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  • br Materials and methods br Results br Discussion The major


    Materials and methods
    Discussion The major findings of the present study are that: 1) the response to inverse agonists may be crossregulated by the activation of either Pyr3 H1 or H2 receptors, 2) histamine H1 and H2 receptor inverse agonists crossdesensitize the receptor´s response to their agonist, and 3) the crointernalization of histamine H1 and H2 receptors is responsible for the crossdesensitization induced by inverse agonists. One hundred and thirty-four GPCRs are targets for drugs approved in the United States and European Union and it is estimated that approximately 35% of approved drugs target GPCRs. Among the receptors with the highest number of approved drugs are the histamine H1 and H2 receptors (Sriram and Insel, 2018). Many of the histamine H1 and H2 receptor antagonists, now reclassified as inverse agonists, have been successfully used in the clinic, achieving the desired effects, although side effects do happen. The concept of inverse agonism arose from experimental observations showing that certain drugs were able to reduce the activity of receptor systems that were active even in the absence of agonists. These ligands bind preferentially and stabilize the inactive conformation of the receptors. However, whether inverse agonism is essential or important for these drugs to exert their medicinal actions has not been clarified yet. It has been described that GPCRs functionally interact with other pathways in several ways. In particular, histamine H1 and H2 receptor agonists interfere with the agonist´s response of the other GPCR. This crossregulation has already been described in recombinant and naïve systems involving GRK2-dependent crossdesensitization and cointernalization/ heterodimerization of both receptors, and has proven to be part of histamine´s fine regulation to achieve a final response (Alonso et al., 2013). Now, we show that the crossdesensitization induced by histamine H1 or H2 receptor agonists also influences the response to inverse agonists. In this way, the activation of whichever of these receptors modifies the response of histamine H1 or H2 receptor inverse agonists in different systems. Thus, the anti-inflammatory effect of histamine H1 receptor inverse agonists evaluated by the reduction in IL-8 and COX-2 gene expression in monocytic U937 cells was less pronounced when histamine H2 receptor was activated by amthamine (Fig. 2C and D). On the other hand, cAMP response triggered by incubation with the histamine H2 receptor inverse agonist was prevented when U937 cells were pretreated with the histamine H1 receptor agonist (Fig. 3A). These results evidence how the efficacy of these inverse agonists is affected by the presence and activation of the other subtype of histaminergic receptor. This crosstalk may explain why histamine H1 and H2 receptor inverse agonists have different efficacies in different tissues. Therefore, when considering targeting histamine H1 or H2 receptors, it would be necessary to take into account not only the selectivity, affinity and residence time of ligands, but also the expression levels of both receptors and their crossregulation in order to accomplish the desired effect. 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 cell cycle and engage with apoptotic processes or proliferate instead (Fig. 5).