Guanosine 5'-(γ-[35S]thio) triphosphate autoradiography allows selective detection of histamine H3 receptor-dependent G protein activation in rat brain tissue sections

Abstract

Histamine elicits its biological effects via three distinct G protein- coupled receptors, termed H1, H2, and H3. We have used guanosine 5'-(γ- [35S]thio)triphosphate (GTPγ[35S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor- dependent GTPγ[35S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 μM), a selective A1 receptor antagonist. Histamine elicited dose-dependent increments in GTPγ[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by N(α)-methylhistamine, (R)-α-methylhistamine, and immepip, three H3-selective agonists. In all regions examined, the GTPγ[35S] signal was reversed with thioperamide and clobenpropit, two potent H3-selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[35S] autoradiography selectively detects H3 receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[35S] autoradiography preferentially reveals responses to G(i/o)-coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to G(i/o), the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPγ[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.