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- W2069974463 abstract "regulating CIE; however, its role in asymmetric CCH-stimulated signal transduction is not well understood. PURPOSE: Since PLD generates phosphatidic acid (PA) that regulates endocytosis, we (1) investigated whether CCH activates BB PLD, (2) identified which PLD isoform is necessary for CCH-inhibition of NHE3 activity, and (3) determined whether PA or PA-derived diacylglycerol (DAG) is required for this effect. METHODS: 12 day postconfluent Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in Caco-2/BBe cells treated with vehicle, isoform specific PLD inhibitors (EVJ = PLD1; JWJ = PLD2, 5WO = dual), or propranolol (to inhibit conversion of PA to DAG) in the presence or absence of 10μM CCH to elevate [Ca]i. PLD activity was determined in total cell lysates from Caco-2/BBe cells treated with vehicle or CCH in the presence and absence of 10nM (blocks PLD1) or 50nM (blocks PLD2) FIPI. RESULTS: CCH induced rapid (1min) elevation of PLD activity in Caco-2/BBe cells, an effect which was blocked by BB, but not BLM, pretreatment with FIPI. Pretreatment of Caco-2/BBe cells with apical 10μM 5WO abolished CCH-mediated inhibition of NHE3 activity suggesting that BB PLD1 and/or PLD2 are necessary for NHE3 inhibition by CCH. This result was confirmed in studies in which cells were pretreated with both EVJ and JWJ as exposure to each inhibitor alone did not prevent CCH-inhibition of NHE3 activity. Propranolol treatment also prevented CCH-inhibition of NHE3 activity suggesting that PA-derived DAG is required for this effect. CONCLUSIONS: In summary, these data suggest that both PLD1 and PLD2 are activated by CCH signaling and are both necessary to inhibit NHE3 activity, which involves production of PA and DAG. Elevated [Ca]i-mediated signal transduction from the BLMM3 cholingergic receptor activates both BB PLD1 and PLD2 as part of an asymmetric signaling pathway that stimulates production of PA-derived DAG that results in the inhibition of BB NHE3 activity." @default.
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- W2069974463 date "2014-05-01" @default.
- W2069974463 modified "2023-09-26" @default.
- W2069974463 title "Mo1757 Trafficking of SLC26A3 (DRA) in Intestinal Epithelial Cells: Effect of EPEC Infection" @default.
- W2069974463 doi "https://doi.org/10.1016/s0016-5085(14)62374-x" @default.
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