ORIGINAL RESEARCH published: 10 March 2015 doi: 10.3389/fphar.2015.00045 Physiological implications of biased signaling at histamine H2 receptors Natalia Alonso 1,2,CarlosD.Zappia2,3, Maia Cabrera 2,3, Carlos A. Davio 2,3,4 , Carina Shayo 1,2, Federico Monczor 2,3 and Natalia C. Fernández 2,3* 1 Laboratorio de Patología y Farmacología Molecular, Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina, 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina, 3 Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina, 4 Instituto de Investigaciones Farmacológicas – Universidad de Buenos Aires – Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina 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 Edited by: Claudio M. Costa-Neto, and duodenal ulcers. H2 ligands rank among the most widely prescribed and over University of São Paulo, Brazil the counter-sold drugs in the world. Recent evidence indicate that some H2R ligands Reviewed by: display biased agonism, selecting and triggering some, but not all, of the signaling Terry Kenakin, pathways associated to the H2R. The aim of the present work is to study whether University of North Carolina Chapel Hill, USA famotidine, clinically widespread used ligand acting at H2R, exerts biased signaling. Our Andre Sampaio Pupo, findings indicate that while famotidine acts as inverse agonist diminishing cAMP basal São Paulo State University, Brazil levels, it mimics the effects of histamine and the agonist amthamine concerning receptor *Correspondence: Natalia C. Fernández, desensitization and internalization. Moreover, the treatment of HEK293T transfected Laboratorio de Farmacología de cells with any of the three ligands lead to a concentration dependent pERK increment. Receptores, Cátedra de Química Similarly in AGS gastric epithelial cells, famotidine treatment led to both, the reduction Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos in cAMP levels as well as the increment in ERK phosphorylation, suggesting that this Aires, Junin 956, PP (1113) Buenos behavior could have pharmacological relevant implications. Based on that, histidine Aires, Argentina [email protected] 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 Specialty section: This article was submitted to cases, the positive regulation was impeded by the MEK inhibitor PD98059, indicating Experimental Pharmacology and Drug that biased signaling toward ERK1/2 pathway is the responsible of such enzyme Discovery, a section of the journal Frontiers in Pharmacology regulation. These results support that ligand bias is not only a pharmacological curiosity Received: 19 December 2014 but has physiological and pharmacological implications on cell metabolism. Paper pending published: Keywords: 7TMR, H R ligands, biased agonism, pluridimensional efficacy, GPCR internalization, ERK, HDC 20 January 2015 2 Accepted: 20 February 2015 Published: 10 March 2015 Citation: Introduction Alonso N, Zappia CD, Cabrera M, Davio CA, Shayo C, Monczor F and Histamine is a biogenic amine synthesized from L-histidine by histidinede carboxylase (HDC). Fernández NC (2015) Physiological It plays an important role in human health and disease by acting through four receptor subtypes implications of biased signaling at histamine H2 receptors. (H1R–H4R) that differ in their expression, molecular characteristics, signal transduction pathways, Front. Pharmacol. 6:45. and function (Hill et al., 1997; Leurs et al., 2009). Nevertheless, all types of histamine receptors doi: 10.3389/fphar.2015.00045 belong to the seven transmembrane spanning family of G-protein coupled receptors (GPCRs). The Frontiers in Pharmacology | www.frontiersin.org 1 March 2015 | Volume 6 | Article 45 Alonso et al. Famotidine biased signaling observation that histamine-evoked gastric acid secretion could transfected HEK293T cells, leading to receptor down-regulation. not be blocked with classical antihistamines, led to the conclu- Furthermore, all H2R ligands tested, famotidine, histamine, and sion that H2 histamine receptors (H2R) were involved in gastric amthamine (H2R agonist) induced ERK1/2 activation not only acid secretion (Black et al., 1972). in HEK293T cells, but also in an endogenous expression system, Until now, one of the most clinically relevant uses of his- human gastric adenocarcinoma cells. Remarkably, ERK phospho- tamine receptor ligands are achieved through the regulation of rylation promoted by all ligands assayed induces HDC expression H2R, which are widely expressed in most tissues (Bakker et al., in AGS cells. 2002). Acid-related diseases affect the quality of life of patients In this work we demonstrate that the H2R inverse agonist and are important causes of morbidity and mortality (Shin et al., famotidine displays positive efficacy regarding receptor desensiti- 2008). Approximately 40% of adults in the USA complain of zation, internalization, and ERK activation. Finally, our findings monthly, 20% of weekly, and approximately 7% of daily heart- may have relevant clinical implications given that famotidine reg- burn (Sandler et al., 2002), making gastroesophageal reflux dis- ulates HDC expression and that this ligand is used clinically in ease (GERD) one the most common gastrointestinal (GI) disor- long term treatments. ders with great monetary costs for patients and public health sys- tem (Cappell, 2005). H2 antagonists have proved to be very active agents for the treatment of Zollinger–Ellison syndrome, duodenal Materials and Methods and gastric ulcers, reflux and esophagitis. They have an excellent safety profile that supports their common use as over-the-counter Materials medications. Over the past decades, there have been important Cell culture medium, antibiotics, isobutylmethyl xanthine advances in the treatment of acid-related disorders. The discov- (IBMX), cAMP, bovine serum albumin (BSA), cycloheximide, ery of proton pumps for controlling gastric acid secretion and amthamine, famotidine, forskolin, and PD98059 were obtained the successful synthesis of proton pump inhibitors in the 1980s, from Sigma Chemical Company (St. Louis, MO, USA). Tiotidine made these drugs to emerge as the treatment of choice for acid- were from Tocris Cookson Inc. (Ballwin, MO, USA). [3H]cAMP, related diseases. However, current guidelines recommend pre- and [3H]tiotidine were purchased from Perkin Elmer Life scribing gastroprotective agents to patients taking non-steroidal Sciences (Boston, MA, USA). Fetal calf serum was from Natocor anti-inflammatory drugs (NSAIDs) to prevent GI ulcers caus- (Argentina). Other chemicals used were of analytical grade and ing a rebirth of the clinical use of H2R antagonists, specifically obtained from standard sources. famotidine. A fixed-dose combination of the NSAID, ibupro- fen and the H2R antagonist, famotidine (ibuprofen/famotidine; Plasmid Constructions DUEXIS ) is available for the symptomatic treatment of arthritic GRK2, -3,-5, and -6 cDNAs were subcloned into the pCEFL symptoms and to reduce the risk of GI ulcers in patients requir- vector (pCEFLGRK2,-3, -5, and -6) and the human H2R was sub- ing ibuprofen therapy. The use of the combination of these two cloned previously in in our laboratory (Shayo et al., 2001)inthe drugs was shown to reduce the risk of ulcers by 50% compared pCEFLHA vector (pCEFLHA-H2R). with ibuprofen alone (Laine et al., 2012). In most tissues, including the parietal cells of the gut, H2R Cell Culture stimulation increases adenylate cyclase activity and induces HEK293T (Human embryonic kidney) and AGS (human gas- cAMP accumulation (Soll and Wollin, 1979). However, increas- tric cancer) cells were cultured in Dulbecco’s modified Eagle’s ing evidence indicates that receptors exist as conformational medium (DMEM) and Kaighn’s Modification of Ham’s F-12 collections where each conformation promotes different down- medium (F12K), respectively, supplemented with 10% fetal calf ˝ stream effects. In this context, a ligand is able to cause differential serum and 5 μg/ml gentamicin at 37 C in humidified atmosphere activation of some signaling events associated to a particular containing 5% CO2. receptor causing receptor bias (Ghanouni et al., 2001). Biased agonism has been mainly studied regarding adrenergic receptors. Transient Transfection It has been described that carvedilol, which is used for certain For transient transfection of HEK293T, cells were grown to 80– cardiovascular diseases, blocks the deleterious effects mediated 90% confluency. cDNA constructs were transfected into cells by β1AR and also presents and additional cardioprotective effect using K2 Transfection System (Biontex, Munich, Germany). The mediated by MAPK activation through a β-arrestin and EGFR transfection protocol was optimized as recommended by the sup- transactivation pathway (Kim et al., 2008). Recent evidence indi- plier. Assays were performed 48 h after transfection and the cates that this phenomenon of biased signaling may be extended expression of the constructs was confirmed