Cannabinoid agonists induce contractile responses through Gi/o-dependent activation of pholipase C in the bovine ciliary muscle

This study was undertaken to investigate the effect of some cannabinoid agonists on the bovine ciliary muscle. Both anandamide and CP 55,940 (cis-3-(2-hydroxy-4-(1,1-dimethyl heptyl) phenyl)-trans-4-(3-hydroxypropyl) cyclohexanol) produced a concentration-dependent contractile response in ciliary muscle. These responses were inhibited by SR 141716A (N-[piperidin-1-yl]-5-(4-cholophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) (0.1 and 1 microM) but not by SR 144528 (N-[1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl] 5-(4-chloro-3-methylphenyl)-1-(4 methoxy benzyl)-pyrazole-3-carboxamide) (1 and 10 microM). A preincubation with G(i/o) protein inhibitor pertussis toxin (500 ng/ml) for 20 min inhibited the contractile action of anandamide and CP 55,940. In addition, the phospholipase C inhibitor U73122 (1[6-[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl] amino] hexyl]-1H-pyrrole-2,5-dione) blocked the anandamide- and CP 55,940-induced contractions, whereas the protein kinase C activator, phorbol 12,13 dibutyrate (PDBu) significantly potentiated the contractions evoked by cannabinoid receptor agonists. We evaluated the binding of [(3)H]CP 55,940, which specifically labelled a single class of cannabinoid sites with affinity in low subnanomolar range (K(d)=0.6 nM) and the maximal number of binding sites of 1243 fmol/mg protein. Binding of [(3)H]CP 55,940 was inhibited by ligands having a major selectivity for cannabinoid (CB(1)) receptors. These findings provide strong evidence of the involvement of cannabinoid CB(1) receptors promoting contraction in the bovine ciliary muscle. Furthermore, the action of cannabinoid receptor agonists appears to be mediated via phospholipase C. These data also contribute to elucidate the cannabinoid CB(1) receptor pivotal role in the modulation of intraocular pressure and to show that cannabinoid receptor agonists may be regarded as potential antiglaucoma agents.