一氧化氮对成年斑马鱼骨骼肌细胞钙瞬态的调控机制研究

Study on regulatory mechanism of nitric oxide on calcium transient state in skeletal muscle cells of adult zebrafish

目的利用高速荧光摄影技术,探究一氧化氮(NO)对成年斑马鱼骨骼肌细胞钙瞬态的调控作用。方法利用成年斑马鱼分离提取其游离骨骼肌细胞,将细胞以Fluo-4,AM荧光探针孵育后,使用高速荧光摄像机记录单个电刺激后斑马鱼游离骨骼肌细胞内钙瞬态的荧光变化,并定量计算细胞内钙瞬态相关的生物物理学参数。实验分组为对照组、S-亚硝基-N-乙酰基-DL-青霉胺(SNAP)组和N-硝基-L-精氨酸甲酯(L-NAME)组,使用NO供体SNAP和非特异性一氧化氮合酶(NOS)抑制剂L-NAME探究NO对成年斑马鱼骨骼肌细胞钙瞬态的调控作用。实验再分组为对照组、N-乙基马来酰亚胺(NEM)组、1H-[1,2,4]恶草灵并[4,3-A]喹喔啉-1-酮(ODQ)组、SNAP组和SNAP+ODQ组,使用sGC-cGMP-PKG通路抑制剂ODQ和S-亚硝基化抑制剂NEM探究NO对成年斑马鱼骨骼肌细胞钙瞬态的调控机制。结果高速荧光摄影技术可以完整记录成年斑马鱼骨骼肌细胞内的钙瞬态荧光变化,并计算出细胞内钙瞬态相关的生物物理学参数。与对照组比较,SNAP组骨骼肌细胞钙瞬态明显降低,而L-NAME组骨骼肌细胞钙瞬态较对照组明显增强。ODQ组骨骼肌细胞钙瞬态明显强于对照组,而NEM组与对照组比较各钙瞬态相关参数差异无统计学意义(P>0.05)。SNAP+ODQ组骨骼肌细胞钙瞬态也明显强于SNAP组。结论NO能够通过sGC-cGMP-PKG途径负向调控成年斑马鱼骨骼肌细胞内的钙瞬态过程。

Objective To use the high-speed fluorescence photography to investigate the regulatory effect of nitric oxide(NO)on calcium transient state in skeletal muscle cells of adult zebrafish.Methods The skeletal muscle cells were separated and extracted from adult zebrafish and then incubated with Fluo-4 and AM fluorescent probe.The fluorescence change of calcium transient state in zebrafish free skeletal muscle cells after single electrical stimulation was recorded by a high-speed fluorescence camera,and the biophysical parameters related to the intracellular calcium transient state were quantitatively calculated.The experimental groups were divided into the control group,S-nitroso-n-acetyl-DL-penicillamine(SNAP)group and n-nitroso-L-arginine methyl ester(L-NAME)group.NO donor SNAP and non-specific nitric oxide synthase(NOS)inhibitor L-NAME were used to investigate the regulatory effect of NO on calcium transient state in adult skeletal muscle cells of zebrafish.The experimental groups were redivided into the control group,N-ethylmaleimide(NEM)group,1H-[1,2,4]dioxalin and[4,3-a]quinoxalin-1-one(ODQ)group,SNAP group and SNAP+ODQ group.The regulatory mechanism of NO on calcium transients state in adult zebrafish skeletal muscle cells was investigated by using sGC-cGMP-PKG pathway inhibitor ODQ and S-nitrosation inhibitor NEM.Results The fluorescence changes of calcium transient state in adult zebrafish skeletal muscle cells could be recorded by high speed fluorescence photography and the biophysical parameters related to intracellular calcium transients state were calculated.Compared to the control group,the calcium transient state of skeletal muscle cells in the SNAP group was significantly decreased,while the calcium transient state of skeletal muscle cells in the L-NAME group was significantly enhanced compared to the control group.The calcium transient state of skeletal muscle cells in the ODQ group was significantly stronger than that in the control group,while there was no statistical difference in the related parameters of calcium transient state between the NEM group and control group.The calcium transient state of skeletal muscle cells in the SNAP+ODQ group was also significantly stronger than that in the SNAP group.Conclusion NO could negatively regulate the process of calcium transient state in adult zebrafish skeletal myocytes by the sGC-cGMP-PKG pathway.