title: 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.
authors.affiliation: Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, CM19 5AW, Essex, UK. martyn_wood-1@sbphrd.com
authors.forename: M
authors.initials: M
authors.lastname: Wood
authors.forename: M
authors.initials: M
authors.lastname: Chaubey
authors.forename: P
authors.initials: P
authors.lastname: Atkinson
authors.forename: D R
authors.initials: DR
authors.lastname: Thomas
mesh_terms: D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000814:Aniline Compounds; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D005456:Fluorescent Dyes; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014162:Transfection / Q000235:genetics; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D010879:Piperazines; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists; D014966:Xanthenes; C104425:serotonin 7 receptor; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases; C015068:1-(3-chlorophenyl)piperazine; D002118:Calcium