Figure 3.

Time-dependent and fully DTT-reversible effects of SCH-202676 on [³⁵S]GTPγS-based G protein activation assays with brain cryostat sections. [³⁵S]GTPγS autoradiography of rat brain sagittal sections was conducted using a three-step protocol with 10⁻⁶ M DPCPX present throughout steps 2 and 3, as detailed in the Methods section. SCH-202676 was present at the indicated concentrations either throughout steps 2 and 3 (60+90 min) or only during the [³⁵S]GTPγS labeling (step3, 90 min). Where indicated, DTT (1 mM) was included during step3. The GPCR agonists 2-methylthio ADP (2MeSADP), lysophosphatidic acid (LPA), and CP-55,940 were present during step 3 to stimulate purinergic P2Y₁₂, lysophosphatidic acid LPA₁, and cannabinoid CB₁ receptors. In the control panel (left), the anatomical loci where the receptor agonists typically activate G proteins at this sagittal plane are indicated. Note the time-dependent increase in the SCH-202676-evoked nonspecific [³⁵S]GTPγS labeling. Note also complete reversal of the thiadiazole action in the presence of DTT. Abbreviations: cc, corpus callosum; Cbm, cerebellar molecular layer; Cbw, cerebellar white matter; Cx, cerebral cortex; fi, fimbria of the hippocampus; Hip, hippocampus; Str, striatum; SN, substantia nigra. Scale bar=2 mm.