期刊
EUROPEAN JOURNAL OF PHARMACOLOGY
卷 396, 期 1, 页码 1-8出版社
ELSEVIER
DOI: 10.1016/S0014-2999(00)00213-2
关键词
5-HT (5-hydroxytryptamine serotonin); 5-HT receptor; FLIPR (flourescent imaging plate reader); fluorescence; meta-chlorophenylpiperazine; 8-OH-DPAT (8-hydroxy-N,N-dipropylaminotetralin)
This study compared the use of adapter G-proteins to link G(s) coupled G-protein receptors to a Ca2+ signal, enabling high throughput functional studies using a fluorescent imaging plate reader (FLIPR, Molecular Devices). The pharmacological profile of the human 5-hydroxytryptamine (5-HT7) receptor was studied using the adapter G-proteins G(alpha 16) 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-HT7(a) receptor were transiently transfected with the adapter G-proteins. Changes in intracellular Ca2+ were monitored using the fluorescent Ca2+-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 Ca2+. The pharmacological profile of agonists at the 5-HT7 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 Ca2+. 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 Ca2+ and provide novel information on mCPP and 8-OH-DPAT. (C) 2000 Elsevier Science B.V. All rights reserved.
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