Environmental Enrichment Promotes Antioxidant Effect in the Ventrolateral Medulla and Kidney of Renovascular Hypertensive Rats / O Enriquecimento Ambiental Promove Efeito Antioxidante no Bulbo Ventrolateral e Rins de Roedores com Hipertensão Renovascular

Abstract Background: Arterial hypertension is a precursor to the development of heart and renal failure, furthermore is associated with elevated oxidative markers. Environmental enrichment of rodents increases performance in memory tasks, also appears to exert an antioxidant effect in the hippocampus of normotensive rats. Objectives: Evaluate the effect of environmental enrichment on oxidative stress in the ventrolateral medulla, heart, and kidneys of renovascular hypertensive rats. Methods: Forty male Fischer rats (6 weeks old) were divided into four groups: normotensive standard condition (Sham-St), normotensive enriched environment (Sham-EE), hypertensive standard condition (2K1C-St), and hypertensive enriched environment (2K1C-EE). Animals were kept in enriched or standard cages for four weeks after all animals were euthanized. The level of significance was at p < 0.05. Results: 2K1C-St group presented higher mean arterial pressure (mmHg) 147.0 (122.0; 187.0) compared to Sham-St 101.0 (94.0; 109.0) and Sham-EE 106.0 (90.8; 117.8). Ventrolateral medulla from 2K1C-EE had higher superoxide dismutase (SOD) (49.1 ± 7.9 U/mg ptn) and catalase activity (0.8 ± 0.4 U/mg ptn) compared to SOD (24.1 ± 9.8 U/mg ptn) and catalase activity (0.3 ± 0.1 U/mg ptn) in 2K1C-St. 2K1C-EE presented lower lipid oxidation (0.39 ± 0.06 nmol/mg ptn) than 2K1C-St (0.53 ± 0.22 nmol/mg ptn) in ventrolateral medulla. Furthermore, the kidneys of 2K1C-EE (11.9 ± 2.3 U/mg ptn) animals presented higher superoxide-dismutase activity than those of 2K1C-St animals (9.1 ± 2.3 U/mg ptn). Conclusion: Environmental enrichment induced an antioxidant effect in the ventrolateral medulla and kidneys that contributes to reducing oxidative damage among hypertensive rats.

Resumo Fundamento: A hipertensão arterial é um precursor para o desenvolvimento da insuficiência cardíaca e renal e, além disso, está associada com o aumento dos marcadores oxidativos. O enriquecimento ambiental dos roedores melhora o desempenho em tarefas de memória, e também parece ter um efeito antioxidante sobre o hipocampo dos ratos normotensos. Objetivos: Avaliar o efeito do enriquecimento ambiental sobre o estresse oxidativo no bulbo ventrolateral, coração, e rins de ratos com hipertensão renovascular. Métodos: Quarenta ratos machos, tipo Fischer (6 semanas de idade), foram divididos em quatro grupos: normotensos em condições padrão (Sham-CP), normotensos em ambiente enriquecido (Sham-AE), hipertensos em condições padrão (2R1C-CP), e hipertensos em ambiente enriquecido (2R1C-AE). Os animais foram mantidos em gaiolas enriquecidas ou padrão durante quatro semanas e, por fim, todos os animais foram eutanasiados. O nível de significância foi p < 0,05. Resultados: O grupo 2R1C-CP apresentou pressão arterial média maior (mmHg) 147,0 (122,0; 187,0) quando comparado com os grupos Sham-CP 101,0 (94,0; 109,0) e Sham-AE 106,0 (90,8; 117,8). Observou-se maior atividade das enzimas superóxido dismutase (SOD) (49,1 ± 7,9 U/mg ptn) e da catalase (0,8 ± 0,4 U/mg ptn) no bulbo ventrolateral do grupo 2R1C-AE, em relação à atividade da SOD (24,1 ± 9,8 U/mg ptn) e da catalase (0,3 ± 0,1 U/mg ptn) no grupo 2R1C-CP. No grupo 2R1C-AE, a oxidação lipídica no bulbo ventrolateral foi menor (0,39 ± 0,06 nmol/mg ptn) quando comparado com o grupo 2R1C-CP (0,53 ± 0,22 nmol/mg ptn). Ademais, foi observada maior atividade das enzimas superóxido dismutase nos rins dos animais 2R1C-AE (11,9 ± 2,3 U/mg ptn) em relação aos animais 2R1C-CP (9,1 ± 2,3 U/mg ptn). Conclusão: O enriquecimento ambiental provocou efeito antioxidante no bulbo ventrolateral e nos rins, o que contribuiu para a redução do dano oxidante nos ratos hipertensos.

Environmental Enrichment Promotes Antioxidant Effect in the Ventrolateral Medulla and Kidney of Renovascular Hypertensive Rats.

BACKGROUND: Arterial hypertension is a precursor to the development of heart and renal failure, furthermore is associated with elevated oxidative markers. Environmental enrichment of rodents increases performance in memory tasks, also appears to exert an antioxidant effect in the hippocampus of normotensive rats. OBJECTIVES: Evaluate the effect of environmental enrichment on oxidative stress in the ventrolateral medulla, heart, and kidneys of renovascular hypertensive rats. METHODS: Forty male Fischer rats (6 weeks old) were divided into four groups: normotensive standard condition (Sham-St), normotensive enriched environment (Sham-EE), hypertensive standard condition (2K1C-St), and hypertensive enriched environment (2K1C-EE). Animals were kept in enriched or standard cages for four weeks after all animals were euthanized. The level of significance was at p < 0.05. RESULTS: 2K1C-St group presented higher mean arterial pressure (mmHg) 147.0 (122.0; 187.0) compared to Sham-St 101.0 (94.0; 109.0) and Sham-EE 106.0 (90.8; 117.8). Ventrolateral medulla from 2K1C-EE had higher superoxide dismutase (SOD) (49.1 ± 7.9 U/mg ptn) and catalase activity (0.8 ± 0.4 U/mg ptn) compared to SOD (24.1 ± 9.8 U/mg ptn) and catalase activity (0.3 ± 0.1 U/mg ptn) in 2K1C-St. 2K1C-EE presented lower lipid oxidation (0.39 ± 0.06 nmol/mg ptn) than 2K1C-St (0.53 ± 0.22 nmol/mg ptn) in ventrolateral medulla. Furthermore, the kidneys of 2K1C-EE (11.9 ± 2.3 U/mg ptn) animals presented higher superoxide-dismutase activity than those of 2K1C-St animals (9.1 ± 2.3 U/mg ptn). CONCLUSION: Environmental enrichment induced an antioxidant effect in the ventrolateral medulla and kidneys that contributes to reducing oxidative damage among hypertensive rats.

Tyrosyl-tRNA synthetase: A potential kyotorphin synthetase in mammals.

Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Km = 1400 µM and 200 µM for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6 mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2 h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.

Restoration of Rostral Ventrolateral Medulla Cystathionine-γ Lyase Activity Underlies Moxonidine-Evoked Neuroprotection and Sympathoinhibition in Diabetic Rats.

We recently demonstrated a fundamental role for cystathionine-γ lyase (CSE)-derived hydrogen sulfide (H2S) in the cardioprotective effect of the centrally acting drug moxonidine in diabetic rats. Whether a downregulated CSE/H2S system in the rostral ventrolateral medulla (RVLM) underlies neuronal oxidative stress and sympathoexcitation in diabetes has not been investigated. Along with addressing this question, we tested the hypothesis that moxonidine prevents the diabetes-evoked neurochemical effects by restoring CSE/H2S function within its major site of action, the RVLM. Ex vivo studies were performed on RVLM tissues of streptozotocin (55 mg/kg, i.p.) diabetic rats treated daily for 3 weeks with moxonidine (2 or 6 mg/kg; gavage), H2S donor sodium hydrosulfide (NaHS) (3.4 mg/kg, i.p.), CSE inhibitor DL-propargylglycine (DLP) (37.5 mg/kg, i.p.), a combination of DLP with moxonidine, or their vehicle. Moxonidine alleviated RVLM oxidative stress, neuronal injury, and increased tyrosine hydroxylase immunoreactivity (sympathoexcitation) by restoring CSE expression/activity as well as heme oxygenase-1 (HO-1) expression. A pivotal role for H2S in moxonidine-evoked neuroprotection is supported by the following: 1) NaHS replicated the moxonidine-evoked neuroprotection, and the restoration of RVLM HO-1 expression in diabetic rats; and 2) DLP abolished moxonidine-evoked neuroprotection in diabetic rats, and caused RVLM neurotoxicity, reminiscent of a diabetes-evoked neuronal phenotype, in healthy rats. These findings suggest a novel role for RVLM CSE/H2S/HO-1 in moxonidine-evoked neuroprotection and sympathoinhibition, and as a therapeutic target for developing new drugs for alleviating diabetes-evoked RVLM neurotoxicity and cardiovascular anomalies.

Microglial number is related to the number of tyrosine hydroxylase neurons in SHR and normotensive rats.

Microglia are ubiquitously distributed throughout the central nervous system (CNS) and play a critical role in the maintenance of neuronal homeostasis. Recent advances have shown that microglia, never resting cells of the CNS, continuously monitor and influence neuronal/synaptic activity levels, by communicating with neurons with the aid of their dynamic processes. The brainstem contains many catecholaminergic nuclei that are key to many aspects of brain function. This includes C1 neurons of the ventrolateral medulla that are thought to play a critical role in control of the circulation. Despite the role of catecholaminergic brainstem neurons in normal physiology, the presence of microglia that surrounds them is poorly understood. Here, we investigate the spatial distribution and morphology of microglia in catecholaminergic nuclei of the brainstem in 3 strains of rat: Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Our data reveal that microglia are heterogeneously distributed within and across different strains of rats. Interestingly, intra-strain comparison of tyrosine hydroxylase-immunoreactive (TH-ir) neuronal and microglial number reveals that microglial number varies with the TH-ir neuronal number in the brainstem. Even though microglial spatial distribution varies across brainstem nuclei, microglial morphology (% area covered, number of end point processes and branch length) does not differ significantly. This work provides the first evidence that even though microglia, in their surveilling state, do not vary appreciably in their morphology across brainstem areas, they do have a heterogeneous pattern of distribution that may be influenced by their local environment.

NO-ERGIC CONTROL OF BLOOD CIRCULATION IN THE MEDULLA OBLONGATA OF RATS WITH EXPERIMENTAL HEMIPARKINSONIZM UNDER EXPOSURE TO CONTINUOUS LIGHT.

The study was conducted on rats with unilateral damage to dopaminergic (DA) neurons in substantia nigra of the midbrain (experimental hemiparkinsonism). Degeneration of dopaminergic (DA) neurons was accompanied by hyperactivity of those neurons that remained intact and responded to apomorphine (Apo) test by rotational movements. Depending on the number of rotations, three groups of animals were defined. In the medulla oblongata of rats with unilateral damage to dopaminergic (DA) neurons, a significant increase in the activity of inducible NO-synthase (iNOS) was observed, while the activity of constitutive NO-synthase (cNOS) tended to decrease compared with that in control rats. An activation of neuronal NO-synthase (nNOS) in those rats by injections of L-arginine in the medullary nuclei was accompanied by weakening of the hemodynamic effects compared to those in control rats. An exposure of animals to continuous light for three weeks was accompanied by increasing the number of damaged DA-ergic neurons in substantia nigra. At that, a significant decrease in cNOS activity in the medulla oblongata was observed, leading to the inhibition of de novo synthesis of nitric oxide (NO). The reduction of NO synthesis in the medulla oblongata neurons of rats with experimental hemiparkinsonism following their exposure to continuous light was also evidenced by the reduction.

Impaired expression of neuronal nitric oxide synthase in the gracile nucleus is involved in neuropathic changes in Zucker Diabetic Fatty rats with and without 2,5-hexanedione intoxication.

These studies examined the influence of 2,5-hexanedione (2,5-HD) intoxication on expression of neuronal nitric oxide synthase (nNOS) in the brainstem nuclei in Zucker Diabetic Fatty (ZDF) vs. lean control (LC) rats. Functional neuropathic changes were also investigated following axonal damage and impaired axonal transport induced by the treatment. Animals were intoxicated by i.p. injection of 2,5-HD plus unilateral administration of 2,5-HD over the sciatic nerve. The mechanical thresholds and withdrawal latencies to heat and cold stimuli on the foot were measured at baseline and after intoxication. The medulla sections were examined by nNOS immunohistochemistry and NADPH-diaphorase histochemistry at the end of the treatments. The mechanical thresholds and withdrawal latencies were significantly decreased while nNOS immunostained neurons and NADPH-diaphorase positive cells were selectively reduced in the gracile nucleus at baseline in ZDF vs. LC rats. NADPH-diaphorase reactivity and nNOS positive neurons were increased in the ipsilateral gracile nucleus in LC rats following 2,5-HD intoxication, but its up-regulation was attenuated in ZDF rats. These results suggest that diabetic and chemical intoxication-induced nNOS expression is selectively reduced in the gracile nucleus in ZDF rats. Impaired axonal damage-induced nNOS expression in the gracile nucleus is involved in neuropathic pathophysiology in type II diabetic rats.

[Effect of reboxetine on activity of carboxypeptidase E in the nerve tissue of rats].

Depression is one of the most common mental disorders, but its etiology is not completely understood. It is assumed that peptidergic system components are involved in the formation of this pathology. Neuropeptides play an important role in the regulation of mental and emotional states. Сarboxypeptidase E is a key enzyme of peptide processing; it regulates neuropeptide levels in the various structures of the nervous system. We have studied effects of a single dose of reboxetine on the activity of carboxypeptidase E in various brain regions and the adrenal glands of rats. The reboxetine injection decreased carboxypeptidase E activity in the pituitary gland (12 h after injection), in the pituitary gland, the quadrigeminal bodies, the medulla oblongata, the hypothalamus, the hippocampus and the amygdala (24 h after injection), in the pituitary gland and striatum (72 h after injection). The enzyme activity in adrenal glands remained basically unchanged. Apparently, the decrease of carboxypeptidase E activity may influence the level of regulatory peptides involved in the pathogenesis of depression.

p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats.

BACKGROUND: Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain. RESULTS: Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1ß over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration. CONCLUSIONS: These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Superoxide Mediates Depressive Effects Induced by Hydrogen Sulfide in Rostral Ventrolateral Medulla of Spontaneously Hypertensive Rats.

Hydrogen sulfide (H2S) plays a crucial role in the regulation of blood pressure and oxidative stress. In the present study, we tested the hypothesis that H2S exerts its cardiovascular effects by reducing oxidative stress via inhibition of NADPH oxidase activity in the rostral ventrolateral medulla (RVLM). We examined cell distributions of cystathionine-ß-synthase (CBS) and effects of H2S on reactive oxygen species (ROS) and mean arterial blood pressure (MAP) in spontaneously hypertensive rats (SHRs). We found that CBS was expressed in neurons of the RVLM, and the expression was lower in SHRs than in Wistar-Kyoto rats. Microinjection of NaHS (H2S donor), S-adenosyl-l-methionine (SAM, a CBS agonist), or Apocynin (NADPH oxidase inhibitor) into the RVLM reduced the ROS level, NADPH oxidase activity, and MAP, whereas microinjection of hydroxylamine hydrochloride (HA, a CBS inhibitor) increased MAP. Furthermore, intracerebroventricular infusion of NaHS inhibited phosphorylation of p47(phox), a key step of NADPH oxidase activation. Since decreasing ROS level in the RVLM reduces MAP and heart rate and increasing H2S reduces ROS production, we conclude that H2S exerts an antihypertensive effect via suppressing ROS production. H2S, as an antioxidant, may be a potential target for cardiovascular diseases.

Co-expression of GAD67 and choline acetyltransferase reveals a novel neuronal phenotype in the mouse medulla oblongata.

GABAergic and cholinergic systems play an important part in autonomic pathways. To determine the distribution of the enzymes responsible for the production of GABA and acetylcholine in areas involved in autonomic control in the mouse brainstem, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurones, combined with choline acetyl transferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurones were observed throughout the brainstem. A small number of cells contained both ChAT-IR and GAD67-GFP. Such double labelled cells were observed in the NTS (predominantly in the intermediate and central subnuclei), the area postrema, reticular formation and lateral paragigantocellular nucleus. All ChAT-IR neurones in the area postrema contained GAD67-GFP. Double labelled neurones were not observed in the dorsal vagal motor nucleus, nucleus ambiguus or hypoglossal nucleus. Double labelled ChAT-IR/GAD67-GFP cells in the NTS did not contain neuronal nitric oxide synthase (nNOS) immunoreactivity, whereas those in the reticular formation and lateral paragigantocellular nucleus did. The function of these small populations of double labelled cells is currently unknown, however their location suggests a potential role in integrating signals involved in oromotor behaviours.

Activation of p38 MAPK in the rostral ventromedial medulla by visceral noxious inputs transmitted via the dorsal columns may contribute to pelvic organ cross-sensitization in rats with endometriosis.

Whether visceral organ cross-sensitization is involved in endometriosis-associated pain remains elusive. Previous studies have shown that visceral noxious stimuli may trigger a cascade of signal transductions in the rostral ventromedial medulla (RVM) via the spinal dorsal column (DC) pathway and the RVM plays a critical role in the descending control of visceral nociception. In the current study, we hypothesized that the p38 mitogen-activated protein kinase (MAPK) activation in the RVM by noxious visceral inputs from ectopic growths via the DC was involved in the development of pelvic organ cross-sensitization in established endometriosis. A rat model of experimental endometriosis was established. To examine ectopic growths-to-colon cross-sensitization, graded colorectal distention (CRD) was performed and abdominal withdrawal reflex (AWR) scores were recorded in female rats at 8weeks after the uterine or fat (control) auto-transplantation. Western blot study was carried out to examine the phosphorylated form and the total level of p38 MAPK protein in the RVM. Our results showed that lesions of bilateral DCs immediately following uterine or fat auto-transplantation in female rats significantly attenuated the later development of ectopic growths-to-colon cross-sensitization and the increased p38 MAPK activation in the RVM, as compared to sham DC lesions. Furthermore, intra-RVM microinjection of a p38 MAPK inhibitor (SB 203580), but not vehicle, in female rats with established endometriosis significantly attenuated ectopic growths-to-colon cross-sensitization and the increased activation of p38 MAPK in the RVM. These findings suggest that the noxious inputs from ectopic growths may activate p38 MAPK in the RVM via the DC, which may contribute to the development of ectopic growths-to-colon cross-sensitization in established endometriosis.

Gene transfer of cystathionine ß-synthase into RVLM increases hydrogen sulfide-mediated suppression of sympathetic outflow via KATP channel in normotensive rats.

Hydrogen sulfide has been shown to have a sympathoinhibitory effect in the rostral ventrolateral medulla (RVLM). The present study examined the function of cystathionine ß-synthase (CBS)/hydrogen sulfide system in the RVLM, which plays a crucial role in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding CBS (AdCBS) or enhanced green fluorescent protein (AdEGFP) were transfected into the RVLM in normotensive rats. Identical microinjection of AdCBS into the RVLM had no effect on systolic blood pressure and heart rate (HR) in conscious rats. Acute experiments were performed at day 7 after gene transfer in anesthetized rats. Microinjection of the CBS inhibitors hydroxylamine (HA) or amino-oxyacetate into the RVLM produced an increase in the renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and HR. There was a potentiation of the increases in RSNA, MAP, and HR because of the CBS inhibitors in AdCBS-injected rats compared with AdEGFP-injected rats. Pretreatment with pinacidil, a ATP-sensitive potassium (KATP) channel activator, abolished the effects of HA in two groups. Microinjection of glibenclamide, a KATP channel blocker, produced increases in RSNA, MAP, and HR in AdCBS-injected rats. No changes in behavior were observed in AdEGFP-injected rats. Furthermore, Western blot analysis indicated an increase in the expression of sulfonylurea receptor 2 and inward rectifier K(+) 6.1 in AdCBS-injected rats. These results suggest that the increase in KATP channels in the RVLM may be responsible for the greater sympathetic outflow and pressor effect of HA in AdCBS-injected rats compared with AdEGFP-injected rats.

Cystathionine-ß-Synthase Gene Transfer Into Rostral Ventrolateral Medulla Exacerbates Hypertension via Nitric Oxide in Spontaneously Hypertensive Rats.

BACKGROUND: Rostral ventrolateral medulla (RVLM) plays a crucial role in the central regulation of cardiovascular functions. Cystathionine-ß-synthase (CBS) is a major hydrogen sulfide (H2S)-generating enzyme that has been identified mainly in the brain. The present study was designed to examine CBS expression and determine its roles and mechanisms of regulating sympathetic outflow and blood pressure (BP) in the RVLM in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: CBS expression was decreased in the RVLM in SHR compared to Wistar-Kyoto (WKY) rats. Accumulating evidences suggest that H2S interacts with nitric oxide (NO) to regulate cardiovascular function. Therefore, we hypothesize that the decrease in CBS expression in the RVLM may be involved in the disorder of l-arginine/NO pathway, which subsequently affects BP in SHR. Overexpression of CBS in the RVLM caused significant increases in BP, heart rate, and urinary norepinephrine excretion in SHR but not in WKY. Acute experiments were carried out at day 7 after gene transfer. NO metabolite levels, neuronal NO synthase, and γ-amino butyric acid were decreased in SHR after CBS gene transfer. Furthermore, pressor responses to microinjection of NG-monomethyl-l-arginine into RVLM were blunt in SHR transfected with AdCBS compared to SHR transfected with AdEGFP. CONCLUSIONS: Overexpression of CBS in the RVLM elicits enhanced pressor responses in SHR, but not in WKY, and the NO system is involved in these effects. The results suggest that alterations of H2S signaling in the brain may be associated with the development of hypertension.

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla.

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.

P2 purinergic receptor activation of neuronal nitric oxide synthase and guanylyl cyclase in the dorsal facial area of the medulla increases blood flow in the common carotid arteries of cats.

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,ß-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.

[DYNAMICS OF GLUTAMINE SYNTHASE ACTIVITY IN RAT BRAIN IN PRENATAL HYPOXIA MODEL].

Prenatal ontogenesis is a period of high sensitivity to stressful impact, so any stressor can lead to changes of physiological, biochemical indicators, behavioral and cognitive functions. The most common and clinically significant stress factor, which the embryo may be exposed during embryonic development, is hypoxia. In this case pathological changes in the central nervous system depend on the duration and severity of hypoxic exposure, individual tolerance and the stage of prenatal development, at each of which in the brain take place the basic histogenetic processes. By activating energetically disadvantageous anaerobic glycolysis hypoxia leads to excess of glutamate emission and cell apoptosis. Glutamine synthase is a basic enzyme that regulates metabolism of glutamate, catalyzing conversion of glutamate to glutamine with ammonia detoxification. The aim of the presented work was to reveal changes in the activity of one of the key enzyme of glutamate metabolism- glutamine synthetase in the brain of offspring of white rats undergone to hypoxia at different stages of prenatal ontogenesis. Hypoxia was created by placing female rats at stages of the pregnancy, corresponding to progestation period of organogenesis and fetal period of prenatal development, in the hypobaric chamber with exposure to 5% oxygen and 95% nitrogen gas mixture during 30 minutes per day. The offspring obtained from females of control and experimental groups were used for biochemical determinations in the age of 1 and 3 month. It has been established that hypoxia exposed to pregnant females during embryonic organogenesis causes significant changes in enzyme activity, particularly pronounced in the cerebral cortex and cerebellum, as compared with progestational and fetal hypoxia. Enzyme activity decreased in a greater degree in one-month-old rats undergone to prenatal hypoxia, than three- month-old animals. Thus, stress during intensive processes of proliferation and migration of cells of the forming brain violates glutamate metabolism of the brain.

Ser/thr phosphatases tonically attenuate the ERK-dependent pressor effect of ethanol in the rostral ventrolateral medulla in normotensive rats.

We recently reported that microinjection of ethanol into the rostral ventrolateral medulla (RVLM) elicits modest increases in local extracellular signal-regulated kinase (ERK) and blood pressure (BP) in conscious normotensive rats. In this study, we tested the hypothesis that RVLM ser/thr phosphatases dampen the ERK-dependent pressor effect of ethanol in normotensive rats. We show that the pressor response elicited by intra-RVLM ethanol (10 µg) was (i) abolished following local ERK inhibition with PD98059 (1 µg) and (ii) associated with significant reduction in local phosphatase activity. Inhibition of the RVLM ser/thr phosphatase activity by okadaic acid (OKA, 0.4 µg) or fostriecin (15 pg) caused significant increases in blood pressure (BP) and potentiated the magnitude and duration of the pressor response as well as the phosphatase inhibition elicited by subsequent intra-RVLM administration of ethanol. Intra-RVLM acetaldehyde (2 µg), the main metabolic product of ethanol, caused no changes in BP or RVLM phosphatase activity but it produced significant increases in BP and inhibition of local phosphatase activity in rats treated with OKA or fostriecin. Together, the RVLM phosphatase activity acts tonically to attenuate the ERK-dependent pressor effect of ethanol or acetaldehyde in normotensive rats.

Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats.

The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in the brain has been suggested to be beneficial to hypertension. This study was designed to determine the effect of ACE2 gene transfer into the RVLM on tonically active glutamatergic input in spontaneously hypertensive rats (SHRs). Lentiviral particles containing enhanced green fluorescent protein (lenti-GFP) or ACE2 (lenti-ACE2) were injected bilaterally into the RVLM. Both protein expression and activity of ACE2 in the RVLM were increased in SHRs after overexpression of ACE2. A significant reduction in blood pressure and heart rate in SHRs was observed 6 wk after lenti-ACE2 injected into the RVLM. The concentration of glutamate in microdialysis fluid from the RVLM was significantly reduced by an average of 61% in SHRs with lenti-ACE2 compared with lenti-GFP. ACE2 overexpression significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity evoked by bilateral injection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nl) into the RVLM in SHRs. Therefore, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced tonically active glutamatergic input in SHRs, which may be an important mechanism underlying the beneficial effect of central ACE2 to hypertension.