EXTRACELLULAR ATP MEDIATES CA2-DEPENDENT MECHANISM( SIGNALING IN CULTURED MYENTERIC NEURONS VIA A PLC)

In the myenteric plexus, ATP is released as a neurotransmitter by ''pu rinergic'' nerves, relaxing visceral smooth muscle. Sire report a sign al transduction mechanism for ATP in cultured myenteric neurons involv ing receptor-mediated release of intracellular Ca2+ stores. Primary cu ltures of myenteric neurons hem guinea pig taenia coli were loaded wit h the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined usin g digital imaging microscopy. Superfusion of single neurons with ATP ( 0.01-1,000 mu M) resulted in concentration-dependent increases in intr acellular Ca2+ concentration ([Ca2+](i)) that were independent of extr acellular Ca2+. Decrements in peak [Ca2+](i) were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agoni sts (100 mu M) was consistent with action at a neuronal P-2y purinocep tor: 2-chloro-ATP = ATP = 2-methylthio-ATP (MeSATP) > ADP > alpha,beta -MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transient s were inhibited dose dependently by suramin, a nonspecific P-2 antago nist, and reactive blue 2, a specific P-2y antagonist. ATP and cyclopi azonic acid (30 mu M) appear to release an identical intracellular Ca2 + store. Preincubation with the aminosteroid U-73122 (10 mu M) inhibit ed ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pret reatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+](i) evoked by ATP was not affected by pr eincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 m u M). These data suggest a signal transduction mechanism for ATP in cu ltured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myoinositol 1,4,5-trisphosph ate-sensitive intracellular Ca2+ stores.