G Protein-Dependent Pharmacology of Histamine H3 Receptor Ligands: Evidence for Heterogeneous Active State Receptor Conformations

• Published in: The Journal of pharmacology and experimental therapeutics 2005-07, Vol.314 (1), p.271-281
• Main Authors: Krueger, Kathleen M, Witte, David G, Ireland-Denny, Lynne, Miller, Thomas R, Baranowski, John L, Buckner, Steve, Milicic, Ivan, Esbenshade, Timothy A, Hancock, Arthur A
• Format: Article
• Language: English
• Subjects: Animals; Cyclic AMP - metabolism; Electric Stimulation; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - genetics; GTP-Binding Proteins - physiology; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guinea Pigs; Histamine Agonists - pharmacology; Histamine Antagonists - pharmacology; Histamine Release - drug effects; Ileum - drug effects; Ileum - metabolism; Imidazoles - pharmacology; Ligands; Male; Membranes - metabolism; Neurotransmitter Agents - metabolism; Protein Conformation; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled - drug effects; Receptors, Histamine H3 - drug effects; Signal Transduction - drug effects; Transfection
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.104.078865

Previously reported pharmacological studies using the imidazole-containing histamine H 3 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 H 3 ligands. Ligands were assessed using assays measuring neurotransmitter release, cAMP, and guanosine 5′- O -(3-[ 35 S]thio)triphosphate ([ 35 S]GTPγS) 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 )-α-methylhistamine in cAMP assays. In [ 35 S]GTPγS 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 [ 35 S]GTPγS binding at the human H 3 receptor. To further examine these ligands, we coexpressed Gα16 or chimeric Gαq/i 5 in human embryonic kidney cells expressing the human H 3 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 Gα16, they exhibited minimal agonism in calcium mobilization assays, whereas imetit showed partial agonism. When Gαq/i 5 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 H 3 receptor depends on the type of G protein used and provide indirect evidence for differential ligand-bound active states that mediate signaling by the H 3 receptor.