Characterization of the human cysteinyl leukotriene 2 receptor.
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The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma. (+info)
Ionophore and arachidonic acid stimulation of airway responses in rhesus monkeys.
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Aerosolized doses of the ionophore, A23187, and arachidonic acid individually resulted in no airway response in rhesus monkeys. When these two agents were given simultaneously, by aerosol, an airway response occurred. The pulmonary function abnormalities that occurred qualitatively simulated those of an antigen-induced airway response. This is the first demonstration in our laboratory of two agents which singly will not produce a response but which are reactive when delivered in combination. Other fatty acids did not produce a similar response. The response to A23187 and arachidonic acid occurred only in rhesus monkeys from our colony which had been demonstrated to have airway responses to aerosolized antigen challenge, a response shown previously to be associated with hyperreactive airways to pharmacologic stimuli. The A23187 and arachidonic acid response was inhibited by aerosolized 5,8,11,14-eicosatetraynoic acid, an inhibitor of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Further, indomethacin, a prostaglandin synthetase inhibitor of the cyclooxygenase pathway, inhibited the response, although previous studies showed that this drug will potentiate an antigen-induced response in this animal model of asthma. The slow-reacting substance of anaphylaxis antagonist, FPL 55712, did not inhibit the A23187-arachidonic acid response under the conditions of these experiments. The mechanism of the A23187-arachidonic acid airway response in rhesus monkeys may or may not be the same as the antigen-induced response. (+info)
Molecular cloning and characterization of a second human cysteinyl leukotriene receptor: discovery of a subtype selective agonist.
(3/477)
The cysteinyl leukotrienes (CysLTs) are potent biological mediators in the pathophysiology of inflammatory diseases, in particular of airway obstruction in asthma. Pharmacological studies have suggested the existence of at least two types of CysLT receptors, designated CysLT(1) and CysLT(2). The CysLT(1) receptor has been cloned recently. Here we report the molecular cloning, expression, localization, and functional characterization of a human G protein-coupled receptor that has the expected characteristics of a CysLT(2) receptor. This new receptor is selectively activated by nanomolar concentrations of CysLTs with a rank order potency of LTC(4) = LTD(4) >> LTE(4). The leukotriene analog BAY u9773, reported to be a dual CysLT(1)/CysLT(2) antagonist, was found to be an antagonist at CysLT(1) sites but acted as a partial agonist at this new receptor. The structurally different CysLT(1) receptor-selective antagonists zafirlukast, montelukast, and MK-571 did not inhibit the agonist-mediated calcium mobilization of CysLT(2) receptors at physiological concentrations. Localization studies indicate highest expression of CysLT(2) receptors in adrenal glands, heart, and placenta; moderate levels in spleen, peripheral blood leukocytes, and lymph nodes; and low levels in the central nervous system and pituitary. The human CysLT(2) receptor gene is located on chromosome 13q14.12-21.1. The new receptor exhibits all characteristics of the thus far poorly defined CysLT(2) receptor. Moreover, we have identified BAY u9773 as a CysLT(2) selective agonist, which could prove to be of immediate use in understanding the functional roles of the CysLT(2) receptor. (+info)
Cysteinyl leukotrienes are involved in angiotensin II-induced contraction of aorta from spontaneously hypertensive rats.
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OBJECTIVE: Non specific lipoxygenase inhibitors have been reported to reduce the in vitro constrictor response and the in vivo pressor effect of angiotensin II in rats. The aim of this study was to assess the role of cysteinyl leukotrienes, in the vascular response to angiotensin II in spontaneously hypertensive rats (SHR). METHODS: Rings of thoracic aorta from SHR and normotensive Wistar-Kyoto rats (WKY) were compared in terms of contractile responses and release of cysteinyl leukotrienes in response to angiotensin II. RESULTS: Pretreatment with the specific 5-lipoxygenase inhibitor AA861 10 microM reduced the efficacy of angiotensin II in intact and endothelium-denuded aorta from SHR (% inhibition vs. control: 65+/-12.6% with endothelium (n=6), P<0.05; 43+/-7.2% without endothelium (n=6), P<0.05) but not in aorta from WKY. In addition, in aorta from SHR, the CysLT(1) receptor antagonist MK571 1 microM reduced by 55+/-6.1% (n=6, P<0.05) the contractile effects of angiotensin II in rings with endothelium but not in endothelium-denuded rings. Angiotensin II induced a 8.6+/-2.1-fold increase in cysteinyl leukotriene production in aorta rings from SHR with endothelium which was prevented by the AT(1) receptor antagonist losartan 1 microM but not by the AT(2) receptor antagonist PD123319 0.1 microM. In aorta rings from WKY, cysteinyl leukotriene production remained unchanged after exposition to angiotensin II. The cysteinyl leukotrienes (up to 0.1 microM) induced contractions in aorta rings from SHR but not from WKY. CONCLUSIONS: These data suggest that cysteinyl leukotrienes, acting at least in part on endothelial CysLT(1) receptors, are involved in the contractile response to angiotensin II in isolated aorta from SHR but not from WKY. (+info)
Slow reacting substance as a preformed mediator from human lung.
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Homogenates from human lung contained a preformed slow reacting substance (pSRS). The pattern of contraction on the guinea-pig ileum by pSRS was indistinguishable from that of SRS-A. The activity of pSRS could not be attributed to the presence of K+, Na+, Ca2+ and Mg2+ ions, or any prostaglandin including PGF2 or its 15-oxo derivative. As with SRS-A, pSRS could be absorbed onto Amberlite XAD-2 and silicic acid. Both were eluted from the former with 80 per cent ethanol and from the latter with a mixture of ethanol, ammonia and water. Both pSRS and SRS-A were resistant to the action of NaOH whereas their activities were destroyed by boiling in HCl. Arylsulphatase II B destroyed the activities of both pSRS and SRS-A. An antagonist of SRS-A, FPL55712, inhibited the action of pSRS at comparable concentrations to that of SRS-A. These experiments suggest that pSRS and SRS-A are identical. Thus SRS joins histamine and ECF-A as a preformed mediator. Although SRS was present in a preformed state the amount of material extractable was more than doubled by the anaphylactic reaction. The extraction of slow reacting substance from human lung without apparent requirement for antigen or antibody points to a possible role of this mediator in inflammatory reactions evoked by mechanisms independent of IgE and other tissue-sensitizing antibodies. (+info)
An alternative pathway for metabolism of leukotriene D(4): effects on contractions to cysteinyl-leukotrienes in the guinea-pig trachea.
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Contractions of guinea-pig tracheal preparations to cysteinyl-leukotrienes (LTC(4), LTD(4) and LTE(4)) were characterized in organ baths, and cysteinyl-leukotriene metabolism was studied using radiolabelled agonists and RP-HPLC separation. In the presence of S-hexyl GSH (100 microM) the metabolism of [(3)H]-LTC(4) into [(3)H]-LTD(4) was inhibited and the LTC(4)-induced contractions were resistant to CysLT(1) receptor antagonism but inhibited by the dual CysLT(1)/CysLT(2) receptor antagonist BAY u9773 (0.3 - 3 microM) with a pA(2)-value of 6.8+/-0.2. In the presence of L-cysteine (5 mM), the metabolism of [(3)H]-LTD(4) into [(3)H]-LTE(4) was inhibited and the LTD(4)-induced contractions were inhibited by the CysLT(1) receptor antagonist ICI 198,615 (1 - 10 nM) with a pA(2)-value of 9.3+/-0.2. However, at higher concentrations of ICI 198,615 (30 - 300 nM) a residual contraction to LTD(4) was unmasked, and this response was inhibited by BAY u9773 (1 - 3 microM). In the presence of the combination of S-hexyl GSH with L-cysteine, the LTD(4)-induced contractions displayed the characteristics of the LTC(4) contractile responses, i.e. resistant to CysLT(1) receptor antagonism, increased maximal contractions and slower time-course. This qualitative change of the LTD(4)-induced contraction was also observed in the presence of S-decyl GSH (100 microM), GSH (10 mM) and GSSG (10 mM). S-hexyl GSH, S-decyl GSH, GSH and GSSG all stimulated a formation of [(3)H]-LTC(4) from [(3)H]-LTD(4). In conclusion, GSH and GSH-related compounds changed the pharmacology of the LTD(4)-induced contractions by stimulating the conversion of LTD(4) into LTC(4). Moreover, the results indicate that, in addition to the metabolism of LTC(4) into LTD(4) and LTE(4), also the formation of LTC(4) from LTD(4) may regulate cysteinyl-leukotriene function. (+info)
A comparison of the anti-anaphylactic activities of salbutamol and disodium cromoglycate in the rat, the rat mast cell and in human lung tissue.
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1 Salbutamol and disodium cromoglycate were compared for anti-anaphylactic activity against passive anaphylaxis in rat skin and peritoneum in vivo and in rat mast cells and human lung fragments in vitro.2 Salbutamol administered intravenously to rats inhibited cutaneous anaphylaxis, but also inhibited cutaneous responses to histamine and 5-hydroxytryptamine. Salbutamol administered intraperitoneally inhibited the release of slow reacting substance of anaphylaxis (SRS-A) but not the release of histamine in the peritoneum. It was a very weak inhibitor of histamine release from rat mast cells in vitro.3 Disodium cromoglycate administered intravenously to rats inhibited cutaneous anaphylaxis. Disodium cromoglycate administered intraperitoneally to rats inhibited the release of histamine and, to a lesser extent, SRS-A in the peritoneum. It was an effective but short-acting inhibitor of histamine release from rat mast cells in vitro.4 Salbutamol was a potent inhibitor of the anaphylactic release of histamine and SRS-A from fragments of human lung.5 Disodium cromoglycate was a weak inhibitor of the anaphylactic release of histamine and SRS-A from fragments of human lung. The inhibition was variable and not dose-related.6 The concentration of salbutamol required to inhibit anaphylaxis in human lung is of the same order as that required to relax human bronchial muscle. It is suggested that salbutamol may be more effective in allergic asthma if given in a prophylactic regimen. (+info)
Cysteinyl leukotrienes modulate angiotensin II constrictor effects on aortas from streptozotocin-induced diabetic rats.
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Angiotensin II (Ang II) is a vasopressor peptide involved in the pathogenesis of cardiovascular diseases associated with diabetes mellitus. We have previously reported that the 5-lipoxygenase-derived products, particularly the cysteinyl leukotrienes (CysLTs), are involved in Ang II-induced contraction. In this study, we demonstrated that CysLTs contribute to the contraction elicited by Ang II in isolated aortas from streptozotocin-induced diabetic (SS) rats but not from insulin-treated diabetic rats, fructose-fed rats, or control rats. In an organ bath, pretreatment with the 5-lipoxygenase inhibitor (AA861, 10 micromol/L) reduced by 37.6+/-8.2% and 30.1+/-10.9% the Ang II-induced contractions in intact and endothelium-denuded aortic rings, respectively, from SS rats. In contrast, the CysLT(1) receptor antagonist (MK571, 1 micromol/L) or the dual CysLT(1)/CysLT(2) receptor antagonist (BAY-u9773, 0.1 micromol/L) did not affect Ang II-induced contraction. In addition, Ang II induced a 6.2+/-1.5-fold increase in CysLT release through the stimulation of the Ang II type 1 receptor. Furthermore, the urinary excretion of leukotriene E(4) was increased in SS rats (leukotriene E(4), 13.7+/-2.9 ng/24 h [SS rats, n=10] versus 1.5+/-0.5 ng/24 h [control rats, n=6]; P<0.0004). These data suggest the activation of the 5-lipoxygenase pathway in SS rats and the involvement of 5-lipoxygenase-derived products, particularly the CysLTs, in Ang II-induced contraction in aortas from SS rats through stimulation of CysLT receptors different from the well-characterized CysLT(1) or CysLT(2) receptor. (+info)