GR127935 acts as a partial agonist at recombinant human 5-HT1D alpha, and 5-H1D beta receptors

In this study we have investigated the functional activity of GR127935 (2-methyl-4-(5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-[4- carboxylic acid 4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]-amide) at human 5-HT1D alpha and 5-HT1D beta receptors which have been expressed in a Chinese Hamster Ovary (CHO) cell line. Using [S-35]GTP gamma S binding to cell membranes as a measure of receptor-G protein coupling, GR127935 showed partial agonist activity in both 5-HT1D alpha and 5-HT1D beta receptor expressing cells (E(max): 29 and 31% above basal control; pEC(50): 8.6 and 9.7, respectively). GR127935 also acted as a potent antagonist at the 5-HT1D alpha (app. pA(2) 8.5) and 5-HT1D beta (app. pA(2) 9.1) receptors. From studies measuring cAMP accumulation in cultured CHO cells GR127935 also displayed partial agonism, as well as acting as a potent antagonist at the 5-HT1D alpha receptors which stimulate cAMP levels and 5-HT1D beta receptors which inhibit cAMP levels (app. pA(2) 8.6 and 9.7, respectively). The 5\-HT1-like receptor antagonist methiothepin showed negative intrinsic activity at both receptors in the [S-35]GTP gamma S binding assay only. From studies using the receptor alkylating agent EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline) the 5-HT1D alpha cell line displayed a lack of receptor reserve but it was evident in the 5-HT1D beta cell line. In previous studies we have also shown that agonist stimulation of 5-HT1D alpha receptors increases cAMP levels which may be due to high receptor expression. Further investigation using up to 1 mu M EEDQ to reduce 5-HT1D alpha receptor number did not reveal an underlying inhibitory adenylyl cyclase response. In conclusion, GR127935 acts as a partial agonist, as well as a potent antagonist, at the human 5-HT1D alpha and 5-HT1D beta receptors when expressed in CHO cells.