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Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Karpurapu, Nie, Chung, Yan, Dougherty, Pannu, Wisler, Harkless, Parinandi, Berdyshev, Pei and Christman.)

Background: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction., Methods: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease., Results: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease., Conclusions: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo., Competing Interests: Disclosures T.A. McKinsey is on the scientific advisory boards of Artemes Bio and Eikonizo Therapeutics, received funding from Italfarmaco for an unrelated project, and has a subcontract from Eikonizo Therapeutics for a small business innovation research grant from the National Institutes of Health (HL154959). The other authors report no conflicts.

Sustained high levels of activated polymorphonuclear leukocytes (PMNs) and PMN-derived proteases in the microenvironment of chronic venous leg ulcers (CVLUs) are linked to chronic inflammation and delayed healing. Uncontrolled PMN activity eventually destroys newly developed tissue and degrades critical growth factors. The bioactive components of fish oil (n-3 eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) have strong inflammation-resolving actions and have been shown to assuage PMN activity, but have not been tested in CVLU patients. This randomized controlled study compared the effectiveness of oral EPA + DHA therapy to a placebo for reducing PMN activation in CVLU microenvironments. At Days 0, 28, and 56, markers of PMNs (CD15) and activated PMNs (CD66b), and levels of PMN-derived proteases human neutrophil elastase and matrix metalloproteinase-8 were measured in CVLU fluid from patients receiving standard compression therapy and (1) EPA + DHA therapy (n = 16) or (2) placebo (n = 19). By Day 56, the EPA + DHA Group had a significantly lower percentage of CD66b+ cells in CVLU fluid compared to Day 0 (p = 0.02) and to Day 28 (p = 0.05). Importantly, there were downward trends in levels of both matrix metalloproteinase-8 and human neutrophil elastase over time in the EPA + DHA Group, which also demonstrated greater reductions in wound area by Day 28 (57% reduction) and Day 56 (76% reduction) than the Control Group (35% and 59%, respectively). Moreover, reductions in wound area had significant negative relationships with CD15+ cells in wound fluid at Days 28 (p = 0.008) and 56 (p < 0.001), and CD66b+ cells at Days 28 (p = 0.04) and 56 (p = 0.009). The collective findings provide supplemental evidence that high levels of activated PMNs in CVLU microenvironments inhibit healing, and suggest that EPA + DHA oral therapy may modulate PMN activity and facilitate healing of CVLUs when added to standard care regimens., (© 2017 by the Wound Healing Society.)

Key thioredoxin (Trx) system components are nicotinamide adenine dinucleotide phosphate (NADPH), Trx reductase (TrxR), and Trx. TrxR catalyzes disulfide reduction in Trx with NADPH as cofactor. Because Trx is an antioxidant, oxidative stress results in an increase in Trx, which has a reduced disulfide component. If Trx is suppressed, oxidative stress in higher. In contrast a decrease in oxidative stress is associated with low Trx levels. Trx is involved in inflammation, apoptosis, embryogenesis, and cardiovascular disease (CVD). This review focuses on the Trx system in CVD. Abnormal Trx binding occurs in mouse familial combined hyperlipidemia; however, this has not been confirmed in humans. Congestive heart failure is a manifestation of many CVDs, which may be improved by attenuating oxidative stress through the suppression of Trx and decreased reactive oxygen species. Angiotensin II is associated with hypertension and other CVDs, and its receptor blockade results in decreased oxidative stress with reduced Trx levels. Inflammation is a major causative factor of CVDs, and myocarditis as an example, is associated with increased Trx levels. Vascular endothelial dysfunction has an association with CVD. This dysfunction is alleviated by hormone replacement therapy, which involves decreased oxidative stress and Trx levels. Diabetes mellitus has a major association with CVDs; increase in Trx levels may reflect insulin resistance. Identification of Trx system abnormalities may lead to innovative approaches to treat multiple CVDs and other pathologies.

Cutaneous leishmaniasis (CL) manifests as localized skin lesions, which lead to significant tissue destruction and disfigurement. In the Yucatan Peninsula, Mayan traditional healers use Pentalinon andrieuxii Muell.-Arg. (Apocynaceae) roots for the topical treatment of CL. Here, we studied the effect of P. andrieuxii root hexane extract (PARE) on the parasites and host cells in vitro and examined its efficacy in the topical treatment of CL caused by Leishmania mexicana. PARE exhibited potent antiparasitic activity in vitro against promastigotes as well as amastigotes residing in macrophages. Electron microscopy of PARE-treated parasites revealed direct membrane damage. PARE also activated nuclear factor kappaB and enhanced interferon-γ receptor and MHC class II expression and TNF-α production in macrophages. In addition, PARE induced production of the Th1 promoting cytokine IL-12 in dendritic cells as well as enhanced expression of the co-stimulatory molecules CD40, CD80, and CD86. In vivo studies showed that L. mexicana-infected mice treated by topical application of PARE resulted in the significant reduction in lesion size and parasite burden compared to controls. These findings indicate that PARE could be used as an alternative therapy for the topical treatment of CL., (Copyright © 2013 John Wiley & Sons, Ltd.)

Study Objectives: The effects of intermittent hypoxia (IH) on pancreatic function in the presence of diabetes and the underlying mechanisms are unclear. We hypothesized that IH would exacerbate pancreatic β-cell dysfunction and alter the fatty acids in the male Tallyho/JngJ (TH) mouse, a rodent model of type 2 diabetes., Design: TH mice were exposed for 14 d to either 8 h of IH or intermittent air (IA), followed by an intraperitoneal glucose tolerance test (IPGTT) and tissue harvest. The effect of IH on insulin release was determined by using a β3-adrenergic receptor (AR) agonist., Measurements and Results: During IH, pancreatic tissue pO2 decreased from 20.4 ± 0.9 to 5.7 ± 2.6 mm Hg, as determined by electron paramagnetic resonance oximetry. TH mice exposed to IH exhibited higher plasma glucose levels during the IPGTT (P < 0.001) while the insulin levels tended to be lower (P = 0.06). Pancreatic islets of the IH group showed an enhancement of the caspase-3 staining (P = 0.002). IH impaired the β-AR agonist-mediated insulin release (P < 0.001). IH increased the levels of the total free fatty acids and saturated fatty acids (palmitic and stearic acids), and decreased levels of the monounsaturated fatty acids in the pancreas and plasma. Ex vivo exposure of pancreatic islets to palmitic acid suppressed insulin secretion and decreased islet cell viability., Conclusions: Intermittent hypoxia increases pancreatic apoptosis and exacerbates dysfunction in a polygenic rodent model of diabetes. An increase in free fatty acids and a shift in composition towards long chain saturated fatty acid species appear to mediate these effects.

Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-κB dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cholesterol secoaldehyde (ChSeco), a putative product of the reaction of ozone with cholesterol in aqueous environments, has been shown to induce apoptosis in H9c2 cardiomyoblasts. This study further investigated the involvement of apoptotic-related proteins and gene expression using RT-PCR, Western blot, and appropriate biochemical assays. The RT-PCR analysis revealed that ChSeco activates the expression of genes involved in the death receptor (extrinsic) pathway. The significance of this pathway was also evident from the increased activity of caspase-8. The overexpression of Apaf-1, loss of mitochondrial transmembrane potential, release of cytochrome c, and increased activity of caspase-9 provide further evidence for the involvement of a mitochondrial (intrinsic) pathway. Time-course analysis of ChSeco-exposed H9c2 cells showed an upstream increase in the generation of reactive oxygen species (ROS) and an associated decrease in the intracellular glutathione. N-acetyl-L-cysteine and Trolox significantly attenuated the ChSeco-induced ROS formation and cytotoxicity and also down-regulated the expression of the genes of all the players in either pathway. This study clearly shows that ChSeco induces apoptosis in H9c2 cells through ROS generation and the activation of both the intrinsic and the extrinsic pathway.

Study Objectives: There are few data on the effects of continuous positive airway pressure (CPAP) therapy on the structural and functional characteristics of the right heart in patients with obstructive sleep apnea (OSA). We sought to leverage the advantages of cardiac magnetic resonance imaging (CMR) and hypothesized that CPAP treatment would improve right ventricular (RV) function in a group of patients with OSA who were free of other comorbid conditions., Methods: Patients with severe (apnea-hypopnea index > or = 30/h) untreated OSAwere prospectively enrolled. CMR included 3-dimensional measurement of biventricular size and function, and rest/stress myocardial perfusion and was performed at baseline and after 3 months of CPAP therapy., Results: Fifteen patients with mild to moderate desaturation were enrolled; 2 could not undergo CMR due to claustrophobia and obesity. There were significant decreases in the Epworth Sleepiness Scale score (p < 0.0001) and RV end-systolic and RV end-diastolic volumes (p < 0.05) with CPAP. There was a trend toward improvement in RV ejection fraction, but the improvement did not reach statistical significance. Other measures such as left ventricular volumes, left ventricular ejection fraction, myocardial perfusion reserve index, and thickness of the interventricular septum and ventricular free wall did not change significantly., Conclusions: This preliminary study found that CPAP treatment decreases RV volumes in patients with severe OSA who are otherwise healthy. CMR offers a novel technique to determine the effects of CPAP on ventricular structure and function in patients with OSA. A randomized controlled study is needed to confirm the results of our study.

Obstructive sleep apnea (OSA), characterized by cyclic intermittent hypoxia (IH) during sleep, is an independent risk factor for cardiovascular disease. Adiponectin (APN), an adipocytokine secreted exclusively by adipocytes, possesses antiatherogenic properties. Low levels of APN, particularly the high-molecular-weight (HMW) form, are associated with an increased risk of cardiovascular disease. Here, we hypothesized that IH would result in the dysregulation of APN expression and secretion. 3T3-L1 adipocytes were exposed to IH at 12 cycles/h for 6 h/d to simulate the IH condition similar to that encountered in OSA. Control adipocytes were exposed to 21% O(2) under identical conditions. After 48 h of incubation, IH caused a decrease in the secretion of total and HMW APN in spite of a significant upregulation of APN mRNA expression by adipocytes. This study suggested a novel mechanism of how the cyclic hypoxemia in OSA predisposes OSA patients to cardiovascular disease through the dysregulation of secretion of APN by adipocytes. Further studies are needed to determine the exact molecular mechanism how IH reduces the release of APN by adipocytes.

Mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. This study identified the critical temperature, resulting in a loss of contractile function in isolated diaphragm (thermal tolerance), and then tested the hypotheses 1) that increased reactive oxygen species (ROS) production contributes to the loss of contractile function at this temperature, and 2) eicosanoid metabolism plays an important role in preservation of contractile function in hyperthermia. Contractile function and passive force were measured in rat diaphragm bundles during and after 30 min of exposure to 40, 41, 42 or 43 degrees C. Between 40 and 42 degrees C, there were no effects of hyperthermia, but at 43 degrees C, a significant loss of active force and an increase in passive force were observed. Inhibition of ROS with the antioxidants, Tiron or Trolox, did not inhibit the loss of contractile force at 43 degrees C. Furthermore, treatment with dithiothreitol, a thiol (-SH) reducing agent, did not reverse the effects of hyperthermia. A variety of global lipoxygenase (LOX) inhibitors further depressed force during 43 degrees C and caused a significant loss of thermal tolerance at 42 degrees C. Cyclooxygenase (COX) inhibitors also caused a loss of thermal tolerance at 42 degrees C. Blockage of phospholipase with phospholipase A(2) inhibitors, bromoenol lactone or arachidonyltrifluoromethyl ketone failed to significantly prevent the loss of force at 43 degrees C. Overall, these data suggest that ROS do not play an apparent role in the loss of contractile function during severe hyperthermia in diaphragm. However, functional LOX and COX enzyme activities appear to be necessary for maintaining normal force production in hyperthermia.

Nitric oxide synthase is inhibited by NG-methylated derivatives of arginine whose cellular levels are controlled by dimethylarginine dimethylamino-hydrolase (DDAH). DDAH-1 is a Zn(II)-containing enzyme that through hydrolysis of methylated l-arginines regulates the activity of NOS. Herein, we report the kinetic properties of hDDAH-1 and its redox-dependent regulation. Kinetic studies using recombinant enzyme demonstrated Km values of 68.7 and 53.6 microM and Vmax values of 356 and 154 nmols/mg/min for ADMA and L-NMMA, respectively. This enzymatic activity was selective for free ADMA and L-NMMA and was incapable of hydrolyzing peptide incorporated methylarginines. Subsequent studies performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching >or=100 microM were needed to confer significant inhibition of DDAH activity. However, exposure of DDAH to the lipid oxidation product, 4-HNE, dose-dependently inhibited DDAH activity with 15% inhibition observed at 10 microM, 50% inhibition at 50 microM, and complete inhibition at 500 microM. Mass spectrometry analysis demonstrated that the mechanism of inhibition resulted from the formation of Michael adducts on His 173, which lies within the active site catalytic triad of hDDAH-1. These studies were performed with pathophysiologicaly relevant concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Because DDAH is the primary enzyme involved in methylarginine metabolism, the loss of activity of this enzyme would result in impaired NOS activity and reduced NO bioavailability.

Aerobic life has evolved a dependence on molecular oxygen for its mere survival. Mitochondrial oxidative phosphorylation absolutely requires oxygen to generate the currency of energy in aerobes. The physiologic homeostasis of these organisms is strictly maintained by optimal cellular and tissue-oxygenation status through complex oxygen-sensing mechanisms, signaling cascades, and transport processes. In the event of fluctuating oxygen levels leading to either an increase (hyperoxia) or decrease (hypoxia) in cellular oxygen, the organism faces a crisis involving depletion of energy reserves, altered cell-signaling cascades, oxidative reactions/events, and cell death or tissue damage. Molecular oxygen is activated by both nonenzymatic and enzymatic mechanisms into highly reactive oxygen species (ROS). Aerobes have evolved effective antioxidant defenses to counteract the reactivity of ROS. Although the ROS are also required for many normal physiologic functions of the aerobes, overwhelming production of ROS coupled with their insufficient scavenging by endogenous antioxidants will lead to detrimental oxidative stress. Needless to say, molecular oxygen is at the center of oxygenation, oxidative phosphorylation, and oxidative stress. This review focuses on the biology and pathophysiology of oxygen, with an emphasis on transport, sensing, and activation of oxygen, oxidative phosphorylation, oxygenation, oxidative stress, and oxygen therapy.

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS(-/-)) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS(-/-) compared with wild-type (WT) mice. In contrast, contractions to the alpha1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS(-/-) and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS(-/-) compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS(-/-) compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS(-/-) compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS(-/-) aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS(-/-) compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS(-/-) mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.

Both in vivo models of ischemia/reperfusion and in vitro models of hypoxia (H)/reoxygenation (R) have demonstrated the crucial role of the Rac1-regulated NADPH oxidase in the production of injurious reactive oxygen species (ROS) by vascular endothelial cells (ECs). Since membrane lipid peroxidation has been established as one of the mechanisms leading to cell death, we examined lipid peroxidation in H/R-exposed cultured human umbilical vein ECs (HUVECs) and the role of Rac1 in this process. H (24 h at 1% O2)/R (5 min) caused an increase in intracellular ROS production compared to a normoxic control, as measured by dichlorofluorescin fluorescence. Nutrient deprivation (ND; 24 h), a component of H, was sufficient to induce a similar increase in ROS under normoxia. Either H(24 h)/R (2 h) or ND (24 h) induced increases in lipid peroxidation of similar magnitude as measured by flow cytometry of diphenyl-1-pyrenylphosphine-loaded HUVECs and Western blotting analysis of 4-hydroxy-2-nonenal-modified proteins in cell lysates. In cells infected with a control adenovirus, H (24 h)/R (2 h) and ND (24 h) resulted in increases in NADPH-dependent superoxide production by 5- and 9-fold, respectively, as measured by lucigenin chemiluminescence. Infection of HUVECs with an adenovirus that encodes the dominant-negative allele of Rac1 (Rac1N17) abolished these increases. Rac1N17 expression also suppressed the H/R- and ND-induced increases in lipid peroxidation. In conclusion, ROS generated via the Rac1-dependent pathway are major contributors to the H/R-induced lipid peroxidation in HUVECs, and ND is able to induce Rac1-dependent ROS production and lipid peroxidation of at least the same magnitude as H/R.

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).

The precise nature of band 3 protein and its involvement in oxalate exchange in the red blood cells (RBCs) of renal failure patients has not been studied in detail. Therefore, here we studied the oxalate exchange and binding by band 3 protein in RBCs of humans with conditions of acute and chronic renal failure (ARF and CRF). The RBCs of ARF and CRF patients exhibited abnormal red cell morphology and an increased resistance to osmotic hemolysis. Further, an increase in the cholesterol content and decrease in the activities of Na(+)-K(+)-, Ca(2+)-, and Mg(2+)-ATPases of membranes were observed in the RBCs of ARF and CRF patients. A decrease in the oxalate flux was observed in the RBCs of ARF and CRF patients. The oxalate-binding activities of the RBC membranes were significantly lower in ARF (20 pmoles/mg protein) and CRF (5.3 pmoles/mg protein) patients as compared to that in the normal subjects (36 pmoles/mg protein). DEAE-cellulose and Sephadex G-200 column chromatography purification profiles revealed a distinctive shift in oxalate-binding activity of band 3 protein of RBCs of ARF and CRF patients as compared to that of the normal subjects. It was also observed from the binding studies with a fluorescent dye, eosin-5-maleimide, which specifically binds to band 3 protein, that the RBCs of ARF and CRF patients exhibited only 53 and 32% of abundance of band 3 protein, respectively, as compared to that in the RBCs of the normal subjects, thus revealing a decrease in the band 3 protein content in ARF and CRF patients. These results for the first time showed a decrease in the oxalate exchange in RBCs of patients with ARF and CRF, which was also concomitant with the low levels of abundance of band 3 protein.

We investigated the effect of hyperoxia on phospholipase D (PLD) activation in bovine lung microvascular endothelial cells (BLMVECs). Generation of intracellular reactive oxygen species in BLMVECs exposed to hyperoxia for 2 or 24 h was three-fold higher compared with normoxic cells as measured by dichlorodihydrofluorescein di(acetoxymethyl ester) fluorescence. Exposure of BLMVECs to hyperoxia for 2 or 24 h attenuated 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated PLD activation compared with normoxic cells, however, hyperoxia did not alter basal PLD activity. Antioxidants, such as propyl gallate and pyrrolidine dithiocarbamate, reversed the effect of hyperoxia on TPA-induced PLD activity. Furthermore, the TPA-induced PLD activation was inhibited not only by the protein kinase C inhibitor, Go6976, but also by the tyrosine kinase inhibitor, genistein, and by the Src kinase specific inhibitor, PP-2, suggesting the involvement of protein kinase C and also tyrosine kinases in TPA-induced PLD activation. Western blot analysis of cell lysates from the hyperoxic (2 or 24 h) BLMVECs stimulated with TPA with anti-phosphotyrosine antibody showed an attenuation in overall tyrosine phosphorylation of proteins. In conclusion, we have demonstrated that hyperoxia enhanced the generation of reactive oxygen species in lung microvascular endothelial cells and attenuated TPA-induced protein tyrosine phosphorylation and PLD activation. As protein tyrosine phosphorylation and PLD play important roles in inflammatory responses, this could provide a mechanism for the regulation of endothelial barrier function during hyperoxic lung injury.

Phospholipase D (PLD), a phospholipid phosphohydrolase, catalyzes the hydrolysis of phosphatidylcholine and other membrane phospholipids to phosphatidic acid (PA) and choline. PLD, ubiquitous in mammals, is a critical enzyme in intracellular signal transduction. PA generated by agonist- or reactive oxygen species (ROS)-mediated activation of the PLDI and PLD2 isoforms can be subsequently converted to lysoPA (LPA) or diacylglycerol (DAG) by phospholipase A1/A2 or lipid phosphate phosphatases. In pulmonary epithelial and vascular endothelial cells, a wide variety of agonists stimulate PLD and involve Src kinases, p-38 mitogen activated protein kinase, calcium and small G proteins. PA derived from the PLD pathway has second-messenger functions. In endothelial cells, PA regulates NAD[P]H oxidase activity and barrier function. In airway epithelial cells, sphingosine-1-phosphate and PA-induced IL-8 secretion and ERKI/2 phosphorylation is regulated by PA. PA can be metabolized to LPA and DAG, which function as first- and second-messengers, respectively. Signaling enzymes such as Raf 1, protein kinase Czeta and type I phosphatidylinositol-4-phosphate 5-kinase are also regulated by PA in mammalian cells. Thus, PA and its metabolic products play a central role in modulating endothelial and epithelial cell functions.

We have shown earlier that oxidant-induced activation of phospholipase D (PLD) in vascular endothelial cells (ECs) is regulated by protein tyrosine kinases. To further understand the regulation of oxidant-induced PLD activation, we investigated the role of Src kinase. Treatment of bovine pulmonary artery ECs (BPAECs) with a model oxidant, diperoxovanadate (DPV), at 5 microM concentration, for 30 min, stimulated PLD activity (four- to eightfold), which was attenuated by tyrosine kinase inhibitors and by Src kinase-specific inhibitors PP-1 and PP-2, in a dose- and time-dependent fashion. Furthermore, BPAECs exposed to DPV (5 microM) for 2 min showed activation of Src kinase as observed by increased tyrosine phosphorylation and autophosphorylation in Src immunoprecipitates, which was attenuated by PP-2. Src immunoprecipitates of cell lysates from control BPAECs exhibited PLD activity in cell-free preparations, which was Arf- and Rho-sensitive and was enhanced at 2 min of DPV (5 microM) treatment. Also, Western blots of Src immunoprecipitates of control cells revealed the presence of PLD(1) and PLD(2), suggesting the association of PLD with Src kinase under basal conditions. However, exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) but not PLD(1) with Src. Western blotting of immunoprecipitates of PLD(1) and PLD(2) isoforms of control BPAECs revealed the presence of Src under basal conditions and exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) with Src in PLD(2) immunoprecipitates. Transient expression of a dominant negative mutant of Src in BPAECs attenuated DPV- but not TPA-induced PLD activation. In cell-free preparations, Src did not phosphorylate either PLD(1) or PLD(2) compared to protein kinase Calpha or p38 mitogen-activated protein kinase. These data show for the first time a direct association of Src with PLD in ECs and regulation of PLD in intact cells., ((c)2001 Elsevier Science.)

We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.

Reactive oxygen species (ROS) are implicated in the pathophysiology of several vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing evidence for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H(2)O(2)) enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular proteins, which were determined by immunoprecipitation with specific antibodies focal adhesion kinase (p125(FAK)) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H(2)O(2) (1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H(2)O(2)-induced tyrosine phosphorylation of FAK, and the microfilament disruptor cytochalasin D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAECs with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM attenuated H(2)O(2)-induced increases in intracellular Ca(2+) but did not show any consistent effect on H(2)O(2)-induced tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H(2)O(2)-induction of mitogen-activated protein kinase activity. We conclude that H(2)O(2)-induced increases in FAK tyrosine phosphorylation may be important in H(2)O(2)-mediated endothelial cell activation.

We have investigated oxidant-mediated stimulation of phospholipase D (PLD) activity in bovine pulmonary artery endothelial cells (BPAEC), prelabeled with [32P]orthophosphate or [32P]lysophospholipids. Treatment of cells incubated in Hanks' balanced salt solution (HBSS) containing 0.5% ethanol with hydrogen peroxide (H2O2) or linoleic acid hydroperoxide (18:2-OOH) enhanced the formation of 32P-labeled phosphatidylethanol (PEt) and phosphatidic acid (PA) in a dose- and time-dependent manner, indicating the activation of PLD. The H2O2- and 18:2-OOH-mediated PLD activation was not associated with cytotoxicity as determined by [3H]deoxyglucose release. The addition of ferrous chloride (50 microM) augmented H2O2-induced formation of [32P]PEt and [32P]PA about 2-fold, whereas the addition of the iron chelator desferoxamine blocked the potentiating effect of ferrous chloride. Replacement of the HBSS medium with Medium 199 containing 20% calf serum also potentiated the effect of H2O2-induced PLD activation. In addition to phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were readily hydrolyzed by PLD in response to H2O2 and 18:2-OOH treatment. The substrate specificity for oxidant-stimulated PLD activity differed from that observed in the presence of bradykinin or exhibited by agonist stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) where PC was the major phospholipid hydrolyzed by PLD. The formation of PEt in the presence of H2O2 and 18:2-OOH was not abolished by chelation of either extracellular Ca2+ with EGTA (5 mM) or intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) (25 microM, 30 min). Furthermore, pretreatment of BPAEC with the protein kinase C (PKC) inhibitor staurosporine and down-regulation of PKC by chronic TPA treatment (100 nM, 18 hr) had no effect on H2O2-induced PLD activation, suggesting that PLD activation by H2O2 is independent of PKC activity. It is possible that H2O2- and 18:2-OOH-induced activation of PLD represents an important mechanism to produce PA and diacylglycerol in endothelial cells.

Rat myocardial membranes exposed to the free radical-generating systems, Fe2+/ascorbate, Cu2+/t-butylhydro-peroxide, linoleic acid hydroperoxide, and soybean lipoxygenase (Type I) undergo lipid peroxidation. This is evidenced by the accumulation of thiobarbituric acid-reactive substances and the loss of both extractable phospholipids and their polyunsaturated acyl groups. Lipid peroxidation is accompanied by alterations of membrane proteins including the general loss of polypeptides and accumulation of high-molecular weight material. The most sensitive protein is a polypeptide with a molecular weight of 28 kDa. At low levels of oxidation, this protein moves incrementally to slightly higher apparent molecular weight. At higher oxidant levels or longer periods of oxidation, the protein disappears completely from the SDS-PAGE gel. The "28K reaction" occurs prior to the massive, oxidant-induced lipid alterations and may thus indicate specific adduct formation between this protein and certain peroxidized membrane phospholipids.

Alloxan-diabetic rats and age-matched controls were killed after 6 weeks of diabetes; heart and kidneys were removed and assayed for thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides, lipid phosphorus, total fatty acid composition and glutathione. Tissue homogenates from a second group of diabetic and control rats were incubated in oxygen-saturated buffer with and without the free radical generating system Fe2+/ascorbate (0.1/1.0 mM) and were assayed for lipid peroxidation. Diabetic hearts contained markedly lower levels of TBARS and lipid hydroperoxides (40% and 18%, respectively) than control hearts, whereas differences in TBARS were less pronounced in kidneys (9%). Incubation of homogenates of both organs in the presence or absence of Fe2+/ascorbate for up to 2 h yielded significantly lower levels of TBARS and lipid hydroperoxides with diabetic tissue. Diabetic hearts and kidneys contained higher levels of glutathione (28% and 13% over controls) and both diabetic tissues showed much higher linoleate/arachidonate ratios than did the controls (9.86 vs. 2.56 for heart, 2.01 vs. 0.86 for kidney). We conclude that diabetic tissues develop enhanced defense systems against oxidative stress and we assume tha the lower levels of arachidonate contribute to their resistance to lipid peroxidation as well.

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.

Commercial preparations of bovine cardiolipin (diphosphatidylglycerol) in chloroform solution contain substantial amounts of oxidation products. These oxidized derivatives, characterized by the presence of varying amounts of hydroperoxides and conjugated dienes, can be separated from unoxidized cardiolipin by normal phase high-performance liquid chromatography (HPLC) using UV detection. When purified cardiolipin is subjected to autoxidation in aqueous media, oxidation products of similar HPLC properties are produced. Storage of cardiolipin in chloroform induces both autoxidation and hydrolysis whereas storage in ethanol and other solvents does not. It is recommended not to use chloroform for the long-term storage of cardiolipin.

A long-chain N-acylethanolamine (N-oleoyl-2-aminoethanol) is shown to inhibit the production of thiobarbituric acid-reactive substances in rat heart mitochondria treated with Fe2+ or Fe3+/ADP. The inhibition is concentration-dependent in the range 50-150 microM of the agent and can be nearly complete depending on the type and amount of the free radical-generating system. Structural analogues of N-acylethanolamine are inhibitory as well, but neither oleic acid nor ethanol-amine has measurable effects. N-Oleoyl-2-aminoethanol affects peroxidation of linoleic acid micelles only minimally and has no effect on deoxyribose peroxidation.