Inhibition of adenylyl cyclase by neuronal P2Y receptors

Abstract

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y₁₂. Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 – 100 μM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A_2A or recombinant β₂ receptors. Forskolin‐dependent cyclic AMP production was reduced by 1 μM and enhanced by 10 – 100 μM ADP; this latter effect was turned into an inhibition when A_2A receptors were blocked. The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin. The rank order of agonist potencies for the reduction of cyclic AMP was (IC₅₀ values): 2‐methylthio‐ADP (0.12 nM)=2‐methylthio‐ATP (0.13 nM)>ADPβS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, or adenosine‐3′‐phosphate‐5′‐phosphate, but attenuated by reactive blue 2, ATPαS, and 2‐methylthio‐AMP. RT – PCR demonstrated the expression of P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, but not P2Y₁, receptors in PC12 cells. In Northern blots, only P2Y₂ and P2Y₁₂ were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y₁₂ receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity. British Journal of Pharmacology (2002) 135, 673–684; doi:10.1038/sj.bjp.0704514