Brain Nitric Oxide Synthase Research Articles

English

The involvement of nitrergic mechanisms in the behavioural effects of withaferin A in rats was studied in the elevated plus-maze, open-field and rotarod tests. Administration of the nitric oxide (NO) precursor L-arginine (100 mg/kg, i.p.), assumed to increase the synthesis of NO, abolished the anxiolytic-like effect of withaferin A (40 mg/kg, i.p.) in the elevated plus-maze, whereas the inactive enantiomer D-arginine (100 mg/kg) did not. Neither withaferin A alone nor in combination with L- or D-arginine affected the exploratory activity of animals in the open field. Pretreatment with L-arginine (100 and 200 mg/kg) did not modify the motor impairment of rats after withaferin A (40 mg/kg) in the rotarod test. Withaferin A (40 mg/kg i.p.) did inhibit the brain NO synthase activity measured ex vivo by NOx assay. We conclude that a suppression of NO synthase activity may be important in the anxiolytic-like effect of withaferin A.   Key words: Withaferin A, nitric oxide (NO) synthase, L-Arginine , anxiety, elevated plus maze test.

Mice Brain Nitric Oxide Synthase Response Induced by Anaesthetics and Other Porphyrinogenic Drugs

Porphyrias neuropathophysiology could be related to low levels of heme as a cofactor for nitric oxide synthase (NOS). We examined how anaesthetics and other porphyrinogenic agents affect mice NOS activity and expression. Brain response was differential depending on the cellular fraction analyzed. Most of the drugs diminished cytosolic activity. Instead, isoflurane, enflurane and ethanol increased mitochondrial activity. NOS expression also depended on the drug tested. A comparative study was performed in liver. Our present and previous results indicate the widespread action of porphyrinogenic agents in brain, which could be the reason why it is difficult to establish the onset of acute porphyria neurological manifestations.

Effect of fish oils containing different amounts of EPA, DHA, and antioxidants on plasma and brain fatty acids and brain nitric oxide synthase activity in rats

Background. The interest in n-3 polyunsaturated fatty acids (PUFAs) has expanded significantly in the last few years, due to their many positive effects described. Consequently, the interest in fish oil supplementation has also increased, and many different types of fish oil supplements can be found on the market. Also, it is well known that these types of fatty acids are very easily oxidized, and that stability among supplements varies greatly.Aims of the study. In this pilot study we investigated the effects of two different types of natural fish oils containing different amounts of the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and antioxidants on plasma and brain fatty acids, blood lipids, vitamin E, and in vivo lipid peroxidation, as well as brain nitric oxide synthase (NOS) activity, an enzyme which has been shown to be important for memory and learning ability.Methods. Sprague-Dawley rats were divided into four groups and fed regular rat chow pellets enriched with 5% (w/w) of butter (control group), a natural fish oil (17.4% EPA and 11.7% DHA, referred to as EPA-rich), and a natural fish oil rich in DHA (7.7% EPA and 28.0% DHA, referred to as DHA-rich). Both of the fish oils were stabilized by a commercial antioxidant protection system (Pufanox®) at production. The fourth group received the same DHA-rich oil, but without Pufanox® stabilization (referred to as unstable). As an index of stability of the oils, their peroxide values were repeatedly measured during 9 weeks. The dietary treatments continued until sacrifice, after 10 days.Results. Stability of the oils varied greatly. It took the two stabilized oils 9 weeks to reach the same peroxide value as the unstable oil reached after only a few days. Both the stabilized EPA- and DHA-rich diets lowered the triacylglycerols and total cholesterol compared to control (-45%, P < 0.05 and -54%, P < 0.001; -31%, P < 0.05 and -25%, P < 0.01) and so did the unstable oil, but less efficiently. Only the unstable oil increased in vivo lipid peroxidation significantly compared to control (+40%, P < 0.001). Most of the fatty acids in the plasma phospholipids were significantly affected by both the EPA- and DHA-rich diets compared to control, reflecting their specific fatty acid pattern. The unstable oil diet resulted in smaller changes, especially in n-3 PUFAs. In the brain phospholipids the changes were less pronounced, and only the diet enriched with the stabilized DHA-rich oil resulted in a significantly greater incorporation of DHA (+13%, P < 0.01), as well as total n-3 PUFAs (+13%, P < 0.01) compared to control. Only the stabilized DHA-rich oil increased the brain NOS activity (+33%, P < 0.01).Conclusions. Both the EPA- and DHA-rich diets affected the blood lipids in a similarly positive manner, and they both had a large impact on plasma phospholipid fatty acids. It was only the unstable oil that increased in vivo lipid peroxidation. However, the intake of DHA was more important than that of EPA for brain phospholipid DHA enrichment and brain NOS activity, and the stability of the fish oil was also important for these effects.

Nitric oxide production inhibited by xenobiotic compounds in the protozoan Paramecium primaurelia

Abstract The notable increase in agricultural and industrial activities over the last decades has caused a considerable increase in anthropogenic waste and, consequently, the presence of pollutants in both water and sediments. For this reason, there is great interest in identifying alternative models and bioassays complying with the 3Rs strategy (aimed at Reducing, Refining and Replacing tests on vertebrate organisms in toxicological studies). Protozoa seem to be well suited to this strategy and it is widely accepted that assays with protozoa are relevant to the study of environmental modifications due to the presence of xenobiotic compounds. Recently, we detected the presence of nitric oxide synthase (NOS)-related NADPH-diaphorase activity and neuronal NOS-related molecules, immunologically recognized by the anti-rat brain NOS antibody, in a single-cell freshwater eukaryote, Paramecium primaurelia . In this work we have looked for the basal NO production in living cells of P. primaurelia using the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) and measuring the intracellular NO levels with image analysis. The NO production was sensitive to compounds modulating NOS activity such as: S-methyl-tiocitrulline, an NOS activity inhibitor, l -NAME, an analogue of arginine that inhibits NO production, arginine, an NOS substrate, or sodium nitroprusside, an NO donor. The NO production in P. primaurelia was also shown to be sensitive to μM concentrations of heavy metals (HgCl 2 and CdCl 2 ), or μM concentrations of pesticides (diazinon and AFD 25), thus representing a potential biomarker for environmental biomonitoring. The possible involvement of cellular Ca 2+ concentration, assayed by the fluorescent probe chlortetracycline hydrochloride, in NO production was examined after xenobiotic exposure.

Vasomotion of Intraradicular Microvessels in Rat

This study is to investigate the changes of vasomotion of intraradicular microvessels in vivo. We have observed microvascular corrosion casts of the lumbar nerve root by scanning electron microscopy and used an immunohistochemical technique to investigate the presence and distribution of autonomic and sensory nerve in blood vessels of the nerve root. It is generally considered that the genesis of radiculopathy associated with the degenerative conditions of the spine may result from both mechanical compression and circulatory disturbance. However, the neurogenic control of intraradicular blood flow has received little attention in the past. For three-dimensional observation of intraradicular vessels, we used scanning electron microscopic examination of microvascular corrosion casts in ten Wister rats. To investigate the mechanism of vasomotion of the nerve root, we used immunohistochemical methods. The sections were incubated overnight with antisera to tyrosine hydroxylase, choline acetyl transferase, substance P, calcitonin-gene-related peptide, vasoactive intestinal peptide, somatostatin, neuropeptide Y, leucine-enkephalin, cholecystokinin octapeptide, brain-nitric oxide synthase, and endothelium-nitric oxide synthase. Abidin-biotin complex method was used as the immunohistochemical procedure and the sections were observed under the light microscope. The general view of whole vascular casts of the lumbar spinal cord and nerve roots showed a high density of vessels. Bifurcation or anastomoses of capillaries approximately took place at right angles in a T-shaped pattern and capillaries showed a lot of ring-like compressions. This ring-like compression on the cast may represent a vascular sphincter in the microvessels. This study also reveals the existence of perivascular adrenergic, cholinergic, peptidergic, and nitroxydergic innervation with a possible role in neurogenic regulation of nerve root circulation. Perivascular nerve plexuses around intraradicular microvessels suggest that the autonomic nerves play an important role in intraradicular circulation.

Brain cyclooxygenase and nitric oxide synthase are involved in restraint stress-induced neuronal activation of spinally projecting neurons in rats

Brain cyclooxygenase and nitric oxide synthase are involved in restraint stress-induced neuronal activation of spinally projecting neurons in rats

Effect of fish oils containing different amounts of EPA, DHA, and antioxidants on plasma and brain fatty acids and brain nitric oxide synthase activity in rats

Effect of fish oils containing different amounts of EPA, DHA, and antioxidants on plasma and brain fatty acids and brain nitric oxide synthase activity in rats

Downregulation by lipopolysaccharide of Notch signaling, via nitric oxide

The Notch signaling pathway appears to perform an important function in inflammation. Here, we present evidence to suggest that lipopolysaccharide (LPS) suppresses Notch signaling via the direct modification of Notch by the nitration of tyrosine residues in macrophages. In the RAW264.7 macrophage cell line and in rat primary alveolar macrophages, LPS was found to inhibit Notch1 intracellular domain (Notch1-IC) transcription activity, which could then be rescued by treatment with N(G)-nitro-l-arginine, a nitric oxide synthase (NOS) inhibitor. Nitric oxide (NO), which was produced in cells that stably express endothelial NOS (eNOS) and brain NOS (bNOS), also induced the inhibition of Notch1 signaling. The NO-induced inhibition of Notch1 signaling remained unchanged after treatment with 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylyl-cyclase inhibitor, and was not found to be mimicked by 8-bromo-cyclic GMP in the primary alveolar macrophages. With regards to the control of Notch signaling, NO appears to have a significant negative influence, via the nitration of Notch1-IC, on the binding that occurs between Notch1-IC and RBP-Jk, both in vitro and in vivo. By intrinsic fluorescence, we also determined that nitration could mediate conformational changes of Notch1-IC. The substitution of phenylalanine for tyrosine at residue 1905 in Notch1-IC abolished the nitration of Notch1-IC by LPS. Overall, our data suggest that an important relationship exists between LPS-mediated inflammation and the Notch1 signaling pathway, and that this relationship intimately involves the nitration of Notch1-IC tyrosine residues.

Increasing the availability of nitric oxide (NO) increases sensitivity of nitrous oxide (N2O)‐insensitive inbred mice to N2O‐induced antinociception

N2O‐induced antinociception in mice is dependent on the neuromodulator NO. In contrast to C57BL/6 mice, DBA/2 mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of DBA/2 mice to respond to N2O might result from a deficit of NO function (Ishikawa and Quock, Brain Res. 976:262–263, 2003). Therefore, it was of interest to determine whether increasing the availability of NO might increased sensitivity of DBA mice to N2O. Male DBA/2 mice (20–25 g) were pretreated with sub‐antinociceptive intracerebroventricular doses of the NO donor 3‐morpholinosydnoimine (SIN‐1) or the NO precursor L‐arginine (L‐Arg) then assessed for responsiveness to N2O‐induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of DBA/2 mice to N2O. These results show that the NOS enzyme in DBA/2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O‐induced antinociception may lie in some other component or function related to NO. (Supported in part by NIH Grant GM‐77153).

Amyloid-β (25–35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-β (Aβ) peptide fragment Aβ(25–35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that Aβ(25–35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. Aβ(25–35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. Aβ(25–35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by Aβ(25–35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.

Significance of Experimental Infarct Size as an Indicator of Therapeutic Efficacy in Humans

To the Editor: We would like to make a comment not in particular to the article by Shimamura et al1 but to the use of infarct size in animal experiments as a guide to human cerebrovascular research. Many human studies of treatments with neuroprotective agents for acute ischemic stroke, which had good experimental potential, had disappointing clinical results. This failure can be attributed to many factors.2 One of the main problems is that reliance on infarct size measurement alone in animals can be misleading as an indicator of therapeutic efficacy in humans.3 We know for example that nitric oxide synthase (NOS) inhibitors have provided significant neuroprotection in experimental animals subjected to focal ischemia.4,5 Tirilazad mesylate is a potent inhibitor of lipid peroxidation caused by …

Exposure to enriched environments increases brain nitric oxide synthase and improves cognitive performance in prepubertal but not in young rats

Rats were randomly assigned to enriched (EE) or standard environments (SE) at 21 or 73 days of age, for 17 days. Half of the rats of each rearing condition were trained in a radial maze (RM). At 38 days (pre-pubertal) or 90 days (young), rats were sacrificed and brain cytosolic and mitochondrial nitric oxide synthase (mtNOS) activity was assayed. Western blot analysis of brain mtNOS was conducted. In the pre-pubertal group, EE rats improved their performance in the RM while SE rats did not. In the young group, SE and EE rats showed a random performance in the RM. In SE pre-pubertal rats, training increased brain cytosolic NOS and mtNOS activity by 68% and 82%. In EE non-trained pre-pubertal rats, brain cytosolic NOS and mtNOS activity increased by 80% and 60%, as compared with SE non-trained pre-pubertal rats. In EE pre-pubertal rats that were trained, brain cytosolic NOS and mtNOS activity increased by 70% and 90%, as compared with SE pre-pubertal rats that were not trained. A higher protein expression of brain mtNOS was found in EE rats, as compared with SE animals. Mitochondrial complex I activity was higher in EE than in SE rats. Training had no effect on complex I activity neither in SE nor in EE rats. In young rats, no significant differences in enzyme activities were found between EE and SE rats. These results support the hypothesis that brief exposure to EE and training produce effects on behavioral performance and on biochemical parameters in an age-dependent manner.

Quercetin attenuates spontaneous behavior and spatial memory impairment in d-galactose–treated mice by increasing brain antioxidant capacity

Quercetin, one of the flavonoids in fruits and vegetables, has a powerful antioxidant activity both in vitro and in vivo. To evaluate the antiaging effects of quercetin on the mouse brain, we investigated whether long-term intake of quercetin could improve spontaneous behavior and spatial memory in a mouse aging model. d-Galactose, known to induce age-related biochemical changes of the rodent brain, was used to develop the mouse aging model. In the present study, mice were subcutaneously injected with d-galactose (50 mg/kg) for 8 weeks and were administered quercetin via gastric intubation simultaneously. Animal behavior was observed in the open field and Morris water maze task. The results showed that quercetin significantly prevented changes of spontaneous behavior and spatial memory of d-galactose–treated mice. The antiaging effect of quercetin was found to be related to its ability to inhibit biochemical changes in hippocampi and cortices of d-galactose–treated mice. Quercetin increased brain glutathione level, hydroxy radical scavenging ability, and Na+,K+-adenosine triphosphatase activity, but decreased brain nitric oxide synthase activity and mitochondrial malondialdehyde content in d-galactose–treated mice. In conclusion, quercetin attenuated d-galactose-induced behavioral impairments, suggesting a potential antiaging effect of quercetin.

Brain nitric oxide synthases and mitochondrial function

Nitric oxide is a small signaling molecule, which may act as a neurotransmitter and neuromodulator, exerting a regulatory effect on neuronal function. It can diffuse from its site of synthesis to different intra and extracellular compartments, being therefore present in the pre-synaptic, synaptic and post-synaptic spaces. Recently, a NOS located in the mitochondria (mtNOS) has been observed in different brain regions, responsible for the production of NO in these organelles and identified as nNOS. A regulatory effect of NO on mitochondrial function was described in brain mitochondria, where NO acts mainly by inhibiting cytochrome oxidase activity. Hippocampal mitochondrial dysfunction and decreased mtNOS activity and expression were reported in association with ultrastructural damage in an experimental model of hepatic encephalopathy. Enriched environment exposure preserved the aged animals from spatial cognition impairment; also environment and training modulated neuronal plasticity in pre-pubertal rats through NO-dependent mechanisms. In addition, brain cortical mitochondrial respiration and mtNOS activity and expression were analyzed as function of age. Mitochondrial NO production showed a decreasing tendency as a function of age. These results are in accordance with the protein expression analyzed by Western Blot of mitochondrial fractions which was 6.5 times higher in 1 month aged rats as compared with 14 old animals. Concomitant with these results, a clear increasing oxygen uptake tendency in state 3 respiration was observed, meanwhile only a slight increase was observed in state 4. All these results seems to be clearly related with the reversible and concentration-dependent attenuation of the respiratory chain by NO.

Sarcomas Often Express Constitutive Nitric Oxide Synthases (NOS) But Infrequently Inducible NOS

Nitric oxide (NO) has a dual action in tumors, with both pro-tumor and anti-tumor activities. NO is produced by nitric oxide synthases (NOS). There are three enzyme isoforms: two of them are constitutively produced (neuronal or brain NOS and endothelial NOS), and one is an inducible form (iNOS). NOS expression has been shown in several epithelial tumors, but there is no report addressing NOS expression in sarcomas. The authors evaluated the expression of NOS in 97 cases of various sarcomas spotted in duplicate in a tissue array paraffin block. Eighty-four of the 97 tumor specimens (86.6%) expressed nNOS, and most of them showed a strong expression of the isoenzyme. Only chondrosarcomas and liposarcomas had significant numbers of negative cases, and all pleomorphic sarcomas, alveolar soft part sarcomas, angiosarcomas, gastrointestinal stromal tumors, and synovial sarcomas showed some degree of positivity. Forty-three cases (44.4%) showed eNOS immunostaining, but only 15.5% showed a strong signal, with emphasis on angiosarcomas, chondrosarcomas, alveolar soft part sarcomas, and synovial sarcoma. Strong expression of iNOS was observed in only 9 cases (9.3%), with weak expression in another 26 cases (26.8%). Strong expression of iNOS was found in malignant peripheral nerve sheet tumors, liposarcomas, pleomorphic sarcomas, fibrosarcomas, chondrosarcomas, and synovial sarcomas. Apparently alveolar soft part sarcomas are unusual in their capacity of expression of NOS isoforms, and in a very peculiar pattern. In conclusion, sarcomas in general commonly express constitutive NOS, and only a few types of sarcomas can express iNOS, the isoenzymes capable of releasing large amounts of NO. More comprehensive studies should be performed to better understand the clinical importance of NOS expression and NO production in sarcomas.

Protecting the Brain With eNOS

See related article, pages 1132–1140 Nitric oxide (NO) plays a major role in both vascular and neural biology in health and disease. This concept is exemplified when one examines the role of NO in the brain and the cerebral circulation. NO is a potent vasodilator, and blood vessels in the brain are normally exposed to NO from 2 major sources—endothelium and neurons. Acting as a messenger molecule, NO mediates the majority of endothelium-dependent responses in the brain.1 The source of this NO is the endothelial isoform of NO synthase (eNOS). This prominent role for NO has been observed in a variety of blood vessels from multiple species including humans.1–3 Through this signaling mechanism, NO influences resting vascular tone and mediates responses to varied stimuli, including endothelium-dependent agonists and increases in blood flow (Figure).1,4,5 In addition, eNOS inhibits vasoconstrictor responses and cerebral vasospasm6–8 and can affect permeability of cerebral endothelium, the blood-brain barrier (Figure).9 eNOS normally inhibits cerebral vascular growth or hypertrophy (Figure),10 a structural change that can have functional consequences by impairing of maximal vasodilator capacity. Impairment of NO-mediated signaling is a key underlying mechanism of vascular dysfunction in diverse forms of disease and aging.1,11–13 Impairment of these eNOS-dependent mechanisms in the presence of cardiovascular risk factors may contribute to …

The participation of brain NO synthase in blood pressure control of adult spontaneously hypertensive rats

Increased blood pressure (BP) in genetic hypertension is usually caused by high activity of sympathetic nervous system (SNS) which is enhanced by central angiotensin II but lowered by central nitric oxide (NO). We have therefore evaluated NO synthase (NOS) activity as well as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) protein expression in brainstem and midbrain of adult spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. We also studied possible participation of brain NO in antihypertensive effects of chronic captopril treatment of adult SHR. NOS activity was increased in midbrain of SHR compared to Wistar-Kyoto (WKY) rats. This could be ascribed to enhanced iNOS expression, whereas nNOS expression was unchanged and eNOS expression was reduced in this brain region. In contrast, no significant changes of NOS activity were found in brainstem of SHR in which nNOS and iNOS expression was unchanged, but eNOS expression was increased. Chronic captopril administration lowered BP of adult SHR mainly by attenuation of sympathetic tone, whereas the reduction of angiotensin II-dependent vasoconstriction and the decrease of residual BP (amelioration of structural remodeling of resistance vessels) were less important. This treatment did not affect significantly either NOS activity or expression of any NOS isoform in the two brain regions. Our data do not support the hypothesis that altered brain NO formation contributes to sympathetic hyperactivity and high BP of adult SHR with established hypertension.

Differential up-regulation of hypoxia-inducible vasoactive factors in fetal mouse brain in vivo upon intrauterine hypoxia

Objective: Hypoxia inducible transcription factor-1 (HIF-1) and HIF-1 regulated vasoactive mechanisms are crucial factors in the adaptation to ischemic and non-ischemic hypoxia of the developmental brain. Aims: (i) To investigate the expression of HIF-1α and its target genes vascular endothelial growth factor (VEGF) and inducible NO synthase (iNOS) in fetal mouse brain upon exposure to non-lethal intrauterine hypoxia. In addition, expression of HIF-1-independent but hypoxia-inducible genes (nNOS, caspase-3) was investigated.

Effects of deltamethrin on neurobehavioral development of offspring of intoxicated rats

To investigate the effects of deltamethrin on the filial brain nitric oxide synthase (NOS) activity and neurobehavioral development of the exposed lactational rats. Pregnant rats were randomizedly divided into the treated group and the control group. The treated group was administered orally with 3.35, 6.70 mg/kg deltamethrin every other day from postnatal day (PND) 1 to PND 19 while the control group was administered with the corn oil of same amount in the same period. The activity of NOS of filial brain and neurobehavioral functions of the filial rats were observed. The lactational survival rate (81.80%:78.60%) in both treated groups was decreased significantly (P < 0.01) compared with that in the control group. The body weight of filial rats on PND 10, 21 in 6.70 mg/kg DM treated group [(16.62 +/- 2.2 8), (31.34 +/- 6.94) g] was less than those in the control group (P < 0.05). The delayed time in the filial rats in 6.70 mg/kg group was (3.05 +/- 1.20) s and the positive rates of passive escaping response in 3.35 and 6.70 mg/kg DM treated group were 22.5% and 21.5% respectively. There was the trend of the developmental increase of the activity of filial brain NOS between PND 5 and PND 21 and the NOS activity of rat brain on PND 5 in 6.70 mg/kg group [(0.60 +/- 0.07) U.mg pro(-1).h(-1)] was lower than that in the control group (P < 0.05). Exposure to high dose of deltamethrin in lactational female rats will decrease the activity of NOS of brain and retard the neurobehavioral development of their filial rats.

Immunocytochemical localization of metabotropic (mGluR2/3 and mGluR4a) and ionotropic (GluR2/3) glutamate receptors in adrenal medullary ganglion cells.

The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.