Recent evidence implicates NO (Nitric oxide) as the principal mediator in an erection. To investigate the role of NO in the human erectile function, we studied the distribution pattern of nitroxergic fibers in the corpus cavernosum specimens obtained from 38 men using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry. Diffusely scattered delicate nerve fibers showing blue color reaction after NADPH-d histochemical staining were observed in normal control specimens from potent men. The neurogenic impotence group showed a statistically-significant decrease in the number of positive fibers compared to the normal control group. The number of positive fibers in the non-neurogenic impotence group was decreased compared to the normal control group but was statistically insignificant. With nitric oxide synthase (NOS) immunohistochemical stain, immunoreactive nerve bundles were easily seen in normal control specimens from potent men. NOS immunoreactive nerve bundles were contained within the corpus cavernosa which stained with NADPH-d reaction. Our results suggest that nitric oxide, a potent smooth muscle relaxing neurotransmitter in the autonomic nervous system, plays a physiologic role in erectile function and NADPH-d enzyme histochemical staining on the biopsied corpus cavernosum may be used as an important diagnostic method in the evaluation of neurogenic impotence.
Histocytochemistry ; Human ; Impotence/enzymology* ; Male ; NADPH Dehydrogenase/metabolism ; Nitric-Oxide Synthase/metabolism* ; Penis/enzymology* ; Tissue Distribution

Axotomy of the vagal motor neurons by cervical vagotomy induces NADPH diaphorase staining due to increased nitric oxide synthase expression in both the rat dorsal motor nucleus and nucleus ambiguous; furthermore, cerical vagotomy leads to cell death of the dorsal motor nucleus cells. Subdiaphragmatic vagotomy axotomizes the vagal motor cells further from the brainstem than cervical vagotomy, and cuts the fibers running only to the abdominal viscera. Here we report that subdiaphragmatic vagotomy is sufficient to induce NADPH diaphorase staining in the dorsal motor nucleus but does not induce staining in the nucleus ambiguus. Because the neurons of the dorsal motor nucleus do not undergo cell death after subdiaphragmatic vagotomy and are able to re-enervate the gut, the increased nitric oxide synthase expression after distal axotomy may be related more to regeneration than degeneration.
Animal ; Fourth Ventricle/physiology* ; Fourth Ventricle/enzymology* ; Fourth Ventricle/cytology ; Male ; Motor Neurons/enzymology ; NADPH Dehydrogenase/metabolism* ; Rats ; Rats, Sprague-Dawley ; Vagotomy/methods* ; Vagus Nerve/physiology*

OBJECTIVEBoth of gp91(phox) (an isoform of nicotinamide adenine dinucleotide phosphate-reduced oxidases) and Src (a non-receptor protein tyrosine kinase) play a prominent role in mediating hypoxia/reoxygenation injury of neurons. The present study was designed to investigate the neuroprotective effect of equol, a predominant active metabolite of daidzein, against hypoxia/reoxygenation injury in rat pheochromocytoma cell line (PC12) and explore the underlying mechanisms.

METHODSPC12 cells exposed to hypoxia/reoxygenation injury were examined for reactive oxygen species (ROS) using dihydroethidium and 2', 7'-dichlorofluorescein diacetate and analyzed for changes in lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content. The expression levels of gp91(phox) and phosphorylated Src-Tyr416 (p-Src) were measured using Western blotting.

RESULTSEquol dose-dependently restored the cell viability and decreased LDH activity and MDA content in culture medium of PC12 cells exposed to hypoxia/reoxygenation. Pretreatment of the cells with 10(-5) and 10(-6) mol/L equol inhibited hypoxia/reoxygenation-induced increase of ROS. PC12 cells treated with equol prior to hypoxia/reoxygenation injury showed significant enhancement of the protein levels of gp91(phox) and p-Src.

CONCLUSIONEquol confers neuroprotection against hypoxia/reoxygenation injury in PC12 cells by inhibiting the generation of ROS very likely as a result of down-regulation of gp91(phox) and inhibition of Src phosphorylation.

Animals ; Cell Hypoxia ; Cell Survival ; Down-Regulation ; Equol ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Membrane Glycoproteins ; metabolism ; NADPH Oxidase 2 ; NADPH Oxidases ; metabolism ; Neurons ; drug effects ; metabolism ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Phosphorylation ; Rats ; Reactive Oxygen Species ; metabolism ; src-Family Kinases ; metabolism

This study was conducted to define the molecular mechanism of fasting-induced down-regulation of neuronal nitric oxide synthase (nNOS) expression in the hypothalamic paraventricular nucleus (PVN). Rats were adrenalectomized (ADX), and then either underwent food deprivation or received varying doses of dexamethasone for 48 h. The brain tissues were processed for NADPH-diaphorase (NADPH-d) staining, a histochemical marker of nNOS enzyme activity. Both the ADX and the sham operated rats showed a significant weight loss after 48 h of food deprivation. Food deprivation decreased the number of NADPH-d containing cells in the PVN of sham rats, however, not in the ADX rats. Dexamethasone dose- dependently decreased NADPH-d cells in the PVN of ADX rats. The effect of ADX or dexamethasone was limited to the parvocellular subdivision of PVN. These results suggest that the adrenal glucocorticoids may down-regulate nNOS expression in the PVN during food deprivation.
*Adrenalectomy ; Animals ; Biological Markers ; Dexamethasone/blood/pharmacology ; Down-Regulation/physiology ; Fasting/*physiology ; Food Deprivation/physiology ; Glucocorticoids/blood/pharmacology ; Male ; NADPH Dehydrogenase/*metabolism ; Nitric-Oxide Synthase/*metabolism ; Paraventricular Hypothalamic Nucleus/*enzymology ; Rats ; Rats, Sprague-Dawley ; Support, Non-U.S. Gov't

OBJECTIVETo investigate the significance of NADPH quinine oxidoreductase 1 (NQO1) protein overexpression on prognostic evaluation of head and neck squamous cell carcinoma (HNSCC).

METHODSNQO1 protein was detected in 162 of HNSCC, 45 cases of adjacent nontumor tissues and 26 samples of normal head and neck epithelia using EnVision immunohistochemical. Correlation between NQO1 overexpression and patients prognosis was also analyzed.

RESULTSThe positive rate and strongly positive rate of NQO1 protein were 84.0% (136/162) and 69.8% (113/162) in HNSCC, respectively, and both of which were significantly higher than either those in adjacent nontumor tissues and normal head and neck epithelia (both P < 0.01). NQO1 expression was significantly correlated with the clinical stage, pT and chemoradiotherapy of HNSCC (P < 0.01). Kaplan-Meier survival analysis showed that overall survival and disease-free survival rates were significantly higher in HNSCC patients with high level NQO1 expression than that those with low level of NQO1 expression (Log-rank = 6.625 , P = 0.010;Log-rank = 6.234 , P = 0.013). Additional analysis by Cox proportional hazard regression model showed that high level of NQO1 expression was an independent hazard predictor for overall survival of patients with HNSCC (Wald = 6.626, P = 0.008).

CONCLUSIONSNQO1 expression level is closely correlated with the progression and prognosis of patients with HNSCC. High level of NQO1 expression may be used as an important indicator for patients with poor prognostic HNSCC.

Breast ; enzymology ; Carcinoma, Squamous Cell ; enzymology ; mortality ; pathology ; Disease-Free Survival ; Female ; Head and Neck Neoplasms ; enzymology ; mortality ; pathology ; Humans ; Kaplan-Meier Estimate ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NADH, NADPH Oxidoreductases ; metabolism ; Prognosis ; Proportional Hazards Models

OBJECTIVEElevation of reactive oxygen species (ROS), especially the level of superoxide is a key event in many forms of cardiovascular diseases. To study the mechanism of tea polyphenols against cardiovascular diseases, we observed the expressions of ROS-related enzymes in endothelial cells.

METHODSTea polyphenols were co-incubated with bovine carotid artery endothelial cells (BCAECs) in vitro and intracellular NADPH oxidase subunits p22phox and p67phox, SOD-1, and catalase protein were detected using Western blot method.

RESULTSTea polyphenols of 0.4 microg/mL and 4.0 microg/mL (from either green tea or black tea) down-regulated NADPH oxidase p22phox and p67phox expressions in a dose-negative manner (P < 0.05), and up-regulated the expressions of catalase (P < 0.05).

CONCLUSIONSTea polyphenols regulate the enzymes involved in ROS production and elimination in endothelial cells, and may be beneficial to the prevention of endothelial cell dysfunction and the development of cardiovascular diseases.

Animals ; Camellia sinensis ; chemistry ; Carotid Arteries ; cytology ; Catalase ; biosynthesis ; Cattle ; Cells, Cultured ; Down-Regulation ; Endothelial Cells ; drug effects ; enzymology ; metabolism ; Flavonoids ; isolation & purification ; pharmacology ; Membrane Transport Proteins ; biosynthesis ; NADPH Dehydrogenase ; biosynthesis ; NADPH Oxidases ; Phenols ; isolation & purification ; pharmacology ; Phosphoproteins ; biosynthesis ; Polyphenols ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; biosynthesis ; Superoxide Dismutase-1 ; Up-Regulation

In this study, we examined if glucocorticoids are required for the fasting-induced decrease of neuronal nitric oxide synthase (nNOS) in the magnocellular division of the paraventricular nucleus (PVN). Rats were adrenalectomized, subjected to 48 h of food deprivation with/without dexamethasone (5 mg/ kg, 4 subcutaneous injections with 12 h intervals), and the brain slices were processed for NADPH-diaphorase (NADPH- d) staining, a histochemical marker for nNOS in neuronal cells. In food deprived adrenalectomized rats, but not in free fed intact rats, dexamethasone significantly decreased NADPH-d staining in the magnocellular PVN. We previously reported that food deprivation decreases nNOS in the magnocellular PVN of intact rats. Thus, the present results together with our previous report suggest that although glucocorticoids are required for fasting-induced nNOS down-regulation in the magnocellular PVN, glucocorticoids may not be directly involved and some other molecular signals produced by food deprivation may play a pivotal role over glucocorticoid in the regulatory pathway for nNOS expression in this brain region.
Adrenalectomy ; Animals ; Dexamethasone/pharmacology ; Down-Regulation ; Fasting/*metabolism ; Male ; NADPH Dehydrogenase/*analysis ; Nitric-Oxide Synthase/genetics ; Paraventricular Hypothalamic Nucleus/*enzymology ; Peptides/physiology ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Weight Gain

An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
Amino Acid Sequence ; Base Sequence ; Cell Membrane/metabolism ; Cell-Free System ; DNA Primers ; Flavin-Adenine Dinucleotide/*metabolism ; Humans ; Kinetics ; Membrane Glycoproteins/*metabolism ; Molecular Sequence Data ; NADH Dehydrogenase/metabolism ; *NADPH Oxidase ; Neutrophils/enzymology/metabolism ; Oxidation-Reduction ; Peptide Fragments/chemistry ; Phosphoproteins/*chemistry/*metabolism ; Polymerase Chain Reaction ; Sequence Deletion

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4% acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.
Acetic Acid/toxicity ; Animals ; Colitis/chemically induced/*pathology/*physiopathology ; Colon/drug effects/enzymology/*innervation/pathology ; Indicators and Reagents/toxicity ; Male ; Muscle Contraction/drug effects ; Muscle, Smooth/drug effects/metabolism ; Myenteric Plexus/pathology ; NADPH Dehydrogenase/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Neuromuscular Junction/drug effects/*metabolism ; Nitrergic Neurons/drug effects/*metabolism ; Nitric Oxide/*metabolism ; Peroxidase/metabolism ; Potassium Chloride/pharmacology ; Rats ; Rats, Sprague-Dawley

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on cavernous nerve (CN) regeneration and functional status in a nerve-crush rat model. Twenty-four Sprague-Dawley male rats were randomly divided into three equal groups: eight had a sham operation, eight underwent bilateral nerve crushing with no further intervention and eight underwent bilateral nerve crushing with an immediate application of PRP on the site of injury. Erectile function was assessed by CN electrostimulation at 3 months and nerve regeneration was assessed by toluidine blue staining of CN and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. Three months after surgery, in the group that underwent bilateral nerve crushing with no further intervention, the functional evaluation showed a lower mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure (MAP) with CN stimulation than those in the sham group. In the group with an immediate application of PRP, the mean maximal ICP and maximal ICP/MAP were significantly higher than those in the injured control group. Histologically, the group with the application of PRP had more myelinated axons of CNs and more NADPH-diaphorase-positive nerve fibres than the injured control group but fewer than the sham group. These results show that the application of PRP to the site of CN-crush injury facilitates nerve regeneration and recovery of erectile function. Our research indicates that clinical application of PRP has potential repairing effect on CN and peripheral nerves.
Animals ; Disease Models, Animal ; Electric Stimulation ; Erectile Dysfunction ; pathology ; physiopathology ; therapy ; Male ; NADPH Dehydrogenase ; metabolism ; Nerve Regeneration ; physiology ; Penile Erection ; physiology ; Penis ; innervation ; Peripheral Nerve Injuries ; Peripheral Nerves ; metabolism ; pathology ; Platelet Transfusion ; Platelet-Rich Plasma ; Radiculopathy ; etiology ; pathology ; physiopathology ; Rats ; Rats, Sprague-Dawley