RESUMEN
Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.
Asunto(s)
Activación del Canal Iónico/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Leucotrieno B4/metabolismo , Animales , Calcio/metabolismo , Arterias Cerebrales/citología , Femenino , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Leucotrieno A4/química , Leucotrieno A4/metabolismo , Leucotrieno A4/farmacología , Leucotrieno B4/química , Leucotrieno B4/farmacología , Leucotrieno C4/química , Leucotrieno C4/metabolismo , Leucotrieno C4/farmacología , Leucotrieno D4/química , Leucotrieno D4/metabolismo , Leucotrieno D4/farmacología , Leucotrieno E4/química , Leucotrieno E4/metabolismo , Leucotrieno E4/farmacología , Potenciales de la Membrana/efectos de los fármacos , Microinyecciones , Modelos Moleculares , Estructura Molecular , Células Musculares/citología , Células Musculares/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Unión Proteica , Estructura Terciaria de Proteína , ARN Complementario/administración & dosificación , ARN Complementario/genética , Ratas , Xenopus laevisRESUMEN
Classical Hodgkin lymphoma has unique clinical and pathological features and tumour tissue is characterized by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by inflammatory cells. In the present study, we report that the Hodgkin lymphoma-derived cell line L1236 has high expression of 15-lipoxygenase-1 and that these cells readily convert arachidonic acid to eoxin C(4), eoxin D(4) and eoxin E(4). These mediators were only recently discovered in human eosinophils and mast cells and found to be potent proinflammatory mediators. Western blot and immunocytochemistry analyses of L1236 cells demonstrated that 15-lipoxygenase-1 was present mainly in the cytosol and that the enzyme translocated to the membrane upon calcium challenge. By immunohistochemistry of Hodgkin lymphoma tumour tissue, 15-lipoxygenase-1 was found to be expressed in primary Hodgkin Reed-Sternberg cells in 17 of 20 (85%) investigated biopsies. The enzyme 15-lipoxygenase-1, however, was not expressed in any of 10 biopsies representing nine different subtypes of non-Hodgkin lymphoma. In essence, the expression of 15-lipoxygenase-1 and the putative formation of eoxins by Hodgkin Reed-Sternberg cells in vivo are likely to contribute to the inflammatory features of Hodgkin lymphoma. These findings may have important diagnostic and therapeutic implications in Hodgkin lymphoma. Furthermore, the discovery of the high 15-lipoxygenase-1 activity in L1236 cells demonstrates that this cell line comprises a useful model system to study the chemical and biological roles of 15-lipoxygenase-1.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Enfermedad de Hodgkin/enzimología , Leucotrieno D4/análogos & derivados , Leucotrieno E4/análogos & derivados , Leucotrienos/biosíntesis , Células de Reed-Sternberg/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Araquidonato 15-Lipooxigenasa/análisis , Biopsia , Línea Celular Tumoral , Niño , Preescolar , Femenino , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/patología , Humanos , Leucotrieno D4/biosíntesis , Leucotrieno D4/química , Leucotrieno E4/biosíntesis , Leucotrieno E4/química , Leucotrienos/química , Linfoma no Hodgkin/diagnóstico , Linfoma no Hodgkin/enzimología , Linfoma no Hodgkin/patología , Masculino , Persona de Mediana EdadRESUMEN
The purpose of this study is to investigate the interactions of water adsorption on the surfaces of different crystal forms of the same drug. The energy of interaction between water vapor and the surfaces of the two crystal polymorphs has been investigated as a function of temperature and water activity. One of the adsorbents, the metastable form of the monotropically related pair used here, showed greater adsorptive capacity in terms of both the amount of water uptake as well the integral heat of adsorption. However, the specific heat of adsorption values revealed that even though the surface of the thermodynamically stable crystal adsorbs less water, water molecules are actually more strongly bound when adsorbed on the surface of the stable crystal form. This means that the metastable crystal form adsorbs a greater amount of more weakly bound water. Conversely, the thermodynamically stable form, presents on its surface a smaller number of stronger adsorption sites for water. This study also shows that the crystalline character of the surfaces of the two polymorphs, shown as quantifiable differences in their surface interactions, is maintained despite the presence of any crystal defects incorporated upon milling.
Asunto(s)
Leucotrieno D4/química , Propiedades de Superficie , Agua/química , Adsorción , Cristalización , Heptanos/química , Humedad , Tamaño de la Partícula , Polvos , Estereoisomerismo , Temperatura , TermodinámicaRESUMEN
Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/farmacología , Inflamación/prevención & control , Lipoxinas , Proteínas de la Membrana , Receptores de Superficie Celular/metabolismo , Receptores de Formil Péptido , Receptores de Lipoxina , Animales , Aspirina/farmacología , Unión Competitiva/efectos de los fármacos , Células COS , Línea Celular , Relación Dosis-Respuesta a Droga , Expresión Génica , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inflamación/patología , Leucotrieno D4/química , Leucotrieno D4/metabolismo , Leucotrieno D4/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , ARN/genética , ARN/metabolismo , Receptores de Superficie Celular/genética , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/metabolismo , Proteínas Recombinantes/metabolismo , Estereoisomerismo , TritioRESUMEN
Molecular dynamics simulations of leukotriene C4 (LTC4), leukotriene D4 (LTD4), and leukotriene E4 (LTE4) were carried out, and the data were visualized in an animated video format. Three-dimensional ghost images show the positions of the heavy atoms of all three molecules throughout the simulations. The ghost images can be superimposed to give a single three-dimensional image in which the shapes of the most populated conformers of each molecule are apparent and can be compared. Leukotriene D4 was found to occupy mostly T-shaped conformations, while LTC4 occupied mostly cup-shaped conformations, and LTE4 occupied a wide range of conformations spanning the LTD4 and LTC4 types. Digital filtering and graphing of the internal geometries of the molecules as a function of time revealed differences in dynamic behavior. The results are discussed in light of current knowledge about leukotriene receptors.