RESUMO
The acyl-CoA: cholesterol acyl transferase (ACAT) reaction in macrophages is a critical step in atherosclerotic foam cell formation, but little is known about the reaction's sterol substrate specificity. In this report we examine the macrophage ACAT reactivity of the shellfish sterol, desmosterol, and other sterols found in man because of shellfish ingestion or in association with the foam cell diseases sitosterolemia and cerebrotendinous xanthomatosis (CTX). We first show that the J774 macrophage, a foam cell model with a hyperactive ACAT pathway, synthesizes desmosterol instead of cholesterol and that both endogenous and exogenous desmosterol are substrates and stimulators of the ACAT reaction in these cells. When exogenous desmosterol was added to human monocyte-derived macrophages, ACAT was stimulated 29- and 4-fold compared with control and cholesterol-treated cells, respectively. Steryl ester mass accumulation in desmosterol-treated human macrophages was 10-fold greater than in control cells and 3-fold greater than in cholesterol-treated cells. Another shellfish sterol, 24-methylene cholesterol, also stimulated ACAT in human macrophages, but most of the xanthomatosis-related sterols did not stimulate ACAT. These data suggest that: (a) the shellfish sterols desmosterol and 24-methylene cholesterol may be atherogenic; and (b) the excessive foam cell formation seen in sitosterolemia and CTX cannot be explained by ACAT hyperreactivity of their associated sterols.
Assuntos
Desmosterol/metabolismo , Macrófagos/enzimologia , Esterol O-Aciltransferase/metabolismo , Animais , Linhagem Celular , Colesterol/análogos & derivados , Colesterol/metabolismo , Colesterol/farmacologia , Desmosterol/farmacologia , Esterificação , Células Espumosas/enzimologia , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Sitosteroides/sangue , Xantomatose/metabolismoRESUMO
Polymorphonuclear leukocytes (PMNL) isolated from two patients with chronic granulomatous disease (CGD) were tested for their ability to metabolize arachidonic acid to lipoxygenase products including 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). Analyses of incubations of these PMNL with arachidonic acid and the calcium ionophore A23187 did not differ from simultaneous controls in the production of LTB4, other 5,12-dihydroxy-eicosatetraenoic acids, or monohydroxy-eicosatetraenoic acids. The clinical diagnosis of CGD was confirmed in both cases by determination of PMNL chemiluminescence. Leukocytes from both patients failed to generate active oxygen species in response to either LTB4 or formyl-methionyl-leucyl-phenylalanine. The observation of arachidonic acid oxidation in the absence of superoxide anion precludes a role for the active oxygen species in this metabolic process. These studies clearly dissociate the ionophore-induced leukocyte respiratory burst from the oxidation of arachidonate to the leukotrienes. In addition, the defect of CGD appears to be unrelated to the ability of PMNL to carry out arachidonate oxygenation.
Assuntos
Doença Granulomatosa Crônica/sangue , Leucotrieno B4/sangue , Neutrófilos/metabolismo , Adulto , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Calcimicina/farmacologia , Humanos , Medições Luminescentes , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacosRESUMO
Leukotriene synthesis and metabolism were studied in cultured porcine aortic smooth muscle cells (PSM). Cultures stimulated with calcium ionophore A23187, with or without exogenous arachidonic acid, did not release detectable levels of leukotriene B4, C4, D4 or E4. Those products were assayed by high-performance liquid chromatography, ultraviolet spectrometry and, in some cases, radioimmunoassay. Smooth muscle cultures were able to convert leukotriene A4 to leukotriene C4, indicating the presence of leukotriene C4 synthetase. Although this enzymatic activity has previously been found in cultured porcine aortic endothelial cells, it was not detectable in cardiac myocytes, fibroblasts from several organs or renal epithelial cells. It is known from previous work that inflammatory cells such as polymorphonuclear leukocytes (PMNL) or mast cells release leukotriene A4 when stimulated. Further, increased numbers of these cell-types are found associated with vascular tissue during several pathologic situations. Therefore, the potential for a leukocyte-smooth muscle cell interaction involving the transcellular metabolism of leukotriene A4 was assessed. Stimulation of PMNL suspensions in the presence of PSM resulted in a significant increase in total leukotriene C4 produced in comparison to either cell-type alone (255% of PMNL alone, P less than 0.05). Furthermore, after the intracellular glutathione pool of PSM was prelabelled with 35S, a PSM-PMNL coincubation produced levels of [35S]leukotriene C4 which were significantly greater (P less than 0.05) than those found after coincubating prelabelled PMNL with unlabelled PSM. These data demonstrate a PMNL-PSM interaction in which smooth muscle cell leukotriene C4 synthesis results from the transcellular metabolism of PMNL-derived leukotriene A4. Since leukotriene C4 and its metabolites are vasoconstrictors and cause increased vascular permeability, the biochemical interaction described in this report may be relevant to the pathophysiology of arterial vasospasm, atherogenesis and to the abnormalities of tissue perfusion associated with ischemic or inflammatory disorders.
Assuntos
Ácidos Araquidônicos/metabolismo , Músculo Liso Vascular/metabolismo , SRS-A/biossíntese , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Transporte Biológico , Calcimicina/farmacologia , Células Cultivadas , Epitélio/metabolismo , Glutationa Transferase/metabolismo , Rim/metabolismo , Leucotrieno A4 , Macrófagos/citologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Neutrófilos/citologia , SRS-A/isolamento & purificação , SuínosRESUMO
Leukotriene A4 hydrolase (LTA-H) is a bifunctional protein that has aminopeptidase activity, but also contains an epoxide hydrolase activity that converts leukotriene (LT)A4 to LTB4. The lipid metabolic activity of this enzyme plays a central role in the control of polymorphonuclear leukocyte function and in the development of inflammation. LTA-H is widely spread in many mammalian tissues, although it appears to be inactive in many cases. Regulation of this enzyme's activity by phosphorylation of a serine at residue 415 has recently been described. Since the activation of LTA-H in the presence of activated PMNL would likely lead to a substantial increase in the production of inflammatory lipids, regulation of LTA-H presents a novel potential target for anti-inflammatory therapy. We have now made a series of site-directed mutants at this site to test the importance of this residue to the activity of LTA-H. Replacement of the critical serine with threonine or glutamine has little effect on either the epoxide hydrolase or aminopeptidase activities. However, replacing serine with a negatively charged amino acid (either aspartate or glutamate), intended to mimic phosphorylation at that site, causes significant reduction in epoxide hydrolase activity (50-70%). These mutations have little effect on the aminopeptidase activity of the LTA-H, suggesting that the mutation models the regulatory event and is not simply due to improper folding of the protein.
Assuntos
Epóxido Hidrolases/genética , Serina/química , DNA Complementar/química , Epóxido Hidrolases/química , Epóxido Hidrolases/metabolismo , Escherichia coli/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Sequências Reguladoras de Ácido NucleicoRESUMO
Leukotriene B4 stimulated the formation of cyclic AMP, the release of lysosomal enzyme and generation of superoxide anions by human leukocytes. Dose-response curves have shown that the enzyme release proceeded in parallel with increments in cyclic AMP, suggesting a linkage between cyclic AMP and leukotriene B4-induced leukocyte activation. However, preincubation of the cells with (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid or leukotriene B4 resulted in a dose-dependent inhibition of leukotriene B4-induced degranulation, without causing parallel changes in the levels of cyclic AMP. Both dihydroxy acids also blocked leukotriene B4-induced superoxide anion generation. These results suggest that the leukocyte responses to leukotriene B4 and the concomitant cyclic-AMP increments may be merely coincidental. In addition, the present study further supports the suggestion that (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid may modulate the action of leukotriene B4 in the leukocyte.
Assuntos
AMP Cíclico/metabolismo , Ácidos Hidroxieicosatetraenoicos , Leucócitos/fisiologia , Leucotrieno B4/farmacologia , Superóxidos/metabolismo , Ácidos Araquidônicos/farmacologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/enzimologia , Lisossomos/enzimologiaRESUMO
1. 15-Lipoxygenase (15-LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15-LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol-fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. 2. PD 146176 inhibited rabbit reticulocyte 15-LO through a mixed noncompetitive mode with a Ki of 197 nM. The drug had minimal effects on either copper or 2,2'-azobis(2-amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. 3. Control New Zealand rabbits were fed a high-fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg-1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nM). During the 12 week study, there were no significant differences in weight gain haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. 4. The drug plasma concentrations achieved in vivo did not inhibit low-density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176-treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. 5. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15 +/- 4 to 0% (P < 0.02); esterified cholesterol content was reduced from 2.1 +/- 0.7 to 0 micrograms mg-1 (P < 0.02) in this region. Immunostainable lipid-laden macrophages present in aortic intima of control animals were totally absent in the drug-treated group. 6. Results of these studies are consistent with a role for 15-LO in atherogenesis.
Assuntos
Arteriosclerose/tratamento farmacológico , Dieta , Fluorenos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Animais , Antioxidantes/farmacologia , Arteriosclerose/sangue , Arteriosclerose/etiologia , Lipídeos/sangue , Lipoproteínas/sangue , CoelhosRESUMO
15-Lipoxygenase has been suggested to play a role in atherogenesis. The proposed action of this enzyme is to oxidize low density lipoprotein (LDL) to the extent that LDL becomes a ligand for the macrophage scavenger receptor. 15-Lipoxygenase and oxidized LDL are co-localized in atherosclerotic lesions; antioxidant drugs that block the lipoxygenase also block oxidation of LDL and the progression of experimental atherosclerosis. Biochemical experiments have demonstrated that the lipoxygenase can be induced by cytokines and/or another factor(s) associated with hypercholesterolemia. However, molecular biological work has shown that induction of the enzyme alone is not sufficient to induce lesion formation. Furthermore, the mechanism of action of 15-lipoxygenase in atherogenesis remains unclear. Predictions of the stereochemistry of enzyme-oxidized linoleate products appear to conflict with the available data. In fact, most studies have discovered substantial levels of racemic 13-hydroxyoctadecadienoic acid (13-HODE) in arterial lesions rather than the stereochemically pure 13(S)-HODE expected from purified enzyme. The possibility that the generation of products of 15-lipoxygenase metabolism must occur in a specific cellular location and during a brief time window in the development of the disease has been discussed. It is also possible that the true function of the linoleate metabolites is to modulate gene expression and regulate mitogenesis, and that oxidation of LDL may play a secondary role. The advent of transgenic species that both develop atherosclerosis and either fail to express or overexpress the lipoxygenase presents an opportunity to clarify some of these issues in the near future.
Assuntos
Araquidonato 15-Lipoxigenase/fisiologia , Arteriosclerose/etiologia , Animais , Humanos , Lipoproteínas LDL/metabolismo , EstereoisomerismoRESUMO
The lipoxygenase product 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), stimulates the synaptic response produced by the modulatory transmitter histamine and the neuroactive peptide Phe-Met-Arg-Phe-amide (FMRFamide) in identified neurons of the marine mollusk Aplysia californica. The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. Therefore, we began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. We have identified 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) as a metabolite of 12-HPETE formed by Aplysia nervous tissue. 12-KETE was identified in incubations of the tissue with arachidonic acid using HPLC, UV spectrometry, and gas-chromatography/mass spectrometry. [3H]12-KETE is formed from endogenous lipid stores in nervous tissue, labeled with [3H]arachidonic acid upon stimulation by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. Another metabolite of 12-HPETE, 12(s)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE], is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.
Assuntos
Ácidos Araquidônicos/isolamento & purificação , Gânglios/fisiologia , Sistema Nervoso/análise , Animais , Aplysia , Ácidos Araquidônicos/biossíntese , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Técnicas In Vitro , Sistema Nervoso/metabolismoRESUMO
Nervous tissue of the marine mollusc, Aplysia californica, generates arachidonic acid metabolites in response to neurotransmitters such as histamine or FMRFamide. In addition, identified neurons of Aplysia respond to the pharmacologic application of some of these products, particularly those of the 12-lipoxygenase pathway. We investigated the chirality of the initial Aplysia 12-lipoxygenase product, 12-HPETE, in preparation for more detailed metabolic studies and for the analysis of the physiological activity of the endogenous lipid. Neural homogenates and intact ganglia exclusively generate 12(S)-HPETE as do the better characterized mammalian lipoxygenases. The direct application of 12(S)-HPETE to cultured sensory neurons induced a hyperpolarization which averaged 2.6 mV. We did not find any difference between the response to the naturally-occurring 12(S)-HPETE and its diastereomer, 12(R)-HPETE which is not generated in Aplysia. Both isomers were significantly more effective than 15(S)-HPETE. In contrast, 12(S)-HPETE, but not 12(R)-HPETE, was a potent modulator of the action of the molluscan neuropeptide, FMRFamide. Prior application of 12(S)-HPETE to cultured sensory neurons increased the subsequent response to a submaximal dose of FMRFamide by 60%. On the other hand, 12(R)-HPETE reduced the subsequent response to the peptide by 30%. The lack of stereospecificity in the direct effect of the lipids differs markedly from their stereospecific effects as modulators of FMRFamide action. This suggests that there may be an important neurophysiologic role for these lipid modulators which is distinct from their direct effects, and also indicates that there are multiple sites and mechanisms by which lipid hydroperoxides act on neurons in Aplysia.
Assuntos
Aplysia/enzimologia , Araquidonato 12-Lipoxigenase/metabolismo , Hormônios de Invertebrado/metabolismo , Leucotrienos/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Vasoconstritores/metabolismo , Animais , Araquidonato 12-Lipoxigenase/química , Células Cultivadas/enzimologia , Eletrofisiologia , FMRFamida , Leucotrienos/isolamento & purificação , Potenciais da Membrana/fisiologia , Neurônios Aferentes/enzimologia , Estereoisomerismo , Vasoconstritores/isolamento & purificaçãoRESUMO
Physiologic stimulation of identified neurons in ganglia of the marine mollusk, Aplysia californica, leads to the generation of arachidonic acid metabolites. Using various preparations of Aplysia nervous tissue, we have identified 12-lipoxygenase products including the inactive 12-hydroxyeicosatetraenoic acid (12-HETE) and the biologically active 12-ketoeicosatetraenoic acid (12-KETE) and 8-hydroxy-11(12)-epoxyeicosatrienoic acid (8-HEpETE). Each of these metabolites can be derived from the intermediate 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can itself activate several identified neurons in Aplysia. In spite of conflicting results in studies of mammalian brain 12-lipoxygenase, Aplysia nervous tissue clearly contains an enzymatic activity which generates stereochemically pure 12(S)-HETE. This activity is destroyed by boiling and is sensitive to nonspecific lipoxygenase inhibitors but not to agents specific for other lipoxygenases or the cyclooxygenase enzyme. The Aplysia 12-lipoxygenase is highly enriched in neural tissue and is almost completely absent in the neural sheath, which is composed primarily of connective tissue and muscle. Preliminary purification has shown that, in contrast to the previously characterized 12-lipoxygenases, the Aplysia enzyme is associated with membrane fractions and is not found in the cytosol. Further studies are in progress to determine the kinetic properties and to define the cellular and subcellular distribution of this novel lipoxygenase.
Assuntos
Aplysia/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Animais , Gânglios/metabolismo , Leucotrienos/biossíntese , Leucotrienos/química , Neurônios/metabolismo , Estereoisomerismo , Distribuição TecidualAssuntos
Ácidos Araquidônicos/farmacologia , Grânulos Citoplasmáticos/enzimologia , Neutrófilos/enzimologia , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Citocalasina B/farmacologia , Glucuronidase/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Leucotrieno B4 , Muramidase/metabolismo , Neutrófilos/efeitos dos fármacos , Oxirredução , EstereoisomerismoAssuntos
Comunicação Celular , Endotélio Vascular/fisiologia , Neutrófilos/fisiologia , SRS-A/biossíntese , Animais , Separação Celular/métodos , Células Cultivadas , Técnicas de Cultura/métodos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Leucotrieno A4 , Leucotrienos/metabolismo , Leucotrienos/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , SuínosAssuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato Lipoxigenases/metabolismo , Gânglios/enzimologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animais , Aplysia , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Ácidos Graxos/análise , Gânglios/fisiologia , Histamina/farmacologia , Ácidos Hidroxieicosatetraenoicos/biossíntese , Ácidos Hidroxieicosatetraenoicos/isolamento & purificação , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Neurônios/fisiologiaRESUMO
Endothelial cells do not contain 5-lipoxygenase and thus are unable to generate LTA4 from arachidonate. Nonetheless, endothelial cells may play an important role in leukotriene synthesis by virtue of their ability to metabolize LTA4 derived from activated polymorphonuclear leukocytes (PMNL) and to modulate PMNL 5-lipoxygenase activity. Porcine aortic endothelial cells were found to metabolize exogenous LTA4 to LTC4, and under some conditions human umbilical vein endothelial cells have been found to generate LTB4. Production of LTB4 by these cells appears to be under poorly understood cellular control, and it remains a controversial area of research. Under physiologic conditions, endothelial cells are in constant contact with circulating PMNL, which are known to generate substantial amounts of LTA4. When these two cell types are coincubated in vitro, clear evidence of transcellular metabolism of PMNL-derived LTA4 to LTC4 by endothelial cells is found. Coincubations produce from two to greater than 10 times more LTC4 than either cell alone. In contrast to these findings, when these cells were activated by the receptor-mediated agonist fMLP, evidence for an endothelial cell inhibition of PMNL 5-lipoxygenase was obtained. Rather than augmentation of LTC4 production, as seen with A23187 activation, coincubation activated by fMLP generated significantly less LTC4 (0.23 +/- 0.08 versus 0.75 +/- 0.39 pmol/10(7) cells). The endothelial cell inhibition was removed when these cells were pretreated with aspirin, suggesting that their major cyclooxygenase product, prostacyclin, acts as a feedback regulator of LT synthesis. When cyclooxygenase was blocked, significant transcellular LTC4 synthesis was once again apparent (1.66 +/- 0.44 pmol/10(7) cells).
Assuntos
Endotélio Vascular/imunologia , Inflamação/imunologia , Leucotrienos/biossíntese , Animais , Aspirina/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Epoprostenol/imunologia , Estudos de Avaliação como Assunto , Cobaias , Inflamação/tratamento farmacológico , Leucotrienos/classificação , Leucotrienos/imunologia , Masculino , Neutrófilos/imunologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/imunologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Radioimunoensaio , SuínosRESUMO
Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.
Assuntos
Ácidos Araquidônicos/sangue , Endotélio/metabolismo , Neutrófilos/metabolismo , SRS-A/biossíntese , Animais , Aorta/metabolismo , Ácidos Araquidônicos/biossíntese , Células Cultivadas , Glutationa/metabolismo , Cobaias , Humanos , Leucotrieno A4 , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Masculino , SRS-A/farmacologia , SuínosRESUMO
Unsaturated fatty acids, such as arachidonic acid, have been implicated as second messengers of various cellular responses. In this paper, we demonstrate that the release of arachidonate from human polymorphonuclear leukocytes (PMNL) via a pathway initiated by phospholipase D (PLD) mediates phorbol myristate acetate (PMA)-induced desensitization of leukotriene B4 (LTB4) receptors. PMA caused a delayed release of arachidonic acid from PMNL, which occurred within minutes of the generation of the PLD products, phosphatidic acid and diglyceride, from [3H]lysophosphatidylcholine prelabeled PMNL. Moreover, stimulating PMNL with PMA in the presence of ethanol resulted in the formation of phosphatidylethanol at the expenses of phosphatidic acid, diglyceride and arachidonic acid. The PMA-induced generation of these three PLD products was inhibited by mepacrine which, in parallel, significantly blocked PMA-induced desensitization of [3H]LTB4 binding, which suggested that elevation of one or more of these products played a role in desensitization. In contrast, under basal conditions mepacrine reduced levels of these three lipids in a dose-related manner and, in parallel, increased basal [3H]LTB4 binding to PMNL. The reduction of PMA-induced LTB4 receptor desensitization by mepacrine could be overcome to various degrees by adding back PLD-derived lipids such as arachidonic acid. These data demonstrate that [3H]LTB4 binding to PMNL is decreased by PLD-derived lipids and suggests that intracellular arachidonic acid may modulate PMNL responsiveness to LTB4 activation.
Assuntos
Leucócitos/efeitos dos fármacos , Leucotrieno B4/farmacologia , Fosfolipase D/farmacologia , Ligação Competitiva , Relação Dose-Resposta a Droga , Etanol/farmacologia , Humanos , Quinacrina/farmacologiaRESUMO
Thromboxane A2 (rabbit aorta-contracting substance) is a proaggregatory vasoconstrictive, oxygenated metabolite of arachidonic acid which was originally discovered in guinea pig lung perfusates during antigen-induced anaphylaxis. The specific stimuli which activate synthesis and the cellular source in the lung remain undefined. In order to study pulmonary thromboxane A2 (TXA2) synthesis, a cultured lung cell model has been used. Monolayer cultures of human diploid embryonic lung fibroblast (WI-38) metabolized exogenously supplied [14C]arachidonic acid to TXA2 as well as prostaglandin E2. Both were unequivocally identified by gas chromatography/mass spectrometry. Cellular phospholipids were labeled by preincubating cultures overnight with [14C]arachidonic acid. Release of thromboxane A2 into the culture fluid from these prelabeled cultures was stimulated by two phospholipase activating agents, mellitin and the calcium ionophore A23187. The lung cells also released TXA2 and prostaglandin in a dose-dependent fashion when treated with thrombin but not when exposed to trypsin. Bradykinin, an anaphylactic mediator in vivo, was a potent TXA2 releasing agent in this in vitro system whereas histamine was inactive. In addition, anaphylactic shock perfusates from guinea pig lung were shown to contain a factor (other than bradykinin) which activates fibroblasts TXA2 synthesis in these cultured lung cells. These experiments indicate that the lung fibroblast is probably a source of pulmonary thromboxane in vivo and that the cultured lung cell system described here is a useful model for defining the complex interactions of mediators of anaphylaxis and asthma.
Assuntos
Metabolismo dos Lipídeos , Pulmão/metabolismo , Tromboxano A2/biossíntese , Tromboxanos/biossíntese , Anafilaxia/fisiopatologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Radioisótopos de Carbono , Linhagem Celular , Dinoprostona , Feminino , Fibroblastos/metabolismo , Cobaias , Humanos , Pulmão/embriologia , Prostaglandinas E/biossínteseRESUMO
Canine and human epicardial coronary arteries were tested for their ability to metabolize exogenous arachidonic acid to lipoxygenase products. Unextracted medium from incubations of canine or human arteries with arachidonic acid and the calcium ionophore, A23187, contained a substance which exhibited a leukotriene (LT)-like smooth muscle contracting activity when tested on the superfused guinea pig lung parenchymal strip bioassay. This activity could be blocked by the LT antagonist, FPL-55712. Compounds present in these media were purified by high-performance liquid chromatography and identified as LTC4, LTD4 and LTE4 by liquid scintillation counting, bioassay and radioimmunoassay. In addition, coronary artery rings converted synthetic [3H]LTC4 to [3H]LTD4 with a half-life of 44 +/- 8 min. LTD4 metabolism to LTE4 was also demonstrated. The metabolism of [3H]LTC4 was abolished by incubation of the arterial rings with a gamma-glutamyl transpeptidase inhibitor, serineborate. Production of monohydroxyeicosatetraenoic acids (5-, 12- and 15-HETE) which have been isolated previously from vascular tissue incubations was confirmed. Production of HETE was inhibited by nordihydroguaiaretic acid and unaffected by indomethacin. These findings indicate that coronary vessels can metabolize exogenous arachidonic acid by the lipoxygenase pathway. In addition to HETE, the vessels were shown to synthesize LTC4, LTD4 and LTE4, compounds which possess potent biological actions on the coronary circulation.
Assuntos
Vasos Coronários/metabolismo , SRS-A/biossíntese , Animais , Bioensaio , Cromatografia Líquida de Alta Pressão , Cães , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Indometacina/farmacologia , Masculino , Masoprocol/farmacologiaRESUMO
INTRODUCTION: Both ischemia and reperfusion are associated with ventricular arrhythmias. In both instances, neutrophils migrate into the ischemic zone, are activated by locally released factors, and bind to myocytes. The activated neutrophils liberate platelet activating factor (PAF). We have studied the arrhythmogenic actions of PAF on transmembrane potentials of isolated canine cardiac myocytes. METHODS AND RESULTS: Cardiac myocytes were prepared from normal canine hearts by standard methods and studied in vitro by recording transmembrane potentials under control conditions and during exposure to graded doses of PAF, usually 0.25 to 1.25 micrograms (0.25 to 1.2 microM). Myocytes were superfused with Tyrode's solution (2.0 mL/min), paced at a cycle length of 1000 msec, and maintained at a temperature between 36 degrees and 38 degrees C. PAF caused a consistent and dose-dependent set of alterations in the transmembrane potential, including increased action potential duration, runs of early afterdepolarizations (EADs), and transient arrest of repolarization (PA). In addition, in some myocytes PAF caused intermittent small depolarizations both at the plateau voltage and resting potential. The effects of PAF were transient: only some residual action potential prolongation was noted after Tyrode's washout for 5 minutes. Effects of PAF were blocked in a dose-dependent manner by the PAF receptor antagonist, CV-6209. Both tetrodotoxin (1.2 x 10(-6) M) and xylocaine (5 x 10(-5) M) antagonized the ability of PAF to cause EADs and PA. CONCLUSIONS: PAF consistently exerts arrhythmogenic effects on the membrane of ventricular myocytes. Since PAF is liberated by activated neutrophils and since activated neutrophils migrate into ischemic myocardium on reperfusion, we judge that PAF liberated by such neutrophils is an important arrhythmogenic factor for reperfusion arrhythmias. The same mechanism may be a cause of arrhythmias during the evolution of infarction.