Interaction between 6-chloro PB and CCPA on [35S]GTPγS binding in rat striatum

Brian Nolan, Kathy O’Boyle. UCD School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.

Substantial evidence suggests that adenosine and dopamine receptors antagonise each other at different levels in the CNS (Franco et al, 2000). This study investigates possible interactions between A1 and D1 receptors at the level of [35S] GTPγS binding in rat striatum, a region known to co-express both receptors.

G-protein stimulation was measured by incubating rat striatal membranes with drugs and [35S] GTPγS, as previously described (Roberts et al, 2004), with minor modifications. The A1 agonist CCPA stimulated [35S] GTPγS binding with an EC50 of 15 nM and an EMAX of 63% increase over basal. The effects of CCPA were significantly reduced by the A1 antagonist DPCPX (p<0.01, 1-way ANOVA). The D1 agonist, 6-chloro PB (6-Cl PB), and partial agonist, SKF38393, also stimulated [35S] GTPγS. Their EC50 and EMAX values were: 6-Cl PB: 300 nM and 90%; SKF38393: 598 nM and 63%. However, despite the fact that these agonists behaved in a manner consistent with their relative intrinsic activities at D1 receptors, their effects on [35S] GTPγS binding were not blocked by the D1 antagonist SCH23390. To investigate possible interactions between A1 and D1 receptors, the responses to CCPA and 6-Cl PB were examined in the presence of antagonists and agonists for the other receptor. SCH23390 did not alter the response to CCPA and DPCPX did not alter the response to 6-Cl PB. The combination of 1 nM - 100 μM 6-Cl PB with 1 μM CCPA resulted in a parallel upward shift of the 6-Cl PB concentration-effect curve, consistent with a strictly additive effect. However, combining 1 nM - 100 μM CCPA with 1 μM 6-Cl PB resulted in a non-parallel upward shift of the CCPA concentration effect curve. Analysis of the data by 2-way ANOVA revealed a significant interaction (p<0.001) between treatment and concentration, such that low concentrations (1-10 nM) of CCPA had a greater than expected effect in the presence of the D1 agonist.

These data indicate that the D1 agonist 6-Cl PB stimulates the binding of [35S] GTPγS in rat striatal membranes via an A1- and D1-independent target. When this target is co-activated with A1 receptors the stimulation of G-proteins is greater than additive.

Franco R et al. (2000) Neuropsychopharmacology 23, S50-S59.
Roberts DJ et al. (2004) Mol Pharmacol 66, 1573-1579.