Introduction Histamine Imidazolyl ethylamine is an important

Introduction Histamine [2-(4-Imidazolyl)-ethylamine] is an important mediator of many physiological and pathological processes including inflammation, gastric dextromethorphan hydrobromide secretion, neuromodulation, regulation of immune function, cell proliferation and differentiation, among others. Histamine exerts its biological effects by binding to four different G protein-coupled receptor subtypes (H1-H4) (Panula et al., 2015). Until now, the most clinically relevant uses of histamine receptor ligands are achieved through the interaction with histamine H1 or H2 receptors, which are widely expressed in many tissues. In this regard, histamine H1 receptor antagonists/inverse agonists are used in the treatment of several allergic conditions, such as rhinoconjunctivitis, urticaria, and atopic dermatitis, and are promptly available as prescription and/or as over-the-counter drugs (Church, 2016). On the other hand, histamine H2 receptor antagonists/inverse agonists have proved to be active agents for the treatment of duodenal and gastric ulcers, reflux, esophagitis and Zollinger–Ellison syndrome (Hershcovici and Fass, 2011, Sigterman et al., 2013). Additionally, since histaminergic ligands are low-cost drugs with no patent protection, there is a great interest to facilitate the repurposing of these drugs for other pathologies. Consequently, a deep understanding of their mechanisms of action is needed. The histamine H1 and H2 receptors are coexpressed in most human tissues and cell types, such as neurons, airway and vascular smooth muscle cells, endothelial cells, hepatocytes, epithelial cells, neutrophils, eosinophils, monocytes, dendritic cells, as well as T and B lymphocytes, among others (Jutel et al., 2009, Parsons and Ganellin, 2009). In most tissues, histamine H1 receptor couples to Gαq/11 leading to an increase in phosphoinositide metabolism, whereas histamine H2 receptor couples to Gαs, triggering adenylyl cyclase (AC) activation and cyclic AMP (cAMP) accumulation (Panula et al., 2015). In these systems, the action of endogenous histamine may result from the balance and coordination of the signaling events activated by these, or even more, subtypes of histamine receptors. In this way, previous studies of our laboratory have described the existence of histamine H1 and H2 receptor crossregulation. In native and recombinant systems, both receptors desensitize when cells are exposed to a sustained stimulus with histamine H1 or H2 receptor agonists. Interestingly, this crossdesensitization does not depend on second messengers nor their downstream kinases, PKA or PKC, but on G protein-coupled receptor kinase 2 (GRK2) (Alonso et al., 2013). In addition, upon activation of histamine H1 or H2 receptor, both cointernalize in endosomes and form heteromers. Since these crossregulation mechanisms proved to be critical for the output response to histaminergic stimulation, it would be expected that it also affects the response of the histamine H1 and H2 receptor inverse agonists used in the clinic. Nowadays, it is accepted that ligands which have been classically described as inverse agonists, due to their negative efficacy at modulating the G protein pathway, could also display some positive efficacy regarding receptor desensitization, internalization, or even signaling though another pathway (Kenakin, 2002, Pupo et al., 2016). In this sense, our laboratory has described that several histamine H2 receptor inverse agonists behave as full agonists regarding histamine H2 receptor desensitization and internalization, in spite of diminishing cAMP production (Alonso et al., 2014, Alonso et al., 2015). In the present work, we hypothesized that histamine H1 and H2 receptor inverse agonists also induce the crossdesensitization between histamine H1 and H2 receptors, and that the crossregulation induced by histamine H1 and H2 receptor agonists affects the behavior of histamine H1 and H2 receptor inverse agonists. In order to address this issue, we analyzed the receptor´s crossregulation in U937 cells, which endogenously express histamine H1 and H2 receptors and in cotransfected HEK293 cells. We utilise clinically relevant ligands as mepyramine, chlorpheniramine, triprolidine and diphenhydramine (first-generation histamine H1 receptor inverse agonists), cetirizine (second-generation histamine H1 inverse agonist) and the widely used histamine H2 receptor inverse agonists cimetidine, ranitidine and famotidine.