Cholecystokinin Octapeptide Promotes ANP Secretion through Activation of NOX4–PGC‐1α–PPARα/PPARγ Signaling in Isolated Beating Rat Atria

Figures

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  Figure 1. CCK-8 role in ANP secretion and dynamics and its effects on CCK receptor expressions in isolated beating rat atria. (a, c, e, g) Atrial ANP secretion by radioimmunoassay. (b, d, f, h) Atrial pulse pressure by RM6240BD. (i) The protein expression levels of CCK1R and CCK2R were detected by Western blot. (j, k) The statistics histograms of Western blot were expressed as band density normalized versus β-actin. Cont: control; CCK-8s: sulfated CCK-8; CCK-8d: desulfated CCK-8; Lo: Loxiglumid, an antagonist of CCK1 receptor; YM: YM022, an antagonist of CCK2 receptor. Data were expressed as mean ± SEM. (a–h) n = 6; (i–k) n = 5. ∗∗∗P < 0.001 vs. control period or group; ###P < 0.001 vs. CCK-8s period or group; ns: no significant.

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  Figure 2. Effects of CCK-8s and PLC on cPLA2 expression and its role in ANP secretion and dynamics in isolated beating rat atria. (a) The protein expression levels of p-cPLA2 were detected by Western blot. (b, c) The statistics histograms of Western blot were expressed as band density normalized versus t-cPLA2. (d, f) Atrial ANP secretion by radioimmunoassay. (e, g) Atrial pulse pressure by RM6240BD. Cont: control; CCK-8s: sulfated CCK-8; Lo: Loxiglumid, an antagonist of CCK1 receptor; YM: YM022, an antagonist of CCK2 receptor; U: U-73122, an inhibitor of PLC; CAY: CAY10650, an inhibitor of cPLA2. Data were expressed as mean ± SEM. (a–c) n = 5; (d–g) n = 6. ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control period or group; ##P < 0.01 and ###P < 0.001 vs. CCK-8s period or group.

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  Figure 3. Effects of CCK-8s on NOX4 expression and its role in ANP secretion and dynamics in isolated beating rat atria. (a) The level of AA was tested by the ELISA method. (b) The protein expression levels of NOX4 were detected by Western blot. (c, d) The statistics histograms of Western blot were expressed as band density normalized versus β-actin. (e) The level of H2O2 was tested by ELISA. (f, h, j) Atrial ANP secretion by radioimmunoassay. (g, i, k) Atrial pulse pressure by RM6240BD. Cont: control; CCK-8s: sulfated CCK-8; Lo: Loxiglumid, an antagonist of CCK1 receptor; YM: YM022, an antagonist of CCK2 receptor; U: U-73122, an inhibitor of PLC; CAY: CAY10650, an inhibitor of cPLA2; GLX: GLX351322, an inhibitor of NOX4; Gli: Glibenclamide, an inhibitor of KATP; GAL: GAL-021, a blocker of BKCa. Data were expressed as mean ± SEM. (a–e) n = 5; (f–k) n = 6. ∗∗∗P < 0.001 vs. control period or group; ##P < 0.01 and ###P < 0.001 vs. CCK-8s period or group.

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  Figure 4. Effects of p38 MAPK and Akt on ANP secretion and dynamics induced by CCK-8s in isolated beating rat atria. (a) The protein expression levels of p-p38 MAPK and p-Akt were detected by Western blot. (b) The statistics histograms of Western blot were expressed as band density normalized versus t-p38. (c) The statistics histograms of Western blot were expressed as band density normalized versus t-Akt. (d, f) Atrial ANP secretion by radioimmunoassay. (e, g) Atrial pulse pressure by RM6240BD. Cont: control; CCK-8s: sulfated CCK-8; GLX: GLX351322, an inhibitor of NOX4; SB: SB239063, an antagonist of p38 MAPK; LY: LY294002, an inhibitor of Akt. Data were expressed as mean ± SEM. (a–c) n = 5; (d–g) n = 6. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. control period or group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. CCK-8s period or group.

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  Figure 5. Effects of CCK-8s on PGC-1α expression and its role on ANP secretion and dynamics in isolated beating rat atria. (a) The protein expression levels of PGC-1α were detected by Western blot. (b, c) The statistics histograms of Western blot were expressed as band density normalized versus β-actin. (d) Atrial ANP secretion by radioimmunoassay. (e) Atrial pulse pressure by RM6240BD. Cont: control; CCK-8s: sulfated CCK-8; SB: SB239063, an antagonist of p38 MAPK; LY: LY294002, an inhibitor of Akt; SR: SR-18292, an antagonist of PGC-1α. Data were expressed as mean ± SEM. (a–c) n = 5; (d, e) n = 6. ∗∗∗P < 0.001 vs. control period or group; ##P < 0.01 and ###P < 0.001 vs. CCK-8s period or group.

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  Figure 6. Effects of CCK-8s on PPARα as well as PPARγ expression and its role in ANP secretion and dynamics in isolated beating rat atria. (a, b, f) The mRNA levels of PPARα, PPARγ, and NPPA were tested by RT-qPCR. (c) The protein expression levels of p-PPARα and p-PPARγ were detected by Western blot. (d) The statistics histograms of Western blot were expressed as band density normalized versus t-PPARα. (e) The statistics histograms of Western blot were expressed as band density normalized versus t-PPARγ. (g, h) Atrial ANP secretion by radioimmunoassay. (i, j) Atrial pulse pressure by RM6240BD. Cont: control; CCK-8s: sulfated CCK-8; SR: SR-18292, an antagonist of PGC-1α; Lo: Loxiglumid, an antagonist of CCK1 receptor; YM: YM022, an antagonist of CCK2 receptor; GW64: GW6471, an antagonist of PPARα; GW96: GW9662, an inhibitor of PPARγ. Data were expressed as mean ± SEM. (a–f) n = 5; (g–j) n = 6. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. control period or group; ###P < 0.001 vs. CCK-8s period or group; @@P < 0.01 vs. Lo+CCK-8s group; &&P < 0.01 vs. YM+CCK-8s group.

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  Figure 7. Effects of PPARα and PPARγ on CCK-8s-induced SOD and CAT expressions in isolated beating rat atria. (a) The protein expression levels of SOD, CAT, and GPX were detected by Western blot. (b–d) The statistics histograms of Western blot were expressed as band density normalized versus β-actin. (e–g) The bands of SOD and CAT and their statistics histograms. Cont: control; CCK-8s: sulfated CCK-8; Lo: Loxiglumid, an antagonist of CCK1 receptor; YM: YM022, an antagonist of CCK2 receptor; GW64: GW6471, an antagonist of PPARα; GW96: GW9662, an inhibitor of PPARγ. Data were expressed as mean ± SEM. (a–g) n = 5. ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control group; ###P < 0.001 vs. CCK-8s group.

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  Figure 8. Effects of endogenous ANP on SOD, CAT, and NOX4 expressions under CCK-8s action in isolated beating rat atria. (a, b) The levels of AA and H2O2 were tested by ELISA method. (c) The protein expression levels of NOX4, CAT, and SOD were detected by Western blot. (d–f) The statistics histograms of Western blot were expressed as band density normalized versus β-actin. Cont: control; CCK-8s: sulfated CCK-8; A71: A71915, an antagonist of ANP receptor. Data were expressed as mean ± SEM. (a–f) n = 5. ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. CCK-8s group; &&&P < 0.001 vs. A71+CCK-8s group.