共查询到20条相似文献,搜索用时 31 毫秒
1.
María C. Ortiz María F. Albertoni Borghese Sabrina E. Balonga Agustina Lavagna Ana L. Filipuzzi Rosana Elesgaray María A. Costa Mónica P. Majowicz 《PloS one》2014,9(8)
The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [14C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression. 相似文献
2.
Tomat AL Costa MA Girgulsky LC Veiras L Weisstaub AR Inserra F Balaszczuk AM Arranz CT 《Life sciences》2007,80(14):1292-1302
This study was designed to investigate the effects of moderate zinc deficiency during growth on renal morphology and function in adult life. Weaned male Wistar rats were divided into two groups and fed either a moderately zinc-deficient diet (zinc: 8 mg/kg, n=12) or a control diet (zinc: 30 mg/kg, n=12) for 60 days. We evaluated: renal parameters, NADPH-diaphorase and nitric oxide synthase activity in kidney, renal morphology and apoptotic cells in renal cortex. Zinc-deficient rats showed a decrease in glomerular filtration rate and no changes in sodium and potassium urinary excretion. Zinc deficiency decreased NADPH diaphorase activity in glomeruli and tubular segment of nephrons, and reduced activity of nitric oxide synthase in the renal medulla and cortex, showing that zinc plays an important role in preservation of the renal nitric oxide system. A reduction in nephron number, glomerular capillary area and number of glomerular nuclei in cortical and juxtamedullary areas was observed in zinc deficient kidneys. Sirius red staining and immunostaining for alpha-smooth muscle-actin and collagen III showed no signs of fibrosis in the renal cortex and medulla. An increase in the number of apoptotic cells in distal tubules and cortical collecting ducts neighboring glomeruli and, to a lesser extent, in the glomeruli was observed in zinc deficient rats. The major finding of our study is the emergence of moderate zinc deficiency during growth as a potential nutritional factor related to abnormalities in renal morphology and function that facilitates the development of cardiovascular and renal diseases in adult life. 相似文献
3.
Daniela Amelio Filippo Garofalo Elvira Brunelli Ai May Loong Wai Peng Wong Yuen Kwong Ip Bruno Tota Maria Carmela Cerra 《Nitric oxide》2008,18(1):1-10
African lungfish Protopterus dolloi is an obligatory air-breather, which aestivates in a cocoon during the dry season. Aestivation associates with functional modifications in many tissues and organs, including heart and kidney. Due to its pleiotropic modulatory effects, nitric oxide (NO), generated by nitric oxide synthases (NOSs), may coordinate organ rearrangement, allowing adaptive adjustments under stressful environmental conditions. By immunofluorescence, Western blotting and NADPH-diaphorase, we examined cardiac and renal localization and activity of NOSs isoforms in both freshwater (FW) and aestivating [6 days (6DA) and 40 days (40DA) of estivation] P. dolloi. In heart and kidney endothelial NOS (eNOS) is the major isoform with respect to inducible and neuronal NOS (iNOS and nNOS, respectively). Cardiac eNOS locates in the epicardium, the trabecular endothelial endocardium, and myocardiocytes of both FW and aestivating fish. Western blotting revealed that cardiac eNOS expression increases in 6DA, but decreases in 40DA fish. In FW fish kidney eNOS is present in vascular endothelial cells and in podocytes of renal corpuscles. In tubular epithelial cells it is restricted to the apical pole. With aestivation, both renal localization and expression of eNOS increase. NADPH-diaphorase revealed an enhancement of cardiac and renal NOS activities during aestivation. Results suggest that in P. dolloi NO contributes, in an autocrine-paracrine fashion, to cardiac and renal readjustments during aestivation. Our findings are of evolutionary interest, since they document for the first time the presence of a NOS system in a ancestral fish, indicative of deep phylogenetic roots of NO bio-synthesis. 相似文献
4.
H. M. Young Anna J. O’Brien John B. Furness Daniela Ciampoli James P. Hardwick Timothy J. McCabe Ramani Narayanasami Bettie Sue S. Masters W. Ross Tracey 《Histochemistry and cell biology》1997,107(1):19-29
The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent
enzymes – neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450
reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting
that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue.
However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent
enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry
was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons
and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver,
sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea,
zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase
staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules
and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide
with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate
immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH
diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.
Accepted: 2 September 1996 相似文献
5.
华鲮泌尿系统组织学的初步观察 总被引:5,自引:0,他引:5
2004年3月至9月采用常规组织学方法对华鲮泌尿系统作了研究,结果表明:华鲮泌尿系统包括中肾、输尿管、膀胱。中肾由肾小体和肾小管组成,无皮质、髓质之分,有肾小体聚集现象;肾小管可分为颈段、第一近曲小管、第二近曲小管、远曲小管、集合管。肾中散布有大量淋巴髓样组织,还有甲状腺滤泡和斯坦尼氏小体,显然华鲮的肾脏是一个具有多种生理功能的复合器官。输尿管位于两肾叶腹侧,其上皮为似复层,缺黏膜下层,无纵肌,外膜甚薄。左右输尿管在后端合并,稍微扩大,形成膀胱,膀胱内壁具有发达的绒毛,绒毛表面为变移上皮。 相似文献
6.
The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which--when disrupted--results in PKD. 相似文献
7.
Jones RJ Smith SM Gao YT DeMay BS Mann KJ Salerno KM Salerno JC 《The Journal of biological chemistry》2004,279(35):36876-36883
Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity. 相似文献
8.
Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia 总被引:4,自引:0,他引:4
Shile Huang Hubert H. Kerschbaum Edwin Engel Anton Hermann 《Journal of neurochemistry》1997,69(6):2516-2528
Abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by N G -nitro- l -arginine ( l -NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+ - and NADPH-dependent NOS activity, which was partially inhibited by EGTA and l -NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue. 相似文献
9.
Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an 'input state', with FMN in close contact with FAD. We recently produced two domain 'output state' (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ~4.3 ns. The majority state in holoenzyme has a short lifetime of ~90 ps, probably because of FAD-FMN interactions. In oxyFMN about 25-30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12-15 ?. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle. 相似文献
10.
Premaratne S Xue C McCarty JM Zaki M McCuen RW Johns RA Schepp W Neu B Lippman R Melone PD Schubert ML 《American journal of physiology. Gastrointestinal and liver physiology》2001,280(2):G308-G313
Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion. 相似文献
11.
Using the fixation insensitive NADPH-diaphorase reaction as a histochemical marker for the enzyme nitric oxide synthase (NOS), we investigated the possible sites of putatively NOS-related NADPH-diaphorase in the brain and retrocerebral complex of the cockroach, Diploptera punctata. In the cerebral ganglion, NADPH-diaphorase expression was localized in antennal lobes, optic lobes, mushroom bodies and neurosecretory cells. The highest NADPH activity was detected in the corpora allata (CA). Spectrophotometric quantitation indicated that NADPH-diaphorase activity first increased and then decreased (cycled) in the CA of mated females. In addition, during the first ovarian cycle, NADPH-diaphorase activity fluctuated concurrently with cyclic changes in the size of corpus allatum cells. In virgin females, NADPH-diaphorase activity remained at a low level, but it increased if the neural connectives between CA and brain were severed, indicating that the brain inhibited NADPH-diaphorase expression in the CA. Although nerve terminals were abundant in the CA, NADPH-diaphorase was clearly endogenous and synthesized by glandular cells, as was shown by histochemical staining of the cytosol in all dissociated cells of the CA. We have also demonstrated NADPH-diaphorase activity in the CA of the American cockroach Periplaneta americana, the house cricket Acheta domesticus, the lepidopteran Leucania loreyi, and the fruit fly Drosophila melanogaster, suggesting that NOS occurs in the CA of most, if not all insects. It is therefore possible that corpus allatum cells release NO, along with juvenile hormone, which presumably can function as a messenger molecule. 相似文献
12.
Bhattacharya ST Bayakly N Lloyd R Benson MT Davenport J Fitzgerald ME Rothschild M Lamoreaux WJ Coons LB 《Experimental parasitology》2000,94(2):111-120
We colocalized nitric oxide synthase (NOS) activity in epithelial cells that surround the salivary gland duct in female Dermacentor variabilis with NADPH diaphorase histochemistry and immunohistochemistry using a polyclonal anti-endothelial NOS. Using size-exclusion chromatography, a fraction with a molecular mass of about 185 kDa that had diaphorase activity was eluted from tick salivary gland homogenate. This fraction converted arginine to citrulline with the production of nitric oxide (NO), which was detected by using electron spin resonance spectroscopy. The complete activity of the diaphorase fraction was dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, (CaM), and Ca(2+), but was not dependent on dithiothreitol. The arginine analog N(G)-monomethyl-L-arginine inhibited the activity of this fraction. NO and arginine activated soluble guanylate cyclase to produce cGMP in dopamine-stimulated isolated salivary glands. Dopamine-stimulated isolated salivary glands treated with tick saline containing either EDTA, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, or the calcium/CaM binding inhibitor W-7 showed no increase in cGMP. The NO donor sodium nitroprusside significantly increased cGMP levels in unstimulated isolated salivary glands. A possible function for NO in salivation by this ixodid tick is discussed. 相似文献
13.
Time course of nitric oxide synthase activity in neuronal,glial, and endothelial cells of rat striatum following focal cerebral ischemia 总被引:3,自引:0,他引:3
Mihoko N. Nakashima Kimihiro Yamashita Yasufumi Kataoka Yasuko S. Yamashita Masami Niwa 《Cellular and molecular neurobiology》1995,15(3):341-349
Summary 1. The time course of nitric oxide synthase (NOS) activity in neuronal, endothelial, and glial cells in the rat striatum after middle cerebral artery (MCA) occlusion and reperfusion was examined using a histochemical NADPH-diaphorase staining method.2. In sham-operated rats, neuronal cells of the striatum exhibited strong NADPH-diaphorase activities. When rats were subjected to MCA occlusion for 1 hr, neuronal damage, including neurons with positive NADPH-diaphorase activities, appeared in the striatum at 3 hr after and extended to all areas of the striatum 3–4 days after reperfusion.3. NADPH-diaphorase activities in the endothelial cells increased in the damaged part of striatum from 3 hr after, peaked at 1–2 days after MCA occlusion/reperfusion, then gradually decreased.4. In parallel with the development of neuronal damage, some astrocytes and a high proportion of microglia/macrophages located in the perisite and in the center of the damaged striatum, respectively, exhibited a moderate to high level of NADPH-diaphorase activities. Most of these activities disappeared at 4 days after MCA occlusion.5. These findings provided evidence that an inappropriate activation of NOS in endothelial cells and microglia/macrophages, in response to MCA occlusion/reperfusion, is closely associated with initiation and progression of ischemic neuronal injury in the striatum. 相似文献
14.
Javkhedkar AA Lokhandwala MF Banday AA 《American journal of physiology. Renal physiology》2012,302(1):F47-F51
Angiotensin (ANG) II via ANG II type 1 receptors (AT1R) activates renal sodium transporters including Na-K-ATPase and regulates sodium homeostasis and blood pressure. It is reported that at a high concentration, ANG II either inhibits or fails to stimulate Na-K-ATPase. However, the mechanisms for these phenomena are not clear. Here, we identified the signaling molecules involved in regulation of renal proximal tubular Na-K-ATPase at high ANG II concentrations. Proximal tubules from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were incubated with low concentrations of ANG II (pM), which activated Na-K-ATPase in both the groups; however, the stimulation was more robust in SHR. A high concentration of ANG II (μM) failed to stimulate Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) continued to stimulate Na-K-ATPase, which was sensitive to the AT1R antagonist candesartan. In the presence of N(G)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, ANG II (μM) caused stimulation of Na-K-ATPase in proximal tubules of WKY rats while having no further stimulatory effect in SHR. ANG II (μM), via AT1R, increased proximal tubular NO levels in WKY rats but not in SHR. In SHR, NOS was uncoupled as incubation of proximal tubules with ANG II and l-arginine, a NOS substrate, caused superoxide generation only in SHR and not in WKY rats. The superoxide production in SHR was sensitive to l-NAME. There was exaggerated proximal tubular AT1R-G protein coupling and NAD(P)H oxidase activation in response to ANG II (μM) in proximal tubules of SHR compared with WKY rats. In SHR, inhibition of NADPH oxidase restored NOS coupling and ANG II-induced NO accumulation. In conclusion, at a high concentration ANG II (μM) activates renal NO signaling, which prevents stimulation of Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) overstimulates NADPH oxidase, which impairs the NO system and leads to continued Na-K-ATPase activation. 相似文献
15.
Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes. 相似文献
16.
The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain 总被引:8,自引:0,他引:8
Danielisová V Némethová M Burda J 《Physiological research / Academia Scientiarum Bohemoslovaca》2004,53(5):533-540
The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury. 相似文献
17.
Anna Sobolewska-Stawiarz Nicole G. H. Leferink Karl Fisher Derren J. Heyes Sam Hay Stephen E. J. Rigby Nigel S. Scrutton 《The Journal of biological chemistry》2014,289(17):11725-11738
Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape. 相似文献
18.
目的观察生后小鼠肾脏发育不同阶段神经型一氧化氮合酶(nNOS)的表达,以及新生小鼠与成年小鼠肾脏nNOS表达差异,探讨nNOS在小鼠生后肾脏发育中的意义。方法分别取新生(出生小于2h)、生后3、5、7、14、40d昆明小鼠各8只,共6组。用免疫组织化学及免疫印迹方法对小鼠肾脏内nNOS表达进行定性、定量分析。结果新生小鼠生肾区nNOS呈强阳性表达,肾小管也有表达;成年小鼠肾远端小管,特别是致密斑,nNOS呈强阳性表达,集合管及肾小管均有阳性表达;新生小鼠肾脏nNOS含量最多,随后逐渐减少,成年小鼠nNOS含量最低。结论新生小鼠与成年小鼠肾脏nNOS表达部位不同,且表达含量由新生时最高到成年时降至最低。 相似文献
19.
用还原型辅酶Ⅱ黄递酶组织化学和一氧化氮合酶(NOS)免疫细胞化学技术研究了成年爪蛙(Xenopuslaevis)鼻粘膜NOS的阳性结构。嗅上皮中嗅感觉神经元和支持细胞,以及固有层中的神经束、血管和粘膜下腺均呈还原型辅酶Ⅱ黄递酶阳性染色。在嗅上皮中,未见Ⅰ型或Ⅱ型NOS抗体免疫反应阳性结构,但鼻内侧窦和内侧窦口顶嗅上皮中的嗅感觉神经元见有Ⅲ型NOS强免疫反应。在固有层中,Ⅰ型或Ⅲ型NOS免疫反应性存在于神经束和血管中,未见于粘膜下腺的腺泡中。结果表明,不同异型的NOS存在于爪蛙鼻粘膜中,提示一氧化氮可能参与爪蛙的化学感觉活动。 相似文献
20.
Elesgaray R Caniffi C Savignano L Romero M Mac Laughlin M Arranz C Costa MA 《American journal of physiology. Renal physiology》2012,302(11):F1385-F1394
Atrial natriuretic peptide (ANP) is an important regulator of blood pressure (BP). One of the mechanisms whereby ANP impacts BP is by stimulation of nitric oxide (NO) production in different tissues involved in BP control. We hypothesized that ANP-stimulated NO is impaired in the kidneys of spontaneously hypertensive rats (SHR) and this contributes to the development and/or maintenance of high levels of BP. We investigated the effects of ANP on the NO system in SHR, studying the changes in renal nitric oxide synthase (NOS) activity and expression in response to peptide infusion, the signaling pathways implicated in the signaling cascade that activates NOS, and identifying the natriuretic peptide receptors (NPR), guanylyl cyclase receptors (NPR-A and NPR-B) and/or NPR-C, and NOS isoforms involved. In vivo, SHR and Wistar-Kyoto rats (WKY) were infused with saline (0.05 ml/min) or ANP (0.2 μg·kg(-1)·min(-1)). NOS activity and endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS expression were measured in the renal cortex and medulla. In vitro, ANP-induced renal NOS activity was determined in the presence of iNOS and nNOS inhibitors, NPR-A/B blockers, guanine nucleotide-regulatory (G(i)) protein, and calmodulin inhibitors. Renal NOS activity was higher in SHR than in WKY. ANP increased NOS activity, but activation was lower in SHR than in WKY. ANP had no effect on expression of NOS isoforms. ANP-induced NOS activity was not modified by iNOS and nNOS inhibitors. NPR-A/B blockade blunted NOS stimulation via ANP in kidney. The renal NOS response to ANP was reduced by G(i) protein and calmodulin inhibitors. We conclude that ANP interacts with NPR-C, activating Ca-calmodulin eNOS through G(i) protein. NOS activation also involves NPR-A/B. The NOS response to ANP was diminished in kidneys of SHR. The impaired NO system response to ANP in SHR participates in the maintenance of high blood pressure. 相似文献