It is important to note that histamine H and H
It is important to note that histamine H1 and H2 receptors are coupled to different G proteins and that their crossinterference induced by histamine H1 and H2 receptor inverse agonists depends on the cointernalization mechanism. To date, histamine H1 and H2 receptor inverse agonist have shown to interfere with the response of other GPCRs that share the same signaling pathway. In fact, treatment with these drugs stabilizes a conformation of the receptor that, although it is inactive, it may couple and recruit G-protein making it less available for other unrelated receptors that signal through the same pathway (Monczor et al., 2003, Tubio et al., 2010). Our results show that interference may occur among receptors that do not share the same signaling pathway and determine cell's fate. Thus, the exposure to histamine H1 receptor agonist, alone or in combination with specific histamine H2 receptor inverse agonists, ultimately determines whether U937 cells arrest their Calpeptin and engage with apoptotic processes or proliferate instead (Fig. 5). It is important to mention that the IC50 of cetirizine needed to achieve histamine H2 receptor desensitization was 0.43 µM or 170 ng/ml (Fig. 1B), becoming clinically relevant since pharmacokinetic studies after oral administration of the clinically used dose (10 mg/day) reported a maximal plasma concentration of 311 ng/ml. Among all the tested histamine H1 receptor inverse agonist, cetirizine showed the highest efficacy leaving a residual activity of histamine H2 receptor between 40% and 60% for HEK293-H1R-H2R and U937 cells, respectively (Fig. 1C and F). This interference in histamine H2 receptor response was similar to that achieved by histamine H1 receptor agonist (Alonso et al., 2013), denoting a control of the histamine response through histamine H2 receptor by histamine H1 receptor. Several GPCRs regulate their functions through cointernalization, which explains the signaling crossdesensitization reported in somatostatin 2 A/opioid receptors and adenosine A2A/dopamine D2 receptors (Hillion et al., 2002, Pfeiffer et al., 2002), and can even trigger new intracellular signaling pathways. In this sense, Smith et al. described that the cointernalization of the protease-activated receptor-4/purinergic receptor P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling (Smith et al., 2017). Regarding histamine H1 and H2 receptors, cointernalization and heterodimerization have been described upon histamine H1 or H2 receptor agonist stimulus, although receptor cointernalization is not the only mechanism of desensitization (Alonso et al., 2013). In reference to histamine H2 receptor inverse agonists, cimetidine, ranitidine, and famotidine have shown to elicit histamine H2 receptor internalization as part of their pluripotential efficacy, in an arrestin and dynamin dependent manner (Alonso et al., 2014). This report provides the first evidence that histamine H1 receptor inverse agonists can induce the internalization of their own receptor. In line with these findings, cetirizine showed to induce histamine H1 receptor internalization both, in cells that endogenously express the receptor (U937) and in a recombinant system (HEK293-H1R and HEK293-H1R-H2R) (Fig. 6). Moreover, regarding the crossregulation between histamine H1 and H2 receptor, here we describe a new efficacy for several histamine H1 and H2 receptor inverse agonists, as they induce the cointernalization of both receptors, interfering in the signaling cascade of receptors that have never been challenged to their own ligands. Further investigations are necessary in order to unravel what happens after internalization, whether the receptors heterodimerize in the endosomes and may, eventually, trigger some type of intracellular signaling. Our findings open an interesting field of study related to histamine H1 and H2 receptor inverse agonists.