Effects of BAY 41–2272 on smooth muscle tone, soluble guanylyl cyclase activity and NADPH oxidase activity/expression in corpus cavernosum from wild-type, neuronal and endothelial NOS null mice

2007 ◽  
Vol 7 (S1) ◽  
Author(s):  
Cleber E Teixeira ◽  
Fernanda BM Priviero ◽  
RClinton Webb
Keyword(s):  
Smooth Muscle ◽  
Nadph Oxidase ◽  
Guanylyl Cyclase ◽  
Oxidase Activity ◽  
Muscle Tone ◽  
Corpus Cavernosum ◽  
Soluble Guanylyl Cyclase ◽  
Wild Type ◽  
Nadph Oxidase Activity ◽  
Guanylyl Cyclase Activity

gp91phox Contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

2003 ◽  
Vol 285 (6) ◽  
pp. H2284-H2289 ◽  
Author(s):  
Ali H. Chamseddine ◽  
Francis J. Miller
Keyword(s):  
Smooth Muscle ◽  
Nadph Oxidase ◽  
Smooth Muscle Cells ◽  
Oxidase Activity ◽  
Functional Expression ◽  
Wild Type ◽  
Nadph Oxidase Activity ◽  
Adventitial Cells ◽  
And Function

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91 phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide [Formula: see text] levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in [Formula: see text] levels between adventitial cells and medial SMC in aorta from gp91 phox-deficient (gp91 phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91 phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91 phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91 phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91 phox KO and wild-type FB to account for these observations. Because gp91 phox is associated with membranes, we examined NADPH-stimulated [Formula: see text] production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91 phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91 phox in FB, antisense to gp91 phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91 phox did not alter expression of the gp91 phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91 phox contributes to NADPH oxidase function in vascular FB, but not SMC.

scholarly journals The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle

2005 ◽  
Vol 65 (2) ◽  
pp. 495-504 ◽  
Author(s):  
S ELLMARK ◽  
G DUSTING ◽  
M NGTANGFUI ◽  
N GUZZOPERNELL ◽  
G DRUMMOND
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Nadph Oxidase Activity

Variable correction of host defense following gene transfer and bone marrow transplantation in murine X-linked chronic granulomatous disease

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3738-3745 ◽  
Author(s):  
Mary C. Dinauer ◽  
Mary A. Gifford ◽  
Nancy Pech ◽  
Ling Lin Li ◽  
Patricia Emshwiller
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Gene Transfer ◽  
Nadph Oxidase ◽  
Host Defense ◽  
Marrow Transplantation ◽  
Oxidase Activity ◽  
Granulomatous Disease ◽  
Wild Type ◽  
Nadph Oxidase Activity

Chronic granulomatous disease (CGD) is an inherited immunodeficiency in which the absence of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in recurrent bacterial and fungal infections. A murine model of X-linked CGD (X-CGD) was used to explore variables influencing reconstitution of host defense following bone marrow transplantation and retroviral-mediated gene transfer. The outcomes of experimental infection with Aspergillus fumigatus, Staphylococcus aureus, orBurkholderia cepacia were compared in wild-type, X-CGD mice, and transplanted X-CGD mice that were chimeric for either wild-type neutrophils or neutrophils with partial correction of NADPH oxidase activity after retroviral-mediated gene transfer. Host defense to these pathogens was improved in X-CGD mice even with correction of a limited number of neutrophils. However, intact protection against bacterial pathogens required relatively greater numbers of oxidant-generating phagocytes compared to protection against A fumigatus. The host response also appeared to be influenced by the relative level of cellular NADPH oxidase activity, particularly forA fumigatus. These results may have implications for developing effective approaches for gene therapy of CGD.

Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2386-2386
Author(s):  
Chaekyun Kim ◽  
Mary C. Dinauer
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Superoxide Production ◽  
Wild Type ◽  
Functional Responses ◽  
Nadph Oxidase Activity ◽  
Nadph Oxidase Activation

Abstract Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.

Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems

2007 ◽  
Vol 292 (1) ◽  
pp. C413-C422 ◽  
Author(s):  
Denise C. Fernandes ◽  
João Wosniak ◽  
Luciana A. Pescatore ◽  
Maria A. Bertoline ◽  
Marcel Liberman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Experimental Studies ◽  
Oxidation Products ◽  
Ang Ii ◽  
Specific Product ◽  
Nadph Oxidase Activity

Dihydroethidium (DHE) is a widely used sensitive superoxide (O2•−) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2•−, and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was ∼1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2•− production or NADPH oxidase activity in many vascular experimental studies.

Selective sequestration of nitric oxide by subcellular components of vascular smooth muscle and platelets: relationship to nitric oxide stimulation of the soluble guanylyl cyclase

1993 ◽  
Vol 71 (12) ◽  
pp. 938-945 ◽  
Author(s):  
Zhenguo Liu ◽  
Kanji Nakatsu ◽  
James F. Brien ◽  
E. Danielle Beaton ◽  
Gerald S. Marks ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Catalytic Activity ◽  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Subcellular Fractions ◽  
Cyclase Activity ◽  
Cytosolic Fraction ◽  
Headspace Gas ◽  
Guanylyl Cyclase Activity

Sequestration of nitric oxide (NO) by subcellular fractions isolated from bovine pulmonary arterial medial layer (BPA) and rabbit platelets (RP) was studied utilizing a novel chemiluminescence – headspace gas technique. Sequestration in all fractions was similarly rapid (5 min) and remained constant for at least 30 min. When incubated with 108 pmol of NO, the BPA mitochondrial, microsomal, and nuclear fractions sequestered 22.8 ± 1.9, 20.5 ± 2.2 and 15.2 ± 3.6% of the NO, respectively (n = 14). However, significantly more of the 108 pmol of NO, 36.8 ± 2.8 and 32.9 ± 3.6%, respectively, was sequestered by the BPA homogenate (about 2 mg protein/mL) and BPA cytosolic fraction (about 1 mg protein/mL) (n = 19). Also, RP cytosolic fraction (about 3 mg protein/mL) sequestered a greater amount of NO than any BPA fraction when incubated with 108 pmol of NO (83.0 ± 1.0%; n = 3). Analysis of the binding data obtained for the BPA homogenate and cytosolic fraction was consistent with the existence of two binding sites, one site with a Kd of approximately 100 nM and another with a Kd of approximately 1 μM. Both the BPA homogenate fraction and the cytosolic fraction as well as the RP cytosolic fraction were shown to have soluble guanylyl cyclase activity. The nitrovasodilator sodium nitroprusside (SNP) caused a concentration-dependent increase in the activity of this enzyme in all these fractions. Maximum stimulations caused by 1 mM SNP in BPA homogenate fraction, BPA cytosolic fraction, and RP cytosolic fraction were equivalent to 2-, 4- and 3-fold increases in catalytic activity, respectively. No effect of SNP was observed in BPA mitochondrial, microsomal, or nuclear fraction. Prior incubation of BPA and RP cytosolic fractions with authentic NO significantly stimulated the soluble guanyiyl cyclase activity. In both the BPA and RP cytosolic fractions, maximal stimulation brought about by prior incubation with authentic NO was equivalent to approximately 60% of that caused by 100 μM SNP. Thus, incubation of subcellular fractions from two nitrovasodilator-sensitive tissues with authentic NO resulted in significant sequestration of the free radical in these fractions and a concentration-dependent activation of the soluble guanylyl cyclase. In conclusion, the chemiluminescence – headspace gas technique is a suitable method for the study of NO sequestration in subcellular fractions of various tissues. Also, this study demonstrates that NO is sequestered preferentially by subcellular fractions of BPA and RP that contain soluble guanylyl cyclase activity, and that the sequestration of NO in these fractions stimulates the catalytic activity of this enzyme.Key words: nitric oxide, vascular smooth muscle, platelets, soluble guanylyl cyclase.

Sign in / Sign up

Export Citation Format

Share Document