Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.1 (NADPH-diaphorase)
3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent work has implicated nitric oxide (NO) in several aspects of male genital physiology including erectile function and androgen secretion, as well as in vitro effects on sperm motility and capacitation. The objectives of this study were to characterize the distribution of endothelial nitric oxide synthase (eNOS) in "normal" human testis, epididymis, and vas deferens and in testis pathology. Nitric oxide synthase protein was localized immunohistochemically using an eNOS monoclonal antibody. Endothelial NOS protein co-localized to areas that showed positive NADPH diaphorase activity. Within the testis, eNOS protein was localized to the cytoplasm of Leydig cells and Sertoli cells at all stages of spermatogenesis. Within the epididymis and vas deferens, eNOS was localized to the epithelium. Endothelial NOS was also localized to endothelial cells in all tissues; it was not detectable in normal germ cells. Endothelial NOS and diaphorase activity were, however, detected in degenerating or apoptotic intraepithelial germ cells. In addition, prematurely shed spermatocytes and spermatids had intense eNOS expression. Previous studies have suggested a role for NOS in the contractile, hemodynamic, and hormonal aspects of testicular function as well as in epididymal secretion. The studies reported herein suggest a role for eNOS in spermatogenesis and germ cell degeneration.
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PMID:Immunohistochemical localization of endothelial nitric oxide synthase in human testis, epididymis, and vas deferens suggests a possible role for nitric oxide in spermatogenesis, sperm maturation, and programmed cell death. 890 2

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.
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PMID:Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes. 891 54

The cellular localization and distribution of inducible and constitutive nitric oxide synthase (iNOS/cNOS) was determined in tissue sections from multiple sclerosis (MS) and control brain and spinal cord. Immunocytochemical techniques were applied using specific iNOS- and cNOS-directed antibodies. In addition, NADPH-diaphorase histochemistry was performed. To establish the identity of iNOS-, cNOS- and NADPH-diaphorase-positive cells single and double staining was performed on tissue sections with the macrophage marker KP1 (CD68) and with the astrocyte marker glial fibrillary acidic protein (GFAP). Areas of myelin breakdown and demyelination were determined using a staining for neutral lipids, Oil Red O (ORO). Furthermore, macrophages isolated from active demyelinating MS lesions were stained for iNOS, cNOS, KP1 and ORO. In active MS lesions strong iNOS immunoreactivity was found exclusively in perivascular and parenchymal macrophages distributed within regions of active demyelination. In these active MS lesions immunoreactivity for cNOS was also found in macrophages. Macrophages isolated from active MS lesions also showed immunoreactivity for iNOS and cNOS. Moreover, these isolated macrophages produced nitric oxide (NO; >30 microM) in vitro. NADPH-diaphorase activity was detected in KP1-positive perivascular and parenchymal macrophages and in GFAP-positive reactive astrocytes in active MS lesions and in reactive astrocytes located in the hypercellular rims of chronic active MS lesions. cNOS-positive reactive astrocytes were detected in both active and chronic active MS lesions. Inside chronic active lesions some residual macrophages were weakly iNOS-positive. In control brain and spinal cord no iNOS immunoreactivity could be detected. These results suggests an important role for human macrophages capable of producing the free radical nitric oxide (NO), which may contribute to the cytotoxicity of oligodendrocytes and destruction of myelin in MS brain and spinal cord.
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PMID:Immunocytochemical characterization of the expression of inducible and constitutive isoforms of nitric oxide synthase in demyelinating multiple sclerosis lesions. 899 Jan 25

Nitric oxide (NO) is an important excitatory neurotransmitter in the central nervous system. In the adult rat, both selective and nonselective blockers of constitutive nitric oxide synthase (NOS) induce marked ventilatory reductions during sustained hypoxia, thereby enhancing ventilatory roll-off. Since hypoxic ventilatory depression is greater in developing mammals during the late phases of hypoxic exposure, we hypothesized that limited NOS activity may play a role in the late arm of the ventilatory response. To test our hypothesis, 5-d-, 10-d-, and 15-d-old rat pups underwent a 30-min hypoxic challenge (10% O2) before and after administration of 100 mg/kg N-nitro-L-arginine methyl ester (L-NAME), a competitive NOS inhibitor. Minute ventilation (VE) was measured using whole-body plethysmography. In 5-d-old pups, early VE hypoxic responses were enhanced, and late VE were similar after administration of L-NAME. In contrast, in 15-d-old hypoxic pups, L-NAME administration was associated with smaller early VE increments and significantly larger VE reductions when compared with pretreatment conditions. The role of central nervous system NO in the development of these ventilatory changes was further assessed by Western blots of protein equivalents from the nucleus tractus solitarius (NTS), the first central relay for peripheral chemoreceptor afferent input, which revealed increasing neuronal NOS expression with age. Furthermore, NADPH-diaphorase immunohistochemical staining of neurons in the NTS revealed increased positively labeled neuronal populations within subnuclei of this structure with advancing postnatal age. Current findings suggest that NOS activity mediates both excitatory and inhibitory components of the hypoxic ventilatory response. Furthermore, in brainstem respiratory regions, NO may play a role in modulating the prominent second phase of the biphasic response to hypoxia typically seen in early postnatal life.
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PMID:Nitric oxide modulates ventilatory responses to hypoxia in the developing rat. 915 88

Previous studies showed that different nitric oxide synthase isoforms are present in the uterus of laboratory mammals and that their expression is influenced by ovarian steroids. However, the results of these studies are not univocal, probably owing to the different hormonal treatments and techniques applied to reveal nitric oxide synthases. In this study we investigated the distribution and expression of constitutive and inducible nitric oxide synthase isoforms by immunocytochemistry and their changes following treatment of the mice with 17 beta-estradiol alone or in combination with medroxyprogesterone. Moreover, we compared the immunoreactivities for nitric oxide synthases with the histochemical reaction for NADPH-diaphorase, an enzyme that may be associated with nitric oxide synthase. The results obtained show that the two nitric oxide synthase isoforms are differently expressed in surface epithelium, glands, stromal cells and myometrium and that, as compared with the uteri from mice treated with estrogen alone, those from mice treated with estrogen plus progestin showed enhanced expression of constitutive nitric oxide synthase in the myometrium and of the inducible isoform in surface epithelium, glands, stromal cells and myometrium. The results obtained with NADPH-diaphorase reaction show that there is not a colocalization of nitric oxide synthase isoforms and NADPH-diaphorase, apart from a partial colocalization in part of the stromal cell population and myometrium. This provides evidence that NADPH-diaphorase histochemistry is not a valid technique to localize the sites of nitric oxide synthesis in the mouse uterus and that the use of this technique may generate misleading in the interpretation of the effect of ovarian steroids in regulating nitric oxide production by the different components of the uterine wall.
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PMID:Constitutive and inducible nitric oxide synthases in mouse uterus and their changes following treatment with ovarian steroids. Immunocytochemical and histoenzymological studies. 936 29

To clarify the role of endothelial-derived nitric oxide (EDNO) and its synthase (NOS) in the normal and hypertensive pulmonary vasculature, activity of endothelial NOS in the lungs, ENDO-dependent vasodilating response induced by bradykinin (BK), and cGMP content of lung tissue in normoxic and hypoxic rats were investigated. We also studied the effects of NOS inhibitor-L-NAME on the activity of NOS, cGMP content, mean pulmonary arterial pressure (mPAP) and carotid systolic arterial pressure (CAPs) in both rats. The results were as follows (1) In normoxic rats there was no NOS activity in the endothelium of small vessels (phi < or = 80 microns) and no relaxing response to BK. Long-term administration of L-NAME obviously inhibited the activity of ecNOS and cGMP content in the lungs of normoxic rats, therefore it led to the increment of CAPs but failed to elevate mPAP. (2) After hypoxic exposure for 10 days, NADPH-diaphorase (NADPH-d and ecNOS immunoreactivity turned to be positive in the endothelium of small vessels with diameter less than 80 microns. BK-induced EDNO-dependent vasodilation, the enzyme activity of cNOS and cGMP content in the lungs of hypoxic rats were significantly enhanced as compared with normoxic rats. Long-term administration of L-NAME in hypoxic rats markedly inhibited the enhancement of cNOS enzyme activity, the production of EDNO and cGMP content in rat lungs, consequently it significantly decreased mPAP but elevated CAPs obviously. These results suggest that the role of EDNO in maintaining the low basal tone of normal adult pulmonary circulation remain to be studied more precisely. The increased activity of ecNOS and the enhancement of EDNO synthesis might act to moderate the hypertension. The excess synthesis of EDNO might be toxic to the endothelium of pulmonary vessels, therefore potentiating the development of pulmonary hypertension.
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PMID:[Role of endothelial-derived nitric oxide and its synthase in the development of hypoxic pulmonary hypertension in rat]. 1007 45

To study the role of nitric oxide synthase (NOS) in rat experimental bronchial asthma. 3H-arginine/3H-L-citrulline conversion technique was used to assay NOS activity of rat lung tissue and histochemical staining method for detect NADPH-d diaphorase. The results revealed that there were significant increase in the level of iNOS activity in asthma group from 152.39% to 249.40%, but the cNOS activity reduced from 64.84% to 61.81% (P < 0.05-0.01). Histochemical staining of NADPH-d showed deep staining of trachial and bronchial epithelium in asthma group. These results suggested that NOS plays a role in regulating airway inflammation and bronchial responsiveness. cNOS possesses a down regulatory effect, while iNOS upregulatory. The occurrence of airway inflammation is earlier than that of smooth muscle contraction and endothelial injury.
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PMID:[An experimental study on the effect of nitric oxide synthase in bronchial asthma]. 1043 84

Nitric oxide is an important cellular messenger molecule that has been implicated in a wide range of physiological and pathophysiological actions in cardiovascular, immune, and nervous systems. Nitric oxide synthase (NOS) is the sole and key enzyme responsible for the generation of nitric oxide. The effect of hypoxia-induced pulmonary hypertension on NOS in the lung is controversial. To clarify the effect of hypoxia on distribution and activity of NOS in rat lung, localization of NOS in the lungs of hypoxic and normoxic rats were studied using NADPH-diaphorase histochemical staining techniques, NOS enzyme activity in the lung homogenates was assessed by [3H] arginine to [3H] citrulline conversion. In the normoxic rat, NADPH-d was distributed in the endothelial cells of large pulmonary vessels (ID > 150 microns) and medium-sized (50 microns < ID < 150 microns) vessels but was not detected in the endothelium of small vessels (ID < 50 microns), and there was an absence of NADPH-d staining in the smooth muscle cells of small, medium, and large pulmonary vessels. However, after hypoxic exposure for two weeks, NADPH-d staining increased dramatically in the endothelial cells of large and medium-sized pulmonary vessels, and NADPH-d became markedly positive in the endothelial cells of small vessels. Hypoxia was also found to induce de novo NOS expression in the smooth muscle cells of small, medium-sized, and large pulmonary vessels. The enzyme activity of constitutive NOS(cNOS) was decreased obviously in the hypoxic rat lungs, but that of inducible NOS(iNOS) was increased significantly in the hypoxic rat lungs. These results suggested that the inhibited endothelium-derived relaxing factor (EDRF)/NO-dependent vasodilation after hypoxic exposure might be induced by decreased activity of cNOS in the endothelium of pulmonary vessels, and hypoxia-induced upregulation of iNOS expression and activity in the rat lung might play an important role in the adaptation of pulmonary circulation to hypoxia.
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PMID:[Effect of hypoxia on distribution and activity of nitric oxide synthase in rat lung]. 1045 4

The purpose of this study was to determine whether nitric oxide (NO) is present in clinically normal horses under basal conditions and if it increases secondary to naturally acquired small intestinal strangulation obstruction. Thirty-one horses were used; 20 horses with naturally acquired small intestinal strangulation obstruction and 11 clinically normal horses with no signs of gastrointestinal tract disease. Jugular venous blood, abdominal fluid, and urine were collected for NO quantification. Plasma, abdominal fluid, and urine were stored at -70 degrees C until analyzed for NO using a chemiluminescent method. Biopsy specimens collected from the affected jejunal segment, during anesthesia or after immediately after euthanasia, or from the midjejunum of control horses, were divided into subsections for fixation in zinc formalin and cryopreservation in OCT gel. Nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) diaphorase histochemical stains were performed on cryopreserved tissues and inducible nitric oxide synthase (iNOS) and nitrotyrosine immunohistochemical stains were performed on formalin-fixed, paraffin-embedded tissues. There were significantly greater plasma and abdominal fluid NO concentrations in affected horses as compared with controls, but there were no significant differences between horses for urine NO concentrations. There was a significant decrease in NADPH diaphorase stain in mucosal epithelium, vasculature, and leukocytes, and in submucosal plexi in affected horses compared with control horses. There was a significant increase in iNOS staining in mucosal and submucosal leukocytes and in mucosal leukocyte nitrotyrosine staining of the affected compared with control horses. Endothelial NOS and neuronal NOS are present under basal conditions in the jejunum of horses and probably mediate physiologic or cytoprotective effects. Plasma and abdominal fluid, but not urine, NO concentrations increase subsequent to small intestinal strangulation obstruction; this may be associated with increased mucosal and submucosal iNOS staining in leukocytes, which was likely due to increased expression subsequent to stimuli associated with ischemia. The increased nitrotyrosine staining in mucosal leukocytes of affected horses likely reflects the presence of peroxynitrite subsequent to increased NO and superoxide production and may reflect a cytotoxic role of NO in small intestinal strangulation obstruction in horses.
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PMID:Detection and comparison of nitric oxide in clinically normal horses and those with naturally acquired small intestinal strangulation obstruction. 1053 1

Immature rats receiving equine chorionic gonadotropin (eCG) and human CG (hCG) were used to study the time course changes in nitric oxide synthase (NOS) activity in the ovary during ovulation. To study the role of NO in ovulation, the effects of intrabursal injection of L-N(G)-monomethylarginine (L-NMMA, 125 microg/20 microl/bursa), a NOS inhibitor, on the number of ova shed were also examined. Rats were sacrificed at -48, 0, 3, 6, 9, 12, and 24 h after hCG injection, and the ovaries were collected for the NOS activity assay, Western blotting, NADPH-diaphorase histochemistry and immunohistochemistry. Total NOS and constitutive NOS activities in the ovary increased significantly at 9 h after hCG injection and the values remained high thereafter. Inducible NOS (iNOS) activity was detectable as a small peak at 3 and 6 h after hCG injection. Endothelial NOS (eNOS) protein production increased after hCG injection with a peak at 12 h, whereas iNOS protein production decreased at 12 and 24 h after hCG injection. NADPH-diaphorase positive cells increased at the thecae of growing follicles after hCG injection, appeared at mural granulosa cells before ovulation, and were detected in newly formed corpora lutea, which coincided with the results in eNOS positive cells by immunohistochemistry. L-NMMA given to rats at 5 or 7 h after hCG was most effective in reducing the number of ova shed. These results indicate that the NOS activity and NOS positive cells increased after hCG injection, and that eNOS was likely the main NOS increasing in the ovary during ovulation. It is concluded that NO produced between 5 and 9 h after hCG might play a supportive role in ovulation.
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PMID:Changes in nitric oxide synthase activity in the ovary of gonadotropin treated rats: the role of nitric oxide during ovulation. 1058 Jul 45


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