Inhibition Of Arachidonic Acid Research Articles

Blockade of LTC 4 synthesis caused by additive inhibition of gIV-PLA 2 phosphorylation: Effect of salmeterol and PDE4 inhibition in human eosinophils

Background: Prior investigations have demonstrated that β 2-adrenoceptor stimulation is ineffective in inhibiting synthesis of eicosanoids in human eosinophils. This effect has been postulated to relate to density or structural differences in the β 2-adrenoceptor or its coupled G-protein. However, recent reports indicate that cAMP-specific PDE4 activity in eosinophils is 10-fold that of other inflammatory cells. We postulated that selective blockade of PDE4 in eosinophils would unmask the inhibitory effect of β 2-adrenoceptor stimulation and that this inhibition would result from decreased phosphor-ylation of cytosolic group IV-PLA 2 (gIV-PLA 2). Objective: To determine (a) whether PDE4 inhibition alone with rolipram blocked secretions of arachidonic acid (AA) and leukotriene C 4 (LTC 4) caused by activation of eosinophils with formyl-met-leu-phe plus cytochalasin B (FMLP/B), (b) to determine if PDE4 inhibition plus β 2-adrenoceptor agonist act additively to augment endogenous cAMP concentration, and (c) to determine the mechanism by which additive inhibition of AA and LTC 4 synthesis is regulated by cAMP. Methods: Human eosinophils were pretreated with buffer, salmeterol or rolipram (singly or combination) before FMLP/B activation. Release of AA and LTC 4, intracellular cAMP concentration, and phosphorylation and activation of gIV-PLA 2 were determined. Results: Rolipram unmasked the inhibitory effect of β 2-adrenoceptor stimulation with salmeterol and significantly attenuated the stimulated release of AA and subsequent LTC 4. Inhibition corresponded to increased cAMP production caused by rolipram alone or rolipram plus salmeterol and blocked proportionately the phosphorylation and activation of gIV-PLA 2 in FMLP/B-activated eosinophils. Conclusions: Inhibition of PDE4 by rolipram unmasks β 2-adrenergic blockade of LTC 4 synthesis caused by FMLP/B. (J Allergy Clin Immunol 2003;112:404-10.)

Synthesis and structure-activity relationships of cis-tetrahydrophthalazinone/pyridazinone hybrids: a novel series of potent dual PDE3/PDE4 inhibitory agents.

In this study, the synthesis and in vitro and in vivo pharmacological investigations of a new series of phthalazinone/pyridazinone hybrids with both PDE3 and PDE4 inhibitory activities are described. These compounds combine the pharmacophores of recently discovered 4a,5,8,8a-tetrahydro-2H-phthalazin-1-one-type inhibitors of PDE4 and the well-known 2H-pyridazin-3-one-type PDE3 inhibitors such as the tetrahydrobenzimidazoles. Most of the synthesized compounds are pharmacologically spoken PDE3/PDE4 hybrids. All hybrids show potent PDE4 inhibitory activity (pIC(50) = 7.0-8.7), whereas the pIC(50) values for inhibition of PDE3 vary from 5.4 to 7.5. In general, analogues with a 5-methyl-4,5-dihydropyridazinone moiety exhibit the highest PDE3 inhibitory activities. The highest in vivo antiinflammatory activity is displayed by phthalazinones 43 and 44 showing, at a dose of 30 micromol/kg po, 46% inhibition of arachidonic acid (AA) induced mouse ear edema. No correlation was found between the in vitro PDE3 and/or PDE4 inhibitory activity and the in vivo antiinflammatory capacity after oral dosing.

Cationic amphiphilic drugs and platelet phospholipase A 2 (cPLA 2)

The aim of the study was to verify and compare the effect of cationic amphiphilic drugs (CAD) from different pharmacological groups on activation of platelet phospholipase A 2 (PLA 2)—the essential enzyme of arachidonic pathway in blood platelets. β-Adrenoceptor-blocking (BAB) drugs inhibited platelet aggregation in the rank order of potency: propranolol>alprenolol>metipranolol>atenolol. The higher the inhibition of arachidonic acid (AA) liberation by BAB drugs, the higher the inhibition of aggregation. Similarly did the H 1-histamine antagonists bromadryl (BRO) and dithiaden (DIT) as well as the antimalarial chloroquine (CQ) show antiplatelet effect in vitro in the rank order of potency: DIT>BRO>CQ. Dose-dependent inhibition of aggregation was followed by the inhibition of AA liberation from membrane phospholipids of platelets stimulated either at the receptor site (thrombin) or by a stimulus bypassing membrane receptors (Ca 2+ ionophore A23187). The rank order potency for inhibition of stimulated 3H-AA liberation from membrane phospholipids was: (a) for BAB drugs: propranolol>alprenolol>metipranolol, (b) for other drugs: DIT>BRO>CQ. The investigated drugs' interference with stimulated liberation of AA showed nonspecific inhibition of platelet cytosolic PLA 2 (cPLA 2) by these drugs at intracellular level. The results revealed that besides the inhibition of cyclooxygenase pathway and receptors for adenosine diphosphate (ADP) and glycoproteins Gp II bIII a, the interaction of drugs with cPLA 2 may represent a further site for antiplatelet action.

O-15 Aspirin inhibition of arachidonic acid mediated platelet aggregation is significantly reduced during surgery

O-15 Aspirin inhibition of arachidonic acid mediated platelet aggregation is significantly reduced during surgery

Antiplatelet activity of N-methoxycarbonyl aporphines from Rollinia mucosa

Bioassay-directed fractionation of the stems of Rollinia mucosa led to the isolation of new N-methoxycarbonyl aporphine alkaloids, romucosine A ( 1), romucosine B ( 2), romucosine C ( 3), and romucosine D ( 4), along with the known alkaloid, N-methoxylcarbonyl-nornuciferine ( 5). Alkaloids 1 and 4 exhibited significant inhibition of collagen, arachidonic acid, and platelet activating factor-induced platelet aggregation, and alkaloid 3 also showed an inhibitory effect on arachidonic acid induced platelet aggregation.

Studies on antiplatelet agents from natural safrole: II. Synthesis and pharmacological properties of novel functionalized oxime O-benzylether derivatives

In an ongoing research program aiming at the synthesis and pharmacological evaluation of new possible prototype candidates exploring the molecular hybridation and bioisosterism principles for molecular designing, we describe in this paper the design and synthesis of a series of new functionalized oxime O-benzylethers (4a–b) and (14a–b) as antiplatelet agents based on the inhibition of arachidonic acid (AA) cascade enzymes. For the synthesis of these new bioactive derivatives we used safrole (5), a Brazilian abundant natural product, as starting material. The platelet anti-aggregating evaluation of these oxime O-benzylether compounds (4a–b) and (14a–b) in model induced by ADP, collagen and AA, has permitted to evidence an antithrombotic profile to these new derivatives, being the most active the derivative methyl [[3,4-methylenedioxyphenyl]methylene]amino]oxy]-4-methylenephenylacetic acid (14a).

Identification and characterization of zebrafish thrombocytes.

To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

Synthesis and pharmacological investigation of 9-methyl-1,2,3,4,6,7,12,12b-octahydro-7-oxo-indolo[2,3- a]quinolizine

The title ketone, a supposed metabolite of 9-methyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3- a]quinolizine (MIQ), was prepared and found different on chromatographic comparison from the isolated metabolite M 4 . However, the pharmacological screening of this compound evidenced, among others, some interesting properties as inhibition of arachidonic acid induced platelet aggregation and protection from both hypobaric and cyanide induced hypoxia in mice. These activities suggest a possible cerebroprotective action to be further investigated.

The Effect of Phospholipase A2Inhibitors on Proliferation and Apoptosis of Murine Intestinal Cells

Background.Group II phospholipase A2(PLA2) enzymes, the rate controlling enzymes in arachidonic acid metabolism, have been well characterized and subdivided into secretory 14-kDa PLA2(sPLA2) and cytoplasmic 85-kDa PLA2(cPLA2). Previous research has demonstrated increased PLA2in colorectal tumors. The present study was performed to determine the effect of specific PLA2 inhibitors on the proliferation and induction of apoptosis of intestinal epithelial cells.Methods.A continuously proliferating rat small intestinal cell line (IEC-18) and a mouse colon cancer cell line (WB-2054-M4) were utilized for these experiments. The cells were placed in microwells with serum-free or serum-supplemented media. The effects of serum on proliferation were then evaluated in the presence of the cPLA2inhibitor, methylarachidonyl fluorophosphate (MAFP), or the sPLA2inhibitorp-bromophenacyl bromide (BPB). The sPLA2and cPLA2protein was estimated by Western blotting. Proliferation of intestinal cells was quantitated by incorporation of [3H]thymidine into DNA and PLA2activity was evaluated by quantitating arachidonic acid formation and prostaglandin E2production.Results.Western blotting of IEC-18 and WB-2054 cell protein demonstrated sPLA2and cPLA2enzyme in cells incubated in media containing 10% serum. Spontaneous DNA synthesis was present in both cell lines and serum consistently increased proliferation. In IEC-18 cells [3H]thymidine incorporation stimulated by serum was inhibited by MAFP and BPB, while in the malignant cell line, proliferation was inhibited only by BPB. BPB, but not MAFP, produced a dose-dependent increase in apoptotic ratios in both cell lines. Arachidonic acid and PGE2formation, stimulated by serum, was inhibited by MAFP and BPB.Conclusions.A differential effect on intestinal cell mitogenesis was seen with different PLA2inhibitors. The sPLA2inhibitor, but not the cPLA2inhibitor, significantly inhibited [3H]thymidine incorporation in the malignant cell line. This occurred with an induction of apoptosis. sPLA2inhibitors may be specific inhibitors of growth of malignant cells. The inhibition of arachidonic acid and PGE2production did not correlate with the inhibition of proliferation, suggesting that the two processes may be unrelated.

Two new 7-dehydroaporphine alkaloids and antiplatelet action aporphines from the leaves of Annona purpurea.

Bioactivity-directed fractionation led to the isolation of two new 7-dehydroaporphine alkaloids, 7-hydroxy-dehydrothalicsimidine (1) and 7-formyl-dehydrothalicsimidine (2), along with the five known alkaloids, thalicsimidine (3), norpurpureine (4), N-methyllaurotetanine (5), lirinidine (6) and N-methylasimilobine (7), from the leaves of Annona purpurea. Structural elucidation of these compounds was established by mass and spectroscopic analyses. Among them, 1, 3, 4, 6 and 7 exhibited significant inhibition of collagen, arachidonic acid and platelet activating factor-induced platelet aggregation; 1 also showed inhibition against thrombin-induced platelet aggregation.

Analysis of inhibitory effects of scutellariae radix and baicalein on prostaglandin E2 production in rat C6 glioma cells.

Inhibitory mechanism of the water extract of Scutellariae Radix on prostaglandin E2 (PGE2) release was examined in C6 rat glioma cells. Scutellariae Radix reduced a Ca2+ ionophore A23187-induced PGE2 release by inhibition of arachidonic acid (AA) liberation. Sho-saiko-to and San'o-shashin-to, which contain Scutellariae Radix, also inhibited PGE2 release. A23187 caused phosphorylation of mitrogen-activated protein kinase (MAPK), resulting in activation of cytosolic phospholipase A2 (cPLA2). Scutellariae Radix and baicalein inhibited the phosphorylation of MAPK. Baicalein, but not baicalin, inhibited A23187-induced PGE2 release. These results suggest that baicalein in Scutellariae Radix reduces AA liberation through the inhibition of the MAPK-cPLA2 pathway.

Functional dissociation between glucocorticoid-induced decrease in arachidonic acid release and inhibition of adrenocorticotropic hormone secretion in AtT-20 corticotrophs.

The biochemical basis of the short-term inhibitory effects of glucocorticoids on corticotropin release from pituitary corticotrophs is still obscure. A well-characterized effect of glucocorticoids in several cell types is the inhibition of arachidonic acid (AA) generation by phospholipase A2 (PLA2). Arachidonic acid and its metabolites have been implicated in the secretory process from a number of pituitary cells, such as the corticotrophs. We have thus examined the role of AA in the anti-secretagogue effects of glucocorticoids in a corticotropin-secreting clonal corticotroph line (AtT-20 D16/16). Glucocorticoids decreased AA release induced by melittin, a bee venom protein related to extracellular PLA2. When a possible role of AA in corticotropin release was studied, the following results were obtained: (a) all corticotropin secretagogues tested, including corticotropin-releasing factor (CRF), did not alter AA generation; (b) calcium and guanine nucleotides, which stimulate corticotropin release in permeabilized cells, inhibited the release of AA under the same conditions; (c) administration of melittin or of exogenous AA had no effect on basal and CRF-stimulated corticotropin release; (d) administration of large amounts of exogenous AA was unable to restore the ability to secrete corticotropin under suppression by glucocorticoids. Altogether, the data suggest that whereas glucocorticoids can inhibit both AA generation and corticotropin release, these two effects appear to be causally unrelated.

Biphasic effect of cadmium ions on the secretion of leukotriene B4 in rabbit alveolar macrophages.

One major role of alveolar macrophages is the production of eicosanoids, which modulate immune and inflammatory processes in the lung. In this study, the effects were investigated of cadmium ions on the secretion of leukotriene (LT)B4 and prostaglandin (PG)E2, predominant products of lipoxygenase and cyclooxygenase, respectively. Cd2+ had an inhibitory effect on the secretion of LTB4 and PGE2 in response to A23187 stimulation at concentrations > 3 x 10(-5)M. This effect can be explained by the inhibition of arachidonic acid (20:4) liberation from membrane phospholipids by Cd2+, because Cd2+ inhibits both [3H] arachidonic acid (20:4) liberation from [3H]20:4-prelabeled macrophages and the cytosolic phospholipase A2 activity. At concentrations < 3 x 10(-5)M, Cd2+ had no effect on PGE2 secretion but showed an augmentation of LTB4 secretion. In vitro study using macrophage lysate showed enhanced LTB4 synthesis from arachidonic acid by Cd2+, which could be responsible for the augmentation of LTB4 secretion in cells. These results indicate that Cd2+ may increase inflammation by increasing LTB4 production in lung.

Antiplatelet and Vasorelaxing Actions of Some Aporphinoids

A series of aporphines and oxoaporphines was tested for antiplatelet and vasorelaxing actions. (+)-N-Methylactinodaphnine, (-)-norannuradhapurine x HBr, xylopine, actinodaphnine, and N-methylnandigerine showed strong inhibition of adenosine 5'-diphosphate (ADP)-induced platelet aggregation. Boldine, (+)-N-methylactinodaphnine, (-)-norannuradhapurine x HBr, xylopine, N-acetyllaurolitsine, N-methyllaurotetanine, actinodaphnine, N-methylnandigerine, O-methylbulbocapnine, and liriodenine showed strong inhibition of arachidonic acid (AA)-induced platelet aggregation. (+)-N-Methylactinodaphnine, fissoldine x HCIO4, (-)-norannuradhapurine x HBr, xlylopine, N-methyllaurotetanine, actinodaphnine, N-methylnandigerine, O-methylbulbocapnine, and liriodenine showed strong inhibiton of collagen-induced platelet aggregation. (-)-Norannuradhapurine x HBr, xylopine, N-methyllaurotetanine, and actinodaphnine showed strong inhibition of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-induced platelet aggregation. (+)-N-Methylactinodaphnine, laurotetanine, N-methylactinodaphnine N-oxide, oxoglaucine, boldine, and actinodaphnine showed vasorelaxing action in rat thoracic aorta. The results are discussed on the basis of structure-activity relationships.

Pulmonary Pharmacology of DT-TX 30 SE, a Potent Selective Combined Thromboxane Synthetase Inhibitor and Receptor Antagonist, in Guinea Pigs

A novel chemical compound, DT-TX 30 SE (E-6-(4-(2-(4-chlorobenzenesulphonylamino)-ethyl)phenyl)-6-(3-pyridyl)-hex-5-enoic acid), was studied in various models of guinea pig pulmonary function. The compound was a potent inhibitor (ED50 0.019 mg/kg, i.v.) of bronchospasm induced by the thromboxane receptor agonist U-46619, indicating thromboxane receptor antagonism. At even lower doses (ED50 0.0036 mg/kg, i.v.), it blocked arachidonic acid-induced bronchospasm. Interpretation of the latter results as evidence for additional thromboxane synthetase inhibitory activity was supported by the inhibition of arachidonic acid- or bradykinin-induced thromboxane B2 production in an isolated lung preparation, although prostaglandin E2 and prostaglandin 6-oxo-F1α production measured at the same time were not inhibited. The potency of DT-TX 30 SE was compared with thromboxane receptor antagonists and synthetase inhibitors described in the literature. As a receptor antagonist, DT-TX 30 SE was significantly more potent than BM 13505 and BM 13177 (assessed by antagonism of U-46619-induced bronchospasm), but less potent than SQ 29548, while as a thromboxane synthetase inhibitor, it was significantly more potent than OKY 046 and UK 37248 as assessed by antagonism of arachidonic acid-induced bronchospasm or (OKY 046) inhibition of thromboxane production in isolated lung. The compound was active by the oral route as shown by its ability, at 10 mg/kg, p.o., to significantly reduce the immediate allergic response of sensitized guinea pigs to an ovalbumin aerosol.

Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines.

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.

Platelet Reactivity In Vitro in Relation to Thromboxane in Healthy Pregnancy

There is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy. Previous in vitro studies suggest that platelets from pregnant women show increased sensitivity to agonists, the response to which has a thromboxane dependent component. The aim of this study was to determine whether this is due to increased activity of the thromboxane biosynthetic pathway or to increased platelet sensitivity to the effects of thromboxane. During pregnancy, platelets were more sensitive to the pro-aggregatory effects in vitro of the thromboxane mimetic U46619, in whole blood and in platelet rich plasma, compared to those from non-pregnant controls. The difference in extent of U46619-induced platelet aggregation between groups was abolished in the presence of a high concentration of the specific thromboxane antagonist ICI 192605, but not by prior incubation of blood with aspirin. Platelets from pregnant women were significantly less sensitive to inhibition of arachidonic acid induced activation by the thromboxane synthetase inhibitor dazmegrel, but there was no change in platelet cyclic AMP accumulation under these conditions. Arachidonic acid induced platelet thromboxane B2 production was similar in pregnant and non-pregnant subjects. In conclusion, platelets are more sensitive to the activating effects of thromboxane during pregnancy, but there is no change in the intrinsic reactivity of the thromboxane biosynthetic pathway.

Synthesis and evaluation of coumarin alpha-methylene-gamma-butyrolactones: a new class of platelet aggregation inhibitors.

In a search for new inhibitors of platelet aggregation, certain coumarin-bearing alpha-methylene-gamma-butyrolactones were synthesized and evaluated for inhibitory activity against thrombin (Thr)-, arachidonic acid (AA)-, collagen (Col)-, and platelet-activating factor (PAF)-induced aggregation in washed rabbit platelets. These compounds were efficiently synthesized from commercially available 7-hydroxycoumarin or its derivatives. Among them, 7-[(2,3,4,5-tetrahydro-4-methylene-5-oxo-2-phenyl-2-furanyl) methoxy]-2H-1-benzopyran-2-one (3d) showed the most potent inhibition of AA- and PAF- induced aggregation, with IC50 values of 3.65 and 16.36 microM respectively.

Antiplatelet constituents of formosan Rubia akane.

A known steroid, in addition to triterpenoids, anthraquinones, naphthalenes and a new anthraquinone glycoside, xanthopurpurin 3-O-beta-D-glucoside, were isolated from the roots of Rubia akane grown in Taiwan. Mollugin, a naphthohydroquinone, showed strong inhibition of arachidonic acid (AA)-induced and collagen-induced platelet aggregation. In contrast, 2-methyl-1,3,6-trihydroxyl-9,10-anthraquinone, xanthopurpurin 3-O-beta-D-glucoside, and xanthopurpurin showed mainly strong inhibition of collagen-induced platelet aggregation.

Dietary fish oil supplementation alters LTB 4:LTB 5 ratios but does not affect the expression of acute graft versus host disease in mice

One of the mechanisms by which corticosteroids may modify acute graft vs host disease (GvHD) is via inhibition of arachidonic acid (AA) metabolism. Leukotriene B 4 (LTB 4) is a product of that pathway which may take part in the pathogenesis of GvHD through the stimulation of T-lymphopoiesis and T-lymphocyte activation. LTB 4 is a metabolite of AA (20:4n-6). Alternate dietary sources of polyunsaturated fatty acids (PUFA), specifically eicosapenteinoic acid (20:5n-3) (EPA) and docosahexaenoic acid (22:6n-3) (DHA), shift the LTs formed with a decrease in LTB 4 an increase in LTB 5. LTB 5 is a less potent agonist than LTB 4 and this results in a theoretical decrease of LTB 4 mediated events. Supplementation of in vitro bone marrow cultures with EPA or DHA had no detrimental effect on myeloid colony formation. Dietary EPA/DHA supplementation in mice with induced GvHD appeared to be safe and well tolerated. The LTB 4:LTB 5 ratio shifted from 7.65 ± 1.75 in control-fed animals to 1.03 ± 0.18. Fish-oil-supplementation did not compromise engraftment or stem cell content. Alone, this therapy was unable to modify GvHD.