Sulakhe P V, Vo X T, Phan T D, Morris T E
Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
Mol Cell Biochem. 1997 Oct;175(1-2):98-107.
Myocytes were isolated from rat heart ventricles and then incubated with [32P]-sodium phosphate to label intracellular ATP stores. Incubations of the [32P]-labelled cardiomyocytes with a beta-adrenoceptor agonist isoproterenol (10 microM) and with a plant diterpene forskolin (100 microM) which directly stimulates adenylyl cyclase increased the phosphorylation of an inhibitory subunit of troponin (TN-I) and phospholamban (PLN). Brief exposure (1 min) of labelled myocytes to the hydroxyl radical generating system (H2O2 plus FeCl2) decreased markedly the stimulatory action of isoproterenol and forskolin on TN-I and PLN phosphorylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic acid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory effect on the isoproterenol or forskolin stimulated TN-I and PLN phosphorylation. In contrast exposure of myocytes to low concentrations (< 50 microM) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augmented the stimulatory effect of isoproterenol on TN-I and PLN phosphorylation. The results further showed that brief treatment of myocytes to H2O2 plus FeCl2 markedly decreased isoproterenol-, but not forskolin-, stimulated cyclic AMP accumulation in the myocytes. The stimulatory action of NEM on the isoproterenol-stimulated TN-I and PLN phosphorylation appeared related to greater increase in the isoproterenol-stimulated cyclic AMP accumulation in the NEM-treated cardiomyocytes. The results are consistent with the postulate that hydroxyl radical exposure of cardiomyocytes blunts the beta-adrenoceptor-mediated stimulation of adenylyl cyclase leading to decreased phosphorylation of TN-I and PLN and imply that such alterations account in part the reported depressed rate of relaxation of the myocardium exposed to oxygen free radicals.
从大鼠心室分离出心肌细胞，然后用[32P] - 磷酸钠孵育以标记细胞内ATP储备。将[32P]标记的心肌细胞与β - 肾上腺素能受体激动剂异丙肾上腺素（10μM）以及直接刺激腺苷酸环化酶的植物二萜福斯高林（100μM）一起孵育，可增加肌钙蛋白抑制亚基（TN - I）和受磷蛋白（PLN）的磷酸化。将标记的心肌细胞短暂暴露（1分钟）于羟基自由基生成系统（H2O2加FeCl2），可显著降低异丙肾上腺素和福斯高林对TN - I和PLN磷酸化的刺激作用。将心肌细胞类似地暴露于巯基氧化试剂5 - 5'-二硫代双硝基苯甲酸（DTNB），对异丙肾上腺素或福斯高林刺激的TN - I和PLN磷酸化几乎没有抑制作用。相反，将心肌细胞暴露于低浓度（<50μM）的巯基烷基化试剂N - 乙基马来酰亚胺（NEM），可增强异丙肾上腺素对TN - I和PLN磷酸化的刺激作用。结果还表明，将心肌细胞短暂处理于H2O2加FeCl2可显著降低异丙肾上腺素刺激的，但不是福斯高林刺激的心肌细胞中环磷酸腺苷（cAMP）的积累。NEM对异丙肾上腺素刺激的TN - I和PLN磷酸化的刺激作用似乎与NEM处理的心肌细胞中异丙肾上腺素刺激的cAMP积累的更大增加有关。这些结果与心肌细胞暴露于羟基自由基会减弱β - 肾上腺素能受体介导的腺苷酸环化酶刺激，导致TN - I和PLN磷酸化减少的假设一致，并暗示这种改变部分解释了所报道的暴露于氧自由基的心肌舒张速率降低的原因。
