Abstract. To clarify heterologous regulation of a receptor is important in considering medication. Histamine constricts the airway smooth muscle through the action to the H 1 receptor (H1R), which contributes to asthma. b 2 -Adrenergic receptor (b2R) agonists are widely used in asthmatic therapy for their bronchodilating effects. In this study, we investigated the effect of b2R activation on the H1R function using Chinese hamster ovary cells stably co-expressing human histamine H1R and b2R (CHO-H1/ b2 cell). The stimulation of b2R resulted in the decrease of H1R in the membrane. Heterologous H1R down-regulation was significantly reversed in the presence of the cyclic AMP-dependent protein kinase (PKA) inhibitor KT5720. Since phosphorylation of G protein-coupled receptor (GPCR) by second messenger-dependent kinases, is proposed to be a key step initiating heterologous receptor desensitization, we examined whether heterologous H1R down-regulation was accompanied by H1R phosphorylation. H1R was phosphorylated by b2R stimulation; however, a PKA inhibitor did not inhibit heterologous H1R phosphorylation. Our results suggest that H1R was heterologously regulated by b2R. Not only a direct action of b2R agonist to b2R causing bronchodilation but also indirect action that reduces the number of H1R responsible for bronchoconstriction might contribute to a decrease in the bronchial resistance, which proposes another possible advantage of b2R agonists for asthmatic medication.
Abstract. Heterologous down-regulation of histamine H 1 receptor (H1R) mediated by muscarinic acetylcholine receptor subtype was investigated using five kinds of Chinese hamster ovary (CHO) cells stably co-expressing the human H1R and one of the five (M 1 -M 5 ) muscarinic acetylcholine receptors, CHO-H1/ M1, CHO-H1/ M2, CHO-H1/ M3, CHO-H1/ M4, and CHO-H1/ M5 cells. Among the CHO-H1/ M1, CHO-H1/ M3, and CHO-H1/ M5 cells, carbachol treatment of the CHO-H1/ M3 cells time-dependently led to remarkable down-regulation of the H1R to 60% of the control level. In contrast, stimulation of CHO-H1/ M1 cells by carbachol induced negligible effect on the down-regulation. Stimulation of CHO-H1/ M5 cells by carbachol induced significant but only small H1R down-regulation. M 2 and M 4 muscarinic receptors showed negligible effect on the down-regulation. H1R-mediated accumulation of inositol phosphates in CHO-H1/ M3 cells with long-term expose to carbachol was decreased to 60% compared with non-treated cells. Heterologous phosphorylation of H1R was induced by the stimulation of each muscarinic receptor. H1R was phosphorylated by about twofold from the basal level through five subtypes of muscarinic receptor. The M 3 muscarinic receptor-mediated phosphorylation of H1R was reversed by the inhibition of protein kinase C. In the present study we demonstrated that the M 3 muscarinic acetylcholine receptor mediated remarkable down-regulation of the H1R with decreased receptor signaling.
Abstract. Histamine H 1 receptor (H1R) signaling is regulated by changing its expression level. Two mechanisms are involved in this regulation. One is down-regulation through receptor desensitization. Receptor phosphorylation seemed crucial because stimulation of the mutant H1R lacking five putative phosphorylation sites did not show down-regulation. The phosphorylation level of the mutant receptor was much smaller than that of the wild type ones by several protein kinases. The other is up-regulation through activation of receptor gene expression. Protein kinase C (PKC) signaling was suggested to be involved in this up-regulation. Regulation of H1R expression level was mediated not only through H1R but also autonomic nerve receptors. Stimulation of M 3 muscarinic receptors (M3R) induced both down-regulation and up-regulation of H1R. Down-regulation of M3R-mediated H1R seemed not to be mediated by PKC activation, although PKC activation induced H1R phosphorylation. Elevation of H1R expression was induced by the stimulation of M3Rs. PKC was suggested to be involved in this up-regulation. Stimulation of β 2 -adrenergic receptors induced H1R down-regulation through several mechanisms. One of them is enhanced receptor degradation after desensitization and another is suppression of receptor synthesis that includes the suppression of receptor gene expression and enhanced degradation of the receptor mRNA. Protein kinase A was suggested to be involved in enhanced degradation and the activation of the receptor gene expression. Elevation of both H1R expression and its mRNA was observed in nasal mucosa of nasal hypersensitivity allergy model rat after toluene diisocyanate provocation. These results suggest that activation of H1R gene expression plays an important patho-physiological role in allergy. Elevation of the mRNA was partially but significantly suppressed by antihistamines.
Abstract. Homologous and heterologous phosphorylations of histamine H 1 receptor (H1R) in intact cells were investigated using Chinese hamster ovary cells stably co-expressing c-myctagged human histamine H 1 and muscarinic M 3 receptors. Increase in histamine-induced homologous phosphorylation of H1R was induced in a dose-and time-dependent manner. Maximum phosphorylation of H1R by 8-fold over the basal level was induced 1 min after the stimulation, and the increased phosphorylation level was maintained over 40 min. M 3 receptormediated heterologous phosphorylation of H1R reached maximum by 2-fold over the basal level at 5 min after the stimulation and then rapidly returned to the basal level by 40 min after the stimulation. Histamine-induced phosphorylation of H1R was partially inhibited by three protein kinase inhibitors including Ro-31-8220 for protein kinase C (PKC), KN-93 for calcium / calmodulin-dependent kinase II (CaMKII), and KT5823 for protein kinase G (PKG), while, M 3 -receptor-mediated phosphorylation of H1R was completely inhibited by Ro 31-8220. Protein kinase activators including phorbol 12-myristate 13-acetate (PMA), 8-bromo-cyclic GMP (8-BrcGMP), and 8-bromo-cyclic AMP (8-Br-cAMP) induced increases in H1R phosphorylation. Increased phosphorylation of H1R, by 5-fold over the basal level, induced with a combination of PMA, 8-Br-cGMP, and 8-Br-cAMP was still lower than that with histamine. It was suggested that H1R-mediated H1R phosphorylation involves the activation of PKC, CaMKII, PKG, and other unidentified kinases including G-protein coupled receptor kinases (GRKs) and that PKC is solely involved in M 3 receptor-mediated H1R phosphorylation.
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