The putative "silent" 5-HT1A receptor antagonist, WAY 100635, has inverse agonist properties at cloned human 5-HT1A receptors

Agonist binding to G protein-coupled receptors induces the formation of a receptor-G protein complex and subsequent guanosine 5'-diphosphate/guanosine 5'-triphosphate (GDP/GTP) exchange. Some receptors, however, form receptor-a protein complexes and promote GDP/GTP exchange even when not occupied by agonists. Such receptors preferentially activate pertussis toxin-sensitive G proteins (i.e., G(i)/G(o)), and the interactions of receptors and G proteins are affected by monovalent cations (most notably Na+), both in the occupied and unoccupied state. We investigated the effects of Na+ on the intrinsic activity of 5-hydroxytryptamine,. (5-HT1A) receptor ligands, measured as maximal effect (E-MAX), using guanosine 5'-0-(3-[S-35]thio)-triphosphate ([S-35]GTP gamma S) binding to membranes prepared from human epithelioid carcinoma (HeLa) cells, expressing 500 fmol/mg protein of cloned human 5-HT1A receptor (HA7 cells). A decrease of the NaCl concentration decreased the maximal effect of serotonin, increased basal [S-35]GTP gamma S binding, and increased the negative intrinsic activity of spiperone and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635). This ability of WAY 100635 to decrease basal [S-35]GTP gamma S binding was antagonized by (s)-N-tert-butyl-3-(4-(2-methaxyphenyl)piperazine-1-yl)-2-phenylpropanamide ((s)-WAY 100135) (pA(2) = 7.77). Further, WAY 100635 was able to antagonize carboxamidotryptamine (5-CT)-stimulated [S-35]GTP gamma S binding with a pA(2) of 9.9, in standard NaCl conditions, and of 9.7, in the absence of NaCl. Changes in membrane concentration did not affect the ability of WAY 100635 to decrease [S-35]GTP gamma S binding. WAY 100635 did not affect basal [S-35]GTP gamma S binding to membranes from untransfected HeLa cells. Pertussis toxin (200 ng/ml) prevented WAY 100635 and spiperone to decrease [S-35]GTP gamma S binding, showing that their effects were mediated by G proteins of the G(i)/G(o) family. In conclusion, the constitutive and stimulated activity of human 5-HT1A receptors expressed in HA7 cells is sodium-dependent, which allowed to confirm the 5-HT1A inverse agonist properties of spiperone, and to show that WAY 100635 is an inverse agonist at 5-HT1A receptors that inhibits basal [S-35]GTP gamma S binding to a lesser extent than spiperone. [S-35]GTP gamma S binding to membranes from HA7 cells under low NaCl conditions appears to be especially suitable to evidence and pharmacologically analyze the inverse agonist properties of 5-HT1A receptor ligands. (C) 2000 Elsevier Science B.V. All rights reserved.