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MOLECULAR PHARMACOLOGY 52:623-631 (1997).

5-Hydroxytryptamine_1A and 5-Hydroxytryptamine_1B Receptors Stimulate [³⁵S]Guanosine-5-O-(3-thio)triphosphate Binding to Rodent Brain Sections as Visualized by In Vitro Autoradiography

Christian Waeber and Michael A. Moskowitz

Stroke and Neurovascular Regulation, Neurosurgery Service, Department of Surgery, Neurology Service, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

[³⁵S]Guanosine-5-O-(3-thio)triphosphate ([³⁵S]GTPS) binding to G proteins was measured by in 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-HT_1B/1D receptor antagonist GR-127,935 and in the former two regions by the 5-HT_1A antagonist NAN-190. 5-HT_1B/1D receptor agonists stimulated binding in substantia nigra and in areas containing 5-HT_1A receptors. In guinea pig substantia nigra, 5-(nonyloxy)-tryptamine maximally stimulated [³⁵S]GTPS 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]GTPS labeling stimulated by the 5-HT_1A agonist (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT)]. (R)-8-OH-DPAT, (S)-8-OH-DPAT, and buspirone stimulated [³⁵S]GTPS binding in hippocampus by 340%, 140%, and 78%, with EC₅₀ values of 71, 51, and 132 nM. Enhanced [³⁵S]GTPS binding was not detected in the presence of 5-HT_1F, 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]GTPS binding autoradiography. This study also shows that different 5-HT receptors coupled to these proteins can show a wide range of [³⁵S]GTPS 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.