Involvement of cannabinoid receptors in the intraocular pressure-lowering effects of WIN55212-2.

It is known that marijuana smoking and administration of natural cannabinoids reduce intraocular pressure. However, it has not been established whether the intraocular pressure-lowering effects of cannabinoids are mediated by cannabinoid receptors. Aminoalkylindoles are a new class of cannabimimetics with structures entirely different from those of natural cannabinoids. WIN55212-2, a prototypic aminoalkylindole, has been shown to bind cannabinoid receptors and to exhibit cannabinoid-like activities. The objective of this study was to determine whether aminoalkylindoles lower intraocular pressure and whether the effects of aminoalkylindoles are mediated by ocular cannabinoid receptors. The intraocular pressure of New Zealand White rabbits was measured with the use of applanation pneumatonography. After the measurement of baseline intraocular pressure, drugs were applied topically and the intraocular pressure was monitored. The topical application of WIN55212-2 significantly reduced intraocular pressure in the treated eyes. The intraocular pressure-lowering effects of WIN55212-2 were time and dose dependent, and the maximal reduction was 4.7 +/- 0.5 mm Hg at a dose of 100 microg. In contrast to treated eyes, the intraocular pressure on the contralateral eyes was not significantly affected. The topical application of WIN55212-3, the enantiomer of WIN55212-2, had no effect on intraocular pressure. Furthermore, the intraocular pressure-lowering effects of WIN55212-2 were significantly reduced by topically administered SR141716A, a selective antagonist for the CB1 cannabinoid receptor. The dose-response curve of WIN55212-2 is shifted parallel to the right by SR141716A. These data demonstrate that like natural cannabinoids, WIN55212-2 also reduces intraocular pressure, and the effects of WIN55212-2 are mediated at least in part by the CB1 cannabinoid receptors in the eye.

The difference between the CB(1) and CB(2) cannabinoid receptors at position 5.46 is crucial for the selectivity of WIN55212-2 for CB(2).

It has been reported that WIN55212-2, a prototypic aminoalkylindole, has higher affinity for CB(2) than for CB(1). To explain the selectivity of WIN55212-2 for CB(2), molecular modeling studies were performed to probe the interacting sites between WIN55212-2 and cannabinoid receptors. In TMH5 the position 5.46 is a Phe in CB(2) versus a Val in CB(1). Docking of WIN55212-2 into the models of CB(1) and CB(2) predicts that F5.46 will result in a greater aromatic stacking of CB(2) with WIN55212-2. Using site-directed mutagenesis, this hypothesis was tested by exchanging the amino acids at position 5.46 between CB(1) and CB(2). Two mutations, including a Phe to Val mutation at the position 5.46 in CB(2) (CB2F5. 46V), and a corresponding Val to Phe mutation at the position 5.46 in CB(1) (CB(1)V5.46F), were made. The mutant receptors were transfected into 293 cells, and stable cell lines expressing similar numbers of receptors as wild-type receptors were chosen for additional ligand binding and cAMP accumulation studies. In ligand- binding assays, the CB(2)F5.46V mutation decreased the affinity of WIN55212-2 for CB(2) by 14-fold. In contrast, the CB(1)V5.46F mutation increased the affinity of WIN55212-2 for CB(1) by 12-fold. However, these mutations did not change the affinity of HU-210, CP-55940, and anandamide for CB(1) and CB(2). In cAMP accumulation assays, the changes in EC(50) values of WIN55212-2 were consistent with the changes in its binding affinity caused by the mutations. These results strongly support the hypothesis that the selectivity of WIN55212-2 for CB(2) over CB(1) is attributable to the change from Val in CB(1) at position 5.46 to Phe in CB(2).