CBC is a low (1 μM < Ki < 2 μM) and moderate (Ki∼0.1 μM) affinity ligand for human CB1 and CB2 receptors respectively (V. Di Marzo, unpubl. data). Furthermore, CBC inhibits endocannabinoid reuptake, and thus might indirectly activate – via increased extracellular endocannabinoid levels – the cannabinoid receptors (Ligresti et al., 2006; De Petrocellis et al., 2011). CBC was shown to stimulate descending pathways of antinociception and to cause analgaesia in rats in a manner partly attenuated by a CB1 receptor antagonist (Maione et al., 2011). We have shown here that the inhibitory effect of CBC was mimicked by selective CB1 and CB2 receptor agonists, suggesting that a direct pharmacological activation of such receptors results in inhibition of nitrite production. The ability of direct activation of both CB1 and CB2 receptor to reduce nitrite production in activated macrophages was previously documented (Ross et al., 2000; Aviello et al., 2011). Surprisingly, however, we observed that the inhibitory effect of CBC was further increased by rimonabant and AM251 (two CB1 receptor antagonists), at concentrations that, however, were inactive per se. These results, while confirming that exogenous activation of CB1 reduces NO formation in macrophages, negate the possibility that CBC acts via direct or indirect activation of CB1 receptors. This hypothesis is also supported by the results we obtained in the [35S]GTPγS binding assay performed with hCB1-CHO cell membranes. Thus, we found that CBC, at concentrations that included the one at which it significantly inhibits nitric oxide production (1 μM), did not induce any significant activation of cannabinoid CB1 receptors in this assay. Moreover, using the same assay, we also found that when CBC was administered 30 min after 1 μM AM251 or 0.1 μM rimonabant, it did not significantly affect the Emax of either of these compounds for their inhibition of [35S]GTPγS binding. It might be possible that an endogenous CB1 tone exists, which may couple negatively to the CBC signalling pathway and counteract CBC inhibition of nitrite production. Indeed, we found that LPS enhances anandamide levels in macrophages, and that CBC, instead, only elevates OEA levels. According to some authors, also OEA, but not PEA (the levels of which were not elevated by CBC) is taken up by cells through the same mechanism responsible for anandamide uptake (Hillard et al., 1997; Alhouayek and Muccioli, 2012). It is possible that CBC could not elevate anandamide levels because these were already maximally up-regulated by LPS. OEA, which is chemically related to anandamide, was previously shown to produce anti-inflammatory effects (Lo Verme et al., 2005) and hence, it is possible that a part of the beneficial effect of CBC observed here in macrophages could be due to its ability to increase OEA levels. It is also possible that CBC can merely synergize with rimonabant by unmasking the anti-inflammatory action of a per se inactive dose of this antagonist. In agreement with this hypothesis, rimonabant, but not the CB2 receptor antagonist SR144528, was previously shown to inhibit LPS-induced inflammation in wild-type mice, but not CB1 null mice (Croci et al., 2003). Accordingly, we also found here that SR144528 did not change the inhibitory effect of CBC on nitrite production in LPS-challenged macrophages. In another model of LPS-induced inflammation (LPS-induced paw oedema), DeLong et al. (2010) have recently shown that the anti-inflammatory action of CBC in vivo (LPS-induced paw oedema) was not blocked by either SR144528 or rimonabant.
In order to give further insights into the role of cannabinoid receptors in CBC action, we evaluated the effect of this plant constituent on cannabinoid receptors mRNA expression in macrophages. It was recently demonstrated that CBC alters the mRNA expression of cannabinoid receptors in the inflamed gut (Izzo et al., 2012). In the present study, we have shown that LPS causes up-regulation of CB1 receptors and down-regulation of CB2 receptors and that those changes were not modified by CBC. These results rule against the possibility that CBC could exert anti-inflammatory actions in macrophages by altering cannabinoid mRNA receptor expression.