Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

BACKGROUND: The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. METHODS: nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. RESULTS: In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. CONCLUSIONS: Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

Sex-dimorphic Rostro-caudal Patterns of 5'-AMP-activated Protein Kinase Activation and Glucoregulatory Transmitter Marker Protein Expression in the Ventrolateral Ventromedial Hypothalamic Nucleus (VMNvl) in Hypoglycemic Male and Female Rats: Impact of Estradiol.

The ventromedial hypothalamic nucleus-ventrolateral part (VMNvl) is an estradiol-sensitive structure that controls sex-specific behavior. Electrical reactivity of VMNvl neurons to hypoglycemia infers that cellular energy stability is monitored there. Current research investigated the hypothesis that estradiol elicits sex-dimorphic patterns of VMNvl metabolic sensor activation and gluco-regulatory neurotransmission during hypoglycemia. Rostral-, middle-, and caudal-VMNvl tissue was separately micropunch-dissected from letrozole (Lz)- or vehicle-injected male and estradiol- or vehicle-implanted ovariectomized (OVX) female rats for Western blot analysis of total and phosphorylated 5'-AMP-activated protein kinase (AMPK) protein expression and gluco-stimulatory [neuronal nitric oxide synthase (nNOS); steroidogenic factor-1 (SF1) or -inhibitory (glutamate decarboxylase65/67 (GAD)] transmitter marker proteins after sc insulin (INS) or vehicle injection. In both sexes, hypoglycemic up-regulation of phosphoAMPK was estradiol-dependent in rostral and middle, but not caudal VMNvl. AMPK activity remained elevated after recovery from hypoglycemia over the rostro-caudal VMNvl in female, but only in the rostral segment in male. In each sex, hypoglycemia correspondingly augmented or suppressed nNOS profiles in rostral and middle versus caudal VMNvl; these segmental responses persisted longer in female. Rostral and middle segment SF1 protein was inhibited by estradiol-independent mechanisms in hypoglycemic males, but increased by estradiol-reliant mechanisms in female. After INS injection, GAD expression was inhibited in the male rostral VMNvl without estradiol involvement, but this hormone was required for broader suppression of this profile in the female. Neuroanatomical variability of VMNvl metabolic transmitter reactivity to hypoglycemia underscores the existence of functionally different subgroups in that structure. The regional distribution and estradiol sensitivity of hypoglycemia-sensitive VMNvl neurons of each neurochemical phenotype evidently vary between sexes.

Upregulation of the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor axis by a combination of Yinyanghuo (Herba Epimedii Brevicornus) and Cheqianzi (Semen Plantaginis) improves erectile function in spontaneously hypertensive rats.

OBJECTIVE: To evaluate the effects of a combination of Yinyanghuo (Herba Epimedii Brevicornus) (HEB) and Cheqianzi (Semen Plantaginis) (SP) on erectile dysfunction caused by essential hypertension in spontaneously hypertensive rats (SHRs), and to elucidate the role of the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor (ACE2/Ang [1-7]/Mas receptor) axis in this process. METHODS: A total of 24 SHRs were randomly assigned to three groups: SHR-control, low-dose (12.5 g/kg) and high-dose (25 g/kg) HEB+SP (HEBSP). Eight Wistar-Kyoto rats were used as normal controls. HEBSP was administered by oral gavage for 28 d. Erectile function was measured once a week using the Heaton test. After 4 weeks of treatment, the corpus cavernosum was harvested from each rat to measure nitric oxide (NO), nitric oxide synthase (eNOS) and Ang (1-7) levels, as well as ACE2, Mas receptor and neuronal nitric oxide synthase (nNOS) protein expression. RESULTS: After 4 weeks of treatment, HEBSP significantly increased erectile function in the treated group compared with SHR-control group (P < 0.01). Additionally, HEBSP treatment significantly increased cavernosal levels of Ang (1-7), eNOS and NO. Moreover, HEBSP significantly elevated the expression levels of ACE2, Mas receptor and nNOS. These beneficial effects were elevated in the high-dose HEBSP group. CONCLUSION: HEBSP improved erectile function in SHRs by upregulating the ACE2/Ang (1-7)/Mas receptor axis, eNOS and nNOS pathways.

Evodiamine via targeting nNOS and AMPA receptor GluA1 inhibits nitroglycerin-induced migraine-like response.

ETHNOPHARMACOLOGICAL RELEVANCE: Evodiamine (EVO) is a natural compound derived from Tetradium ruticarpum (A.Juss.) T.G.Hartley used to treat pain and migraine in traditional Chinese medicine. EVO is the primary active ingredient of Tetradium ruticarpum. However, the preventive effect of EVO against migraine remains unexplored. AIM OF THE STUDY: To investigate the preventive effect of EVO against nitroglycerin (NTG)-induced acute migraine in rats. MATERIALS AND METHODS: Male Sprague-Dawley rats were intragastrically administered EVO (45 or 90 mg/kg) for nine days. To establish an acute migraine model, we subcutaneously injected rats with a 10 mg/kg NTG solution. The migraine-like behavior of the rats was evaluated via the formalin test and the warm water tail-withdrawal assay. The periaqueductal gray (PAG) and serum samples were collected from the rats and used to determine the effect of EVO on the levels of serum nitric oxide (NO), CGRP, c-Fos, neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor GluA1. RESULTS: The formalin test and the warm water tail-withdrawal assay showed that EVO inhibited the licking foot/shaking response and reversed the shortened tail-withdrawal latency in NTG-treated rats. Additionally, EVO suppressed serum NO levels and reduced the mRNA/protein expression of c-Fos and nNOS, but not iNOS, in the PAG. Furthermore, EVO suppressed total protein expression of the AMPA receptor GluA1 and its phosphorylation at Ser831 and Ser845. CONCLUSIONS: This study showed that EVO inhibits the migraine-like pain response and that this beneficial effect might be attributed to the regulation of nNOS and suppression of the AMPA receptor GluA1. We suggest that EVO has the potential to treat migraine as a lead compound of natural origin.

Astragalus root extract inhibits retinal cell apoptosis and repairs damaged retinal neovascularization in retinopathy of prematurity.

Since the functions of Astragalus root extract in retinopathy remain to be unraveled, this study is performed to elucidate whether Astragalus root extract functions in retinal cell apoptosis and angiogenesis in retinopathy of prematurity (ROP). Newborn mice were selected for establishing mice models of oxygen-induced retinopathy (OIR), which were treated with high-, medium- or low-Astragalus root extract. Evans Blue (EB) was perfused to detect the blood retinal barrier. Additionally, the vascular morphology, number of endothelial cell nuclei of neovascularization, proliferation of blood vessels, ultrastructural changes were determined via a series of assays. Moreover, levels of reactive oxygen species (ROS), expression of other factors such as VEGF, PEDF, IGF-1, HIF-1α, Bax, Bcl-2, eNOS, nNOS, and iNOS were detected. Astragalus root extract was found to protect blood-retinal barrier in the OIR model mice through repairing the structure and morphology of retina, inhibiting ROS production, retinal cell apoptosis, as well as improving retinal vascular angiogenesis. Astragalus root extract was also found to decrease VEGF and HIF-1α expression, but enhance PEDF and IGF-1 expression in the OIR model mice, thereby protecting retinas in ROP. This study highlights that Astragalus root extract is able to suppress retinal cell apoptosis and repair damaged retinal neovascularization in ROP, which provides basis for ROP therapy.

Ocimum basilicum miRNOME revisited: A cross kingdom approach.

O. basilicum is medicinally important herb having inevitable role in human health. However, the mechanism of action is largely unknown. Present study aims to understand the mechanism of regulation of key human target genes that could plausibly modulated by O. basilicum miRNAs in cross kingdom manner using computational and system biology approach. O. basilicum miRNA sequences were retrieved and their corresponding human target genes were identified using psRNA target and interaction analysis of hub nodes. Six O. basilicum derived miRNAs were found to modulate 26 human target genes which were associated `with PI3K-AKTand MAPK signaling pathways with PTPN11, EIF2S2, NOS1, IRS1 and USO1 as top 5 Hub nodes. O. basilicum miRNAs not only regulate key human target genes having a significance in various diseases but also paves the path for future studies that might explore potential of miRNA mediated cross-kingdom regulation, prevention and treatment of various human diseases including cancer.

Agarwood Essential Oil Ameliorates Restrain Stress-Induced Anxiety and Depression by Inhibiting HPA Axis Hyperactivity.

In our previous investigation, we found that agarwood essential oil (AEO) has a sedative-hypnotic effect. Sedative-hypnotic drugs usually have an anxiolytic effect, where concomitant anxiety and depression are a common comorbidity. Therefore, this study further investigated the anxiolytic and antidepressant effects of AEO using a series of animal behavior tests on a restraint stress-induced mice model. The elevated plus maze (EPM) test, the light dark exploration (LDE) test, and the open field (OF) test demonstrated that AEO has a significant anxiolytic effect. Simultaneously, the tail suspension (TS) test and the forced swimming (FS) test illuminated that AEO has an antidepressant effect with the immobility time decreased. Stress can cause cytokine and nitric oxide (NO) elevation, and further lead to hypothalamic-pituitary-adrenal (HPA) axis hyperactivity. AEO was shown to dose-dependently inhibit the levels of cytokines, including interleukin 1α (IL-1α), IL-1ß, and IL-6 in serum, significantly decrease the mRNA level of neural nitric oxide synthase (nNOS) in the cerebral cortex and hippocampus, and inhibit the nNOS protein level in the hippocampus. Concomitant measurements of the HPA axis upstream regulator corticotropin releasing factor (CRF) and its receptor CRFR found that AEO significantly decreases the gene expression of CRF, and significantly inhibits the gene transcription and protein expression of CRFR in the cerebral cortex and hippocampus. Additionally, AEO dose-dependently reduces the concentrations of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) downstream of the HPA axis, as measured by ELISA kits. These results together demonstrate that AEO exerts anxiolytic and antidepressant effects which are related to the inhibition of CRF and hyperactivity of the HPA axis.

Ling-Yang-Gou-Teng-decoction prevents vascular dementia through inhibiting oxidative stress induced neurovascular coupling dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Vascular dementia (VaD) is the common cognitive disorder derived mainly from lacunar stroke (LS). The oxidative stress induced neurovascular coupling (NVC) dysfunction involves in the pathogenesis of VaD. Currently, there is no specific drug for VaD. Ling-Yang-Gou-Teng -Decoction (LG), a well-known traditional Chinese formula, has been used for preventing VaD in clinic. AIM OF THE STUDY: In this study, we aimed to investigate the underlying mechanism of LG on VaD in rats. MATERIALS AND METHOD: VaD was replicated with autologous micro-thrombi against the background of hypercholesterolemia induced with high fatty diet. PTX (68.90 mg/kg/day), LG with three dosages (2.58, 8.14, 25.80 g/kg/day) was orally administrated to VaD rats, respectively. The NVC sensitivity was defined as the ratio of the microcirculative cerebral blood velocity (CBV) to the electroencephalograph (EEG) before and after penicillin stimulation. Behavioral performance, pathological changes of brain and oxidation related molecules were detected to assess the effects of LG on VaD. RESULTS: LG exhibited beneficial effects on the VaD, which was demonstrated as improved exploratory, learning and memory abilities, relieved vascular or neural pathological changes in cerebral cortex or hippocampus. LG maintained NVC sensitivity, which was confirmed as significantly increased ΔCBV and the elevated ratio of ΔCBV/ΔqEEG. The underlying mechanisms of LG was associated with antioxidant effects, which was confirmed as significantly decreased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression, and increased superoxide dismutase 3 (SOD3) expression. LG also reduced iNOS, increased nNOS and eNOS expression to restore NO bioavailability. CONCLUSIONS: The results suggested that LG prevented VaD may associate with inhibiting oxidative stress, protecting NO bioavailability, and then maintaining NVC sensitivity.

Galanin enhances systemic glucose metabolism through enteric Nitric Oxide Synthase-expressed neurons.

OBJECTIVE: Decreasing duodenal contraction is now considered as a major focus for the treatment of type 2 diabetes. Therefore, identifying bioactive molecules able to target the enteric nervous system, which controls the motility of intestinal smooth muscle cells, represents a new therapeutic avenue. For this reason, we chose to study the impact of oral galanin on this system in diabetic mice. METHODS: Enteric neurotransmission, duodenal contraction, glucose absorption, modification of gut-brain axis, and glucose metabolism (glucose tolerance, insulinemia, glucose entry in tissue, hepatic glucose metabolism) were assessed. RESULTS: We show that galanin, a neuropeptide expressed in the small intestine, decreases duodenal contraction by stimulating nitric oxide release from enteric neurons. This is associated with modification of hypothalamic nitric oxide release that favors glucose uptake in metabolic tissues such as skeletal muscle, liver, and adipose tissue. Oral chronic gavage with galanin in diabetic mice increases insulin sensitivity, which is associated with an improvement of several metabolic parameters such as glucose tolerance, fasting blood glucose, and insulin. CONCLUSION: Here, we demonstrate that oral galanin administration improves glucose homeostasis via the enteric nervous system and could be considered a therapeutic potential for the treatment of T2D.

A cross-domain charge interaction governs the activity of NO synthase.

NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells.

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.

The gentle art of saying NO: how nitric oxide gets things done in the hypothalamus.

The chemical signalling molecule nitric oxide (NO), which freely diffuses through aqueous and lipid environments, subserves an array of functions in the mammalian central nervous system, such as the regulation of synaptic plasticity, blood flow and neurohormone secretion. In this Review, we consider the cellular and molecular mechanisms by which NO evokes short-term and long-term changes in neuronal activity. We also highlight recent studies showing that discrete populations of neurons that synthesize NO in the hypothalamus constitute integrative systems that support life by relaying metabolic and gonadal signals to the neuroendocrine brain, and thus gate the onset of puberty and adult fertility. The putative involvement and therapeutic potential of NO in the pathophysiology of brain diseases, for which hormonal imbalances during postnatal development could be risk factors, is also discussed.

The transition from day-to-night activity is a risk factor for the development of CNS oxygen toxicity in the diurnal fat sand rat (Psammomys obesus).

Performance and safety are impaired in employees engaged in shift work. Combat divers who use closed-circuit oxygen diving apparatus undergo part of their training during the night hours. The greatest risk involved in diving with such apparatus is the development of central nervous system oxygen toxicity (CNS-OT). We investigated whether the switch from day-to-night activity may be a risk factor for the development of CNS-OT using a diurnal animal model, the fat sand rat (Psammomys obesus). Animals were kept on a 12:12 light-dark schedule (6 a.m. to 6 p.m. at 500 lx). The study included two groups: (1) Control group: animals were kept awake and active during the day, between 09:00 and 15:00. (2) Experimental group: animals were kept awake and active during the night, between 21:00 and 03:00, when they were exposed to dim light in order to simulate the conditions prevalent during combat diver training. This continued for a period of 3 weeks, 5 days a week. On completion of this phase, 6-sulphatoxymelatonin (6-SMT) levels in urine were determined over a period of 24 h. Animals were then exposed to hyperbaric oxygen (HBO). To investigate the effect of acute melatonin administration, melatonin (50 mg/kg) or its vehicle was administered to the animals in both groups 20 min prior to HBO exposure. After the exposure, the activity of superoxide dismutase, catalase and glutathione peroxidase was measured, as were the levels of neuronal nitric oxide synthase (nNOS) and overall nitrotyrosylation in the cortex and hippocampus. Latency to CNS-OT was significantly reduced after the transition from day-to-night activity. This was associated with alterations in the level of melatonin metabolites secreted in the urine. Acute melatonin administration had no effect on latency to CNS-OT in either of the groups. Nevertheless, the activity of superoxide dismutase and catalase, as well as nitrotyrosine and nNOS levels, were altered in the hippocampus following melatonin administration. On the basis of these results, we suggest that a switch from diurnal to nocturnal activity may represent an additional risk factor for the development of CNS-OT. Utilizing a diurnal animal model may contribute to our understanding of the heightened risk of developing CNS-OT when diving with closed-circuit oxygen apparatus at night.

Involvement of DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP Pathways in Human Tissue Kallikrein 1 Protecting Erectile Function in Aged Rats.

Our previous studies had reported that Human Tissue Kallikrein 1 (hKLK1) preserved erectile function in aged transgenic rats, while the detailed mechanism of hKLK1 protecting erectile function in aged rats through activation of cGMP and cAMP was not mentioned. To explore the latent mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 18 months old and divided into four groups: young WTR (yWTR) as the control, aged WTR (aWTR), aged TGR (aTGR) and aged TGRs with HOE140 (aTGRH). Erectile function of all rats was evaluated by cavernous nerve electrostimulation method and measured by the ratio of intracavernous pressure/ mean arterial pressure (ICP/MAP) in rats. Expression levels of cAMP and cGMP were assessed, and related signaling pathways were detected by western blot, immunohistochemistry and RT-PCR. Our experiment results showed erectile function of the aWTR group and aTGRH group was lower compared with those of other two groups. Also, expression levels of cAMP and cGMP were significantly lower than those of other two groups. Moreover, expressions of related signaling pathways including DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP were also downregulated in the corpus cavernosum of rats in aWTR group. Our finding revealed hKLK1 played a protective role in age-related ED. The DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP pathways that were linked to the mechanism hKLK1 could increase the levels of cGMP and cAMP, which might provide novel therapy targets for age-related ED.

Naringin regulates glutamate-nitric oxide cGMP pathway in ammonium chloride induced neurotoxicity.

Naringin, plant bioflavonoid extracted mainly from grapefruit and other related citrus species. This study was designed to assess the neuroprotective effect of naringin on ammonium chloride (NH4Cl) induced hyperammonemic rats. Experimental hyperammonemia was induced by intraperitonial injection (i.p) of NH4Cl (100mg/kg body weight (b.w.)) thrice a week for 8 consecutive weeks. Hyperammonemic rats were treated with naringin (80mg/kg b.w.) via oral gavage. Naringin administration drastically restored the levels of blood ammonia, plasma urea, nitric oxide (NO), glutamate, glutamine, lipid peroxidation, lipid profile, activities of liver marker enzymes, antioxidant status and sodium/potassium-ATPase (Na+/K+-ATPase). In addition, naringin supplementation reverted back the pathological changes of liver, brain and kidney tissues, the expressions of Glutamine synthetase (GS), Na+/K+-ATPase, neuronal nitric oxide (nNOS) and soluble guanylate cyclase (sGC) in hyperammonemic rats. Hence, this study suggested that nargingin exhibited their protective effect against NH4Cl induced toxicity via enhancing the activities of antioxidant enzymes and inhibiting the lipid peroxidation process. Take together, this study provides data that naingin effectively reduced neurotoxicity by attenuating hyperammonemia, suggesting that naringin act as a potential therapeutic agent to treat hyperammonemic rats.

Enhanced XOR activity in eNOS-deficient mice: Effects on the nitrate-nitrite-NO pathway and ROS homeostasis.

Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis. Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS-/-) and wildtype (wt) mice. XOR activity was upregulated in eNOS-/- compared with wt, but not in nNOS-/-, iNOS-/- or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS-/- compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS-/- displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS-/-, but not in wt mice. A high dose of dietary nitrate reduced blood pressure in naïve eNOS-/- mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.

[Chinese herbal medicine enhances sexual function and c-Fos/nNOS expression in the nucleus accumbens of orchidectomized rats].

RESULTS: There was a decrease in accessory genital organ weight, plasma testosterone, and sexual behavior, as well as a low number of c-Fos-positive cells and a large nNOS-positive cell area in orchidectomized rats. Administration of the herbal medicine increased accessory genital organ weight, testosterone level, mating behavior, and c-Fos-positive cell number, while it decreased the nNOS-positive cell area in orchidectomized rats. CONCLUSION: An increase of plasma testosterone after administration of "kidney-nourishing" herbal medicine might contribute to the elevated sexual function and activity in orchidectomized rats. In addition, a central nervous system mechanism, such as the functional alteration of NAc, might be involved. Abstract OBJECTIVE: To determine whether the central nervous system is involved in the effect of Chinese herbal medicine on sexual function recovery in orchidectomized rats. METHODS: Orchidectomized rats were administered intragastrically with a decoction of "kidney-nourishing" Chinese herbal medicine once per day for 28 days. Accessory genital organ weight, plasma testosterone, and mating behavior were investigated. The expression of c-Fos and neuronal nitric oxide synthase (nNOS) in neuronal cells in the nucleus accumbens (NAc) was analyzed by immunohistochemistry.

[Effects of Electroacupuncture on Hippocampal nNOS Expression in Rats with Anxiety-like Behavior].

OBJECTIVE: To compare the effects of electroacupuncture (EA) preconditioning and post-conditioning on hippocampal neuronal nitric oxide synthase (nNOS) expression in rats with anxiety-like behavior, so as to explore the reasonable EA intervention time. METHODS: Forty-two SD rats were randomly divided into 6 groups:normal 1, normal 2, anxiety model 1, an-xiety model 2, EA-pre-conditioning and EA-post-conditioning. The anxiety model was established by giving the rats with repeated foot shock stimulation (0.8 mA, 2-25 s/time, 10 times in 5 min) combined with isolation-raising. Before or after modeling, EA stimulation was applied to "Baihui" (GV 20) and "Yintang" (GV 29) for 20 min, once a day for 7 days. Elevated plus maze (EPM) tests were performed to determine the percentages of time spent in the open arms and the percentages of entries into the open arms in 5 min for evaluating the animals' anxiety-like behavioral activities. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect hippocampal nNOS mRNA expression and the immunohistochemical staining adopted to detect the expression of nNOS protein in the hippocampal CA 1 and CA 3 areas, respectively. RESULTS: Compared with their own normal control group 1 and 2, both the percentage of time spent in the open arms and the percentage of entries into the open arms were significantly decreased in model group 1 and 2 (P<0.05, P<0.01). After pre- and post-conditioning of EA, the decreased proportions of time spent and entries into the open arms were considerably increased (P<0.05), suggesting an improvement of anxiety-like behavior activities after EA intervention. The expression levels of hippocampal nNOS mRNA and nNOS protein in the hippocampal CA 3 region were significantly higher in the model group 1 and 2 than in their own normal control group 1 and 2 (P<0.01), but those of nNOS protein in the CA 1 area were markedly lower in the model group 1 and 2 than in their own normal control group 1 and 2 (P<0.01). Following pre- and post-conditioning of EA, the increased expression levels of nNOS mRNA and protein of CA 3 area and the decreased level of nNOS protein of CA 1 area were all notably reversed (P<0.05, P<0.01). CONCLUSIONS: Both pre- and post-conditioning of EA can improve anxiety-like behavior in anxiety rats, which may be associated with its effects in down-regulating hippocampal nNOS expression.

The effects of repetitive vibration on sensorineural function: biomarkers of sensorineural injury in an animal model of metabolic syndrome.

Exposure to hand-transmitted vibration in the work-place can result in the loss of sensation and pain in workers. These effects may be exacerbated by pre-existing conditions such as diabetes or the presence of primary Raynaud's phenomena. The goal of these studies was to use an established model of vibration-induced injury in Zucker rats. Lean Zucker rats have a normal metabolic profile, while obese Zucker rats display symptoms of metabolic disorder or Type II diabetes. This study examined the effects of vibration in obese and lean rats. Zucker rats were exposed to 4h of vibration for 10 consecutive days at a frequency of 125 Hz and acceleration of 49 m/s(2) for 10 consecutive days. Sensory function was checked using transcutaneous electrical stimulation on days 1, 5 and 9 of the exposure. Once the study was complete the ventral tail nerves, dorsal root ganglia and spinal cord were dissected, and levels of various transcripts involved in sensorineural dysfunction were measured. Sensorineural dysfunction was assessed using transcutaneous electrical stimulation. Obese Zucker rats displayed very few changes in sensorineural function. However they did display significant changes in transcript levels for factors involved in synapse formation, peripheral nerve remodeling, and inflammation. The changes in transcript levels suggested that obese Zucker rats had some level of sensory nerve injury prior to exposure, and that exposure to vibration activated pathways involved in injury and re-innervation.

Additive effects of Artemisia capillaris extract and scopoletin on the relaxation of penile corpus cavernosum smooth muscle.

The objective was to investigate the cellular effect and action mechanism of Artemisia capillaris extract (ACE) and its component, scopoletin, on penile corpus cavernosum smooth muscle (PCCSM). In vitro study with PCCSM, the precontracted PCCSM with phenylephrine was treated with ACE or scopoletin. Cyclic nucleotides in the perfusate were measured by radioimmunoassay and expression of protein and mRNA of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase in the perfused PCCSM were measured by western blot and real-time PCR, respectively. The interaction of ACE or scopoletin with udenafil was also evaluated. ACE and scopoletin exerted a significant and concentration-dependent relaxation in PCCSM. The perfusion with ACE or scopoletin significantly increased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and the perfusion with ACE or scopoletin increased the expression of eNOS mRNA and protein. Furthermore, ACE or scopoletin enhanced udenafil-inducing relaxation in PCCSM. ACE and scopoletin relaxed the PCCSM mainly by activating nitric oxide-cGMP system and cAMP pathway and they may be additive therapeutic candidates for ED patients who do not completely respond to udenafil.