The putative <<silent>> 5-HT(1A) receptor antagonist, WAY 100635, has inverse agonist properties at cloned human 5-HT(1A) 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-G 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(1A) (5-HT(1A)) receptor ligands, measured as maximal effect (E(MAX)), using guanosine 5'-0-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to membranes prepared from human epithelioid carcinoma (HeLa) cells, expressing 500 fmol/mg protein of cloned human 5-HT(1A) receptor (HA7 cells). A decrease of the NaCl concentration decreased the maximal effect of serotonin, increased basal [35S]GTPgammaS binding, and increased the negative intrinsic activity of spiperone and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridinyl)cyclohexaneca rboxamide (WAY 100635). This ability of WAY 100635 to decrease basal [35S]GTPgammaS binding was antagonized by (s)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropa namide ((s)-WAY 100135) (pA(2)=7.77). Further, WAY 100635 was able to antagonize carboxamidotryptamine (5-CT)-stimulated [35S]GTPgammaS 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 [35S]GTPgammaS binding. WAY 100635 did not affect basal [35S]GTPgammaS binding to membranes from untransfected HeLa cells. Pertussis toxin (200 ng/ml) prevented WAY 100635 and spiperone to decrease [35S]GTPgammaS 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-HT(1A) receptors expressed in HA7 cells is sodium-dependent, which allowed to confirm the 5-HT(1A) inverse agonist properties of spiperone, and to show that WAY 100635 is an inverse agonist at 5-HT(1A) receptors that inhibits basal [35S]GTPgammaS binding to a lesser extent than spiperone. [35S]GTPgammaS 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-HT(1A) receptor ligands.
Division de Neurobiologie II, Centre de Recherche Pierre Fabre, 17 Ave Jean Moulin, 80106, Castres, France. cristina.cosi@pierre-fabre.com
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D001667:Binding, Competitive / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004274:DNA, Recombinant / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010879:Piperazines / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics; D044263:Receptors, Serotonin, 5-HT1; D012701:Serotonin / Q000031:analogs & derivatives* / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D013134:Spiperone / Q000494:pharmacology; D013462:Sulfur Radioisotopes
D004274:DNA, Recombinant; D010879:Piperazines; D011725:Pyridines; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013462:Sulfur Radioisotopes; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; D013134:Spiperone; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; C047087:5-carboxamidotryptamine