Role Of Cyclooxygenase Metabolites Research Articles

Purification and Characterization of LTC 4 Synthase from Sheep Uterus

Purification and Characterization of LTC 4 Synthase from Sheep UterusPurification and Characterization of LTC 4 Synthase from Sheep UterusEicosanoids, the oxygenated metabolites of eicosapolyenoic fatty acids such as arachidonic acid via the cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase (EPOX) pathways, are generated in response to specific stimuli, elicit the response and are then quickly metabolized. Hence, these are rightly termed as “local hormones” or “autocoids”. They are involved in the regulation of a variety of physiological as well as pathological processes, including reproduction. While there are extensive studies on the role of COX metabolites, such as prostaglandins, in reproduction, not much is known on the role of LOX metabolites in reproduction. Earlier, we have identified abundant LOX activity in sheep uterus and the highly purified enzyme was found to be a homo-dimeric protein with a molecular weight of 66 kDa. When incubated with arachidonic acid, the enzyme showed two lipoxygenase activities producing both 12- and 15-Hydroxyeicosatetraenoic acid (15-HPETEs) at the optimum pH of 5.5. The relative concentration of 12- and 15-HPETEs, however, changed with the pH of the reaction, 12-Hydroxyeicosatetraenoic acid (HETE) being higher in the alkaline range and 15-HETE being the abundant in the acidic range. Furthermore, the enzyme showed the dual lipoxygenase based 14,15-LTA 4 synthase activity as evidenced by the formation of 8,15-diHETEs, the hydrolysis products of 14,15-LTA 4 . In the present study, leukotriene C 4 synthase (LTC 4 S) enzyme was purified on Q-Sepharose column after solubilization of microsomes utilizing a combination of CHAPS and taurocholate. The purified enzyme showed activity with 5, 6-LTA 4 and 14, 15-LTA 4 , with slight preference towards the latter, and converting them to corresponding LTC 4 s. Both methyl esters and free acids of LTA 4 served as substrates, though the activity was more with methyl esters. However, the enzyme showed no activity with I-chloro-2, 4-dinitrobenzene (CDNB), the conventional substrate of glutathione S-transferases. Western blot analysis of sheep uterine microsomal proteins with LTC 4 synthase specific-peptide as well as whole protein antibodies showed strong cross reactivity with two closely migrating 70 kDa proteins. While showing similarity with the known LTC 4 synthases, sheep uterine LTC 4 synthase thus appears to be quite different in terms of molecular weight, as most LTC 4 synthases reported are 18 kDa proteins. In view of its association with the microsomal membranes and involvement in eicosanoid and glutathione metabolism, sheep uterine LTC 4 synthase may form a member of MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily.

Endothelial dysfunction and improvement of the angiotensin II-reactivity in hypercholesterolemic rabbits: Role of cyclooxygenase metabolites

The aim of this paper was to study the effect of high cholesterol diet on endothelial function and vascular reactivity to angiotensin II and to test the role of vasoconstrictor cyclooxygenase metabolites in this experimental condition. Rabbits were fed with either normal chow or a diet containing 1% cholesterol for 6–7-week. Isometric contractions were measured in rubbed or unrubbed aortic rings. Arteries were contracted with noradrenaline and then exposed to one cumulative dose–response curve to acetylcholine in absence (control) or in presence of indomethacin, ( N-[2-cyvlohexyloxy)-4-nitrophenyl]-methanesulfonamide) (NS 398) or 4-hydroxy-2,2,6,6-tetraethylpiperidine- N-oxyl (tempol). After washing the arteries, one cumulative dose–response curve to angiotensin II was constructed in absence or presence of indomethacin, NS 398, [1 S-[1 alpha,2 beta (5 Z),3 beta,4 alpha]-7-[3-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid (SQ29548) or 17-octadecynoic acid (17-ODYA). In other group, resting potential was recorded in basal and angiotensin II-stimulated conditions. Indomethacin, NS 398 or 17-ODYA were added to the bath before angiotensin II-stimulation. Rabbits fed on a diet enriched with cholesterol showed higher plasma levels of total cholesterol and LDL. Hypercholesterolemic diet impaired acetylcholine relaxation. Indomethacin normalized endothelium-dependent relaxation whereas NS 398 and tempol had no effect on this phenomenon. Angiotensin II-reactivity was increased in endothelium intact hypercholesterolemic aortic rings and indomethacin, SQ29548 or 17-ODYA blocked this effect. The resting potential of unrubbed hypercholesterolemic arteries was significantly less negative to control after angiotensin II-stimulation. 17-ODYA but not indomethacin prevented angiotensin II-depolarization. High cholesterol diet caused endothelial dysfunction and increased the angiotensin II-reactivity. Both effects were cyclooxygenase1-dependent. Deficit in the NO-production might improve 20-hydroxyeicosatrienoic acid availability, which induces depolarization and angiotensin II-sensitization. In addition, 20-hydroxyeicosatrienoic acid would be metabolized by cyclooxygenase1 to 20-endoperoxides which act through thromboxane A 2/prostaglandin H 2 receptors contributing to angiotensin II-reactivity increase.

Cyclosporine A Preparations and Their Vehicles Induce Contraction of the Guinea Pig Gallbladder in vitro: The Role of Cyclooxygenase Metabolites

The aim of this study was to establish whether prostanoids play a role in the contraction induced by cyclosporine A (CyA) preparations in the guinea pig isolated gallbladder strips. It was also aimed to study the effects of the preparations and their solvents on the acetylcholine-evoked rhythmic contractions of the guinea pig isolated sphincter of Oddi (SO). Isometric contractions were recorded. CyA parenteral and oral preparations and their vehicles, Cremophor-EL and Labrafil caused concentration-dependent and sustained contractions (74.2 ± 6.2, 58.4 ± 6.3, 88.9 ± 4.9 and 47.5 ± 6.2% of maximum KCl contraction, respectively) of gallbladder strips, but not of SO. Quinacrine, indometacin and ridogrel inhibited the contraction induced by CyA preparations and their vehicles in gallbladder strips (for CyA parenteral preparation, 34.7 ± 6.7, 1.4 ± 0.9, 19.0 ± 6.4% of maximum KCl contraction, respectively). The drug and its vehicles changed neither the initial contraction nor the amplitude and frequency of the phasic contractions induced by acetylcholine in SO preparations. The results indicate that the drug is able to contract the gallbladder strips and the vehicles contribute to the contracting effect of CyA. Prostanoids may be responsible for the CyA-induced contraction of the gallbladder.

Role of cyclooxygenase metabolites in mediating platelet-induced baroreceptor dysfunction.

The goal of the study was to determine the role of cyclooxygenase metabolites in mediating platelet-induced suppression of baroreceptor activity. Exposure of the isolated carotid sinus of rabbits to thrombin-activated rabbit platelets (3 x 10(8) cells/ml Krebs buffer) decreased baroreceptor activity (P < 0.05) without significantly altering the slope of the pressure-activity relation (gain). The platelet-induced suppression of activity was not blocked but instead was even more pronounced after inhibition of cyclooxygenase with indomethacin; both maximum baroreceptor activity and gain were decreased markedly. The exacerbation of platelet-induced baroreceptor dysfunction contrasted with equivalent carotid vasoconstrictor responses to platelets before and after indomethacin. Furthermore, the stable thromboxane (TxA2) mimetic U-46619 caused similar vasoconstriction as platelets but did not influence baroreceptor gain or maximum activity. In contrast to indomethacin, the selective TxA2 synthesis inhibitor and receptor blocker CGS-22652 failed to influence platelet-induced suppression of activity. In summary, 1) rabbit platelet aggregating in carotid sinus suppress baroreceptor activity, which cannot be explained by the vasoconstriction, and 2) the suppression of activity is not mediated by TxA2 from platelets and is opposed by prostacyclin (PGI2) or other prostanoids produced in carotid sinus. The combination of impaired formation of PGI2 and platelet activation in atherosclerotic and thrombotic states may lead to profound baroreceptor dysfunction.

Bronchopulmonary responses to endothelin-1 in sensitized and challenged guinea-pigs: role of cyclooxygenase metabolites and platelet-activating factor.

The effect of phosphoramidon on the increase in pulmonary inflation pressure (PIP) induced by endothelin-1 (ET-1) administered by aerosol in ovalbumin (OA)-sensitized and challenged guinea-pigs was investigated after pretreatment or not of the animals with the neutral endopeptidase inhibitor, phosphoramidon, the cyclooxygenase inhibitor, indomethacin or the platelet activating factor (PAF) antagonist, BN 50730. When guinea-pigs were pretreated by phosphoramidon (0.1 mM, aerosol for 15 min), a significant enhancement of PIP was observed after administration of ET-1 (1 or 3 micrograms ml-1, aerosol for 2 min), whereas these doses of the peptide were only slightly active in control animals. In sensitized and unchallenged guinea-pigs, ET-1 (1 or 3 micrograms.ml-1, aerosol for 2 min) induced, as in controls, a moderate increase in PIP. In contrast, aerosol exposure of OA in sensitized guinea-pigs developed an increased PIP following ET-1 (1 and 3 micrograms.ml-1, aerosol for 2 min) administration, that was non significantly affected by pretreatment of the animals with phosphoramidon after the dose of 3 micrograms ml-1 ET-1. Guinea-pigs exposed to phorphoramidon and treated with indomethacin (10 mg kg-1, i.v.) or BN 50730 (25 mg kg-1, per os) significantly reduced the increase in PIP upon administration of ET-1 (3 micrograms.ml-1, aerosol for 2 min). No inhibitory effect of indomethacin was noted when ET-1 (3 micrograms.ml-1, aerosol for 2 min) was administered to sensitized and OA-exposed guinea-pigs, pretreated or not with phosphoramidon. In contrast, BN 50730 significantly reduced the increase in PIP induced by ET-1 observed in sensitized and OA-exposed guinea-pigs. Moreover, this drug was moderately active in reducing the increase in PIP induced by ET-1, when the animals were pretreated by phosphoramidon. These results suggest that a phosphoramidon-sensitive endopeptidase-like enzyme, present in the airway tissue modulates the effect of ET-1. Furthermore, the increase in PIP to ET-1 observed in aerosol-sensitized and antigen-exposed guinea-pigs appears to be mediated by PAF rather than cyclooxygenase metabolites, even though the participation of other mediators in this process is open.

Suppression of the development of tumoricidal function in gamma interferon-treated human peripheral blood monocytes by lipopolysaccharide: the role of cyclooxygenase metabolites.

Bacterial lipopolysaccharide (LPS) is generally regarded as one of the most potent macrophage activators. Thus, LPS has been used as an obligatory second signal to stimulate macrophage cytotoxic function against a wide array of bacterial and neoplastic targets. In this study, however, we define conditions under which LPS can suppress the development of cytotoxic function in normal human peripheral blood monocytes. When monocytes were treated with a priming dose of gamma interferon (gamma-INF), followed 18-24 hr later by a triggering dose of LPS, significant cytotoxic function developed. However, when monocytes were treated with even minimal amounts of LPS during priming with interferon, the development of cytotoxic function following stimulation with a second, triggering dose of LPS was virtually abolished. This effect could be produced from 0 to 14 hr following the addition of gamma-INF. The inhibition of monocyte cytotoxicity which was produced by LPS treatment during priming was dose dependent and could not be overcome by modifying either the priming dose of gamma-IFN or the triggering dose of LPS. The suppression was largely overcome, however, by treatment with the cyclooxygenase inhibitor, indomethacin. The possibility that LPS-induced suppression of monocyte cytotoxicity was mediated by products of the cyclooxygenase pathway was supported further in this study by demonstrating that LPS stimulated the production of significant amounts of prostaglandin E2 (PGE2) from monocytes and that this was facilitated by gamma-IFN. In kinetics studies, it appeared that LPS suppression of monocyte activation was correlated temporally with a heightened sensitivity to suppression by exogenously added PGE2, a condition which was reduced greatly by the end of the priming phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin-Detectable Effects of Localized Pneumonia on Airway Mucous Cell Populations: Role of Cyclooxygenase Metabolites

We examined the airway secretory apparatus of adult sheep with experimental pneumonia to look for morphologic and lectin-binding correlates of increased mucus production. The animals were inoculated in the right caudal lobar bronchus either with starch broth containing Pasteurella haemolytica (INF, n = 6), starch broth alone (SHAM, n = 6), or with P. haemolytica and subsequently treated (INF/T, n = 5) with 2 mg/kg indomethacin, subcutaneously three times daily for 6 days. In the INF and INF/T groups, a localized pneumonic infiltrate containing P. haemolytica organisms was present. The bronchi (18-23rd generation) adjacent to the pneumonic lesion had an increased gland volume fraction (6.3 +/- 3.7% in INF, 11.3 +/- 2.4% in INF/T, and 3.1 +/- 1.9% in SHAM, p less than 0.05 among the three). The mean population densities of BSA-reactive (identifying alpha-D-gal) cells were 41.9 +/- 2.7% in the INF, 40.1 +/- 5.6% in the INF/T, versus 14.3 +/- 1.5% in the SHAM group (p less than 0.05), while the corresponding values for PNA-reactive [identifying beta-D-gal(1----3)-D-galNAc] cells were 28.8 +/- 5.1%, 0%, and 0%, respectively. Nor morphologic abnormalities were seen in the trachea, but BSA staining was shifted to morphologically different mucous cells in the INF and INF/T. We conclude that in localized P. haemolytica pneumonia in sheep (1) there are morphologic changes of the airway secretory apparatus adjacent to the lesion, (2) the glycoconjugate profile of secretory cells adjacent to and remote from the lesion is altered, and (3) cyclooxygenase products influence the chemical composition of secretory cells.

Pulmonary vascular response to platelet-activating factor in awake sheep and the role of cyclooxygenase metabolites.

We examined the effects of platelet activating factor (PAF) (1.0 microgram/kg infusion for 15 min) on pulmonary hemodynamics and lung fluid balance and the role of cyclooxygenase metabolites in mediating these responses in unanesthetized sheep prepared with lung lymph fistulas. Platelet activating factor infusion resulted in immediate and transient increases in pulmonary artery pressure, pulmonary vascular resistance, and pulmonary lymph flow. The lymph-to-plasma protein concentration ratio did not change significantly from baseline. Circulating platelet and leukocyte counts decreased immediately after PAF infusion; the leukopenia was the result of a rapid decrease in both the neutrophil and mononuclear leukocyte counts. Arterial thromboxane B2 (TxB2) concentration increased after the PAF infusion, but the 6-keto prostaglandin F1 alpha (a prostacyclin degradation product) concentration did not change from baseline. A chemically similar substance, Lyso-PAF, had no effect on the pulmonary hemodynamic, lymph, and hematologic parameters or the TxB2 generation. Administration of cyclooxygenase inhibitors, meclofenamate or indomethacin, prior to PAF infusion prevented thromboxane B2 generation and attenuated the pulmonary hemodynamic response. The initial pulmonary lymph flow and transvascular protein clearance (lymph flow times lymph-to-plasma protein concentration) responses to PAF were attenuated after cyclooxygenase inhibition. However, there were time-dependent increases in pulmonary lymph flow and transvascular protein clearance in the cyclooxygenase-inhibited groups. These results indicate that PAF induces pulmonary vasoconstriction mediated by cyclooxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

C3a-induced contraction of guinea pig lung parenchyma: Role of cyclooxygenase metabolites

The complement anaphylatoxin C3a from human or porcine serum contracts isolated peripheral airways from guinea pig in a manner which is independent of histamine release. In order to evaluate the role of arachidonic acid metabolites in the C3a response, dose-response curves for C3a-induced contractions of guinea pig lung strips were compared in the presence and absence of several inhibitors which block metabolism of arachidonic acid at relatively specific steps in the pathways. The inhibitor of leukotriene activity, FPL 55712 ant the lipoxygenase inhibitor, nordihydroguiretic acid (NDGA), had no significant effect on the tissue response to C3a alone or in combination with anthistamine. The cyclooxygenase inhibitors, indomethacin and aspirin, however, both resulted in significant inhibition, causing a shift in the C3a dose-response curve to higher concentrations. When the antihistamine, pyrilamine, was included with either indomethacin or aspirin, the C3a response was inhibited further, although pyrilamine alone had no effect. Thus, C3a-induced contractions of isolated lung parenchyma are mediated primarily by cyclooxygenase metabolites of arachidonic acid. This result is in contrast to the finding that C5a anaphylatoxin mediates lung tissue contraction by release of lipoxygenase metabolites of arachidonic acid.