Inhibition of adenylyl cyclase by neuronal P2Y receptors

Abstract

1 P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identi®ed as P2Y 12 . Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. 2 ADP and ATP (0.1 ± 100 mM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A 2A or recombinant b 2 receptors. Forskolindependent cyclic AMP production was reduced by 41 mM and enhanced by 10 ± 100 mM ADP; this latter e ect was turned into an inhibition when A 2A receptors were blocked. 3 The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin. 4 The rank order of agonist potencies for the reduction of cyclic AMP was (IC 50 values): 2-methylthio-ADP (0.12 nM)=2-methylthio-ATP (0.13 nM)4ADPbS (71 nM)4ATP (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, ATPaS, and 2-methylthio-AMP. 5 RT ± PCR demonstrated the expression of P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 12 , but not P2Y 1 , receptors in PC12 cells. In Northern blots, only P2Y 2 and P2Y 12 were detectable. Di erentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged.

Activity-Dependent Autocrine-Paracrine Activation of Neuronal P2Y Receptors

“…Stimulation of PC12 cells with the adenosine A 2A receptor agonist CGS 21680 caused a reproducible increase in these values of cAMP, but the extent of basal and stimulated cAMP synthesis may vary between different preparations (Unterberger et al, 2002). Therefore, the values of cAMP obtained in the presence of CGS 21680 were normalized to the values obtained in its absence within the same preparation (normalized to basal).…”

“…The accumulation of cAMP in PC12 cell cultures was determined as described previously (Unterberger et al, 2002). After labeling of cellular purines with tritiated adenine (2.5 Ci/ml for 12 hr), the medium was replaced by the buffer described above supplemented with 100 M of the phosphodiesterase inhibitor RO 20-1724 and 1 U/ml adenosine deaminase.…”

“…The incubation was terminated by exchanging the buffer for 1 ml of 2.5% perchloric acid containing 100 M nonlabeled cAMP followed by a 20 min incubation at 4°C. Subsequently, cAMP was separated from the other purines by a chromatographic procedure described previously (Unterberger et al, 2002). Finally, radioactivity within the samples obtained was determined by liquid scintillation counting.…”

“…2C), but not for P2Y 4 (data not shown). Previously, transcripts for P2Y 4 and P2Y 6 receptors, but none for P2Y 1 receptors, had been detected in PC12 cells by RT-PCR (Arslan et al, 2000;Unterberger et al, 2002). Thus, nondifferentiated PC12 cells express high levels of P2Y 2 , but of no other phospholipase C-linked P2Y receptor.…”

“…These and all subsequent values were obtained in the presence of the phosphodiesterase inhibitor RO 20-1724 (100 M). Therefore, any druginduced alteration will reflect changes in adenylyl cyclase activity rather than alterations in cAMP degradation (Unterberger et al, 2002). Exposure of PC12 cells to 1 M of the A 2A adenosine receptor agonist CGS 21680 for 15 min increased cellular cAMP by ϳ20-fold (Fig.…”