BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein-Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B-4 (K-d value of similar to 0.4 nM) and were expressed at 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 receptor 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)14Z)-12-Hydroxyeicosatetraen-1-oic acid) > 20-COOH-leukotriene B-4 > U75302 (6-(6-(3-hydroxy-1E, 5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol) >> leukotriene 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 to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca2+ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression 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 guinea pig and human BLT receptors. U-75302 a leukotriene B-4 analogue which possesses both agonistic and antagonistic properties behaved as a full agonist 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 comparison of the intrinsic potencies of leukotriene B-4 receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders. (C) 1999 Elsevier Science B.V. All rights reserved.
