Abstract
[35S]Guanosine-5′-O-(3-thio)triphosphate ([35S]GTPγS) binding to G proteins was measured byin vitro autoradiography in guinea pig and rat brain sections after activation by 5-hydroxytryptamine (5-HT) receptor agonists. 5-Carboxamidotryptamine stimulated binding strongly in hippocampus and lateral septum and weakly in substantia nigra. This effect was blocked in the substantia nigra by the 5-HT1B/1Dreceptor antagonist GR-127,935 and in the former two regions by the 5-HT1A antagonist NAN-190. 5-HT1B/1D receptor agonists stimulated binding in substantia nigra and in areas containing 5-HT1A receptors. In guinea pig substantia nigra, 5-(nonyloxy)-tryptamine maximally stimulated [35S]GTPγS binding by 54%, with an EC50 value of 62 nm; at 100 μm, this agonist increased binding by ∼200% in hippocampus (with a 2-fold weaker EC50 value). The distribution of [3H]8-OH-DPAT binding sites was identical to that of the [35S]GTPγS labeling stimulated by the 5-HT1A agonist (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT)]. (R)-8-OH-DPAT, (S)-8-OH-DPAT, and buspirone stimulated [35S]GTPγS binding in hippocampus by 340%, 140%, and 78%, with EC50 values of 71, 51, and 132 nm. Enhanced [35S]GTPγS binding was not detected in the presence of 5-HT1F, 5-HT2, 5-HT4, and 5-HT7 receptor agonists. Because activation of μ-opioid, muscarinic M2, histamine H3, and cannabinoid receptors was also visualized successfully, these data suggest that only receptors coupled to pertussis toxin-sensitive G proteins can be seen by [35S]GTPγS binding autoradiography. This study also shows that different 5-HT receptors coupled to these proteins can show a wide range of [35S]GTPγS binding stimulation. Although the functional significance of these variations is unclear, this technique offers advantages over receptor autoradiography because it does not require high affinity radioligands and provides a measure of agonist efficacies in various brain regions.
Footnotes
- Received March 3, 1997.
- Accepted June 30, 1997.
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Send reprint requests to: Dr. Christian Waeber, Massachusetts General Hospital, 149 13th Street, CNY149, Rm. 6403, Charlestown, MA 02129. E-mail: waeber{at}helix.mgh.harvard.edu
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This work was supported by National Institutes of Health Grant 1-P01-NS35611–01 (M.A.M.). C.W. is the recipient of a Research Fellowship of the Migraine Trust, and M.A.M. is the recipient of a Bristol-Myers Unrestricted Research Award in Neuroscience.
- The American Society for Pharmacology and Experimental Therapeutics
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