Biased agonism at histamine H1 receptor: Desensitization, internalization and MAPK activation triggered by antihistamines.

Histamine H1 receptor ligands used clinically as antiallergics rank among the most widely prescribed and over-the-counter drugs in the world. They exert the therapeutic actions by blocking the effects of histamine, due to null or negative efficacy towards Gαq-phospholipase C (PLC)-inositol triphosphates (IP3)-Ca2+ and nuclear factor-kappa B cascades. However, there is no information regarding their ability to modulate other receptor responses. The aim of the present study was to investigate whether histamine H1 receptor ligands could display positive efficacy concerning receptor desensitization, internalization, signaling through Gαq independent pathways or even transcriptional regulation of proinflammatory genes. While diphenhydramine, triprolidine and chlorpheniramine activate ERK1/2 (extracellular signal-regulated kinase 1/2) pathway in A549 cells, pre-treatment with chlorpheniramine or triprolidine completely desensitize histamine H1 receptor mediated Ca2+ response, and both diphenhydramine and triprolidine lead to receptor internalization. Unlike histamine, histamine H1 receptor desensitization and internalization induced by antihistamines prove to be independent of G protein-coupled receptor kinase 2 (GRK2) phosphorylation. Also, unlike the reference agonist, the recovery of the number of cell-surface histamine H1 receptors is a consequence of de novo synthesis. On the other hand, all of the ligands lack efficacy regarding cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) mRNA regulation. However, a prolonged exposure with each of the antihistamines impaires the increase in COX-2 and IL-8 mRNA levels induced by histamine, even after ligand removal. Altogether, these findings demonstrate the biased nature of histamine H1 receptor ligands contributing to a more accurate classification, and providing evidence for a more rational and safe use of them.

MRP4/ABCC4 expression is regulated by histamine in acute myeloid leukemia cells, determining cAMP efflux.

Intracellular cAMP (i-cAMP) levels play an important role in acute myeloid leukemia (AML) cell proliferation and differentiation. Its levels are the result of cAMP production, degradation, and exclusion. We have previously described histamine H2 receptors and MRP4/ABCC4 as two potential targets for AML therapy. Acting through histamine H2 receptors, histamine increases cAMP production/synthesis, while MRP4/ABCC4 is responsible for the exclusion of this cyclic nucleotide. In this study, we show that histamine treatment induces MRP4/ABCC4 expression, augmenting cAMP efflux, and that histamine, in combination with MRP inhibitors, is able to reduce AML cell proliferation. Histamine, through histamine H2 receptor, increases i-cAMP levels and induces MRP4 transcript and protein levels in U937, KG1a, and HL-60 cells. Moreover, histamine induces MRP4 promoter activity in HEK293T cells transfected with histamine H2 receptor (HEK293T-H2 R). Our results support that the cAMP/Epac-PKA pathway, and not MEK/ERK nor PI3K/AKT signaling cascades, is involved in histamine-mediated upregulation of MRP4 levels. Finally, the addition of histamine potentiates the inhibition of U937, KG1a, and HL-60 cell proliferation induced by MRP4 inhibitors. Our data highlight that the use of a poly-pharmacological approach aimed at different molecular targets would be beneficial in AML treatment.

Involvement of histamine H1 and H2 receptor inverse agonists in receptor's crossregulation.

Histamine [2-(4-Imidazolyl)-ethylamine] modulates different biological processes, through histamine H1 and H2 receptors, and their respective blockers are widely used in treating allergic and gastric acid-related disorders. Histamine H1 and H2 receptor crossdesensitization and cointernalization induced by its agonists have been previously described. In this study, we show how this crosstalk determines the response to histamine H1 and H2 receptor inverse agonists and how histamine H1 and H2 receptor inverse agonists interfere with the other receptor's response to agonists. By desensitization assays we demonstrate that histamine H1 and H2 receptor inverse agonists induce a crossregulation between both receptors. In this sense, the histamine H1 receptor inverse agonists desensitize the cAMP response to amthamine, a histamine H2 receptor agonist. In turn, histamine H2 receptor inverse agonists interfere with histamine H1 receptor signaling. We also determine that the crossdesensitization induced by histamine H1 or H2 receptor agonists alters the histamine inverse agonists receptor response: activation of histamine H1 receptor affects cAMP response induced by histamine H2 receptor inverse agonists, whereas histamine H2 receptor agonist induces a negative regulation on the anti-inflammatory response of histamine H1 receptor inverse agonists. Binding studies revealed that histamine H1 and H2 receptors cointernalize after stimulus with histamine receptor inverse agonists. In addition, the inhibition of the internalization process prevents receptor crossregulation. Our study provides new insights in the mechanisms of action of histamine H1 and H2 receptors that explain the effect of histamine H1 and H2 receptor inverse agonists and opens up new venues for novel therapeutic applications.

PI3K pathway is involved in ERK signaling cascade activation by histamine H2R agonist in HEK293T cells.

BACKGROUND: Histamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them. METHODS: Phosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor. RESULTS: H2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor. CONCLUSIONS: These results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation. GENERAL SIGNIFICANCE: This work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.