Mechanisms of α,β-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 α,β-methylene ATP (α,β-MeATP), ATP, ADP and UTP. α,β-Methylene ATP and ATP were the only agonists that caused a concentration-dependent inhibition of carbachol-precontracted smooth muscle. The inhibitory effect of α,β-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 α,β-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 α,β-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^-7mol/L), pertussis toxin (10^-6mol/L) N(G)-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 α,β-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 α,β-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 Ca²⁺ stores.