Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A(1 )and P2Y(2 )receptors in rat brain

BACKGROUND: 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 G(i/o)-coupled adenosine A(1 )receptor (A(1)R) and G(q/11)-coupled P2Y(2 )receptor (P2Y(2)R) form a heteromeric complex with unique pharmacology in co-transfected human embryonic kidney cells (HEK293T). However, the heteromeric interaction of A(1)R and P2Y(2)R in situ in brain is still largely unknown. FINDINGS: In the present study, we visualized the surface expression and co-localization of A(1)R and P2Y(2)R 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 A(1)R and P2Y(2)R at the neurons in cortex, cerebellum, and particularly cerebellar Purkinje cells, also supported by co-immunoprecipitation study. CONCLUSION: The results suggest that evidence for the existence of homo- and hetero-dimers of A(1)R and P2Y(2)R, 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.