br Materials and methods br Results br Discussion Main

Materials and methods
Results
Discussion Main results obtained in this work are presented in Table 1 and can be summarized as follows: 1) AEA inhibits FcεRI receptor-induced degranulation with the participation of CB2 and GPR55 receptors by a PTX-insensitive mechanism; 2) AEA does not block early FcεRI-induced phosphorylation events, but prevents calcium-dependent cytokine production; 3) BMMCs express CB1, CB2 and GPR55 receptor mRNAs. When CB2 and GPR55 receptors are stimulated separately, they mimic the inhibitory actions of AEA on degranulation; 4) specific antagonism of CB2 and GPR55 shows that both receptors play an important role in the inhibitory effects of AEA, HU308 and LPI on degranulation. As to FcεRI-induced [Ca2+] rise: 5) AEA, HU308 and LPI inhibit this parameter; 6) neither CB2 receptor blockage nor GPR55 antagonism prevent AEA inhibitory effects; 7) ML193, but not AM630, blocks the inhibitory actions of HU308; and 8) both AM630 and ML193 partially prevent LPI-induced inhibition of [Ca2+] rise. Cannabinoids have been proven to be important regulators of the immune system [4]. Together with the control of immune cell proliferation and differentiation, they exert important effects on chronic and acute inflammatory reactions [12]. Although the effects of those compounds have been widely documented and their use is considered a promising therapeutic strategy for inflammation-related diseases [53], the molecular mechanisms by which they exert their actions are not fully understood. In the present study, AEA inhibited IgE/Ag-dependent degranulation in a concentration-dependent fashion without altering the viability of the omadacycline or the general secretory machinery of MCs. AEA is an important endocannabinoid produced in response to nerve and immune cell stimulation [54]. It is a partial agonist of CB1 (Ki = 61–543 nM) and CB2 receptors (Ki = 279–1940 nM) [55], as well as a GPR55 receptor agonist at low nanomolar concentrations [56]. CB1 and CB2 cannabinoid receptors are GPCRs coupled to PTX-sensitive Gi/o proteins [56,57], while the GPR55 receptor couples to the PTX insensitive G12/13 proteins. The inhibition on degranulation caused by AEA observed in the present study was the same when PTX was added to the cells; therefore, neither CB1 nor CB2 receptors mediate this effect. Our data are in line with those reported previously, where inhibitory actions of endocannabinoids were observed using distinct models of MCs activation. For example, palmitoylethanolamide (PEA) blocks the PMA-induced secretion of nerve growth factor [58] and 2-AG inhibits antigen-dependent histamine release in sensitized guinea pig MCs [59]. However, the sensitivity to PTX was not addressed in those studies. The insensitivity to PTX found in our study could be interpreted as the result of direct GPR55 activation by AEA. However, our findings suggest that CB2 receptors are also involved. This is based on the fact that both HU308 (a highly selective CB2 receptor agonist) [60] and LPI (GPR55 endogenous agonist) prevented MC degranulation. In support to this idea, GPR55 is expressed in our BMMCs and is functional because selective blockage of this receptor with ML193 inhibits AEA actions. To the best of our knowledge, this is the first time that an inhibitory action of GPR55 on the FcεRI signaling system is reported. Our results are in line with those reported by Cantarella and coworkers, who observed that GPR55 activation prevents the release of nerve growth factor stimulated by PMA in the human cell line HMC-1 [58]. Our data also add to the notion that LPI is an important lipid mediator capable of modulating the intensity of inflammatory reactions through GPR55 activation [61]. In this regard, it has been shown that LPI levels increase in different inflammatory conditions [62,63]. Also, GPR55 stimulation has been proposed as a therapeutic strategy for pathologies related to intestinal contractility, colonic motility and vasoconstriction [61] where MCs contribute to tissue damage. We hope that our finding of the inhibitory role of GPR55 on MCs would open new avenues in the search for molecules able to limit diseases related to MCs.