Physiological implications of biased signaling at histamine H2 receptors

Abstract

Histamine mediates numerous functions acting through its four receptor subtypes all belonging to the large family of seven transmembrane G-protein coupled receptors. In particular, histamine H2 receptor (H2R) is mainly involved in gastric acid production, becoming a classic pharmacological target to treat Zollinger-Ellison disease and gastric and duodenal ulcers. H2 ligands rank among the most widely prescribed and over the counter-sold drugs in the world. Recent evidence indicate that some H2R ligands display biased agonism, selecting and triggering some, but not all, of the signaling pathways associated to the H2R. The aim of the present work is to study whether famotidine, clinically widespread used ligand acting at H2R, exerts biased signaling. Our findings indicate that while famotidine acts as inverse agonist diminishing cAMP basal levels, it mimics the effects of histamine and the agonist amthamine concerning receptor desensitization and internalization. Moreover, the treatment of HEK293T transfected cells with any of the three ligands lead to a concentration dependent pERK increment. Similarly in AGS gastric epithelial cells, famotidine treatment led to both, the reduction in cAMP levels as well as the increment in ERK phosphorylation, suggesting that this behavior could have pharmacological relevant implications. Based on that, histidine decarboxylase expression was studied by quantitative PCR in AGS cells and its levels were increased by famotidine as well as by histamine and amthamine. In all cases, the positive regulation was impeded by the MEK inhibitor PD98059, indicating that biased signaling toward ERK1/2 pathway is the responsible of such enzyme regulation. These results support that ligand bias is not only a pharmacological curiosity but has physiological and pharmacological implications on cell metabolism.

Keywords: 7TMR; ERK; GPCR internalization; H2R ligands; HDC; biased agonism; pluridimensional efficacy.

FIGURE 1

Negative efficacy of famotidine. (A) H2R transfected HEK293T cells pretreated (■) or not (∙) with 25 μM forskolin were exposed for 9 min to increasing concentrations of famotidine at 37^∘C in the presence of 1 mM IBMX. (B) Cells were exposed for 9 min to 10 μM amthamine (A), 10 μM famotidine (F) or 100 μM histamine (HA) at 37^∘C in the presence of 1 mM IBMX. (C) Cells were pretreated for 6 h with (black bars) or without (open bars) 100 ng/ml pertussis toxin (PTX) and exposed to 10 μM famotidine (F) for 9 min, in the presence of 1 mM IBMX. ^∗∗∗p < 0.001 with respect to basal (B); ns, no significant difference. (A–C) Cyclic AMP levels were determined as detailed under Experimental Procedures. Data were calculated as the means ± SD of assay duplicates. Similar results were obtained in at least three independent experiments. Error bars are not visible when their size is smaller than the symbol.

FIGURE 2

Famotidine induced H2R desensitization. (A) H2R-transfected HEK293T cells were exposed to 10 μM famotidine (∙) or 10 μM amthamine (■) for different time periods, then washed, and cAMP response to amthamine was determined. (B) Cells were transfected with H2R or co-transfected with different GRKs. (Left) Western blot analysis of the expression of the different GRKs. (Right) Cells were pretreated for 10 min with 10 μM famotidine (black bars) or not (open bars), washed and exposed for 9 min to 10 μM amthamine in the presence of 1 mM IBMX. (A,B) Cyclic AMP levels were determined as detailed under Experimental Procedures and expressed as the difference between the stimulus to the agonist and basal cAMP levels respect to the response of control cells without treatment. Data were calculated as the means ± SD of assay triplicates. Similar results were obtained in at least four independent experiments. Error bars are not visible when their size is smaller than the symbol.

FIGURE 3

Famotidine induced H₂R internalization. (A) H₂R-transfected HEK293T cells were exposed or not (∙) to 10 μM famotidine for 30 min (■), 60 min (▲), or 120 min (∘) and H2R binding sites were determined by saturation assays as described under Experimental Procedures. (B) Data represent the percentage maximal bound value fitted by non-linear regression of [³H]Tiotidine saturation assay. Data were calculated as the means ± SD of assay duplicates. Similar results were obtained in at least three independent experiments. ^∗∗p < 0.01; ^∗∗∗p < 0.001 with respect to untreated cells. Error bars are not visible when their size is smaller than the symbol.

FIGURE 4

Internalization and recovery of H2R membrane sites. [³H]Tiotidine saturation assays were performed in H2R-transfected HEK293T cells treated for 90 min with 10 μM famotidine, washed, and further incubated for 60 min in fresh medium. Data represent the percentage of maximal bound value fitted by non-linear regression of [³H]Tiotidine saturation assay, calculated as the means ±SE (n = 3). Assays were carried out in the absence (∙) or presence of cycloheximide 50 μM (■). 100% correspond to untreated cells. ^∗∗p < 0.01; ^∗∗∗p < 0.001; ns, no significant difference with respect to untreated cells. Error bars are not visible when their size is smaller than the symbol.

FIGURE 5

Famotidine promoted ERK phosphorylation. (A) H2R-transfected HEK293T cells were treated with 10 μM famotidine (F), 10 μM amthamine (A), or 100 μM Histamine (HA) for 5 min, lysed, and equal amounts of proteins were subjected to SDS-PAGE and analyzed by Western Blot. (B) Cells were exposed for 5 min to increasing concentrations of famotidine and western blot analysis were performed as mentioned above. (Right) Densitometric analysis of ERK phosphorylation at 5 min of treatment, normalized to the corresponding ERK total levels, obtained with the Scion Image Program. Data are expressed as times over basal p-ERK levels. Data are expressed as means ± SE (n = 3). ^∗∗∗p < 0.001; ^∗∗p < 0.01 respect to basal levels. (C) cAMP was determined in untreated cells (B) or following exposure to 10 μM amthamine (A) or 10 μM famotidine (F) for 5 min.

FIGURE 6

Biased signaling of famotidine. (A) AGS cells were exposed to 10 μM amthamine (A), 10 μM famotidine (F), or 100 μM histamine (HA) for 9 min, in the presence of 1 mM IBMX. Cyclic AMP levels were determined as detailed under Experimental Procedures. Data were calculated as the means ± SD of assay duplicates. Similar results were obtained in at least three independent experiments. (B) Histidine decarboxylase (HDC) gene expression was determined by quantitative real time PCR in AGS cells treated for 24 h with 10 μM amthamine (A), 10 μM famotidine, or 100 μM histamine (HA) in the presence (black bars) or absence of MEK inhibitor PD98059 (white bars). Relative HDC mRNA quantification was performed using β-actin as housekeeping gene. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; respect to basal.

FIGURE 7

Web of efficay. The scheme represents the efficacies of amthamine (▼), cimetidine (■), ranitidine (∙), tiotidine (♢), and famotidine (x), on cAMP accumulation, pERK levels, receptor desensitization and internalization. Values are percentages relatives to amthamine efficacy for each receptor behavior. The web represents Emax values on a scale from -100 (center) to 250 (exterior line), with intervals of 50.