Chronic tempol treatment attenuates the renal hemodynamic effects induced by a heme oxygenase inhibitor in streptozotocin diabetic rats

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in protecting the kidney from oxidant-mediated damage in the streptozotocin (STZ) rat model of type-1 diabetes mellitus (DM-1). HO-derived metabolites, presumably carbon monoxide (CO), mediate vasodilatory influences in the renal circulation, particularly in conditions linked to elevated HO-1 protein expression or diminished nitric oxide (NO) levels. We tested the hypothesis that diabetes increases oxidative stress and induces HO-1 protein expression, which contributes to regulate renal hemodynamics in conditions of low NO bioavailability. Two weeks after the induction of diabetes with STZ (65 mg/kg iv), Sprague-Dawley rats exhibited higher renal HO-1 protein expression, hyperglycemia, and elevated renal nitrotyrosine levels than control normoglycemic animals. In anesthetized diabetic rats, renal vascular resistance (RVR) was increased, and in vivo cortical NO levels were reduced (P < 0.05) compared with control animals. Acute administration of the HO inhibitor Stannous mesoporphyrin (SnMP; 40 μmol/kg iv) did not alter renal hemodynamics in control rats, but greatly decreased glomerular filtration rate and renal blood flow, markedly increasing RVR in hyperglycemic diabetic rats. Chronic oral treatment with the SOD mimetic tempol prevented the elevation of nitrotyrosine, the HO-1 protein induction, and the increases in RVR induced by SnMP in the diabetic group, without altering basal NO concentrations or RVR. Increasing concentrations of a CO donor (CO-releasing molecule-A1) on pressurized renal interlobar arteries elicited a comparable relaxation in vessels taken from control or diabetic animals. These results suggest that oxidative stress-induced HO-1 exerts vasodilatory actions that partially maintain renal hemodynamics in uncontrolled DM-1.     

Early oxidative stress in the diabetic kidney: effect of DL-alpha-lipoic acid

Oxidative stress is implicated in the pathogenesis of diabetic nephropathy. The attempts to identify early markers of diabetes-induced renal oxidative injury resulted in contradictory findings. We characterized early oxidative stress in renal cortex of diabetic rats, and evaluated whether it can be prevented by the potent antioxidant, DL-alpha-lipoic acid. The experiments were performed on control rats and streptozotocin-diabetic rats treated with/without DL-alpha-lipoic acid (100 mg/kg i.p., for 3 weeks from induction of diabetes). Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats vs. controls (p <.01) and this increase was partially prevented by DL-alpha-lipoic acid. F(2) isoprostane concentrations (measured by GCMS) expressed per either mg protein or arachidonic acid content were not different in control and diabetic rats but were decreased several-fold with DL-alpha-lipoic acid treatment. Both GSH and ascorbate (AA) levels were decreased and GSSG/GSH and dehydroascorbate/AA ratios increased in diabetic rats vs. controls (p <.01 for all comparisons), and these changes were completely or partially (AA) prevented by DL-alpha-lipoic acid. Superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione transferase, and NADH oxidase, but not catalase, were upregulated in diabetic rats vs. controls, and these activities, except glutathione peroxidase, were decreased by DL-alpha-lipoic acid. In conclusion, enhanced oxidative stress is present in rat renal cortex in early diabetes, and is prevented by DL-alpha-lipoic acid.     

Renal protective effect of chronic inhibition of COX-2 with SC-58236 in streptozotocin-diabetic rats

The induction of renal cyclooxygenase-2 (COX-2) in diabetes has been implicated in the renal functional and structural changes in models where hypertension or uninephrectomy was superimposed. We examined the protective effects of 3 mo treatment of streptozotocin-diabetic rats with a highly selective COX-2 inhibitor (SC-58236) in terms of albuminuria, renal hypertrophy, and the excretion of TNF-α and TGF-β, which have also been implicated in the detrimental renal effects of diabetes. SC-58236 treatment (3 mg·kg(-1)·day(-1)) of diabetic rats resulted in reduced urinary excretion of PGE(2), 6-ketoPGF(1α), and thromboxane B(2), all of which were increased in the diabetic rat compared with age-matched nondiabetic rats. However, serum thromboxane B(2) levels were unchanged, confirming the selectivity of SC-58236 for COX-2. The renal protective effects of treatment of diabetic rats with the COX-2 inhibitor were reflected by a marked reduction in albuminuria, a reduction in kidney weight-to-body weight ratio, and TGF-β excretion and a marked decrease in the urinary excretion of TNF-α. The protective effects of SC-58236 were independent of changes in plasma glucose levels or serum advanced glycation end-product levels, which were not different from those of untreated diabetic rats. In an additional study, the inhibition of COX-2 with SC-58236 for 4 wk in diabetic rats resulted in creatinine clearance rates not different from those of control rats. These results confirm that the inhibition of COX-2 in the streptozotocin-diabetic rat confers renal protection and suggest that the induction of COX-2 precedes the increases in cytokines, TNF-α, and TGF-β.     

Ebselen reduces nitration and restores voltage-gated potassium channel function in small coronary arteries of diabetic rats

Small coronary arteries (SCA) from diabetic rats exhibit enhanced peroxynitrite (ONOO(-)) formation and concurrent impairment of voltage-dependent potassium (K(v)) channel function. However, it is unclear whether ONOO(-) plays a causative role in this impairment. We hypothesized that functional loss of K(v) channels in coronary smooth muscle cells (SMC) in diabetes is due to ONOO(-) with subsequent tyrosine nitration of K(v) channel proteins. Diabetic rats and nondiabetic controls were treated with or without ebselen (Eb) for 4 wk. SCA were prepared for immunohistochemistry (IHC), immunoprecipitation (IP) followed by Western blot (WB), videomicroscopy, and patch-clamp analysis. IHC revealed excess ONOO(-) in SCA from diabetic rats. IP and WB revealed elevated nitration of the K(v)1.2 alpha-subunit and reduced K(v)1.2 protein expression in diabetic rats. Each of these changes was improved in Eb-treated rats. Protein nitration and K(v)1.5 expression were unchanged in SCA from diabetic rats. Forskolin, a direct cAMP activator that induces K(v)1 channel activity, dilated SCA from nondiabetic rats in a correolide (Cor; a selective K(v)1 channel blocker)-sensitive fashion. Cor did not alter the reduced dilation to forskolin in diabetic rats; however, Eb partially restored the Cor-sensitive component of dilation. Basal K(v) current density and response to forskolin were improved in smooth muscle cells from Eb-treated DM rats. We conclude that enhanced nitrosative stress in diabetes mellitus contributes to K(v)1 channel dysfunction in the coronary microcirculation. Eb may be beneficial for the therapeutic treatment of vascular complications in diabetes mellitus.     

The effects of resveratrol on cyclooxygenase-1 and cyclooxygenase-2 mRNA and protein levels in diabetic rat kidneys

Cyclooxygenase (COX), which have the isoforms of COX-1 and COX-2, is the key enzyme of prostaglandins biosynthesis. Especially, COX-2 is induced in inflammatory disease such as Diabetes Mellitus (DM). Resveratrol (RSV), a natural antioxidant, has a beneficial role in prevention of inflammatory disease. We investigated the changes of COX-1 and COX-2 mRNA expression and protein level in diabetic rat kidney after RSV treatment. Three months-old, 44 Wistar albino male rats, which were divided into six groups such as control group, sodium citrate buffer (sham control) group, diabetic group (DM), Dimethyl Sulfoxide induced control group, RSV treated sham control group (RSV) and RSV treated diabetic group (DM + RSV) were used for the study. Experimental diabetes was induced by intraperitoneal injection of 55 mg/kg Streptozotocin. After the induction of chronic diabetes 10 mg/kg per day RSV was administered intraperitoneally for 4 weeks. In this study. RSV has no significant effect on COX-1 mRNA expression in diabetic rat kidney (P > 0.05). Immunohistochemical study showed that COX-1 expression was slightly inhibited in RSV group and was not significantly supressed in DM + RSV group. When comparing control and treated groups, there were no significant differences in COX-2 mRNA or protein levels (P > 0.05). In conclusion, our results indicate that resveratrol do not significantly affect COX gene and protein expression. Therefore, different therapy strategies such as combination with other antidiabetic drugs may tried in STZ induced animal model for reducing diabetic symptoms and altering COX-1 and COX-2 mRNA or protein levels.     

Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.     

Time course of NADH oxidase, inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in the BBZ/WOR rat.

This study investigated the time course of NADH oxidase, a source of superoxide in the vascular endothelium, inducible nitric oxide synthase (iNOS), and peroxynitrite (ONOO(-)) in the BBZ/Wor rat, a spontaneous model of noninsulin dependent diabetes (NIDDM). Colloidal gold-labeled immunocytochemical studies of iNOS and nitrotyrosine, a marker for OONO(-), were done on sections of retinas from male BBZ/Wor rats in which NADH oxidase was localized by cerium derived cytochemistry at three time points: pre-diabetes (prior to the onset of hyperglycemia); new onset diabetes (2-6 days after onset of hyperglycemia); and chronic diabetes (4-18 months after onset of hyperglycemia). Control retinas were from age matched non-diabetic BB(DR)/Wor rats. The percentage of blood vessels positive for NADH oxidase increased significantly (P = 0.05) in new onset (64.2 +/- 6.5%) and chronic diabetes (83.2 +/- 11.4%), as compared to pre-diabetes (25.8 +/- 5.6%) and nondiabetic controls (33.6 +/- 15.9%). The percentage of blood vessels positive for iNOS immunoreactivity was significantly higher in new onset diabetic retinas (69.6 +/- 5.88%, P = 0.0001; 8.9 +/- 3.29 colloidal gold particles (cgp) /50 microm(2)) than in chronic diabetic retinas (49.9 +/- 9.75%; 7.9 +/- 5.12 cgp) and both were significantly higher (P = 0.0001) than in prediabetic (3.7 +/- 0.81%; 0.4 +/- 0.56 cgp) and nondiabetic control retinas (8.7 +/- 4.66%; 1.2 +/- 1.40 cgp). In new onset diabetes, levels of nitrotyrosine immunoreactivity (60.8 +/- 16.91 cgp) were significantly higher (P = 0.0001) than those in chronic diabetes (29.5 +/- 4.31 cgp); both were significantly higher (P = 0.0001) than those in prediabetic (8.2 +/- 1.70 cgp) and nondiabetic retinas (9.0 +/- 1.87 cgp). There was no cumulative increase in nitrotyrosine in the chronic diabetic retinas as a function of time. In rats with diabetes there was disruption of the inner blood-retinal barrier. These results suggest that iNOS and ONOO(-) may contribute to retinal damage in diabetes from the onset of hyperglycemia in NIDDM.     

Renal Response to L-Arginine in Diabetic Rats. A Possible Link between Nitric Oxide System and Aquaporin-2

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.     

The effects of resveratrol on cyclooxygenase-1 and -2, nuclear factor kappa beta, matrix metalloproteinase-9, and sirtuin 1 mRNA expression in hearts of streptozotocin-induced diabetic rats

Resveratrol (RSV) has a beneficial role in the prevention of diabetes and alleviates some diabetic complications, such as cardiomyopathy. We investigated cyclooxygenase-1 (COX-1), COX-2, nuclear factor κB (NF-κB), matrix metalloproteinase-9 (MMP-9), and sirtuin 1 (SIRT1) mRNA expression levels in heart tissue after RSV treatment in streptozotocin (STZ)-induced diabetic rats. After induction of chronic diabetes with STZ, 10 mg RSV/kg per day was administered to DM and DM+RSV groups for four weeks. At the end of the experiment, all rats were sacrificed and heart tissues were stored at -80°C; mRNA expression levels of COX-1, COX-2, NF-κB, MMP-9, and SIRT1 genes were analyzed with quantitative real-time PCR. We did not find any significant effect of RSV on MMP-9, COX-1, COX-2, or NF-κB mRNA levels among the groups. However, SIRT1 mRNA levels decreased in the DM group compared to controls and increased in the DM+RSV group when compared to the DM group. SIRT1 is activated by RSV treatment in diabetic heart tissue. Activation of SIRT1 by RSV may lead to a new therapeutic approach for diabetic heart tissue. We conclude that RSV treatment can alleviate heart dysfunction by inhibiton of inflammatory gene expression such as SIRT1.     

Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kappa B-dependent genes

Induction of NF-kappaB-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease (ALD). Curcumin, a phenolic antioxidant, inhibits the activation of NF-kappaB. We determined whether treatment with curcumin would prevent experimental ALD and elucidated the underlying mechanism. Four groups of rats (6 rats/group) were treated by intragastric infusion for 4 wk. One group received fish oil plus ethanol (FE); a second group received fish oil plus dextrose (FD). The third and fourth groups received FE or FD supplemented with 75 mg. kg(-1). day(-1) of curcumin. Liver samples were analyzed for histopathology, lipid peroxidation, NF-kappaB binding, TNF-alpha, IL-12, monocyte chemotactic protein-1, macrophage inflammatory protein-2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitrotyrosine. Rats fed FE developed fatty liver, necrosis, and inflammation, which was accompanied by activation of NF-kappaB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation. Treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol. Because endotoxin and the Kupffer cell are implicated in the pathogenesis of ALD, we investigated whether curcumin suppressed the stimulatory effects of endotoxin in isolated Kupffer cells. Curcumin blocked endotoxin-mediated activation of NF-kappaB and suppressed the expression of cytokines, chemokines, COX-2, and iNOS in Kupffer cells. Thus curcumin prevents experimental ALD, in part by suppressing induction of NF-kappaB-dependent genes.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes

Studies in streptozotocin (STZ)-induced diabetic rats have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure (MABP) and heart rate (HR), endothelial dysfunction, and attenuated pressor responses to vasoactive agents. We investigated whether these abnormalities are due to diabetes-associated activation of inducible nitric oxide synthase (iNOS). In addition, the effect of the duration of diabetes on these abnormalities was also evaluated. Diabetes was induced by administration of 60 mg/kg STZ via the tail vein. One, 3, 9, or 12 wk after STZ injection, MABP, HR, and endothelial function were measured in conscious unrestrained rats. Pressor response curves to bolus doses of methoxamine (MTX) and angiotensin II (ANG II) were constructed in the presence of N-[3(aminomethyl)benzyl]-acetamidine, dihydrochloride (1400W), a specific inhibitor of iNOS. Depressed MABP and HR and impairment of endothelial function were observed as early as 3 wk after induction of diabetes. Acute inhibition of iNOS with 1400W (3 mg/kg i.v.) restored the attenuated pressor responses to both MTX and ANG II without affecting the basal MABP and HR. Immunohistochemical and Western analysis blot studies in cardiovascular tissues revealed decreased expression of endothelial nitric oxide synthase (eNOS) concomitant with increased expression of iNOS and nitrotyrosine with the progression of diabetes. Our findings suggest that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to the depressed pressor responses to vasoactive agents and potentially to endothelial dysfunction.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

IL-1β is upregulated in the diabetic retina and retinal vessels: cell-specific effect of high glucose and IL-1β autostimulation

Many molecular and cellular abnormalities detected in the diabetic retina support a role for IL-1β-driven neuroinflammation in the pathogenesis of diabetic retinopathy. IL-1β is well known for its role in the induction and, through autostimulation, amplification of neuroinflammation. Upregulation of IL-1β has been consistently detected in the diabetic retina; however, the mechanisms and cellular source of IL-1β overexpression are poorly understood. The aim of this study was to investigate the effect of high glucose and IL-1β itself on IL-1β expression in microglial, macroglial (astrocytes and Müller cells) and retinal vascular endothelial cells; and to study the effect of diabetes on the expression of IL-1β in isolated retinal vessels and on the temporal pattern of IL-1β upregulation and glial reactivity in the retina of streptozotocin-diabetic rats. IL-1β was quantified by RealTime RT-PCR and ELISA, glial fibrillar acidic protein, α2-macroglobulin, and ceruloplasmin by immunoblotting. We found that high glucose induced a 3-fold increase of IL-1β expression in retinal endothelial cells but not in macroglia and microglia. IL-1β induced its own synthesis in endothelial and macroglial cells but not in microglia. In retinal endothelial cells, the high glucose-induced IL-1β overexpression was prevented by calphostin C, a protein kinase C inhibitor. The retinal vessels of diabetic rats showed increased IL-1β expression as compared to non-diabetic rats. Retinal expression of IL-1β increased early after the induction of diabetes, continued to increase with progression of the disease, and was temporally associated with upregulation of markers of glial activation. These findings point to hyperglycemia as the trigger and to the endothelium as the origin of the initial retinal upregulation of IL-1β in diabetes; and to IL-1β itself, via autostimulation in endothelial and macroglial cells, as the mechanism of sustained IL-1β overexpression. Interrupting the vicious circle triggered by IL-1β autostimulation could limit the progression of diabetic retinopathy.     

Peroxynitrite mediates TNF-alpha-induced endothelial barrier dysfunction and nitration of actin

We tested the hypothesis that tumor necrosis factor (TNF)-alpha induces a peroxynitrite (ONOO(-))-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated beta-actin (NO(2)-beta-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. beta-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted beta-actin was quantified in terms of its nitrotyrosine/beta-actin ratio with anti-nitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO(2)-beta-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with beta-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/beta-actin ratio at 0.5 h with minimal association of the NO(2)-beta-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/beta-actin ratio and permeability were prevented by the anti-ONOO(-) agent Urate. The data indicate that TNF induces an ONOO(-)-dependent barrier dysfunction, which is associated with the generation of NO(2)-beta-actin.