title: G protein-dependent pharmacology of histamine H3 receptor ligands: evidence for heterogeneous active state receptor conformations.
abstract: Previously reported pharmacological studies using the imidazole-containing histamine H3 receptor ligands GT-2331 (Cipralisant) and proxyfan resulted in a range of classifications (antagonist, agonist, and protean) for these compounds. We examined the role that the signaling system, with particular emphasis on the type of G protein, had on the pharmacology observed for H3 ligands. Ligands were assessed using assays measuring neurotransmitter release, cAMP, and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. Whereas clobenpropit and ciproxifan were consistently antagonists, GT-2331, proxyfan, and imetit exhibited differential activity. Although GT-2331 and proxyfan exhibited little agonist activity in neurotransmitter release assays, both demonstrated full agonism relative to (R)-alpha-methylhistamine in cAMP assays. In [35S]GTPgammaS binding assays, GT-2331 and proxyfan demonstrated partial agonism. Imetit showed full agonism in most assays, but it was slightly less efficacious in a neurotransmitter release assay and in [35S]GTPgammaS binding at the human H3 receptor. To further examine these ligands, we coexpressed G alpha16 or chimeric G alpha q/i5 in human embryonic kidney cells expressing the human H3 receptor and assayed intracellular calcium and cAMP levels. GT-2331, proxyfan, and imetit demonstrated full agonism in all assays of cAMP activity. However, in cells expressing G alpha16, they exhibited minimal agonism in calcium mobilization assays, whereas imetit showed partial agonism. When G alpha q/i5 was used, the activity of both GT-2331 and proxyfan increased, whereas imetit became a full agonist. These results demonstrate that GT-2331 and proxyfan's differential pharmacology at the H3 receptor depends on the type of G protein used and provide indirect evidence for differential ligand-bound active states that mediate signaling by the H3 receptor.
authors.affiliation: Neurosciences Research, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064-6125, USA. kathleen.m.krueger@abbott.com
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Krueger
authors.forename: David G
authors.initials: DG
authors.lastname: Witte
authors.forename: Lynne
authors.initials: L
authors.lastname: Ireland-Denny
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Miller
authors.forename: John L
authors.initials: JL
authors.lastname: Baranowski
authors.forename: Steve
authors.initials: S
authors.lastname: Buckner
authors.forename: Ivan
authors.initials: I
authors.lastname: Milicic
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Esbenshade
authors.forename: Arthur A
authors.initials: AA
authors.lastname: Hancock
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004558:Electric Stimulation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006168:Guinea Pigs; D017442:Histamine Agonists / Q000494:pharmacology; D006633:Histamine Antagonists / Q000494:pharmacology; D006636:Histamine Release / Q000187:drug effects; D007082:Ileum / Q000187:drug effects / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D008297:Male; D008566:Membranes / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects; D018100:Receptors, Histamine H3 / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: C114969:4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole; D017442:Histamine Agonists; D006633:Histamine Antagonists; D007093:Imidazoles; D008024:Ligands; D018377:Neurotransmitter Agents; D043562:Receptors, G-Protein-Coupled; D018100:Receptors, Histamine H3; C417965:proxyfan; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D019204:GTP-Binding Proteins