. Similarly, cerebroventricular administration of another NOS inhibitor, N-omega-nitro-L-arginine methylester (L-NAME), but not D-NAME or saline, increased BDNF content in the neocortex. In the opposite direction, however, BDNF appeared to function as a positive regulator for NO synthesis. Addition of BDNF upregulated the neuronal NOS expression as well as NO production in neocortical culture. In agreement, BDNF knock-out mice exhibited significant impairment of neuronal NOS expression in the neocortex. Taken together, these observations suggest that the trans-synaptic signalling molecules, NO and BDNF, influence the production of each other and mutually regulate the strength of their intercellular communications.     

Involvement of nitric oxide in phencyclidine-induced place aversion and preference in mice

The present study investigated the involvement of nitric oxide (NO) in phencyclidine (PCP)-induced place aversion and preference in the place conditioning paradigm. PCP-induced place aversion in naive mice was dose-dependently attenuated by administration of N(G)-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, during the conditioning. The NOS activity and dopamine (DA) turnover in the hippocampus in mice showing PCP-induced place aversion were decreased, such changes being restored by administration of L-NAME during the conditioning. On the other hand, PCP-induced place preference in mice pretreated with PCP for 28 days was not attenuated by administration of L-NAME during the conditioning. Although NOS activity was not changed, the DA turnover in the cerebral cortex was increased in mice showing PCP-induced place preference. In mice pretreated with L-NAME and PCP for 28 days before the place conditioning paradigm, PCP neither induced place preference, nor changed the NOS activity or DA turnover. These results suggest that NO is involved in the acquisition of PCP-induced aversive effects, and in the development of PCP-induced preferred effects. Further, the functional change of the DAergic neuronal system mediated by NO in the hippocampus and cerebral cortex may be necessary for the expression of aversive effects and development of preferred effects, respectively, induced by PCP.     

Protective effects of lipopolysaccharide preconditioning against nitric oxide neurotoxicity

We have characterized lipopolysaccharide (LPS) preconditioning-induced neuroprotective mechanisms against nitric oxide (NO) toxicity. Pretreatment of rat cortical cultures with LPS attenuated neurotoxicity of NO donors, including sodium nitroprusside (SNP) and diethylamine NONOate (NONOate). A transiently increased expression of endothelial nitric oxide synthase (eNOS) accompanied by an increase in NO production was observed during LPS preconditioning. Application of NOS inhibitors including L-N(5)-(1-iminoethyl)-ornithine (L-NIO) and L-nitroarginine methylester (L-NAME) abolished LPS-dependent protection against SNP toxicity. The LPS effect was also blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). Consistently, application of 8-bromo-cyclic GMP (8-Br-cGMP), a slowly degradable cGMP analogue capable of PKG activation, was neuroprotective. LPS preconditioning resulted in a heightened neuronal expression of Bcl-2 protein that was abolished by L-NAME and KT5823, the respective inhibitors of NOS and PKG. Together, our results reveal the signaling cascade of "LPS --> eNOS --> NO --> cGMP/PKG --> Bcl-2" that might have contributed to the LPS protective effects in cortical neurons.     

Role of nitric oxide in rat locus coeruleus in hypoxia-induced hyperventilation and hypothermia

The locus coeruleus modulates the ventilatory and thermoregulatory response to hypoxia and contains nitric oxide synthase. Therefore, we examined the effects of L-NAME unilaterally microinjected into the locus coeruleus on hypoxic hyperventilation and hypothermia. Ventilation and body temperature were measured before and after microinjection of L-NAME (100 nmol/0.5 microl) into the locus coeruleus, followed by hypoxia. Control rats received microinjection of D-NAME (an inactive enantiomer of L-NAME). Under normoxia, L-NAME treatment did not affect ventilation or body temperature. D-NAME did not affect hypoxia-induced hyperventilation and hypothermia. L-NAME treatment reduced the ventilatory response to hypoxia but did not affect hypoxia-induced hypothermia. These data suggest that nitric oxide in the locus coeruleus is involved in the ventilatory response to hypoxia, exercising an inhibitory modulation on the locus coeruleus neurons, but plays no role in hypoxia-induced hypothermia.     

Effect of low doses of L-NAME on methamphetamine-induced dopaminergic depletion in the rat striatum

Summary. The toxic dose of methamphetamine (METH) (5 mg/kg, s.c., ×4, 2 hr intervals) decreased contents of dopamine, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in striatum, and decreased contents of serotonin (5-HT) in both striatum and nucleus accumbens. Administration of low doses of a non-selective endothelial and neuronal nitric oxide synthase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (5 and 10 mg/kg, i.p., ×1) intensified the METH-induced decreases in contents of dopamine and its metabolites in striatum. NO substrate, L-arginine (500 mg/kg, i.p., ×4) reversed these effects of L-NAME on the METH-neurotoxicity. L-NAME did not change the METH-induced hyperthermia. These findings, which are contrary to our previous study with a high dose of L-NAME, suggest that the inhibition of endothelial or neuronal NOS-mediated NO production by low doses of L-NAME enhanced the METH-induced neurotoxicity. The finding that L-NAME can have opposite effects on the METH-neurotoxicity according to the dosing is important, however, additional experiments should be performed to clarify which type of NOS is related to these effects. Received February 2, 2001; accepted June 27, 2001     

Neonatal capsaicin-treatment in mice: effects on pancreatic peptidergic nerves and 2-deoxy-D-glucose-induced insulin and glucagon secretion.

It is not known whether sensory nerves are involved in the insulin, glucagon or glucose responses to autonomic nerve activation induced by 2-deoxy-D-glucose (2-DG). We therefore treated mice neonatally with capsaicin which permanently destroys sensory afferent nerve fibers. Immunohistochemistry of the pancreas at 13-14 weeks of age revealed a substantial reduction of calcitonin gene-related peptide (CGRP)-immunoreactive nerves and a partial reduction of substance P-immunoreactive nerves. In contrast, no effect was observed on galanin-immunoreactive nerves. At age 10-12 weeks, the mice were injected intravenously with 2-DG (500 mg/kg). In controls, 2-DG stimulated insulin and glucagon secretion and induced hyperglycemia (P less than 0.01). Capsaicin treatment partially reduced the glucose and glucagon responses to 2-DG (P less than 0.01). In contrast, the insulin response to 2-DG was not affected by capsaicin. It is concluded that the mouse pancreas contains capsaicin-sensitive sensory CGRP- and substance P-immunoreactive nerve fibers, whereas the galanin-immunoreactive nerve fibers are not sensitive to capsaicin. Furthermore, capsaicin-sensitive sensory nerve fibers are partially involved in 2-DG-induced glucagon secretion and hyperglycemia, whereas sensory nerves are not involved in 2-DG-induced insulin secretion.     

Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37°C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin=0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α+IL-1β intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid (tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both L-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α+IL-1β have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.     

Nitric oxide synthase inhibition during development: effect on apoptotic death of dopamine neurons

Naturally occurring cell death via apoptosis occurs in the substantia nigra pars compacta (SNc) during rat development, culminating during the perinatal period. We previously showed that lipid peroxidation-mediated oxidative stress is not involved in this cell death process. Nitric oxide (NO) has been proposed to be critical for many developmental processes in brain and has been shown to mediate cell death in neurotoxin models of neurodegenerative disorders. Here, we reported that in vivo pre- and postnatal treatment with the non-specific NO synthase (NOS) inhibitor, L-NAME (60 mg/kg), or with the neuronal NOS inhibitor, 7-NI (30 mg/kg), dramatically decreased the NOS activity as well as the NADPH-diaphorase staining in brain. However, those treatments did not rescue dopamine neurons from developmental death, suggesting that NO is not involved in vivo in developmental death of these neurons or in the overall development of the SNc.     

Local nitric oxide synthase activity in a model of neuropathic pain

A local inflammatory reaction may play an important role in the development of neuropathic pain following peripheral nerve injury. One important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (NO). In this work, we examined physiological and morphological evidence for nitric oxide synthase (NOS) activation in the chronic constriction injury model of neuropathic pain in rats. Physiological evidence of local NO action was provided by studying NO-mediated changes in local blood flow associated with the injury site. Immunohistochemistry was used to localize isoforms of NOS that might generate NO. Sciatic nerve injury associated with behavioural evidence of neuropathic pain had substantial rises in local blood flow. The NOS inhibitor N^G-nitro-l -arginine methyl ester (L-NAME), but not N^G-nitro-d -arginine methyl ester (D-NAME), reversed the hyperaemia in a dose-dependent fashion proximal to the constriction at 48 h and distally at 14 days post-operation when applied systemically or topically. Aminoguanidine, a NOS inhibitor with relatively greater selectivity for the inducible NOS (iNOS) isoform, reversed nerve hyperaemia distal to the constriction only at 14 days. NOS-like immunoreactivity of the neuronal and endothelial isoforms was identified just proximal to the constriction at 48 h. iNOS-like immunoreactivity was observed at 7 and 14 days at the constriction and distal sites, respectively. This work provides evidence for local NOS expression and NO action in the chronic constriction injury model of neuropathic pain. NO has local physiological actions that include vasodilatation of microvessels and that may be important in the development of pain sensitivity.