Figure 5

Pretreatment of rat cerebellar membranes with MAFP concomitantly potentiates 2-AG-stimulated G-protein activity (a) and prevents enzymatic degradation of 2-AG to AA more efficiently than PMSF (b). Membranes were pretreated with MAFP (10⁻⁵ M), PMSF (10⁻³ M) or the vehicle (DMSO) as a control for 30 min at +25°C in the presence of 0.5% BSA. In (a), membranes were used for [³⁵S]GTPγS-binding assay to determine G-protein activation in response to 2-AG concentrations, producing near half-maximal (10⁻⁶ M) or maximal stimulation (10⁻⁴ M). In (b), pretreated membranes were used for HPLC to assess enzymatic hydrolysis of 2-AG (5 × 10⁻⁵ M) under incubation conditions closely mimicking G-protein activation assay. By HPLC analysis, initial purity of 2-AG was 98% with the rest of the material (2%) representing 1(3)-AG. For (a), the data represent the mean±s.e.m. of [³⁵S]GTPγS binding from basal and, for (b), the mean±s.e.m. of relative (%) peak areas, each from at least three independent experiments performed in duplicate. n.d.: not detectable. An asterisk (^*) denotes the statistically significant (P<0.05) difference from the respective control; # indicates statistically the significant (P<0.05) difference between MAFP and PMSF treatment.