Nitric oxide synthesis stimulated by arachidonic acid accumulation via PPARα in acetylcholine-stimulated gastric mucous cells.

New findings The central question of this study. Aarachidonic acid (AA) stimulates NO production in antral mucous cells without any increase in intracellular Ca2+ concentration ([Ca2+ ]i ). Since intracellular AA concentration is too low to measure, the relationship between AA accumulation and NO production still remains uncertain. The central question is whether or not AA accumulation is a key step for NO production. The main finding and its importance. This study demonstrated that AA accumulation is a key step for NO production. Amounts of AA released could be measured using a F-HPLC. Intracellular AA concentration is maintained below 1 μM. NO is produced by AA accumulation in antral mucous cells, not direct effect of [Ca2+ ]i . Abstract The present study demonstrated that NO production is stimulated by an accumulation of arachidonic acid (AA) mediated via PPARα and that NO produced enhances Ca2+ -regulated exocytosis in acetylcholine (ACh) -stimulated antral mucous cells. Amounts of AA released from antral mucosa which were measured by a fluorescence high-performance liquid chromatography (F-HPLC) were increased by an addition of ionomycin (IM, 10 μM) or acetylcholine (ACh), suggesting AA accumulation is stimulated by an increase in intracellular Ca2+ concentration ([Ca2+ ]i ). The AA production was inhibited by an inhibitor of cytosolic phospholipase A2 (cPLA2-inhα). GW6471 (a PPARα inhibitor) and cPLA2-inhα inhibited NO synthesis stimulated by ACh. Moreover, indomethacin (IDM), an inhibitor of cyclooxygenase (COX), stimulates AA accumulation and also NO production. However, acetylsalicylic acid (ASA) did not stimulate AA production and NO synthesis. An analogue of AA (AACOCF3) alone stimulates NO synthesis, which inhibited by GW6471. In antral mucous cells, IDM enhanced Ca2+ -regulated exocytosis by increasing NO via PPARα, and the enhancement was abolished by GW6471 and cPLA2-inhα. Thus, AA produced via PLA2 activation is the key step for NO synthesis in ACh-stimulated antral mucous cells and plays important roles in maintaining antral mucous secretion, especially in the Ca2+ -regulated exocytosis. This article is protected by copyright. All rights reserved.