title: Homologous desensitization of human histamine H₃ receptors expressed in CHO-K1 cells.
abstract: 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 [(35)S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [(3)H]-N-methyl-histamine ([(3)H]-NMHA) to intact cells and modified the sub-cellular distribution of H₃Rs, as detected by sucrose density gradients and [(3)H]-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 °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.
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360 México, D.F., Mexico.
authors.forename: Angélica
authors.initials: A
authors.lastname: Osorio-Espinoza
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360 México, D.F., Mexico.
authors.forename: Juan
authors.initials: J
authors.lastname: Escamilla-Sánchez
authors.affiliation: Unidad de Investigación en Enfermedades Oncológicas, Hospital Infantil de México Federico Gómez, Dr. Márquez 162, Col. Doctores, 06720 México, D.F., Mexico.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Aquino-Jarquin
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360 México, D.F., Mexico. Electronic address: jaarias@fisio.cinvestav.mx.
authors.forename: José-Antonio
authors.initials: JA
authors.lastname: Arias-Montaño
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D018100:Receptors, Histamine H3 / Q000378:metabolism*
chemical_list: D018100:Receptors, Histamine H3; D006632:Histamine; D000242:Cyclic AMP
keywords: CHO-K1 cells; Desensitization; G protein-coupled receptor; GPCR; GPCR kinase; GRK; H(3) receptor; H(3)R; Histamine; Neuromodulation; R-α-methylhistamine; RAMH; histamine H(3) receptor