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PubMed2004

Integration of multiple downstream signals determines the net effect of insulin on MAP kinase vs. PI 3'-kinase activation: potential role of insulin-stimulated H(2)O(2).

Cellular insulin stimulation generates a burst of H(2)O(2) that modulates protein-tyrosine phosphorylation in the insulin action pathway, in part by the inhibition of redox-sensitive protein-tyrosine phosphatases [J. Biol. Chem. 276 (2001) 21938]. Blocking the insulin-induced rise in H(2)O(2) with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) strongly attenuated the activation of phosphatidylinositol 3' (PI 3')-kinase, Akt and GLUT4 translocation by insulin in 3T3-L1 adipocytes; however, under identical conditions, we observed a paradoxical increase in the activation of p42/p44 mitogen-activated protein (MAP) kinase. DPI inhibited the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1/2, and also reduced the association of Grb2 with IRS-1, suggesting that the effect of DPI on MAP kinase activation occurred downstream of the IR and IRS proteins. DPI increased the insulin-stimulated phosphorylation of p42/p44 MAP kinase with no change in basal, and increased insulin-stimulated MAP kinase kinase (MEK) activity by a similar degree. DPI enhanced basal Grb2-Sos binding and reduced the effect of insulin to potentiate the dissociation of the Grb2-Sos complex, suggesting that the effect of DPI was mediated upstream of Raf-1. Cell treatment with dibutyryl cAMP significantly reduced the enhancement of MAP kinase activation in the presence of DPI. However, forskolin, acting in a PKA-independent manner, increased the insulin stimulation of MAP kinase and MEK, but fully abrogated the effect of DPI to enhance these insulin responses. PLCgamma inhibition with U73122 blocked the insulin stimulation of MAP kinase and MEK as well as the enhancing effect of DPI on these responses. PKC activation strongly stimulated MAP kinase and MEK activation, even in the presence of U73122, consistent with PKC acting downstream of PLCgamma. These data show that the insulin-stimulated oxidant signal differentially affects the two major downstream components of the insulin signaling pathway, PI 3'-kinase and MAP kinase, and cross-talk between insulin action, PLCgamma and, to a lesser extent, PKA modulates the net cellular effects of insulin-stimulated cellular H(2)O(2).

3T3-L1 Cells↗
PubMed2005

Serum withdrawal kills U937 cells by inducing a positive mutual interaction between reactive oxygen species and phosphoinositide 3-kinase.

Reactive oxygen species (ROS) can be generated following cell stimulation and function as intracellular signaling molecules. To determine signaling components involved in ROS induction, human U937 blood cells grown in 10% serum were exposed to serum-free media. It was previously reported that serum withdrawal (SW) killed cells by elevating cellular ROS levels. This study showed that SW activates phosphoinositide 3-kinase (PI3K). PI3K activation was evident after the ROS levels began increasing, and an antioxidant blockade of this increase resulted in PI3K activation suppression. Interestingly, the inhibition of PI3K activity/activation using either its specific inhibitor or dominant-negative mutant attenuated the subsequent additional increase in the ROS levels. These results suggest that SW-induced ROS activate PI3K, which in turn promotes the process leading to ROS accumulation. The present study also revealed that both ROS and PI3K support SW-induced cell death by activating stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Overall, it appears that SW triggers a positive mutual interaction between ROS and PI3K, which amplifies signals required for the induction of an SAPK-dependent death pathway.

Catalase↗
PubMed2005

NOX4 as an oxygen sensor to regulate TASK-1 activity.

When oxygen sensing cells are excited by hypoxia, background K+ currents are inhibited. TASK-1, which is commonly expressed in oxygen sensing cells and makes a background K+ current, is inactivated by hypoxia. Thus TASK-1 is a candidate molecule responsible for hypoxic excitation. However, TASK-1 per se cannot sense oxygen and may require a regulatory protein that can. In the present study, we propose that the NADPH oxidase NOX4 functions as an oxygen-sensing partner and that it modulates the oxygen sensitivity of TASK-1. Confocal imaging revealed the co-localization of TASK-1 and NOX4 in the plasma membrane. In HEK293 cells expressing NOX4 endogenously, the activity of expressed TASK-1 was moderately inhibited by hypoxia, and this oxygen response was significantly augmented by NOX4. Moreover, the oxygen sensitivity of TASK-1 was abolished by NOX4 siRNA and NADPH oxidase inhibitors. These results suggest a novel function for NOX4 in the oxygen-dependent regulation of TASK-1 activity.

Biosensing Techniques↗
PubMed2006

Degradation of tetraphenylphosphonium bromide at high pH and its effect on radionuclide solubility.

Recently, tetraphenylphosphonium bromide (TPPB) has been used to remove technetium from some radioactive waste streams. However, before TPPB could be approved for use it was necessary to show that TPPB and its degradation products would not have a significant detrimental effect on post-closure performance of a radioactive waste repository. TPPB is known to be stable at neutral pH, however, under alkaline conditions it degrades by an alkaline hydrolysis mechanism to triphenylphosphonium oxide (TPPO). Degradation can also occur by radiolysis to produce triphenylphosphine (TPP). The kinetics of the alkaline hydrolysis degradation of TPPB is described and the solubility of europium, iodine, nickel, technetium(VII) and uranium(VI) in aqueous solutions of TPPB and its degradation products is reported. These results were used to support the use of TPPB in removing technetium from some waste streams.

Hydrolysis↗
PubMed2004

Propofol scavenges reactive oxygen species and inhibits the protein nitration induced by activated polymorphonuclear neutrophils.

Activated polymorphonuclear neutrophils may damage tissues through the release of biochemical mediators. Among them, peroxynitrite is responsible for hydroxylation reactions and nitration of proteins, or is metabolised into nitrate. We investigated the effect of propofol on the production of reactive oxygen species, the nitration of proteins and the formation of nitrate by activated human polymorphonuclear neutrophils. Propofol dose-dependently inhibited chemiluminescence, nitration of proteins and nitrate production in a concentration range from 10(-3) to 10(-6) mM. A significant correlation was observed between the logarithm of propofol concentration and the intensity of chemiluminescence (r2=0.90), the nitration of proteins (r2=0.67) and the production of nitrate (r2=0.79). Those results are consistent with the scavenging effect of propofol on peroxynitrite and could confer a protective property to propofol in pathological situations involving polymorphonuclear neutrophils activation.

Dose-Response Relationship, Drug↗
PubMed2005

Effect of sildenafil citrate and a nitric oxide donating sildenafil derivative, NCX 911, on cavernosal relaxation and superoxide formation in hypercholesterolaemic rabbits.

Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and negation of nitric oxide (NO) bioactivity in cavernosal tissue. The source of superoxide has not been clearly defined, however. Sildenafil (Viagra), the standard therapy for erectile dysfunction, may also be rendered more effective by the presence of an NO donor. One drug that intrinsically fulfils this criterion is sildenafil nitrate (NCX 911), an NO donating derivative of sildenafil. The objective of this study, therefore, was to determine the source of superoxide and its effect on erectile function in corpus cavernosum from hypercholesterolaemic rabbits and to determine whether NCX 911 confers an improvement over sildenafil citrate in this model. Hypercholesterolaemia elicited an increase in superoxide formation by rabbit cavernosal tissue and a reduction of carbachol-stimulated relaxation both of which were reversed by diphenylene iodonium chloride and apocynin (NADPH oxidase inhibitors). In response to sodium nitroprusside, hypercholesterolaemia also caused an attenuation of cavernosal relaxation which was not reversed with NADPH oxidase inhibitors. Both sildenafil citrate and NCX 911 significantly reversed impaired carbachol-stimulated relaxation and inhibited superoxide formation by cavernosal tissue from hypercholesterolaemic rabbits, NCX 911 being more potent. NCX 911 also augmented cavernosal cGMP levels, an effect blocked by the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo {4,3-a}quinoxalin-1-one (ODQ). These data demonstrate that hypercholesterolaemia promotes erectile dysfunction through an augmentation of superoxide derived from NADPH oxidase in cavernosal tissue. It also indicates that NO donating sildenafil may be therapeutically more beneficial than conventional sildenafil in treating erectile dysfunction with an oxidative stress-related aetiology.

Acetophenones↗
PubMed2006

Penicillamine administration reverses the inhibitory effect of hyperhomocysteinaemia on endothelium-dependent relaxation and superoxide formation in the aorta of the rabbit.

Although hyperhomocysteinaemia is a risk factor for cardiovascular disease, the mechanisms underlying this association have not been elucidated. It has been demonstrated, however, that copper augments the inhibitory effect of homocysteine on nitric oxide (NO)-mediated relaxation of the rat aorta through increased superoxide formation, which reacts with NO thereby reducing the bioavailability of NO. Since it follows that the administration of a copper chelator may blunt the pathogenic impact of hyperhomocysteinaemia, in vivo, the effect of penicillamine administration on NO-dependent relaxation and superoxide formation in the aortae of hyperhomocysteinaemic rabbits was studied. New Zealand White rabbits were fed a methionine-rich (20 g/kg chow) diet for 1 month+/-penicillamine administered orally (10 mg/kg/day) and aortic relaxation elicited with acetylcholine and superoxide measured. The role of NADPH oxidase was also studied using a range of inhibitors and western analysis of gp47(phox) (a catalytic subunit of NADPH oxidase). The methionine-rich diet markedly increased plasma total homocysteine levels. In hyperhomocysteinaemic rabbits there was a marked reduction of acetylcholine-stimulated relaxation and an increase in superoxide formation that were both inhibited with superoxide dismutase and apocynin, an NADPH oxidase inhibitor. Gp47(phox) expression was also increased in aortae from methionine fed rabbits. Penicillamine administration significantly reduced plasma total copper in methionine-fed rabbits compared to controls. Impaired acetylcholine-stimulated relaxation, increased superoxide formation and increased gp47(phox) expression in aortae from methionine-fed rabbits was reversed by penicillamine administration. These data indicate that hyperhomocysteinaemia augments the formation of arterial superoxide through an increase in NADPH oxidase expression/activity which in turn reduces NO bioavailability. Since these effects were reversed by penicillamine, these data consolidate the hypothesis that copper plays a role in mediating homocysteine-induced vasculopathy.

Acetophenones↗
PubMed2006

Reactive oxygen species mediate phorbol ester-stimulated cAMP response in human eosinophils.

Recently, we showed that phorbol 12-myristate 13-acetate (PMA) can cause a direct, PKC-dependent, stimulation of intracellular cAMP in human eosinophils. Since PMA also stimulates the release of reactive oxygen species in these cells, we have investigated whether reactive oxygen species are involved in the cAMP response. Provided eosinophils were incubated for <20 min at 37 degrees C before stimulation, PMA potently stimulated cAMP generation that surpassed that of histamine. Pre-treatment of the cells with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI) and apocynin, strongly inhibited the cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O(2)(-)). The cAMP response was also inhibited by pre-treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or NG-nitro-l-arginine methyl ester (L-NAME), thus, suggesting the possible involvement of a peroxide rather than O(2)(-) or nitric oxide (NO). These results reveal a novel involvement of intracellular reactive oxygen species in protein kinase C (PKC)-dependent stimulation of cAMP production in human eosinophils.

Acetophenones↗
PubMed2006

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells.

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.

Acetophenones↗
PubMed2004

Source of early reactive oxygen species in the apoptosis induced by transforming growth factor-beta in fetal rat hepatocytes.

Transforming growth factor-beta (TGF-beta) induces an oxidative stress process in hepatocytes that mediates its apoptotic activity. To determine the cellular source of the early reactive oxygen species (ROS) generated by fetal rat hepatocytes in response to TGF-beta, we used inhibitors that block different ROS-producing systems. Diphenyleneiodonium, which inhibits NADPH oxidase and other flavoproteins, completely blocked the increase in ROS induced by TGF-beta, coincidently with an impairment of caspase-3 activation and cell death. Rotenone, an inhibitor of the NADH dehydrogenase in mitochondrial complex I, attenuated, but did not completely inhibit, ROS-production, caspase activation, and cell death mediated by TGF-beta. No significant protection was observed with inhibitors of other ROS-producing systems, such as cytochrome P450 (metyrapone), cyclooxygenase (indomethacin), and xanthine oxidase (allopurinol). Additional experiments have indicated that two different mechanisms could be involved in the early ROS production by TGF-beta. First, an inducible (cycloheximide-inhibited) NADPH oxidase-like system could account for the extramitochondrial production of ROS. Second, TGF-beta could increase ROS by a rapid downregulation of antioxidant genes. In particular, intramitochondrial ROS would increase by depletion of MnSOD. Finally, glutathione depletion is a late event and it would be more the consequence than the cause of the increase in ROS induced by TGF-beta.

Animals↗
PubMed2004

Redox activity associated with the maturation and capacitation of mammalian spermatozoa.

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.

4-Chloromercuribenzenesulfonate↗
PubMed2004

Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species.

Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways.

Catalase↗
PubMed2004

Cadmium induces caspase-independent apoptosis in liver Hep3B cells: role for calcium in signaling oxidative stress-related impairment of mitochondria and relocation of endonuclease G and apoptosis-inducing factor.

Cadmium-induced cellular toxicity has been related to necrosis and/or caspase-dependent apoptosis. In the present study, we show that, on cadmium exposure, the human hepatocarcinoma Hep3B cells undergo caspase-independent apoptosis associated with nuclear translocation of endonuclease G and apoptosis-inducing factor, two mitochondrial apoptogenic proteins. Release of these proteins is likely related to calcium-induced alteration of mitochondrial homeostasis. Indeed, it was first preceded by a rapid and sustained increase in cytoplasmic calcium and then by a coincident loss in mitochondrial membrane potential and production of reactive oxygen species. Bapta-AM (acetoxymethyl ester of 5, 5'-dimethyl-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), a calcium chelator, blocked all these events and prevented cadmium-induced apoptosis. Production of reactive oxygen species was inhibited by ruthenium red and rotenone, two mitochondrial inhibitors, and by diphenyleneiodonium, a flavoprotein inhibitor, which also prevented both loss in mitochondrial membrane potential and apoptosis. In addition, Bapta-AM and diphenyleneiodonium were found to almost totally block decreased expression of the mitochondrial anti-apoptotic nuclear factor-kappaB-regulated bcl-x(L) protein in cadmium-treated cells. Taken together, our results show that cadmium induces Hep3B cells apoptosis mainly by calcium- and oxidative stress-related impairment of mitochondria, which probably favors release of apoptosis-inducing factor and endonuclease G.

Active Transport, Cell Nucleus↗
PubMed2004

Reactive oxygen species mediate Endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 signaling, as well as protein synthesis, in vascular smooth muscle cells.

Reactive oxygen species (ROS) have been shown to mediate the effects of several growth factors and vasoactive peptides, such as epidermal growth factor, platelet-derived growth factor, and angiotensin II (AII). Endothelin-1 (ET-1) is a vasoactive peptide which also exhibits mitogenic activity in vascular smooth muscle cells (VSMCs), and is believed to contribute to the pathogenesis of vascular abnormalities such as atherosclerosis, hypertension, and restenosis after angioplasty. However, a possible role for ROS generation in mediating the ET-1 response on extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB), and protein tyrosine kinase 2 (Pyk2), key components of the growth-promoting and proliferative signaling pathways, has not been examined in detail. Our aim was to investigate the involvement of ROS in ET-1-mediated activation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs. ET-1 stimulated ERK1/2, PKB, and Pyk2 phosphorylation in a dose- and time-dependent manner. Pretreatment of A-10 VSMCs with diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase, attenuated ET-1-enhanced ERK1/2, PKB, and Pyk2 phosphorylation. In addition, in parallel with an inhibitory effect on the above signaling components, DPI also blocked ET-1-induced protein synthesis. ET-1 was also found to increase ROS production, which was suppressed by DPI treatment. N-Acetylcysteine, a ROS scavenger, exhibited a response similar to that of DPI and inhibited ET-1-stimulated ERK1/2, PKB, and Pyk2 phosphorylation. These results demonstrate that ROS are critical mediators of ET-1-induced signaling events linked to growth-promoting proliferative and hypertrophic pathways in VSMCs.

Animals↗
PubMed2005

Involvement of UVB-induced reactive oxygen species in TGF-beta biosynthesis and activation in keratinocytes.

TGF-beta produced by keratinocytes in response to UVB (290-320 nm) is a potential mediator for effects of acute and chronic solar radiation on skin. This study was designed to determine whether reactive oxygen species (ROS) mediate UVB-induced TGF-beta biosynthesis in keratinocytes and the subsequent activation of the latent TGF-beta complex. UVB irradiation elevated both total (latent plus active) and active TGF-beta in the keratinocyte supernatants, with a greater increase in the active form. UVB irradiation induced up to a 30% increase in ROS, and the ROS were detected up to 90 min after irradiation. NAC and Trolox, cytoplasmic ROS scavengers, abolished the UVB-induced TGF-beta and intracellular ROS, suggesting that UVB-induced ROS are involved in TGF-beta regulation. Inhibitors of NADPH oxidase activity, DPI and apocynin, decreased UVB-induced ROS. The increase in NADPH oxidase activity was mediated by EGFR activation. UVB-induced ROS also activated latent TGF-beta complex by stimulating MMP-2 and -9 activities. In summary, physiological doses of UVB increase intracellular ROS, which upregulate TGF-beta biosynthesis and activation of TGF-beta through increased activity of MMPs.

ErbB Receptors↗
PubMed2005

Redox response of the endogenous calcineurin inhibitor Adapt 78.

Adapt 78 (DSCR 1/calcipressin/MCIP 1) is a potent natural inhibitor of calcineurin, an important intracellular phosphatase that mediates many cellular responses to calcium. We previously reported two major cytosolic isoforms (1 and 4) of Adapt 78, and that isoform 4 is an oxidative and calcium stress-response protein. Using a higher cell culture density and new antibody, we again observed that both major isoforms localized to the cytosol, but a significant level of isoform 4 (but not isoform 1) was also detected in the nucleus where it was present in the non-soluble region and not associated with RNA. Exposure of cells to hydrogen peroxide led to the significant loss of isoform 4 from the nucleus with a moderate increase in cytosolic localization. The change in isoform 4 phosphorylation state in response to oxidative stress, characterized by a loss of the lesser (hypo) phosphorylated Adapt 78, was not due to accelerated degradation, although general Adapt 78 degradation was proteosome mediated. Finally, stimulation of Jurkat and primary T-lymphocyte signaling led to isoform 4 induction. This induction was BAPTA, diphenylene iodonium, and N-acetylcysteine inhibitable, and accompanied by induction of the classic immune response mediator and calcineurin-pathway-stimulated interleukin-2. These studies reveal new redox-related activities for Adapt 78 isoform 4, which may contribute to its known calcineurin-regulating and cytoprotective activities, and further suggest that Adapt 78 plays a role in basic T-cell response.

Acetylcysteine↗
PubMed2005

Alterations in redox homeostasis and prostaglandins impair endothelial-dependent vasodilation in euglycemic autoimmune nonobese diabetic mice.

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.

Acetophenones↗
PubMed2005

Coronary microvascular endothelial cell growth regulates expression of the gene encoding p22-phox.

The mRNA expression of p22-phox, a membrane-bound component of NAD(P)H oxidase, is elevated in proliferating compared to quiescent rat coronary microvascular endothelial cells (CMEC). However, the underlying molecular mechanisms of this increase and its relevance to cell confluence remain unknown. This study shows greater levels of p22-phox mRNA/protein expression, NAD(P)H oxidase activity, and superoxide anion (O(2)(-)) production in proliferating versus fully confluent and growth-arrested 50% confluent CMEC. Both enhanced transcription of the p22-phox gene and enhanced mRNA half-life were shown to contribute to the increase in p22-phox mRNA levels as demonstrated by nuclear run-on studies and Northern analyses after actinomycin D transcriptional arrest, respectively. Suppression of O(2)(-) availability by a cell-permeable superoxide dismutase mimetic or inhibition of NAD(P)H oxidase activity via its specific inhibitors (apocynin, diphenyleneiodinium, phenylarsine oxide, and 4-(2-aminoethyl)bezenesulfonyl fluoride) or antisense p22-phox cDNA attenuated cell growth independent of changes in intracellular antioxidant glutathione levels. Inhibition of nascent protein synthesis by a translation inhibitor, cycloheximide, significantly reduced p22-phox mRNA stability in proliferating but not in confluent CMEC. These findings suggest that expression of p22-phox mRNA in CMEC is upregulated by growth through a mechanism that involves increases in transcriptional activity of the p22-phox gene and stability of the p22-phox message after its formation.

Acetophenones↗