Differential stimulatory effects of cannabinoids on VIP release and NO synthase activity in synaptosomal fractions from rat ileum

Cannabinoid-1 (CB1) and CB2 receptors are present on neurons of the enteric nervous system. Our aim was to study whether cannabinoid receptor activation is involved in the regulation of VIP release and NO synthesis in isolated fractions of nerve terminals from rat ileum. VIP was measured by RIA and NO synthesis was analyzed using a l-[3H]arginine assay. Anandamide stimulated VIP release (basal: 245.9 ± 12.4 pg/mg, 10−6 M: 307.6 ± 11.7 pg/mg, [n = 6, P < 0.05], 10−7 M: 367.0 ± 26.1 pg/mg, [n = 6, P < 0.01]). The cannabinoid receptor agonist WIN 55,212-2 had similar effects (basal: 250.5 ± 37.4 pg/mg, 10−6 M: 320.9 ± 34.7 pg/mg; [n = 4, P < 0.05]). The stimulatory effect of anandamide was blocked by the selective CB2 receptor antagonist, SR144528 (10−7 M) (anandamide 10−6 M: 307.6 ± 11.7 pg/mg; +SR144528: 249.0 ± 26.3 pg/mg, [n = 6, P < 0.05]), whereas the selective CB1 receptor antagonist SR141716 A had no effect. NO synthesis was stimulated by anandamide ([fmol/mg/min] basal: 0.08 ± 0.01, 10−6 M: 0.16 ± 0.03; 10−7 M: 0.13 ± 0.02, n = 4, P < 0.05) and WIN 55,212-2 ([fmol/mg/min] basal: 0.05 ± 0.01, 10−6 M: 0.1 ± 0.02, n = 4, P < 0.05). The anandamide reuptake inhibitor, AM 404 increased basal NOS activity ([fmol/mg/min] control: 0.1 ± 0.04, 10−6 M: 0.28 ± 0.08, n = 7, P < 0.05). The stimulatory effect of anandamide on NO synthase was not antagonized by antagonists at the CB1, CB2 or TRPV1 receptor, respectively. In conclusion, in enteric nerves anandamide stimulates VIP release by activation of a CB2 receptor specific pathway, while the stimulation of NO production suggests the existence of an additional type of cannabinoid receptor in the enteric nervous system.