Abstract
Functional characterization of the recombinant human 5-hydroxytryptamine7(a) (h5-HT7(a)) receptor isoform was performed using stably transfected LM(tk−) cells. Expression levels of the h5-HT7(a) receptor determined from saturation studies using either a labeled agonist ([3H]5-HT) or antagonist ([3H]LSD) were very similar (Bmax = 160–190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]i) with an EC50 value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine ≥ 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC50 values) to stimulate [cAMP]i were more than 25-fold lower relative to their respective binding affinities (Ki values) obtained in [3H]5-HT competition assays. In contrast, antagonist potencies (Kb values) to block 5-HT-stimulated [cAMP]i were in close agreement with their correspondingKi values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]i, indicating that the 5-HT7(a) subtype directly interacts with Gαs protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT7 receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT7 receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.
Footnotes
-
Send reprint requests to: Dr. Nika Adham, Synaptic Pharmaceutical Corporation, 215 College Road, Paramus, NJ 07652.
-
1 Current address: Wyeth Ayerst Research, CNS Department, Monmouth Junction, NJ 08852.
- Abbreviations:
- [cAMP]i
- intracellular cAMP concentrations
- CRC
- concentration-response curve
- EC50
- concentration of agonist required to produce 50% maximal response
- Emax
- maximal response
- h5-HT7(a)
- human 5-hydroxytryptamine7(a)
- Kb
- apparent antagonist dissociation constant
- Ki
- affinity constant
- RT-PCR
- reverse transcriptase polymerase chain reaction
- 5-CT
- 5-carboxamidotryptamine
- 5-HT
- 5-hydroxytryptamine
- 5-MeOT
- 5-methoxytryptamine
- DP-5-CT
- N,N-dipropyl-5-carboxamidotryptamine
- 5-MeO-DMT
- N,N-dimethyl-5-methoxytryptamine
- 2-Me-5-HT
- 2-methyl-5-hydroxytryptamine
- α-Me-5-HT
- α-methyl-5-hydroxytryptamine
- 8-OH-DPAT
- 8-hydroxy N,N-dipropyl aminotetralin
- 1-NP
- 1-naphthylpiperazine
- Received March 6, 1998.
- Accepted June 23, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|