Androgen receptors are required for full masculinization of the ventromedial hypothalamus (VMH) in rats

The ventromedial hypothalamus (VMH) is one of several sexually dimorphic nuclei that regulate mating behavior, and is rich in steroid hormone receptors and aromatase activity. We looked at the contribution of the androgen receptor (AR) to the volume of the VMH in rats by measuring each of the four subdivisions of the VMH in 90 day old male, female, and XY male rats carrying a mutant AR allele (tfm), which renders animals largely unresponsive to androgens. Confirming published reports, total VMH volume was greater in wild-type males than in females (P<0.01). The mean total volume of the VMH in TFM males was intermediate, but not significantly different from either females or males (Ps>0.10). The sex difference in VMH volume was primarily accounted for by the ventrolateral subdivision (VMHvl), which in both females and TFM males was significantly smaller than in wild-type males (Ps<0.005). There was no significant sex difference in the volume of the other three subdivisions of the VMH. Neuronal somata were larger in males than females in VMHvl, central VMH (VMHc) and the dorsomedial VMH (VMHdm), with TFM males having feminine neuronal somata in the VMHdm and VMHc. These data suggest that AR plays a role during sexual differentiation of the VMH, imparting its greatest effect in the VMHvl. ARs may regulate aromatase expression or activity to affect estrogen receptor activation, or may act independently of estrogen receptors to influence VMH morphology.     

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion

The origin of nitric oxide synthase-containing nerve fibers in rat celiac-superior mesenteric ganglion was examined using retrograde tracing techniques combined with the immunofluorescence method. Fluoro-Gold was injected into the celiac-superior mesenteric ganglion. Neuronal cell bodies retrogradely labeled with Fluoro-Gold in the thoracic spinal cord, the dorsal root ganglia at the thoracic level, the nodose ganglion, and the intestine from the duodenum to the proximal colon were examined for nitric oxide synthase immunoreactivity. About 60% of sympathetic preganglionic neurons in the intermediolateral nucleus projecting to the celiac-superior mesenteric ganglion were immunoreactive for nitric oxide synthase, as were approximately 27% of nodose ganglion neurons and about 65% of dorsal root ganglion neurons projecting to the cceliac-superior mesenteric ganglion. Neurons projecting to the celiac-superior mesenteric ganglion were found in the myenteric plexus of the small and large intestine. In the proximal colon, about 23% of such neurons were immunoreactive for nitric oxide synthase. However, in the small intestine, no immunoreactivity was found in these neurons.     

脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用

在乌拉坦麻醉的成年ＳＤ大鼠上,用玻璃微电极细胞外记录的方法,观察了脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用。结果发现：脑室内注射一氧化氮供体硝普钠对下丘脑室旁核中的加压素神经元产生剂量依赖性抑制作用;脑室内注射一氧化氮合酶抑制剂对加压素神经元也产生抑制作用。上述两种药物对催产素神经元均无作用。这些结果提示：一氧化氮可能在调节加压素和催产素神经元活动中起着不同的作用。     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Upregulation of neuronal nitric oxide synthase, edema and cell injury following heat stress are reduced by pretreatment with EGB-761 in the rat

Rats exposed to 4 h heat stress (HS) at 38°C exhibited marked upregulation of neuronal nitric oxide synthase (nNOS) in the brain regions exhibiting blood–brain barrier (BBB) breakdown, brain edema and cell damage. Pretreatment with an anti-oxidant compound EGB-761 (an extract of Gingko biloba) administered 50 mg/kg, per os for 5 days, significantly attenuated nNOS expression, BBB disruption, brain edema and cell injury. These results suggest that EGB-761 is neuroprotective in heat stress and this effect of the compound is related with the inhibition of NOS expression, not reported earlier.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.     

Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development

Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from l-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.     

Development of immune cellular biosensing system for assessing chemicals on inducible nitric oxide synthase signaling activator

An immune cellular biosensing system has been constructed to assess immunomodulating effects of chemicals. Production of nitric oxide in the immune cellular biosensing system was used as readout of an immune cellular response for assessing the immunomodulating effects of chemicals. The macrophage-like cell line RAW264.7, which has signaling pathways of inducible nitric oxide synthase, was employed in the cellular biosensing system. The immune cellular biosensing system consisted of a Pt counter electrode, an Ag/AgCl reference electrode, and a gold electrode onto which a polyion complex layer was coated to allow adherence of the RAW264.7 cells. As the results of evaluating effects of a polyion complex layer on cell viabilities by using WST-8 assay, the polyion complex layer did not affect RAW264.7 cells. The polyion-coated gold electrode could measure NO without the drawback of electrochemical interference that occurs with differential pulse voltammetry. The detection limit of the immune cellular biosensing system was 4.2 nM released NO as measured by double potential step chronoamperometry. The potent immune activating abilities of lipopolysaccharide and interferon-gamma could be assessed by the cellular biosensing system; NO release from cells was detected within 600 ms.     

Osteogenic differentiation of mesenchymal stem cells from osteopenic rats subjected to physical activity with and without nitric oxide synthase inhibition

Physical activity has potent and complex effects on bones. We hypothesized that physical activity has a positive effect upon osteopenic rat bones because it stimulates osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We also postulated that local nitric oxide concentrations mediate the effects of physical activity on bones. The objective of this study was to investigate the osteogenic differentiation in vitro of MSCs from osteopenic female rats subjected to physical activity with and without nitric oxide synthase inhibition. We used MSCs from the femurs of Wistar female rats divided into six groups: Group 1, sham-operated (control); Group 2, sedentary osteopenic; Group 3, active osteopenic; Group 4, sham-operated with L-NAME; Group 5, sedentary osteopenic with L-NAME; and Group 6, active osteopenic with L-NAME. The cells were cultured at 37 °C and 5% CO₂. Cells were phenotypically characterized with anti-CD45, anti-CD90, anti-CD73, and anti-CD54 using a FACScan cytometer. MSCs were cultured in osteogenic medium for 7, 14 and 21 days. Alkaline phosphatase activity, the capacity of dimethylthiazol conversion in formazan crystals, collagen synthesis and the number of mineralized nodules were analyzed. The means of all of the variables were compared using the SNK test. MSCs did not express CD45 in 96.94% of the cells, but there was expression of CD73, CD54 and CD90 in 93.99%, 95.10% and 86.77% of the cells, respectively. MSCs from osteopenic rats showed less osteogenic differentiation. Surprisingly, physical activity increased the osteogenic differentiation of MSCs in osteopenic rats. Inhibition of nitric oxide synthase in vivo had a negative effect upon the osteogenic potential of MSCs from normal rats and from osteopenic rats subjected to physical activity. Our results suggest that nitric oxide stimulates MSCs osteogenic differentiation and that nitric oxide mediates the beneficial effects of physical activity upon MSCs osteogenic differentiation.     

Endothelial nitric oxide synthase genotype and haplotype are not associated with diabetic retinopathy in diabetes type 2 patients

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene have been associated with the development of diabetic retinopathy (DR) in patients with type 1 diabetes mellitus (T1DM), but not with T2DM. However, no previous study has analyzed combinations of genetic markers (haplotypes), which can be more informative. We studied three eNOS genetic polymorphisms: a single nucleotide polymorphism in the promoter region (T(-786)C), in exon 7 (Glu298Asp), and a variable number of tandem repeats in intron 4 (b/a) in 103 healthy controls, and in 170 patients with T2DM (without DR, N=114; with DR, N=56). We also examined the association of eNOS gene haplotypes with T2DM and with DR. No differences were found in the frequencies of genotypes and alleles of the three polymorphisms among the three groups of subjects. However, the "C-Glu-b" haplotype was more common in healthy controls (24%) than in T2DM patients (7%) (P=0.0001). Finally, no significant difference in the distribution of eNOS haplotypes frequencies was found when T2DM patients with or without DR were compared (P=0.7372). These findings suggest no association between DR and individual eNOS haplotypes in T2DM patients. The "C-Glu-b" haplotype, however, may have a protective effect against T2DM. Further studies should be conducted to address the molecular basis for such an effect.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Effects of moderate hypothermia on constitutive and inducible nitric oxide synthase activities after traumatic brain injury in the rat

We investigated the effects of therapeutic hypothermia (30 degrees C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague-Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury. In normothermic rats (37 degrees C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5+/-68.9% of contralateral value; mean+/-SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8+/-17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30+/-8.4 and 28.6+/-20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30 degrees C) preserved cNOS activity at 3 and 7 days (69.5+/-23.3 and 78.6+/-7.6% of contralateral value, respectively; mean+/-SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021+/-0.06 and 0.23+/-0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature-dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model.     

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.     

Effects of doxazosin on blood flow and mRNA expression of nitric oxide synthase in the spontaneously hypertensive rat genitourinary tract

Hypertension may impact pelvic arterial blood flow resulting in reduction of nitric oxide synthase (NOS) levels. Although doxazosin, an alpha(1)-adrenoceptor antagonist, has been shown to improve erectile dysfunction as well as benign prostatic hyperplasia (BPH) and hypertension, it is not clear whether these improvements using doxazosin are primarily due to direct actions on the prostate, urinary bladder and penis, possibly via inhibition of vascular alpha(1)-adrenoceptors, or other sites of actions. Therefore, we investigated effects of doxazosin to the spontaneously hypertensive rat (SHR) on blood flow and NOS levels in the genitourinary tract. Four groups of rats were assessed: group 1, SHRs treated with doxazosin (30 mg/kg/day) for 4 weeks; group 2, SHRs treated with nifedipine (30 mg/kg/day) for 4 weeks; group 3, untreated SHRs; and group 4, untreated Wistar-Kyoto (WKY) rats. Blood flow to the ventral prostate, dorsolateral prostate, urinary bladder and penis was determined using a fluorescent microsphere infusion technique. Expression levels of nNOS and eNOS mRNAs were quantified by real-time RT-PCR using SYBR Green I. Blood flow to the ventral prostate, dorsolateral prostate, urinary bladder and penis was significantly lower in untreated SHRs than WKY rats. Treatment with doxazosin increased blood flow to each tissue studied in SHRs. RT-PCR data indicated that untreated SHRs had lower mRNA expression levels of nNOS in the bladder and penis and eNOS in the penis than WKY rats and that administration of doxazosin to the SHR caused an increase in expression levels of these genes, i.e., up-regulation of nNOS in the bladder and penis and eNOS in the penis. However, nifedipine had no significant effects on blood flow and NOS levels in the SHR genitourinary tract. Our data demonstrate that doxazosin treatment causes differential alterations in blood flow and NOS levels in the SHR genitourinary tract. These findings may provide insight into the beneficial effects of alpha(1)-adrenoceptor antagonists, on prostate, bladder and penile function, when used to treat symptoms of BPH and elevated blood pressure.     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.