Apelin peptides have recently been identified to be the endogenous ligands for the G protein-coupled receptor APJ. However, little is known about the physiological roles of this ligand-receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure-activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C-terminal apelin peptide, apelin-13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre-proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT-PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co-localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.

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1 The novel 5-HT 7 receptor antagonist, SB-269970-A, potently displaced [ 3 H]-5-CT from human 5-HT 7(a) (pK i 8.9+0.1) and 5-HT 7 receptors in guinea-pig cortex (pK i 8.3+0.2). 2 5-CT stimulated adenylyl cyclase activity in 5-HT 7(a) /HEK293 membranes (pEC 50 7.5+0.1) and SB-269970-A (0.03 ± 1 mM) inhibited the 5-CT concentration-response with no signi®cant alteration in the maximal response. The pA 2 (8.5+0.2) for SB-269970-A agreed well with the pK i determined from [ 3 H]-5-CT binding studies. 3 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC 50 of 8.4+0.2) was inhibited by SB-269970-A (0.3 mM) with a pK B (8.3+0.1) in good agreement with its antagonist potency at the human cloned 5-HT 7(a) receptor and its binding anity at guinea-pig cortical membranes. 4 5-HT 7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. 5 SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min 71 kg 71). Following a single dose (3 mg kg 71 ) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested.

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