Background: We demonstrated previously that striatal adenosine modulates ethanol-induced motor incoordination (EIMI) via adenosine A1 receptors coupled to pertussis toxin (PT)-sensitive G protein and adenylyl cyclase-cyclic adenosine monophosphate (cAMP). Additionally, intrastriatal (IST) PT antagonizes EIMI and its potentiation by the adenosine A1 agonist N6-cyclohexyladenosine; it also inhibits cAMP concentration.
Methods: Guide cannulas were stereotaxically implanted for IST pretreatment with PT followed 5 days later by IST of N6-cyclohexyladenosine and intraperitoneal ethanol. The adenosine diphosphate (ADP) ribosylation reaction involved PT-catalyzed [32P]nicotinamide adenine dinucleotide (NAD) labeling of rat striatal membranes. Antagonism of EIMI (Rotorod method) after IST microinfusion of PT was investigated to determine whether it was due to a decrease in the functional activity of G proteins due to ADP ribosylation of the Giα subunit caused it.
Results: Striatal membranes from IST PT (0.5 μg)–treated animals exhibited significantly attenuated (up to 90%) in vitro ADP ribosylation with [32P]NAD. Striatal membranes from animals injected with ethanol (1.5 g/kg intraperitoneally) exhibited statistically significant increase (11%) in in vitro ADP ribosylation. Similarly, ethanol (50 mM) added to striatal membranes from untreated animals produced significant stimulation of in vitro ADP ribosylation. The decrease in the functional activity of G proteins due to ADP ribosylation of the Giα subunit after IST PT was functionally correlated with marked attenuation in EIMI, as observed previously. This finding suggests a blockade of functional activity of PT-sensitive striatal Gi/Go proteins (i.e., fewer available sites for labeled NAD incorporation). The in vivo ethanol results indicate that it must have caused an increase in the ribosylation capacity of Giα in vivo (i.e., increased Gi activity). Increased ADP ribosylation by in vitro ethanol increases Gi/Go activity, consistent with EIMI, as previously reported.
Conclusions: The results provide biochemical evidence of an ethanol-induced increase in ADP ribosylation of Giα causing a decrease in the functional activity of G proteins coupled via Gi/Go to adenylyl cyclase-cAMP. These results confirm the previously observed antagonism of EIMI by PT (IST).