Homologous desensitization of human histamine H₃ receptors expressed in CHO-K1 cells

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• 作者：Ang茅lica Osorio-Espinoza ; Juan Escamilla-S谩nchez ; Guillermo Aquino-Jarquin ; Jos茅-Antonio Arias-Monta帽o
• 关键词：Histamine ; H3 receptor ; Desensitization ; GRK ; CHO-K1 cells ; Neuromodulation
• 刊名：Neuropharmacology
• 出版年：February, 2014
• 年：2014
• 卷：77
• 期：Complete
• 页码：387-397
• 全文大小：2015 K

文摘

Histamine H₃ receptors (H₃Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H₃Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H₃R (hH₃R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [³⁵S]-GTP纬S binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60聽min with a maximum at 90聽min. Agonist exposure resulted in decreased binding sites for the radioligand [³H]-N-methyl-histamine ([³H]-NMHA) to intact cells and modified the sub-cellular distribution of H₃Rs, as detected by sucrose density gradients and [³H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4聽掳C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl₂ and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH₃Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles.