2 DISTINCT P-2 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+ co ncentration ([Ca2+](i)) in mouse pineal gland tumor (PGT-beta) cells. Studies of the [Ca2+](i) rise using nucleotides and ATP analogues esta blished the following potency order: ATP, adenosine 5'-O-(3-thiotripho sphate) greater than or equal to UTP > 2-chloro-ATP > 3'-O-(4-benzoyl) benzoyl ATP, GTP greater than or equal to 2-methylthio ATP, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) > CTP. AMP, adenosine, alpha,bet a-methyleneadenosine 5'-triphosphate, beta,gamma-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 AT P- 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-2Y purinoceptor-selective agonist ADP beta S induce inositol 1,4,5-trisphosphate generation in a concentrat ion-dependent manner with maximal effective concentrations of similar to 100 mu M. In sequential stimulation, UTP and ADP beta S do not inte rfere with each other in raising the [Ca2+](i). Costimulation with UTP and ADP beta S results in additive inositol 1,4,5-trisphosphate gener ation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 4 5-55%, whereas it has no effect on the ADP beta S response. Treatment with 1 mu M phorbol 12-myristate 13-acetate inhibits the ADP beta S-in duced [Ca2+](i) rise more effectively than the ATP- and UTP-induced re sponses. These results suggest that P-2U and P-2Y purinoceptors coexis t on PGT-beta cells and that both receptors are linked to phospholipas e C.