Antagonist activity of meta-chlorophenylpiperazine and partial agonist activity of 8-OH-DPAT at the 5-HT(7) receptor

Abstract

This study compared the use of adapter G-proteins to link G(s) coupled G-protein receptors to a Ca(2+) signal, enabling high throughput functional studies using a fluorescent imaging plate reader (FLIPR, Molecular Devices). The pharmacological profile of the human 5-hydroxytryptamine (5-HT(7)) receptor was studied using the adapter G-proteins G(alpha16) and G(qs5) and compared to previously published adenylyl cyclase and receptor binding data. Human embryonic kidney (HEK) 293 cells stably expressing the human 5-HT(7(a)) receptor were transiently transfected with the adapter G-proteins. Changes in intracellular Ca(2+) were monitored using the fluorescent Ca(2+)-indicator Fluo-4.5-Carboxamidotryptamine (5-CT) induced an increase in fluorescence in transfected cells only, which was attenuated by N-ethylmalaeimide and abolished by thapsigargin, consistent with a G-protein mediated mobilisation of intracellular Ca(2+). The pharmacological profile of agonists at the 5-HT(7) receptor was similar using either adapter G-protein. Agonist potency estimates were similar to that reported in binding studies but were greater than that seen in adenylyl cyclase studies. 8-Hydroxy-N, N-dipropylaminotetralin (8-OH-DPAT) and tryptamine acted as partial agonists using the adapter G-proteins, but were full agonists in recombinant systems using adenylyl cyclase. meta-Chlorophenylpiperazine (mCPP) and trifluoro-methylphenyl piperazine (TFMPP) were antagonists on intracellular Ca(2+). Antagonist pharmacological profiles were similar between adapter G-proteins, receptor binding, and adenylyl cyclase studies. These results show that adapter G-proteins can be used to study G(s)-linked receptors using the high throughput FLIPR system measuring changes in intracellular Ca(2+) and provide novel information on mCPP and 8-OH-DPAT.