Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A1 and P2Y2 receptors in rat brain

Purines such as adenosine and ATP are now generally recognized as the regulators of many physiological functions, such as neurotransmission, pain, cardiac function, and immune responses. Purines exert their functions via purinergic receptors, which are divided into adenosine and P2 receptors. Recently, we demonstrated that the Gi/o-coupled adenosine A1 receptor (A1R) and Gq/11-coupled P2Y2 receptor (P2Y2R) form a heteromeric complex with unique pharmacology in co-transfected human embryonic kidney cells (HEK293T). However, the heteromeric interaction of A1R and P2Y2R in situ in brain is still largely unknown. Findings: In the present study, we visualized the surface expression and co-localization of A1R and P2Y2R in both transfected HEK293T cells and in rat brain by confocal microscopy and more precisely by immunogold electron microscopy. Immunogold electron microscopy showed the evidence for the existence of homo- and hetero-dimers among A1R and P2Y2R at the neurons in cortex, cerebellum, and particularly cerebellar Purkinje cells, also supported by co-immunoprecipitation study.

The results suggest that evidence for the existence of homo- and hetero-dimers of A1R and P2Y2R, not only in co-transfected cultured cells, but also in situ on the surface of neurons in various brain regions. While the homo-dimerization ratios displayed similar patterns in all three regions, the rates of hetero-dimerization were prominent in hippocampal pyramidal cells among the three regions.