Selective inhibition of β₂-adrenergic receptor-mediated cAMP generation by activation of the P2Y₂ receptor in mouse pineal gland tumor cells

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y₂ receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y₁ and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E₂, or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y₂ and β₂-adrenergic receptors. The binding of [³H]CGP12177 to β₂-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca²⁺, however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca²⁺ and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G_i-protein. We conclude that one of the functions of P2Y₂ receptors on the pineal gland is the selective inhibition of β-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.