Patricia K. Tithof

Polycyclic aromatic hydrocarbons present in cigarette smoke cause endothelial cell apoptosis by a phospholipase A2-dependent mechanism.

Smoking is a major risk factor for endothelial cell injury and subsequent coronary artery disease. Epidemiological studies implicate the phospholipase A2/arachidonic acid cascade in the mechanism by which smoking causes heart disease. However, specific components of cigarette smoke that activate this pathway have not been identified. The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons contained in cigarette smoke on phospholipase A2 (PLA2) activity and apoptosis of human coronary artery endothelial cells. 1methylanthracene (1‐MA), phenanthrene (PA), and benzo(a)pyrene (B(a)P) caused significant release of 3H‐arachidonate from endothelial cells. 1‐MA and PA, but not B(a)P, also caused significant release of 3H‐linoleic acid. Release of fatty acids from membrane phospholipids preceded the onset of apoptosis. 3H‐arachidonate release and apoptosis induced by 1‐MA, B(a)P, and PA were inhibited by methylarachidonoyl‐fluorophosphonate, an inhibitor of Groups IV and VI PLA2s. Bromoenol lactone, an inhibitor of Group VI enzymes, inhibited both 3H‐arachidonate release and apoptosis induced by 1‐MA and PA, but not B(a)P. MJ33, an inhibitor of the acidic calcium‐independent PLA2, attenuated 3H‐arachidonate release and apoptosis by PA, but not 1MA or B(a)P. The presence of Groups IV and VI and the acidic iPLA2 in endothelial cells was demonstrated by reverse transcriptase‐polymerase chain reaction and Western analysis. These data suggest that 1‐MA, B(a)P and PA induce apoptosis of endothelial cells by a mechanism that involves activation of these three distinct isoforms of PLA2.

Phospholipase A2 is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls

Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs), activates neutrophils to produce superoxide anion (O2-) by a mechanism that involves phospholipase C-dependent hydrolysis of membrane phosphoinositides; however, subsequent signal transduction mechanisms are unknown. We undertook this study to determine whether phospholipase A2-dependent release of arachidonic acid is involved in PCB-induced O2- production. We measured O2- production in vitro in glycogen-elicited, rat neutrophils in the presence and absence of the inhibitors of phospholipase A2: quinacrine, 4-bromophenacyl bromide (BPB), and manoalide. All three agents significantly decreased the amount of O2- detected during stimulation of neutrophils with Aroclor 1242. Similar inhibition occurred when neutrophils were activated with the classical stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate. The effects of BPB and manoalide were not a result of cytotoxicity or other nonspecific effects, although data suggest that quinacrine is an O2- scavenger. Significant release of 3H-arachidonic acid preceded O2- production in neutrophils stimulated with Aroclor 1242 or fMLP. Manoalide, at a concentration that abolished O2- production, also inhibited the release of 3H-arachidonate. Aspirin, zileuton, or WEB 2086 did not affect Aroclor 1242-induced O2- production, suggesting that eicosanoids and platelet-activating factor are not needed for neutrophil activation by PCBs. Activation of phospholipase A2 and O2- production do not appear to involve the Ah receptor because a congener with low affinity, but not one with high affinity for this receptor, stimulated the release of arachidonic acid and O2-. These data suggest that Aroclor 1242 stimulates neutrophils to produce O2- by a mechanism that involves phospholipase A2-dependent release of arachidonic acid. ImagesFigure 1. AFigure 1. BFigure 2.Figure 3.Figure 4. AFigure 4. BFigure 4. CFigure 5. AFigure 5. BFigure 5. CFigure 6. AFigure 6. BFigure 6. CFigure 6. D

Mediators of anaphylaxis but not activated neutrophils augment cholinergic responses of equine small airways

Neutrophilic inflammation in small airways (SA) and bronchospasm mediated via muscarinic receptors are features of chronic obstructive pulmonary disease in horses (COPD). Histamine, serotonin, and leukotrienes (LTs) are reported to be involved in the exacerbation of COPD, and currently, histamine has been shown to increase tension response to electrical field simulation (EFS) in equine SA. We tested the effects of these mediators and the effects of activated neutrophils on the cholinergic responses in SA. Histamine, serotonin, and LTD4 had a synergistic effect on EFS responses and only an additive effect on the tension response to exogenous ACh or methacholine. Atropine and TTX entirely eliminated the EFS-induced tension response in the presence of all three inflammatory mediators, indicating that augmentation of the EFS response applies only to the endogenous cholinergic response. Neutrophils isolated from control and COPD-affected horses were activated by zymosan, producing 18.1 +/- 2.3 and 25.0 +/- 2.3 nmol superoxide. 10(6) cells-1. 30 min-1, respectively. However, in contrast to the profound effect of mediators, incubation of SA for over 1 h in a suspension of up to 30 x 10(6) zymosan-treated neutrophils/ml did not significantly affect EFS responses of SA isolated from either control or COPD-affected horses. We conclude that in equine SA 1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not affect cholinergic responses in SA; and 3) in acute bouts of equine COPD, histamine, LTD4, and serotonin (mediators primarily associated with type I allergic reaction) rather than mediators derived from neutrophils most likely contribute to increased cholinergic airway tone.Neutrophilic inflammation in small airways (SA) and bronchospasm mediated via muscarinic receptors are features of chronic obstructive pulmonary disease in horses (COPD). Histamine, serotonin, and leukotrienes (LTs) are reported to be involved in the exacerbation of COPD, and currently, histamine has been shown to increase tension response to electrical field simulation (EFS) in equine SA. We tested the effects of these mediators and the effects of activated neutrophils on the cholinergic responses in SA. Histamine, serotonin, and LTD4 had a synergistic effect on EFS responses and only an additive effect on the tension response to exogenous ACh or methacholine. Atropine and TTX entirely eliminated the EFS-induced tension response in the presence of all three inflammatory mediators, indicating that augmentation of the EFS response applies only to the endogenous cholinergic response. Neutrophils isolated from control and COPD-affected horses were activated by zymosan, producing 18.1 ± 2.3 and 25.0 ± 2.3 nmol superoxide ⋅ 106cells-1 ⋅ 30 min-1, respectively. However, in contrast to the profound effect of mediators, incubation of SA for over 1 h in a suspension of up to 30 × 106 zymosan-treated neutrophils/ml did not significantly affect EFS responses of SA isolated from either control or COPD-affected horses. We conclude that in equine SA 1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not affect cholinergic responses in SA; and 3) in acute bouts of equine COPD, histamine, LTD4, and serotonin (mediators primarily associated with type I allergic reaction) rather than mediators derived from neutrophils most likely contribute to increased cholinergic airway tone.

Distinct phospholipase A2 enzymes regulate prostaglandin E2 and F2alpha production by bovine endometrial epithelial cells

BackgroundThe rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.MethodsBovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.ResultsConstitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.ConclusionResults from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.

Dirofilaria immitis: Do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta?

Endothelial cells modulate the function of their underlying smooth muscle. Thus, altered endothelial behavior could be important in the pathogenesis of vascular and lymphatic diseases, including human and animal filariasis. Endothelium-dependent relaxation is depressed in both in vivo canine femoral artery of dogs infected with Dirofilaria immitis and in vitro rat aorta exposed to adult D. immitis. The experiments reported here were designed to determine if filarial cyclooxygenase products could depress endothelium-dependent relaxation in vitro. Pretreatment of the parasites, but not the vascular ring, with either indomethacin or aspirin, prevented filarial-induced depression of relaxation. Analysis of heartworm-conditioned medium by gas chromatography--mass spectrometry revealed two peaks in the biologically active medium that were not present in the control. One peak had a retention time and chromatographic profile characteristic of derivatized PGD2 standard, and the other was not identified. Incubation of the vascular ring with PGD2 mimicked filarial-induced depression of endothelium-dependent relaxation at low, but not high, concentrations of acetylcholine. Thus, filarial PGD2 may be involved in altered endothelium-dependent relaxation seen in heartworm-infected dogs.

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production

BackgroundProstaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production.MethodsCells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates.ResultsBEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity.ConclusionResults from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production.

Depression of endothelium-dependent relaxation in aorta from rats with Brugia pahangi lymphatic filariasis.

A role for altered endothelial cell function is emerging in the pathogenesis of disease. We have previously demonstrated that Dirofilaria immitis, the canine heartworm, depresses endothelium-dependent responses and alters the mechanism of relaxation in the in vivo femoral artery of infected dogs. Exposure of rat aorta to the parasite or parasite-conditioned medium selectively depresses endothelium-dependent relaxation. D. immitis is closely related to the major human filarial pathogens. This study was designed to examine the effect of chronic infection with the filarial nematode Brugia pahangi on endothelium-mediated responses of the rat aorta in vitro. We tested the hypothesis that endothelium-dependent responses are depressed in the aorta from rats infected with B. pahangi. Rings of thoracic and abdominal aorta were suspended in muscle baths for measurement of isometric tension. Dose-response relations to norepinephrine, endothelium-dependent dilators (acetylcholine, histamine, and A23187), and nitroglycerin were done. In some experiments, inhibitors of cyclooxygenase (indomethacin and aspirin), guanylate cyclase (methylene blue), and nitric oxide formation (N-nitro-L-arginine methyl ester; L-NOARG) were used. No differences in vascular reactivity were detected in the thoracic aorta. In contrast, endothelium-dependent responses in abdominal aorta of Brugia-infected rats were significantly depressed when compared with control aorta from noninfected rats. Acetylcholine relaxation was further depressed by indomethacin and aspirin. After L-NOARG, acetylcholine relaxation in control abdominal aorta was completely abolished; however, in abdominal aorta of Brugia-infected rats, acetylcholine still caused relaxation. Methylene blue inhibited acetylcholine relaxation in both control and Brugia-infected abdominal aorta; however, relaxation in Brugia-infected aorta was significantly greater than control. This study demonstrates that endothelium-dependent relaxation can be altered by chronic experimental filarial infection in the absence of direct contact between the blood vessel and the parasite. The mechanism of relaxation in the Brugia-infected abdominal aorta appears to be altered when compared with control, suggesting that parasites are capable of modulating vascular reactivity by inducing changes in endothelial cell behavior. The mechanism may involve parasite-induced local inflammation or alterations in endothelial cell metabolism. Understanding how chronic experimental filarial infection alters vascular reactivity may enhance our understanding of the pathogenesis of human filariasis.

Gestation Age-Related Increase in 50-kDa Rat Uterine Calcium-Independent Phospholipase A2 Expression Influences Uterine Sensitivity to Polychlorinated Biphenyl Stimulation

Abstract Phospholipase A2 (PLA2) enzymes catalyze the rate-limiting step in eicosanoid production by liberating arachidonic acid from membrane phospholipids. There is limited information regarding the expression pattern and activity of uterine PLA2 enzymes during pregnancy. Polychlorinated biphenyls (PCBs) are a group of persistent environmental toxicants previously associated with decreased gestation length that are capable of activating PLA2. The purpose of the present study was to determine whether uterine sensitivity to PCB stimulation is dependent on PLA2 expression, comparing rat uterine PLA2 expression in Gestational Day (gd) 10 versus gd20. Western blot analysis revealed a significant increase in the expression of calcium-dependent PLA2G2A and a 50-kDa protein immunoreactive to calcium-independent PLA2G6 antibody in gd20 compared to gd10 rat uterine tissue. The increased expression of the 50-kDa PLA2G6 was associated with a gestational age-related increase in endometrial calcium-independent PLA2 activity that was sensitive to inhibition by bromoenol lactone (P < 0.05). Longitudinal uterine strips isolated from gd10 or gd20 rat were suspended in muscle baths to evaluate uterine contractions following exposure to the ortho substituted congener PCB 50. Exposure to 50 and 100 μM PCB 50 significantly increased the frequency of gd20, but not gd10, uteri compared to solvent (dimethyl sulfoxide) controls (P < 0.05). Pharmacologic inhibition of PLA2G6, but not PLA2G2A, attenuated PCB-induced stimulation of gd20 uterine contractions (P < 0.05). These data suggest that PCB 50 stimulates uterine contractions by activating endometrial PLA2G6. Furthermore, gestation age-related sensitivity to PCB is associated with an increase in the expression of a previously unidentified 50-kDa PLA2G6 in rat uterus.

Activation of group IVC phospholipase A2 by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells

Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A2 (PLA2) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA2 in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA2 inhibited both 3H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA2. Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA2.