Mechanisms of alpha,beta-methylene atp-induced inhibition in rat ileal smooth muscle: involvement of intracellular Ca2+ stores in purinergic inhibition.
1. In order to investigate purinergic effects on rat ileal smooth muscle, we used alpha,beta-methylene ATP (alpha,beta-MeATP), ATP, ADP and UTP. alpha,beta-Methylene ATP and ATP were the only agonists that caused a concentration-dependent inhibition of carbachol-precontracted smooth muscle. The inhibitory effect of alpha,beta-MeATP was completely blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) mol/L), a selective antagonist of the P2X > > P2Y receptor. 2. Using reverse transcription-polymerase chain reaction we demonstrated the presence of both, P2X and P2Y receptor mRNA within the rat ileal longitudinal muscle/myenteric plexus layer preparation. 3. The alpha,beta-MeATP-induced inhibition was blocked in a concentration-dependent manner in the presence of the K+ channel blocker apamin, but was unaffected by other K+ channel blockers, such as charybdotoxin (10(-7) mol/L), 4-aminopyridine (10(-4)mol/L), glibenclamide (10(-5) mol/L) and tetraethylammonium (10(-3) mol/L). 4. The alpha,beta-MeATP-induced inhibition was unaffected by pretreatment with atropine (10(-6) mol/L), phentolamine (10(-6) mol/L), propranolol (10(-6) mol/L), nitrendipine (10(-7) mol/L), pertussis toxin (10(-6) mol/L) NG-nitro-L-arginine (3 x 10(-4) mol/L) and tetrodotoxin (10(-6) mol/L), excluding an involvement of adrenergic, cholinergic, neural, nitrinergic or G-protein involvement in purinergic-mediated inhibition. 5. In order to investigate whether the internal Ca2+ stores participated in the inhibitory effect observed, we depleted internal Ca2+ stores with cyclopiazonic acid, a specific Ca2+-ATPase inhibitor. The inhibitory effect of alpha,beta-MeATP was completely abolished after depletion of the intracellular Ca2+ stores. 6. This is in contrast with the effects seen for neurotensin, where neurotensin-induced inhibition was unchanged after depletion of intracellular Ca2+ stores, suggesting at least two different pathways of apamin-sensitive non-adrenergic, non-cholinergic inhibition in rat ileal smooth muscle. 7. According to our results, the inhibitory effect of alpha,beta-MeATP in rat ileum longitudinal smooth muscle is mediated via a P2 purinoceptor, most likely a P2X receptor, involves G-protein-independent activation of an apamin-sensitive K+ channel and requires filled intracellular Ca+ stores.
Storr
M
M
Department of Internal Medicine, Technical University of Munich, Germany. martin.storr@lrz.tum.de
Franck
H
H
Saur
D
D
Schusdziarra
V
V
Allescher
H D
HD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007082:Ileum / Q000187:drug effects* / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002121:Calcium Channel Blockers; D017931:DNA Primers; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium