Differential coupling of 5-HT1A receptors occupied by 5-HT or 8-OH-DPAT to adenylyl cyclase

Summary Human serotonin (5-hydroxytryptamine, 5-HT)-1A receptors have been transfected in NIH-3T3 cells, and their coupling to adenylyl cyclase was analysed depending on (1) the number of receptor expressed, (2) the experimental conditions used, (3) the nature of the agonists. Two monoclonal cell lines were used, expressing low (45 fmol/mg) and high (500 fmol/mg) levels of 5-HT_1A receptor. Two methods were tested to study the negative coupling of the transfected 5-HT_1A receptors to adenylyl cyclase: (1) measurement of CAMP production in intact cells, (2) measurement of adenylyl cyclase activity in vitro on membrane preparations. Studies on intact cells revealed that an increase in the receptor concentration was followed by (1) an increase in the efficacies of 5-HT, 5-CT (5-carboxamidotryptamine) and 8-hydroxy2-(di-n-propylamino)tetralin (8-OH-DPAT), (2) a 2 to 3-fold increase in the potency of 5-CT and 8-OH-DPAT, but no change in the potency of 5-HT. In membrane preparations, 8-OH-DPAT dose-response curve was shifted leftwards when the receptor concentration became higher whereas the corresponding shift was smaller for 5-HT and absent for 5-CT. Surprisingly, on membrane preparations, 8-OH-DPAT was a partial agonist relative to 5-HT. The relative efficacy of 8-OH-DPAT was lower in the clone expressing the lowest level of receptor. This partial agonist behavior of 8-OH-DPAT could be modulated by the ionic conditions under which the adenylyl cyclase activity was measured. When physiological intracellular concentrations of Na⁺, Mg²⁺ and K⁺ were used, 8-OH-DPAT became an almost full agonist relative to 5-HT.These data indicate that (1) the classical pharmacological models do not exactly fit with characteristics of the negative coupling between transfected 5-HT_1A receptors and adenylyl cyclase; (2) on membranes, the experimental procedures (ionic conditions) can modify this coupling differently depending on the nature of the agonist.Abbreviations 5-HT 5-hydroxytryptamine (serotonin) - 8-OHDPAT 8-hydroxy-2-(di-n-propylamino)-tetraline - 5-CT 5-carboxamidotryptamine - IBMX isobutyl methyl xanthine - BSA bovine serum albumine - EC₅₀ half maximal efficacy - DXM dexamethasone - PTX Bordetella pertussis toxin - G protein GTP-binding proteinCorrespondence to A. Varrault at the above address