Race/Sex Interactions and HIV Testing Among College Students.

OBJECTIVES: The primary aim of the study was to examine race/sex interactions and other behavioral and demographic covariates of past-year HIV testing among college students attending a racially diverse historically black university. The relationship between race/sex interactions and engaging with multiple sex partners during the past year was also examined. PARTICIPANTS: The sample included 566 students who identified as Black or White and engaged in vaginal, oral, or anal sex during the past 12 months. METHODS: A total of 113 undergraduate classes were randomly selected, stratified by time of day. Surveys were administered by project team members who were assisted by peer health educators to increase the cultural competency of the study. The response rate was 94 %. RESULTS: The sample of 566 participants included Black women (54 %), Black men (26 %), White women (13 %), and White men (7 %). The mean age was 25 (median = 22 years), and 42 % resided on campus. Nearly half (46 %) reported two or more sex partners in the past year, and 9 % of participants were diagnosed with STD in the past year. Sixty-nine percent reported being tested for HIV, 58 % had been tested in the past year, and 18 % had been tested for HIV on two or more occasions during the past year. In multivariable analysis, Black men (AOR = 0.43; 95 % CI = 0.27, 0.69), White women (AOR = 0.25; 95 % CI = 0.14, 0.47), and White men (AOR = 0.22; 95 % CI = 0.10, 0.49) were significantly less likely than Black women to be tested for HIV in the past year. Residing off campus (AOR = 1.88; 95 % CI = 1.18, 2.99) and engaging with two or more sex partners in the past year (AOR = 2.59; 95 % CI = 1.70, 3.95) significantly increased the likelihood of HIV testing in the past year. Students who engaged only with heterosexual partners (AOR = .25; 95 % CI = 0.09, 0.76) or were female and bisexual (AOR = 0.17; 95 % CI = 0.04, 0.69) were less likely to be tested for HIV in the past year compared to men who have sex with men/men and women. In a separate model, Black men (AOR = 1.87; 95 % CI = 1.18, 2.97) were significantly more likely than Black women to engage with two or more sex partners during the previous year. Compared to Black women, White women (AOR = 0.51; 95 % CI = 0.26, 0.98) were less likely to report two or more sex partners in the past year. Students involved in a relationship during the past 30 days (AOR = 0.33; 95 % CI = 0.22, 0.49) were less likely than other students to engage with two or more sex partners in the past year. CONCLUSIONS: Over half (58 %) of the students had been tested for HIV in the past year-a promising outcome. However, 42 % of sexually active students had not been tested. Campus prevention initiatives need to reinforce the importance of frequent HIV testing. In particular, targeted prevention efforts need to be focused on heterosexual Black male college students.

The intracerebroventricular injection of rimonabant inhibits systemic lipopolysaccharide-induced lung inflammation.

We investigated the role of intracerebroventricular (ICV) injection of rimonabant (500ng), a CB1 antagonist, on lipopolysaccharide ((LPS) 5mg/kg)-induced pulmonary inflammation in rats in an isolated perfused lung model. There were decreases in pulmonary capillary pressure (Ppc) and increases in the ((Wet-Dry)/Dry lung weight)/(Ppc) ratio in the ICV-vehicle/LPS group at 4h. There were decreases in TLR4 pathway markers, such as interleukin receptor-associated kinase-1, IκBα, Raf1 and phospho-SFK (Tyr416) at 30min and at 4h increases in IL-6, vascular cell adhesion molecule-1 and myeloperoxidase in lung homogenate. Intracerebroventricular rimonabant attenuated these LPS-induced responses, indicating that ICV rimonabant modulates LPS-initiated pulmonary inflammation.

Stigma as a Barrier to HIV-Related Activities Among African-American Churches in South Carolina.

South Carolina has one of the highest HIV/AIDS prevalence rates in the United States. More than 70% of those infected are African American. Traditionally, Black churches have been one of the primary sources of health outreach programs in Southern African-American communities. In this research, we explored the role of HIV-related stigma as a barrier to the acceptance of HIV-related activities in Black churches. A survey of African-American adults in South Carolina found that the overall level of stigma associated with HIV/AIDS was comparable to what has been found in a national probability sample of people in the United States. Consistent with the stigma-as-barrier hypothesis, the degree to which survey respondents endorsed HIV-related stigma was related to less positive attitudes concerning the involvement of Black churches in HIV-related activities.

Inhibition of GSK3α/ß promotes increased pulmonary endothelial permeability to albumin by reactive oxygen/nitrogen species.

Glycogen synthase kinase 3α/ß (GSK3α/ß) is a serine/threonine kinase that participates in numerous processes in many cell types. Importantly, the role of GSK3α/ß in homeostatic maintenance of the pulmonary endothelial cell barrier to protein is not known. We tested the hypothesis that GSK3α/ß regulates endothelial barrier function by measuring the permeability to albumin of a rat pulmonary microvessel endothelial cell monolayer (PMECM) treated with and without the selective GSK3α/ß inhibitor SB 216763 (1.0, 5.0 and 10 uM) for 1 h. The treatment with the inhibitor SB 216763 caused a dose dependent decrease in phospho-ß-catenin-Ser(33/37) levels indicating effective suppression of GSK3α/ß. SB216763 caused an increase in both permeability to albumin and DCFDA (6-Carboxy-2',7'-Dichlorodihydrofluorescein Diacetate, Di(Acetoxymethyl Ester)) oxidation that were prevented by co-treatment with the anti-oxidant tiron or the nitric oxide synthase inhibitor L-NAME (Nω-nitro-l-arginine-methyl ester). In separate studies PMECMs were treated with the Akt inhibitor triciribine (12.5 uM) for 1 h to unmask Akt dependent constitutive suppression of GSK3α/ß. Triciribine decreased phospho-GSK3α/ß-Ser(21)/9 (i.e., the product of Akt) which was associated with an increase in phospho-ß-catenin-Ser(33/37) (i.e., the product of GSK3α/ß) indicating constitutive activity of Akt for GSK3α/ß-Ser(21/9). The data indicates GSK3α/ß inhibition causes increased endothelial monolayer protein permeability which is mediated by reactive oxygen/nitrogen species.

The effect of partial outlet obstruction on calpain and phospholipase-2 activities: analyzed by severity and duration.

In an attempt to better understand the two pathways that lead to bladder decompensation following partial obstruction in rabbits one of which is caused by calcium-activated enzymes and the other by oxidative stress, calpain and phospholipase A2 (PLA2) biochemical assays were conducted to see how bladder decompensation is mediated by these two calcium-activated enzymes. Partial outlet obstructions of varying durations (4, 8, and 12 weeks plus controls) were performed on 32 New Zealand white rabbits. The rabbits were also grouped by severity: control, mild, intermediate, and severe. The activities of Calpain and PLA2 on the muscle tissue of the bladders were analyzed. A stronger correlation was seen between activities and severities as opposed to between activities and durations for both PLA2 and calpain. The activity for PLA2 increased dramatically from control to mild and then stayed constant for both intermediate and severe obstructions. Calpain activity increased steadily from control to mild to intermediate to severe. Based on the increase in levels of the calcium-dependent enzymes, it was clearly shown that calcium levels increased in all stages of bladder decompensation most notably with the mild obstructions. Based on previous studies in which nitrotyrosine and dinitrophenol levels did not increase in mildly obstructed rabbits, the calcium overload pathway may predominate in mild decompensation because cells in mildly obstructed bladders are better able to cope with oxidative stress than increased calcium levels.

Lipoteichoic acid from Staphylococcus aureus induces lung endothelial cell barrier dysfunction: role of reactive oxygen and nitrogen species.

Tunneled central venous catheters (TCVCs) are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. Staphylococcus aureus) biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA), a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2). The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from Staphylococcus aureus induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM) that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam((3))CSK((4)) induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS) activation (as measured by the p-eNOS(ser1177):p-eNOS(thr495) ratio). The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.

Tumor necrosis factor-α induces increased lung vascular permeability: a role for GSK3α/ß.

We tested the hypothesis that glycogen synthase kinase 3α/ß (GSK3α/ß) modulates tumor necrosis factor-a (TNF) induced increased lung vascular permeability. Rats were treated with TNF (i.v., ~100ng/ml) or vehicle 0.5h, 4.0h and 24.0h prior to lung isolation. Rats were co-treated with the GSK3α/ß inhibitors SB216763 (0.6mg/kg) or TDZD-8 (1.0mg/kg). After TNF, the isolated lung was assessed for hemodynamics, wet-dry/dry weight (W-D/D) and extravascular albumin. Extravascular albumin significantly increased at TNF-24h compared to Control. In the GSK3α/ß-inhibited+TNF groups, extravascular albumin was similar to the Control and respective SB216763 and TDZD-8 groups. In separate studies, to assess GSK3α/ß-activity, lung lysate was assessed for phospho-GSK3α/ß-Ser(21/9), total GSK3α/ß, un-phospho-ß-catenin-Ser(33/37) and total ß-catenin. In the TNF-4.0h group, there was no change in GSK3α/phospho-GSK3α-Ser(21) but there was an increase in GSK3ß/GSK3ß-Ser(9) compared to Control, indicating GSK3ß activation at TNF-4.0h. GSK3ß activation was verified because there was a decrease in un-phospho-ß-catenin-Ser(33/37)/ß-catenin in the TNF-4.0 group, a specific outcome for GSK3ß activation. In the SB216763+TNF group, un-phospho-ß-catenin-Ser(33/37) was similar to Control, indicating prevention of TNF-induced GSK3ß activation. In the TNF-24h group, there were increases in the biomarkers of inflammation phospho-eNOS-Ser (1117) and oxidized protein, which did not occur in the SB216763+TNF-24h and TDZD-8+TNF-24h groups. In the SB216763+TNF-24h and TDZD-8+TNF-24h groups, un-phospho-ß-catenin-Ser(33/37) was greater than in the Control, indicating continued inhibition of GSK3ß. The data indicates that pharmacologic inhibition of GSK3ß inhibits TNF induced increased endothelial permeability associated with lung inflammation.

TNF-induced activation of pulmonary microvessel endothelial cells: a role for GSK3beta.

The hypothesis tested was PKCalpha mediates the phosphorylation of glycogen synthetase kinase 3beta (GSK3beta) and that the GSK3beta inhibition modulates the response to tumor necrosis factor-alpha (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 h (100 ng/ml) or vehicle. First, to assess the role of PKCalpha in the phosphorylation of GSK3beta (i.e., an indicator of GSK3beta inhibition), PMEC were pretreated with 1) nonsense-RNA-PKCalpha, 2) siRNA-PKCalpha, and 3) the PKC inhibitor Gö6983. In the nonsense RNA-PKCalpha+TNF and TNF groups, there was increased phosphorylated GSK3beta-Ser9 that did not occur in the Gö6983+TNF group. In the TNF groups, there was a significant correlation between PKCalpha protein and phosphorylated GSK3beta-Ser9 that did not occur in the groups without TNF. Second, to assess the role of GSK3beta in beta-catenin activity, PMEC were pretreated with 1) wild-type (w) GSK3beta plasmid to enhance GSK3beta activity, 2) kinase dead (kd)-GSK3beta plasmid, and 3) the GSK3beta inhibitor SB-216763. In the TNF group, there was increased unphosphorylated beta-catenin-Ser37/33 compared with the control group. In the GSK3beta-inhibited groups (i.e., SB-216763 and kdGSK3beta) +/- TNF, the unphosphorylated beta-catenin-Ser37/33 was similar to the TNF group. In the GSK3beta-enhanced group +/- TNF, the unphosphorylated beta-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a beta-catenin-dependent promoter luciferase reporter, or the mutant construct FOPflash, 2 days before treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared with the control group. In the GSK3beta-inhibited groups (i.e., SB-216763 and kdGSK3beta) +/- TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the GSK3beta-enhanced group +/- TNF, the TOPflash/FOPflash activity ratio was similar to the control. The data indicate that TNF induces endothelial activation that is modulated by a PKCalpha-dependent inhibition of GSK3beta.

The effect of chronic partial bladder outlet obstruction on corpus cavernosum smooth muscle and Rho-kinase in rabbits.

AIM: The goal of this study was to investigate the effect of different severities in bladder dysfunction on corpus cavernosum physiology, morphology and expression of Rho-kinase in rabbits. METHODS: Male New Zealand rabbits were divided into control, 2 and 8 weeks of partial bladder outlet obstruction (PBOO) groups. Isolated cavernosal strips from all groups were precontracted with phenylephrine and the relaxant responses to electrical field stimulation (EFS), ATP, acetylcholine, and sodium nitroprusside (SNP) were determined. Histological and molecular studies were performed. RESULTS: Corpus cavernosum smooth muscle (CCSM) from 8 weeks obstruction rabbits showed significant decreases in the contractile response to phenylephrine and further decreased relaxation responses to EFS in comparison to 2 weeks group. Relaxation induced by ATP, acetylcholine, and SNP were all significantly diminished at both 2 and 8 weeks obstruction equally. The ratio of smooth muscle to collagen decreased at 2 weeks and further dropped at 8 weeks obstruction. Expression of both isoforms of Rho-kinase were increased in both obstruction groups at 2 weeks obstruction and decreased significantly (from the 2 week obstructed values) at 8 weeks while remaining above control values. CONCLUSION: The present study indicated that severe bladder dysfunction secondary to chronic PBOO induced significant physiological dysfunctions of CCSM as well as morphological changes. Activities of both ROK isoenzymes showed increases at 2- and 8-week obstructions. Increase in Rho-kinase expression/activity would be expected to make the CCSM more difficult to relax and also contribute to reduction of EFS-induced relaxation of CCSM after chronic PBOO.

Protein oxidation as a novel biomarker of bladder decompensation.

OBJECTIVE: To measure the degree to which partial bladder outlet obstruction (PBOO) results in oxidative bladder damage, which subcellular components of the bladder are affected and whether these changes correlate with bladder function. MATERIALS AND METHODS: In all, 32 rabbits were divided into four groups. Each group underwent PBOO for 1, 2, 4, and 8 weeks, respectively. Bladder tissue from each group was homogenized and separated into subcellular fractions via differential centrifugation. The carbonyl content within the subcellular fractions, including the nuclear, mitochondrial, and microsomal pellets, was then quantified by dot blot analysis. RESULTS: Total bladder oxidation increased with duration of obstruction across all subcellular fractions. The largest increase in total oxidation occurred between 4 and 8 weeks. Protein oxidation density in the nuclear and microsomal fractions both showed increases at 2 weeks obstruction, decreases at 4 weeks, and then large increases at 8 weeks. The increase in protein oxidation density between 4 and 8 weeks obstruction was most pronounced in the microsomal fraction. CONCLUSIONS: Overall bladder protein oxidation increased with the duration of obstruction and increased at a greater rate during the transition to decompensation. Furthermore, the subcellular fraction that exhibited the most oxidation was the microsomal pellet. The amount of protein oxidation correlated with the functional changes in the bladder.

The effect of low-dose estrogen therapy on ovariectomized female rabbit bladder.

OBJECTIVES: To determine whether low-dose estrogen supplementation is as effective as high-dose supplementation in increasing bladder contractile function and mediating bladder hypertrophy and angiogenesis. METHODS: Sixteen New Zealand white female rabbits were separated into four groups of 4 rabbits each. Group 1 served as the control, and groups 2 to 4 underwent ovariectomy. The group 2 rabbits were studied 7 days after ovariectomy. The rabbits in groups 3 and 4 were medicated with 17-beta estradiol at a dose of 0.1 mg/kg/day and 1.0 mg/kg/day, respectively, for 7 days. At the end of the experiment each rabbit was anesthetized and the bladder removed for contractile, morphologic, and biochemical studies. RESULTS: Low- and high-dose estrogen administration resulted in similarly significant increases in the contractile responses to field stimulation, adenosine triphosphate, and potassium chloride. Similarly, both doses of estrogen mediated significant hypertrophy of the smooth muscle and decrease in collagen, similar levels of angiogenesis, and similar increases of citrate synthase activity. CONCLUSIONS: Low-dose estrogen produces similar physiologic, morphologic, and biochemical effects on the bladder as have been shown for high-dose estrogen.

Changes in the smooth muscle of the corpora cavernosum related to reversal of partial bladder outlet obstruction in rabbits.

Previous studies have demonstrated that partial bladder outlet obstruction (PBOO) in the rabbit induces an increase in corpus cavernosum smooth muscle (CCSM) tone, which may make it difficult for the CCSM to relax. Thus, to determine whether the corpus cavernosum restores relaxation after reversal of PBOO, we investigated the physiologic, histologic, and cell biology in penises obtained from rabbits 4 weeks and 8 weeks after reversal of PBOO. CCSM from bladder outlet-obstructed and obstruction-reversed rabbits showed significant decreases in the contractile responses to phenylephrine. The relaxation responses to electrical field stimulation (EFS), ATP, acetylcholine, and sodium nitroprusside (SNP) were decreased in obstructed and reversed for 4 weeks groups. By 8 weeks of reversal, the relaxation of CCSM was increased gradually in response to EFS, SNP, and acetylcholine. However, the response to ATP did not result in the relaxation of CCSM to control levels. The ratio of SM to collagen decreased after obstruction and remained low after reversal. Expression of both isoforms of Rho kinase (ROK) was increased in obstruction groups. At 4 weeks of reversal, the expression of ROK alpha remained at obstruction level, whereas ROK beta expression decreased in comparison with the obstruction group. By 8 weeks of reversal, expression of both ROK alpha and beta significantly decreased when compared with the obstruction group. These results suggested that the poor relaxation response at reversal of 4 weeks was associated with incomplete decreased expression of both isoforms of ROK, whereas the incomplete recovery of the CCSM relaxation response at reversal of 8 weeks may be associated with structural alterations in the CC and irreversible damage from PBOO.

Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit.

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.

Tumor necrosis factor-alpha causes barrier dysfunction mediated by tyrosine198 and tyrosine218 in beta-actin.

We tested the hypothesis that tumor necrosis factor-alpha (TNF) induces barrier dysfunction of pulmonary microvessel endothelial monolayers (PMEM) mediated by specific tyrosine residues in beta-actin. PMEM were transfected with a wild-type, mutant [tyrosine(198) to phenylalanine(198) (Y198F)], mutant Y218F, or mutant Y306F beta-actin construct tagged with enhanced yellow fluorescent protein (EYFP-beta-actin). The cellular compartmentalization of wild-type and mutant EYFP-beta-actin was displayed using EYFP fluorescence of the tagged beta-actin. beta-Actin was quantified for the EYFP-tagged and native beta-actin using Western blot assay. The effect of the EYFP-beta-actin on a cell junction protein was assessed by association of EYFP-beta-actin with beta-catenin using confocal microscopy and coimmunoprecipitation. The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The cellular compartmentalization of wild-type and mutant EYFP-beta-actin was similar to the native beta-actin. Incubation of PMEM with TNF (100 ng/ml) for 0.5 h resulted in increases in permeability to albumin and a decrease in association of the EYFP-beta-actin with beta-catenin. However, the expression of the EYFP-Y198F beta-actin and EYFP-Y218F beta-actin prevented the effect of TNF on beta-catenin and barrier function. The vehicle, wild-type EYFP-beta-actin, and mutant Y306F beta-actin had no affect on the response to TNF. The data indicate that TNF induces an increase in endothelial permeability that is dependent on tyrosine(198) and tyrosine(218) in beta-actin.

L-NAME, a nitric oxide synthase inhibitor, diminishes oxidative damage in urinary bladder partial outlet obstruction.

Partial bladder outlet obstruction (PBOO) results in cellular damage due to ischemia and reperfusion injury. Our study seeks to establish how early this damage can occur and the role that nitric oxide may play in its pathophysiology. Surgical PBOO (1, 3, and 7 days) were performed on male New Zealand White rabbits. Half of the animals were premedicated for 3 days with N(G)-nitro-l-arginine methyl ester(l-NAME), an inhibitor of nitric oxide synthase before obstruction. Bladder weight increased with duration of PBOO but was significantly lower at 3 and 7 days in animals treated with l-NAME compared with their untreated counterparts. Contractile function decreased progressively with PBOO duration. At 1 day postobstruction, bladder contractility was significantly lower in the l-NAME rabbits than in the untreated rabbits. At 3 and 7 days, contractility of the l-NAME bladders was equal or higher than the untreated bladders. The level of hypoxia at 1 day after obstruction was significantly higher in the l-NAME-treated animals than in the untreated controls but equal at 3 and 7 days obstruction. Increased nitrotyrosine was seen by Western blot in all obstructed animals. However, the amount was significantly less in the l-NAME-treated animals at 3 and especially at 7 days. Nerve density decreased progressively after obstruction; however, it decreased to a significantly lesser degree in the l-NAME-treated bladders than in the untreated groups. These results suggest that l-NAME pretreatment enhanced ischemic damage at 1 day after obstruction but protected the bladder from nitric oxide-generated free radical damage at the later time periods by inhibiting the generation of nitrotyrosine.

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.

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder.

PURPOSE: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). MATERIALS AND METHODS: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. RESULTS: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. CONCLUSIONS: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction.

Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk.

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid that can be generated by pathological activities. We investigated the hypothesis that LPC signals increase in endothelial permeability. Stimulation of human dermal microvascular endothelial cells and bovine pulmonary microvascular endothelial cells with LPC (10-50 microM) induced decreases (within minutes) in transendothelial electrical resistance and increase of endothelial permeability. LPC activated (within 5 min) membrane-associated PKC phosphotransferase activity in the absence of translocation. Affinity-binding analysis indicated that LPC induced increases (also by 5 min) of GTP-bound RhoA, but not Rac1 or Cdc42. By 60 min, both signaling pathways decreased toward baseline. Inhibition of RhoA with C3 transferase inhibited approximately 50% of LPC-induced resistance decrease. Pretreatment with PKC inhibitor Gö-6983 (concentrations selective for classic PKC), PMA-induced depletion of PKCalpha, and transfection of antisense PKCalpha oligonucleotide each prevented 40-50% of the LPC-induced resistance decrease. Furthermore, these three PKC inhibition strategies inhibited 60-80% of the LPC-induced GTP-bound RhoA. These results show that LPC directly impairs the endothelial barrier function that was dependent, at least in part, on cross talk of PKCalpha and RhoA signals. The evidence indicates that elevated LPC levels can contribute to the activation of a proinflammatory endothelial phenotype.

TNF-alpha induces a decrease in eNOS promoter activity.

We determined whether TNF-alpha induces a decrease in activity of the promoter for the endothelial nitric oxide synthase (eNOS) gene in pulmonary microvessel endothelial monolayers (PMEM). eNOS promoter activity was assessed in PMEM transfected with plasmids coding the wild-type (F1: -1600 nt from transcription start site) and truncated (F2: -1189, F4: -779, F5: -494, F6: -166) human eNOS promoters linked to a luciferase reporter. PMEM lysates were analyzed for the luciferase/galactosidase ratio (Luc/Gal) after incubation with TNF-alpha (50 ng/ml) for 0.5 or 4 h. TNF-alpha caused a decrease in the Luc/Gal ratio in the PMEM transfected with wild-type F1 and truncated F2, F4, and F5 plasmids but not with truncated F6 plasmid. Truncated-promoter analysis indicated the response elements (-370)CACCC, (-231)GATA, and (-186)CACCC may regulate the effect of TNF-alpha on the eNOS promoter. DNA-binding activity of (32)P-labeled oligonucleotide probes that span the GATA-binding site ((-239)-[(-231)GATA]-(-219)) and the two different CACCC-binding regions ((-379)-[(-370)CACCC]-(-358) and (-196)-[(-186) CACCC]-(-176)) were assessed using EMSA. In response to TNF-alpha treatment for 4 h, nuclear protein binding to (32)P oligonucleotides was characterized as: 1) a significant increase in binding to (-370)CACCC, 2) a significant decrease in binding to (-231)GATA, and 3) no change in (-186)CACCC binding. EMSA supershift analysis indicated that the transcription factor protein GATA-4 bound to the (-231)GATA site, and Sp3 bound to the (-370)CACCC site. Our data indicate TNF causes a decrease in eNOS promoter activity that may be mediated by GATA-4 and Sp3.

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha.

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.