In the myenteric plexus, ATP is released as a neurotransmitter by "purinergic" nerves, relaxing visceral smooth muscle. We report a signal transduction mechanism for ATP in cultured myenteric neurons involving receptor-mediated release of intracellular Ca2+ stores. Primary cultures of myenteric neurons from guinea pigs taenia coli were loaded with the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined using digital imaging microscopy. Superfusion of single neurons with ATP (0.01-1,000 microM) resulted in concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were independent of extracellular Ca2+. Decrements in peak [Ca2+]i were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agonists (100 microM) was consistent with action at a neuronal P 2y purinoceptor: 2-chloro-ATP = ATP = 2-methyl-thio-ATP (MeSATP) > ADP > alpha, beta-MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transients were inhibited dose dependently by suramin, a nonspecific P2 antagonist, and reactive blue 2, a specific P 2y antagonist. ATP and cyclopiazonic acid (30 microM) appear to release an identical intracellular Ca2+ store. Preincubation with the aminosteroid U-73122 (10 microM) inhibited ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pretreatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+]i evoked by ATP was not affected by preincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 microM). These data suggest a signal transduction mechanism for ATP in cultured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myo-inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores.