Burton K P, Morris A C, Massey K D, Buja L M, Hagler H K
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235-9071.
J Mol Cell Cardiol. 1990 Sep;22(9):1035-47. doi: 10.1016/0022-2828(90)91043-7.
Oxygen-derived free radicals have been implicated in damage to membrane phospholipids leading to alterations in membrane function. The purpose of this study was to investigate alterations in intracellular ionic calcium (Ca2+) levels and Ca2+ transients, cellular morphology, conjugated diene levels, arachidonate release, and lactate dehydrogenase release resulting from the exposure of cultured neonatal rat ventricular myocytes to a xanthine oxidase catalyzed free radical generating system capable of producing superoxide and hydroxyl radicals. The ability of alpha-tocopherol to prevent alterations due to free radical exposure was investigated. For measurements of Ca2+, myocytes grown on coverslips for 3-4 days were loaded with fura-2/AM and studied by microspectrofluorometry. Control myocytes superfused with a physiological buffer or buffer containing purine and iron-loaded transferrin exhibited Ca2+ transients associated with spontaneous contractions. For control, buffer perfused myocytes (n = 4), the fura-2 340/380 ratios were 0.5 +/- 0.1 (mean +/- S.E.) and 1.6 +/- 0.03 at the minimum and maximum, respectively, of the Ca2+ transient, after 1 h of perfusion. Exposure to the free radical generating solution (n = 14) altered intracellular Ca2+. The 340/380 minimum ratio was 639% of the control value after approximately 30-70 mins with cessation of normal Ca2+ transients. Bleb development was associated with increased Ca2+. Myocytes reperfused with control medium continued to exhibit an elevated minimum fura-2 ratio at 687% of control. Myocytes pretreated with 10 microM alpha-tocopherol (n = 13) for 18-24 h and exposed to free radicals did not exhibit increases in intracellular Ca2+, having a minimum 340/380 ratio of 0.5 +/- 0.1 after 60-90 mins, and although myocytes often ceased contracting, they resumed spontaneous Ca2+ transients with control medium reperfusion and also maintained normal structure. Exposure of myocyte cultures to free radical generating solutions resulted in increased levels of conjugated dienes and increased release of [3H]arachidonate and lactate dehydrogenase compared to control values after 1 h. alpha-Tocopherol treatment attenuated the increase in conjugated diene levels, and the release of [3H]arachidonate and lactate dehydrogenase. Thus, free radicals alter intracellular Ca2+, conjugated dienes and membrane structure indicating their ability to induce altered ionic homeostasis in association with myocardial membrane damage. alpha-Tocopherol decreased free radical mediated injury.
氧衍生的自由基与膜磷脂损伤有关，进而导致膜功能改变。本研究的目的是调查培养的新生大鼠心室肌细胞暴露于能够产生超氧化物和羟基自由基的黄嘌呤氧化酶催化的自由基生成系统后，细胞内离子钙（Ca2+）水平和Ca2+瞬变、细胞形态、共轭二烯水平、花生四烯酸释放以及乳酸脱氢酶释放的变化。研究了α-生育酚预防自由基暴露所致变化的能力。为了测量Ca2+，将在盖玻片上生长3 - 4天的心肌细胞用fura-2/AM加载，并通过显微分光荧光测定法进行研究。用生理缓冲液或含嘌呤和铁负载转铁蛋白的缓冲液灌注的对照心肌细胞表现出与自发收缩相关的Ca2+瞬变。对于对照，灌注缓冲液的心肌细胞（n = 4），在灌注1小时后，Ca2+瞬变的最小值和最大值时，fura-2 340/380比值分别为0.5±0.1（平均值±标准误）和1.6±0.03。暴露于自由基生成溶液（n = 14）会改变细胞内Ca2+。在正常Ca2+瞬变停止后约30 - 70分钟，340/380最小比值为对照值的639%。小泡形成与Ca2+增加有关。用对照培养基再灌注的心肌细胞在687%对照时仍表现出升高的最小fura-2比值。用10μMα-生育酚预处理18 - 24小时（n = 13）并暴露于自由基的心肌细胞未表现出细胞内Ca2+增加，60 - 90分钟后最小340/380比值为0.5±0.1，尽管心肌细胞常停止收缩，但在用对照培养基再灌注后恢复了自发Ca2+瞬变，并且也维持了正常结构。与对照值相比，将心肌细胞培养物暴露于自由基生成溶液1小时后，共轭二烯水平升高，[3H]花生四烯酸和乳酸脱氢酶释放增加。α-生育酚处理减弱了共轭二烯水平以及[3H]花生四烯酸和乳酸脱氢酶释放的增加。因此，自由基改变细胞内Ca2+、共轭二烯和膜结构，表明它们有能力诱导与心肌膜损伤相关的离子稳态改变。α-生育酚减少了自由基介导的损伤。
