RESUMO
Human polymorphonuclear (PMN) leukocytes bound [3H]leukotriene B4 ([3H]-LTB4) specifically, as assessed by the displacement of 88% or more of the bound radioactivity by a 15,000-fold higher concentration of nonradioactive LTB4 or by micromolar concentrations of structural isomers of LTB4. The specific binding of [3H]LTB4 by PMN leukocytes was characterized by rapid association and dissociation, and was saturable at 800 nM LTB4. The results of computer analyses of the concentration dependence of binding of [3H]LTB4 were consistent with the expression of two classes of receptors having respective mean affinities of 3.9 X 10(-10) M and 6.1 X 10(-8) M and mean densities of 4.4 X 10(3) and 2.7 X 10(5) per PMN leukocyte. Structural isomers of LTB4 inhibited the binding of [3H]LTB4 to PMN leukocytes at concentrations similar to those required to elicit chemotaxis, while chemotactic peptides did not inhibit binding. PMN leukocytes that were deactivated by prior exposure to LTB4 lost high affinity binding sites selectively and concurrently with a reduction in the chemotactic response to LTB4. Chemotactic deactivation altered, but did not eliminate, the low affinity receptors for LTB4 and reduced only minimally the lysosomal degranulation elicited by LTB4. The high affinity receptors for LTB4 on normal human PMN leukocytes appear to transduce the chemotaxis evoked by LTB4 without substantially modifying lysosomal degranulation.
Assuntos
Quimiotaxia de Leucócito , Ácidos Hidroxieicosatetraenoicos , Neutrófilos/metabolismo , Receptores de Superfície Celular/análise , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Ácidos Araquidônicos/farmacologia , Ligação Competitiva , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Cinética , Lisossomos/enzimologia , Neutrófilos/fisiologia , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/efeitos dos fármacos , Receptores do Leucotrieno B4RESUMO
Chemotactic factors stimulate a rapid increase in the cytosolic concentration of intracellular calcium ions ([Ca2+]in) in human polymorphonuclear leukocytes (PMNL), which may be an event that is critical to the expression of chemotaxis and other PMNL functions. Treatment of PMNL with pertussis toxin catalyzes ADP-ribosylation of a protein similar or identical to the inhibiting regulatory protein of adenylate cyclase, Gi, and suppresses the increase in [Ca2+]in elicited by leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine. Chemotactic migration and lysosomal enzyme release elicited by chemotactic factors were inhibited by pertussis toxin with a concentration-dependence similar to that for inhibition of the increase in [Ca2+]in, without an effect on lysosomal enzyme release induced by the ionophore A23187 and phorbol myristate acetate. Activated pertussis toxin catalyzed the [32P]ADP-ribosylation of a 41 kD protein in homogenates of PMNL. The extent of [32P]ADP-ribosylation of this protein was reduced 59% by pretreatment of intact PMNL with pertussis toxin. Pertussis toxin selectively decreased the number of high-affinity receptors for LTB4 on PMNL by 60% without altering the number or binding properties of the low-affinity subset of receptors. Pertussis toxin modification of a membrane protein of PMNL analogous to Gi thus simultaneously alters chemotactic receptors and attenuates the changes in cytosolic calcium concentration and PMNL function caused by chemotactic factors.
Assuntos
Toxinas Bacterianas/farmacologia , Cálcio/sangue , Fatores Quimiotáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Adenosina Difosfato Ribose/sangue , Toxina Adenilato Ciclase , Quimiotaxia/efeitos dos fármacos , Citosol/metabolismo , Proteínas de Ligação ao GTP/sangue , Glucuronidase/sangue , Humanos , Técnicas In Vitro , Neutrófilos/fisiologia , Toxina Pertussis , Receptores Imunológicos/efeitos dos fármacos , Receptores do Leucotrieno B4 , Fatores de Virulência de BordetellaRESUMO
The synthesis of large quantities of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) relative to 1-alkyl-2-acetyl-GPC (PAF; platelet-activating factor) has been demonstrated in several inflammatory cells. The present study has examined agonist and antagonist activities of 1-acyl-2-acetyl-GPC in the human neutrophil. 1-Acyl-2-acetyl-GPC induced a rapid increase in cytosolic calcium in the neutrophil; this effect was detected at 2 x 10(-9) M and was maximal at 10(-6) M. The peak response induced by 1-acyl-2-acetyl-GPC was similar to that induced by PAF although the potency of 1-acyl-2-acetyl-GPC was 300-fold lower than that of PAF. The dose response curves for both 1-acyl-2-acetyl-GPC and PAF were shifted in a parallel fashion by L-652,731 (10(-6) M), a PAF receptor antagonist, suggesting that both 1-acyl-2-acetyl-GPC and PAF act on the same receptor. High concentrations of 1-acyl-2-acetyl-GPC (10(-5) M) induced the release of beta-glucuronidase and lysozyme from the human neutrophil. The percent release of lysozyme induced by 1-acyl-2-acetyl-GPC was consistently higher than that of beta-glucuronidase. Prior stimulation of neutrophils with 1-acyl-2-acetyl-GPC dose-dependently inhibited the increase in cytosolic calcium induced by a subsequent challenge with an optimal concentration of PAF. Similarly, preincubation of neutrophils with 1-acyl-2-acetyl-GPC dose-dependently inhibited beta-glucuronidase and lysozyme release induced by a subsequent stimulation with PAF. The inhibitory effect on degranulation could not be surmounted even by concentrations of PAF 10-fold higher than that of 1-acyl-2-acetyl-GPC. The inhibition appeared to be selective for PAF since 1-acyl-2-acetyl-GPC did not affect f-met peptide-induced degranulation. This study suggests that 1-acyl-2-acetyl-GPC may act as a naturally-occurring specific inhibitor of PAF-induced activation of the human neutrophil.
Assuntos
Neutrófilos/efeitos dos fármacos , Fator de Ativação de Plaquetas/análogos & derivados , Glicoproteínas da Membrana de Plaquetas , Receptores Acoplados a Proteínas G , Cálcio/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Furanos/farmacologia , Glucuronidase/metabolismo , Humanos , Muramidase/metabolismo , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Estimulação QuímicaAssuntos
Leucotrieno B4/metabolismo , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Cálcio/metabolismo , Quimiotaxia de Leucócito , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Proteína Quinase C/metabolismo , Receptores do Leucotrieno B4 , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.
Assuntos
Ácidos Araquidônicos/metabolismo , Quimiotaxia de Leucócito , Neutrófilos/metabolismo , Receptores de Superfície Celular/metabolismo , Humanos , Cinética , Leucotrieno B4 , Receptores do Leucotrieno B4 , Relação Estrutura-AtividadeRESUMO
Human polymorphonuclear leukocytes express two classes of receptors specific for leukotriene B4 (LTB4). The binding of LTB4 to the high-affinity, low-capacity receptors is critical in eliciting a chemotactic response, as assessed by the stereospecificity of binding and the loss of expression of high-affinity receptors after chemotactic deactivation. The stereospecificity and concentration dependence of LTB4 stimulation of granular enzyme release suggest that the low-affinity, high-capacity receptors mediate this response. Although the LTB4 receptors exhibit properties similar to those of the receptors for peptide chemotactic stimuli, the expression of both classes of receptors on intact polymorphonuclear leukocytes will permit a more complete assessment of the coupling of receptor occupancy to functional activation and the delineation of LTB4 receptor-level defects in human disease states.
Assuntos
Leucotrieno B4/sangue , Neutrófilos/fisiologia , Receptores de Superfície Celular/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Cinética , Leucotrieno B4/farmacologia , Neutrófilos/efeitos dos fármacos , Receptores do Leucotrieno B4 , TrítioRESUMO
The course of pregnancy in patients with systemic lupus erythematosus is not known. The Hopkins Lupus Pregnancy Center has followed 64 patients (74 pregnancies) prospectively since 1987. Patients are seen monthly and clinical and pregnancy-related data collected, with particular emphasis on the occurrence of lupus flare. Flare rate during pregnancy was 1.63 per person-year, compared to 0.64-0.65 after delivery or in non-pregnant patients. Flare did not influence pregnancy outcome. Low serum C3 or C4 and high anticardiolipin antibody predicted pregnancy loss, and prednisone dose, aspirin use, diastolic second trimester blood pressure, C3 at first visit, and race predicted preterm birth. Maternal flare and preterm birth are important risks in lupus pregnancy. The latter can be predicted from maternal pregnancy data.
Assuntos
Lúpus Eritematoso Sistêmico/epidemiologia , Complicações na Gravidez/epidemiologia , Resultado da Gravidez/epidemiologia , Aborto Espontâneo/epidemiologia , Aborto Espontâneo/etiologia , Síndrome Antifosfolipídica/epidemiologia , Baltimore/epidemiologia , Biomarcadores , Feminino , Humanos , Recém-Nascido , Trabalho de Parto Prematuro/epidemiologia , Razão de Chances , Gravidez , Estudos Prospectivos , Fatores de Risco , alfa-Fetoproteínas/análiseRESUMO
Rat hepatocyte homogenates converted 5-hydroperoxyeicosatetraenoic acid into leukotriene B4 (LTB4). The reaction was dependent on time and protein and substrate concentration, did not require NADPH or oxygen, and was not supported by heat-inactivated hepatocyte homogenates. The authenticity of the biologically generated LTB4 that eluted at the position of synthetic LTB4 during high performance liquid chromatography was established by UV spectrophotometry, mass spectral analysis, radioimmunoassay, and a LTB4 receptor displacement assay. In addition, a leukotriene bioassay is described in which transient increases in cytosolic Ca2+ within human neutrophils are measured by means of fura-2 fluorescence. Biologically generated LTB4 was 40, 40, and 33% as active as synthetic LTB4 in the radioimmunoassay, receptor displacement assay, and cytosolic calcium bioassay, respectively. This activity is consistent with the biologically derived LTB4 being an epimeric mixture of (5S),(12R)-LTB4 and the much less active (5S),(12S)-LTB4. The formation of LTB4 was inhibited by 5,8,11,14-eicosatetraynoic acid (1 mM), 5,6-dehydro-arachidonic acid (50 microM), propanethiol (1 mM), and O2 (100%) to the extent of 53, 42, 48, and 66%, respectively. No inhibition was observed in the presence of diethylcarbamazine (1 mM) and desferal (1 mM). A possible contribution towards LTB4 formation by contaminating Kupffer cells was excluded (less than 0.2%). These results suggest that hepatocytes can convert lipid peroxides into potent chemoattractants that may alter the homeostasis of immunomediators within the liver.
Assuntos
Ácidos Araquidônicos/metabolismo , Leucotrieno B4/biossíntese , Leucotrienos , Fígado/metabolismo , Animais , Dietilcarbamazina/farmacologia , Técnicas In Vitro , Leucotrieno A4 , Inibidores de Lipoxigenase , Masculino , Espectrometria de Massas , Ratos , Ratos Endogâmicos , Receptores Imunológicos/análise , Receptores do Leucotrieno B4RESUMO
Human platelet factor 4 (PF4) and a substituent dodecapeptide designated PF4(59-70) elicited human neutrophil and monocyte chemotaxis with a similar concentration-dependence and maximal responses equal to that attained by chemotactic fragments of C5 (C5fr). At maximally chemotactic concentrations, PF4(59-70) stimulated the secretion by neutrophils of approximately 40% and 60% of the respective quantities of beta-glucuronidase and beta-glucosaminidase released by 10(-6) M N-formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast to the deactivation of chemotaxis achieved by preincubation of neutrophils with other chemotactic factors, prior exposure to 10(-6)M PF4(59-70) for 2 min, or 20 min at 37 degrees, enhanced by 1.5- to 2-fold the chemotactic responses of neutrophils evoked by optimal concentrations of fMLP, C5fr, leukotriene B4, and PF4(59-70). Concentrations of PF4(59-70) which enhanced neutrophil chemotaxis inhibited the rate of receptor-mediated internalization of [3H]fMLP at 37 degrees and 18 degrees, but at 0 degrees failed to alter the binding affinity or the number of receptors for [3H]fMLP. Preincubation of neutrophils at 37 degrees with concentrations of PF4(59-70) which enhanced neutrophil chemotaxis also did not affect the subsequent binding of [3H]fMLP at 0 degrees. The inhibition by PF4(59-70) of the receptor-mediated internalization of [3H]fMLP was not mimicked by other positively charged compounds. The specific inhibition of receptor-mediated internalization of fMLP may explain the enhanced chemotactic responsiveness of neutrophils preincubated with PF4(59-70).
Assuntos
Quimiotaxia de Leucócito , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Fragmentos de Peptídeos/imunologia , Fator Plaquetário 4/imunologia , Receptores Imunológicos/metabolismo , Acetilglucosaminidase/metabolismo , Glucuronidase/metabolismo , Humanos , Monócitos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
The functionally predominant constituents of the slow-reacting substance of anaphylaxis (SRS-A), designated leukotrienes C4 and D4 (LTC4 and LTD4), as well as the leucocyte chemotactic factor leukotriene B4 (LTB4) enhance the adherence of human neutrophils to Sephadex G-25. Enhancement of neutrophil adherence was significant at leukotriene concentrations of 3 X 10(-9) M -3 X 10(-7) M, and reached a maximum level for each of the leukotrienes that was similar in magnitude to that evoked by the neutrophil chemotactic peptide N-formyl-methionyl-leucylphenylalanine (FMLP). The leukotrienes and FMLP elicited optimum increases in neutrophil adherence within 1-2 min at 37 degrees. Indomethacin inhibited the increase in neutrophil adherence evoked by LTC4 and LTD4 and the concurrent elevation in the concentration of endogenous thromboxane B2. The smooth muscle contractile and vasoactive factors LTC4 and LTD4, which lack chemotactic activity for leucocytes, are as active as LTB4 in stimulating human neutrophil adherence, and the effect may be mediated in part by neutrophil-derived thromboxane A2.
Assuntos
Leucotrieno B4/farmacologia , Neutrófilos/efeitos dos fármacos , SRS-A/farmacologia , Ácidos Araquidônicos/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Dextranos , Relação Dose-Resposta a Droga , Géis , Humanos , Indometacina/farmacologia , N-Formilmetionina/análogos & derivados , N-Formilmetionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/metabolismo , Oligopeptídeos/farmacologia , Tromboxano B2/metabolismo , Fatores de TempoRESUMO
Leukotriene B4 exhibited 10- to 30-fold greater chemotactic potency for human neutrophils than eicosapentaenoic acid-derived leukotriene B5, as assessed in modified Boyden micropore filter chambers. In contrast, leukotrienes B4 and B5 were equipotent stimuli of human neutrophil lysosomal degranulation in vitro, as quantified by the release of beta-glucosaminidase. Analyses of competitive inhibition of the binding of [3H] leukotriene B4 to neutrophils indicated that leukotriene B4 binds with a 500-fold greater association constant than leukotriene B5 to a subclass of high-affinity receptors, which appears to transduce chemotactic responses efficiently, while leukotrienes B4 and B5 bind equally well to low-affinity receptors.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Leucotrieno B4/farmacologia , Neutrófilos/fisiologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Ácido Eicosapentaenoico , Ácidos Graxos Insaturados , Humanos , Isomerismo , Cinética , Leucotrieno B4/sangue , Lisossomos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (NaDodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed three predominant membrane proteins of approximate molecular weight 94 000 (MP-1), 68 000 (MP-2), and 40 000 (MP-3), of which MP-2 accounted for 74--93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2--0.3 and mean KA values of 9 x 10(8) and 2 x 10(7) M-1, respectively, and low-affinity sites with a valence of 0.3--0.5 and mean KA values of 3 x 10(7) and 2 x 10(6) M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepaharose column to which had been coupled chemotactic fragments of the fifth component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides.
Assuntos
Fatores Quimiotáticos/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Neutrófilos/química , Receptores de Superfície Celular/isolamento & purificação , Aminoácidos/análise , Membrana Celular/química , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Peso Molecular , N-Formilmetionina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina , Oligopeptídeos , Receptores de Formil PeptídeoRESUMO
The recent definition of the pathways of generation and structures of diverse products of the lipoxygenation of arachidonic acid has established the identity of a new family of mediators of hypersensitivity and inflammation. Studies of the effects of these mediators have shown that leukotrienes C, D, and E, the constitutents of the slow-reacting substance of anaphylaxis (SRS-A), are extremely potent smooth muscle contractile and vasoactive factors. Leukotriene B is a highly active stimulus of neutrophil and eosinophil functions and suppresses the immunological capabilities of T lymphocytes. The development of specific and sensitive radioimmunoassays has permitted the detection of elevated concentrations of leukotrienes in tissues or exudates in several diseases, including asthma, diverse allergic states, adult respiratory distress syndrome, psoriasis, spondyloarthritis, and gout. The application of selective inhibitors and antagonists of leukotrienes will clarify their pathogenetic contributions in human diseases and may yield new therapeutic approaches.
Assuntos
Ácidos Araquidônicos/fisiologia , Leucotrieno B4/fisiologia , SRS-A/análogos & derivados , SRS-A/fisiologia , Araquidonato Lipoxigenases , Ácidos Araquidônicos/biossíntese , Ácidos Araquidônicos/metabolismo , Artrite/metabolismo , Asma/metabolismo , Fibrose Cística/metabolismo , Humanos , Hipersensibilidade/metabolismo , Leucotrieno A4 , Leucotrieno B4/biossíntese , Leucotrieno B4/metabolismo , Leucotrieno E4 , Lipoxigenase/metabolismo , Psoríase/metabolismo , SRS-A/biossíntese , SRS-A/metabolismo , Lágrimas/análiseRESUMO
Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Complemento C5/metabolismo , Complemento C5a , Endotoxinas/farmacologia , Leucotrieno B4/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/efeitos dos fármacos , Coelhos , Receptores de Complemento/metabolismo , Receptores de Formil PeptídeoRESUMO
Attempts to evaluate the role of kinins in airway inflammation in humans using the bradykinin receptor antagonist [DArg0-Hyp3-DPhe7]-bradykinin (NPC 567) were unsuccessful, possibly because of the low potency and poor stability of this compound. Recently, [DArg0-Hyp3-Thi5-DTic7-Oic8]-bradykinin (Hoe 140), a novel antagonist that seems to overcome these weaknesses, has been developed. The present study was performed to compare the potency and efficacy of Hoe 140 to those of NPC 567 and another antagonist, [DArg0-Hyp3-DPhe7-Ile8]-bradykinin (B7418), on kinin receptors on guinea pig tracheal epithelial cells. Radioligand binding studies showed the presence of two types of B2 kinin receptors on guinea pig tracheal epithelial cells: a high-affinity site with a Kd of 0.44 nM and Bmax of 12.1 fmol/10(6) cells (4000 sites/cell), and a lower affinity site with a Kd of 10 nM and Bmax of 16 fmol/10(6) cells (9600 sites/cell). Bradykinin-induced prostaglandin E2 production seemed to be associated primarily with the lower affinity site. All three B2 receptor antagonists displaced labeled bradykinin from both classes of binding sites and inhibited bradykinin-induced prostaglandin E2 production, but Hoe 140 was up to 40-fold more potent than NPC 567 and showed an affinity comparable to that of bradykinin for both binding sites. This higher potency of Hoe 140, and its stability against peptidases, suggests that this compound will be useful in evaluating the role of bradykinin in inflammatory diseases of the airways.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/antagonistas & inibidores , Bradicinina/farmacologia , Receptores de Neurotransmissores/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Animais , Bradicinina/metabolismo , Células Cultivadas , Dinoprostona/biossíntese , Células Epiteliais , Epitélio/efeitos dos fármacos , Cobaias , Cininas/metabolismo , Masculino , Receptores da Bradicinina , Receptores de Neurotransmissores/fisiologiaRESUMO
The effects of histamine on lung macrophages have been studied by both biologic and radioligand experiments. After overnight adherence, lung macrophages spontaneously released beta-glucuronidase (beta-G) at a rate of approximately 7 nmol of hydrolyzed substrate/h/million cells. Histamine at low concentrations (10(-9) to 10(-8) M) resulted in a consistent potentiation of this release. The concentration-effect curve of histamine was bell-shaped, reaching an optimum at 10(-9) M, with concentrations greater than 10(-8) M having no significant effect. At a maximally effective concentration (10(-9) M), histamine evoked a 135 +/- 9.6% (mean +/- SE; n = 8, P less than 0.001) potentiation in the total amount of beta-G released during the first 60 min of incubation. This increase in beta-G release represented both a slight increase in beta-G synthesis as well as an increase in the percentage of beta-G released. When the secreted beta-G is expressed as a percentage of total content, histamine (10(-9) M) evoked a 125 +/- 3.2% (mean +/- SE; n = 27, P less than 0.0005) enhancement. The potentiation of beta-G release by histamine was evident after 45 min of incubation and persisted for up to 6 h. The potentiation of beta-G by histamine was sensitive to inhibition by pyrilamine (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glucuronidase/metabolismo , Histamina/farmacologia , Pulmão/citologia , Macrófagos/enzimologia , Receptores Histamínicos H1/fisiologia , Adulto , Ligação Competitiva , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Feminino , Histamina/administração & dosagem , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Pirilamina/metabolismo , Pirilamina/farmacologia , Receptores Histamínicos H1/efeitos dos fármacos , Fumar/metabolismoRESUMO
The distinctive characteristics of human polymorphonuclear (PMN) leukocyte receptors for leukotriene B4 (LTB4) have been elucidated by studies of binding of [3H]LTB4, the structure of protein constituents of the receptors isolated from plasma membranes, and the effects of antireceptor antibodies. A high-affinity class of 4400 receptors with a KD of 0.4 nM mediates chemotaxis and increased adherence of PMN leukocytes, whereas a low-affinity class of 270,000 receptors with a KD of 61 nM mediates the release of lysosomal enzymes and increases in oxidative metabolism. The low-affinity receptors are composed of a 60,000-dalton protein-binding unit. The high-affinity receptors are composed of the same binding unit in association with a 40,000-dalton guanine nucleotide-binding protein. That antireceptor antibodies as well as LTB4 distinguish the two classes of receptors with different functional consequences suggests the possibility of unique approaches to the regulation of leukocyte function at the receptor level.
Assuntos
Leucotrieno B4/metabolismo , Neutrófilos/análise , Receptores Imunológicos/isolamento & purificação , Anticorpos Monoclonais/imunologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Inflamação , Modelos Biológicos , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores do Leucotrieno B4RESUMO
Bradykinin (BK) is a weak stimulus for prostaglandin E2 (PGE2) release in untreated human synovial cells, but a potent stimulus in interleukin-1 (IL-1) pretreated cells. The mechanism(s) by which IL-1 induces responsiveness of synovial cells to BK appears to be multifactorial. IL-1 not only upregulates the number of kinin receptors on these cells, but may also upregulate a calcium-dependent process involved in the synthesis of prostaglandins.
Assuntos
Artrite Reumatoide/metabolismo , Bradicinina/metabolismo , Bradicinina/farmacologia , Cálcio/metabolismo , Dinoprostona/metabolismo , Interleucina-1/farmacologia , Receptores de Neurotransmissores/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Cinética , Receptores da Bradicinina , Proteínas Recombinantes/farmacologia , Líquido Sinovial/citologia , Líquido Sinovial/fisiologiaRESUMO
Bradykinin has been implicated in the pathogenesis of inflammatory arthritis by virtue of its potent proinflammatory properties. We have previously shown bradykinin to be a potent stimulus for the release of prostanoids from interleukin-1 (IL-1)-treated, but not untreated, human synovial cells. We hypothesize that one mechanism by which IL-1 induces responsiveness to bradykinin is by upregulation of number or affinity of kinin receptors on human synovial cells. We performed [3H]bradykinin binding studies in intact human synovial tissue and in cultured human synovial cells. Specific, saturable [3H]bradykinin binding sites in intact synovia were identified by autoradiographic localization and were present in much higher density in rheumatoid, than in osteoarthritis, synovia. In untreated human synovial cells in culture, a single (B2) class of kinin binding sites with a Kd of 2.3 nM and Bmax of 58 +/- 9 fmol/10(6) cells was demonstrated. In matched experiments, IL-1 treatment enhanced specific [3H]bradykinin binding 1.5- to 2.0-fold above that observed in untreated cells. This enhancement was attributable to an increase in Bmax (53 +/- 4 vs. 105 +/- 24 fmol/10(6) cells in untreated and IL-1-treated cells, respectively), rather than an alteration in Kd (1.7 and 1.4 nM, respectively). The potencies of a series of kinin analogs and antagonists and unrelated peptides in displacing [3H]bradykinin from IL-1-treated cells correlated well with their abilities to induce prostanoid release. These studies provide novel information regarding the nature of kinin receptors in intact human synovia and in cultured human synovial cells, their regulation by IL-1 and their role in IL-1-treated cells in kinin-mediated prostaglandin E2 production.
Assuntos
Interleucina-1/farmacologia , Receptores de Neurotransmissores/fisiologia , Membrana Sinovial/ultraestrutura , Regulação para Cima/efeitos dos fármacos , Autorradiografia , Sítios de Ligação , Bradicinina/metabolismo , Humanos , Cininas/metabolismo , Receptores da Bradicinina , Receptores de Neurotransmissores/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/fisiologia , TrítioRESUMO
During a 2-min incubation of leukotriene A4 (LTA4) with human liver microsomes, 1.7 mol% was converted into leukotriene B4 (LTB4). The reaction was dependent on protein concentration, time, and substrate concentration, was not supported by heat-inactivated microsomes, and did not require NADPH. Kinetic analysis of the reaction revealed apparent Michaelis-Menten type behavior (app Km approximately 20 microM). Production rates varied widely among three patients examined. Piperonyl butoxide, propanethiol, and cyclohexene oxide (1 mM) inhibited LTB4 formation by microsomal LTA4-hydrolase by 52, 40, and 60%, respectively. The latter two compounds were shown not to inhibit cytosolic LTA4-hydrolase activity. The activity of microsomal and cytosolic LTA4-hydrolase was decreased in the presence of 100% O2 by 45 and 64%, respectively. Direct chemical ionization mass spectrometry was used to obtain a mass spectrum of 50 ng of underivatized synthetic LTB4 free acid and show that this spectrum is identical with that of 10 ng of the product isolated from LTA4 hydrolysis by human liver microsomes. The authenticity of the biologically generated LTB4 was confirmed by functional characterization in a receptor displacement assay. Displacement of [3H]LTB4 from the high affinity receptors of LTB4 on human neutrophils revealed KD50 values of 8.2 and 5.1 nM for human liver microsome derived and synthetic LTB4, respectively. The nearly two-fold higher KD50 of the microsomally generated LTB4 is suggested to result from an epimeric mixture of the active 5(S),12(R)- and the less active 5(S),12(S)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid.