Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism.
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.
Kimball
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Department of Surgery, University of Michigan, Ann Arbor 48109-0331, USA.
Yule
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Mulholland
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D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism*; D006168:Guinea Pigs; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009197:Myenteric Plexus / Q000166:cytology / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D037342:Pertussis Toxin; D011983:Receptors, Purinergic / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002121:Calcium Channel Blockers; D011983:Receptors, Purinergic; D010566:Virulence Factors, Bordetella; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D009543:Nifedipine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium