Two distinct P₂ purinergic receptors, P(2Y) and P(2U), are coupled to phospholipase C in mouse pineal gland tumor cells

We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺](i)) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺](i) rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5'-O-(3- thiotriphosphate) ≤ UTP > 2-chloro-ATP > 3'-O-(4-benzoyl)benzoyl ATP, GTP ≤ 2-methylthio ATP, adenosine 5'-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5'-triphosphate, β,γ- methyleneadenosine 5'-triphosphate, and UMP had little or no effect on the [Ca²⁺](i) rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺](i) rise, because the responses depend on the ATP^4- and UTP⁴ concentrations, respectively. The P(2U) purinoceptor-selective agonist UTP and the P(2V) purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ~100 μM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺](i). Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45-55%, whereas it has no effect on the ADPβS response. Treatment with 1 μM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺](i) rise more effectively than the ATP- and UTP-induced responses. These results suggest that P(2U) and P(2Y) purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.