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背景神经肽 Y(NPY)是中枢神经及末梢神经重要的调节剂,作用广泛,包括调节心脏及血管正常的生理活动,并参与许多心血管疾病的发生发展。研究表明,急性的 NPY 刺激可促进心肌细胞钙活动,对心肌产生正性肌力作用。而持续 NPY 刺激对心肌钙活动的影响及其机制,目前还未见报道。目的观察 NPY 刺激对大鼠心肌细胞胞浆钙和肌浆网(SR)内钙分布的影响,以及 Ca~(2+)/CaM 依赖的蛋白激酶Ⅱ(CaMKⅡ)在其中的作用。方法用100 nmol/L NPY 刺激 Sprague Dawley 乳鼠心肌细胞24 h,CaMKⅡ特异性抑制剂 KN-93干预。应用荧光染料 Fluo-4 AM 负载胞浆钙;Fluo-5N AM 负载肌浆网内游离钙离子,所有钙影像均由激光共聚焦显微镜记录。应用Western-blot 法和免疫荧光法检测 Ca~(2+)-ATP 酶(SERCA2a)和 ryanodine 受体(RyR_2)两种蛋白的分布及蛋白量的变化。结果经100 nmol/L NPY 刺激24 h 后,与对照组相比,心肌细胞胞浆游离钙浓度明显升高(65.3±6.2 vs50.7±4.1,P<0.05),心肌细胞肌浆网内游离钙含量明显低于对照组(67.6±8.3 vs 85.5±6.0,P<0.05),而KN-93可抑制上述效应;NPY 可增加 SERCA2a 和 RyR_2的蛋白表达(SERCA2a:2.4±0.7 vs 对照组:1.4±0.3;RyR_2:2.3±0.4 vs 对照组:1.2±0.4),KN-93可抑制上述作用。结论 CaMKⅡ通过影响 SERCA2a 和 RyR_2,调控 SR 的钙转运,进而介导 NPY 刺激下的细胞内钙重分布效应。
Background Neuropeptide Y (NPY) is an important regulator of the central and peripheral nerves and has a wide range of roles, including regulating the normal physiological activities of the heart and blood vessels and participating in the development of many cardiovascular diseases. Studies have shown that acute NPY stimulation can promote myocardial calcium activity, the positive cardiac muscle produce a positive effect. The continuous NPY stimulation of myocardial calcium activity and its mechanism has not been reported. Objective To observe the effect of NPY stimulation on the distribution of calcium in cytoplasm and sarcoplasmic reticulum (SR) in rat cardiomyocytes and the role of Ca ~ (2 +) / CaM-dependent protein kinase Ⅱ (CaMKⅡ) in it. Methods Sprague Dawley neonatal rat cardiomyocytes were stimulated with 100 nmol / L NPY for 24 h and intervention with CaMKⅡ specific inhibitor KN-93. The fluorescent dye Fluo-4 AM was used to load cytoplasmic calcium. Fluo-5N AM was loaded on the sarcoplasmic reticulum with free calcium ions. All calcium images were recorded by laser scanning confocal microscopy. Western blot and immunofluorescence were used to detect the distribution and protein level of Ca ~ (2 +) - ATPase (SERCA2a) and ryanodine receptor (RyR_2). Results After stimulated with 100 nmol / L NPY for 24 h, the cytosolic free calcium concentration in cardiomyocytes was significantly increased (65.3 ± 6.2 vs 50.7 ± 4.1, P <0.05) compared with the control group. The intracellular sarcoplasmic reticulum (67.6 ± 8.3 vs 85.5 ± 6.0, P <0.05), while KN-93 inhibited the above effects. NPY increased the expression of SERCA2a and RyR_2 (SERCA2a: 2.4 ± 0.7 vs 1.4 ± 0.3; RyR_2: 2.3 ± 0.4 vs control group: 1.2 ± 0.4), KN-93 could inhibit these effects. Conclusion CaMKⅡ regulates the calcium transport of SR by affecting SERCA2a and RyR_2, and then mediates the intracellular calcium redistribution effect induced by NPY.