This study is the first to report that VDM11, a selective anandamide membrane transporter inhibitor, dose-dependently and significantly reduced the number of capsaicin-induced coughs. This antitussive effect of VDM11 was significantly antagonized by pretreatment with SR141716A, a cannabinoid CB1 receptor antagonist. These results suggest that the antitussive effect of VDM11 is mediated by the activation of cannabinoid CB1 receptors. We previously demonstrated that WIN 55212-2, a cannabinoid receptor agonist, had an antitussive effect through the activation of cannabinoid CB1 receptors . Furthermore, we also suggested that the antitussive effect of WIN 55212-2 may depend mainly on central mechanisms . In the present study, we observed that the antitussive effect of VDM11 was antagonized by systemic intraperitoneal or inhaled pretreatment with SR141716A. However, when SR141716A was injected intracerebroventricularly, the antitussive effect of VDM11 was not antagonized. These results suggest that VDM11 exerts its antitussive effect through an activation of peripheral CB1 receptors.
However, it is not reasonable to consider that VDM11 directly activates CB1 receptors. Giuffrida et al  reported that systemic administration of AM404, an anandamide membrane transporter inhibitor, caused a gradual increase in anandamide in rat plasma. Nemeth et al  reported that a low concentration of anandamide inhibited the release of substance P from peripheral capsaicin-sensitive sensory nerve terminals via the activation of cannabinoid CB1 receptors. Recently, we reported that NS-398, a selective cyclooxygenase-2 inhibitor, exerted peripheral antitussive effects by inhibiting the release of substance P from capsaicin-sensitive afferent C-fibers in airways . Thus, it seems likely that inhibition of the release of substance P from capsaicin-sensitive sensory C-fibers may cause inhibition of the cough reflex. It has been reported that capsaicin binds to the cytosolic domain of TRPV1 receptors because of its lipophilic nature . Substance P, which is contained in afferent C-fiber endings within the airway epithelium and smooth muscle layer, is released by activation of afferent C- fibers. Bonham et al  reported that substance P stimulates rapidly adapting receptors in guinea pigs. The stimulation of the rapidly adapting receptors by substance P is a potential link between the two airway defense systems, both of which elicit bronchoconstriction, mucus secretion and cough. Such a link, whereby C-fiber-receptor stimulation lead to the release of substance P and the subsequent stimulation of rapidly adapting receptors, has been previously proposed to explain the overlap of stimuli and reflex effects of both afferent systems . Namely, that activation of TRPV1 receptors by capsaicin causes the release of substance P and the subsequent stimulation of rapidly adapting receptors, which may enhance cough reflexes. Based on these findings, it is possible that VDM11 accumulates endogenous anandamide, which results in the stimulation of cannabinoid CB1 receptors and the subsequent inhibition of substance P release from capsaicin-nensitive afferent C-fibers, which may produce the antitussive effect.
The above mentioned possibilities are further supported by the results of the present study. We observed that anandamide alone produced a biphasic modulation of the cough reflex. Indeed, the number of coughs induced by the inhalation of lower dose of anandamide (0.03 mg/ml) was significantly less than those induced by the inhalation of vehicle alone. On the other hand, the number of coughs induced by the inhalation of higher doses of anandamide (3 mg/ml) was significantly greater than those induced by the inhalation of vehicle alone. We also observed that when the animals were pretreated with AM251, a selective CB1 receptor antagonist, the number of coughs induced by the inhalation of lower dose of anandamide (0.03 mg/ml) was increased to the level of those induced by the inhalation of vehicle alone. This result suggests that activation of CB1 receptors by anandamide causes an inhibitory modulation of the cough reflex. Furthermore, it is possible that higher doses of anandamide may cause the activation of the cytosolic domain of TRPV1 receptors, since anandamide-induced enhancement of the number of coughs was abolished by pretreatment with either capsazepine, a selective TRPV1 receptor antagonist, or VDM11.
In conclusion, the present results suggest that anandamide, an endogenous cannabinoid ligand, may modulate cough sensitivity and anandamide transporters play an important role in this modulation. Furthermore, these findings indicate that inhibition of the uptake of anandamide produced a potent antitussive effect and suggest that the anandamide transporter is a potential target for peripherally acting antitussive drugs.