Characterization of the cloned guinea pig leukotriene B-4 receptor: comparison to its human orthologue

BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respective ly. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein-Bar Vir us Nuclear Antigen) cells showed that both receptors displayed high affinit y for leukotriene B-4 (K-d value of similar to 0.4 nM) and were expressed a t high levels (B-max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [ H-3]leukotriene B-4 specific binding at the recombinant guinea pig BLT rece ptor is leukotriene B-4 > 20-OH-leukotriene B-4 > 12(R)-HETE ((5Z,8Z,10E,12 (R)14Z)-12-hydroxyeicosatetraen-1-oic acid) > 12(S)-HETE ((52,8Z,10E,12(S)1 4Z)-12-Hydroxyeicosatetraen-1-oic acid) > 20-COOH-leukotriene B-4 > U75302 (6-(6-(3-hydroxy-1E, 5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol) >> leuko triene C-4 = leukotriene D-4 = leukotriene E-4. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B-4 and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used t o demonstrate that the guinea pig and human BLT receptors can couple to bot h the cAMP inhibitory and intracellular Ca2+ mobilization signaling pathway s. However, in the case of the aequorin-expressing HEK cells (designated AE Q17-293) transfected with either the guinea pig or human BLT receptor, expr ession of G(alpha 16) was required to achieve a robust Ca2+ driven response . Leukotriene B-4 was a potent agonist in functional assays of both the gui nea pig and human BLT receptors. U-75302 a leukotriene B-4 analogue which p ossesses both agonistic and antagonistic properties behaved as a full agoni st of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comp arison of the intrinsic potencies of leukotriene B-4 receptor antagonists u sed in guinea pig in vivo models of allergic and inflammatory disorders. (C ) 1999 Elsevier Science B.V. All rights reserved.