Ganote C E, Worstell J, Kaltenbach J P
Am J Pathol. 1976 Aug;84(2):327-50.
The effects of 5 mM potassium cyanide (KCN) on creatine phosphokinase (CPK) release and cellular morphology were studied. Rat hearts were perfused with substrate-deficient media gassed with O2 or N2 (O2 medium, N2 medium) at 37 C, and effluent was collected for creatine phosphokinase analysis. Tissue fixation was with glutaraldehyde for light and electron microscopy. Experiments included the following: a) continuous perfusion with O2- or N2-medium in the presence of KCN; b) 45 or 60 minutes of perfusion with N2-medium followed by O2-medium for 15 or 180 minutes, respectively; c) 45 minutes of perfusion with N2-medium with KCN added 15 minutes before reoxygenation with O2-medium plus KCN; (4) 60 minutes of N2-medium plus KCN followed by O2-medium plus KCN for 180 minutes; d) as a control for irreversible injury, 21 minutes of perfusion with calcium-free O2-medium followed by 2.5 mM calcium-O2-medium ("calcium paradox"). The following results were seen: a) Initial CPK release occurred about 30 minutes later from hearts perfused with O2-medium plus KCN than from hearts perfused with N2-medium plus KCN. b) Upon reoxygenation after either 45 or 60 minutes of anoxia, hearts had a sudden peak of oxygen-induced CPK release. Most irreversibly injured cells were massively swollen and had sarcolemmal defects and contraction bands. Reversibly injured cells in the same hearts resembled normal myocardium. A previously unrecognized third population of cells is described. These cells were characterized by contraction bands but were not swollen, had intact sarcolemma, and contained both normal and damaged mitochondria with intramatrical calcium accumulation granules. It could not be determined if these cells were reversibly injured or in an early stage of irreversible injury. c) KCN added 15 minutes before reoxygenation of hearts after 45 minutes of anoxia inhibited the sudden peak of oxygen-induced CPK release but not a slow sustained release. Small to moderate numbers of cells in these hearts contained contraction bands. d) After 60 minutes, KCN completely inhibited both oxygen-induced CPK release and contraction band formation. e) Addition of calcium to calcium-free hearts caused both massive CPK release and contraction band formation. It is concluded that: the beginning of CPK release from oxygenated KCN-inhibited hearts requires about 30 minutes longer than from anoxic hearts; KCN can inhibit both oxygen-induced CPK release and contraction bands in irreversibly injured rat myocardial cells; sudden contracture of myocardial cells as occurs in the calcium paradox can result in massive CPK release; contraction bands occur in nonswollen cells, hence contraction bands can occur independently of massive cell swelling or membrane rupture. It is postulated that there may be two stages of irreversible myocardial injury; a) loss of control of contraction and b) progressive loss of mitochondrial and membrane integrity.
研究了5 mM氰化钾（KCN）对肌酸磷酸激酶（CPK）释放及细胞形态的影响。将大鼠心脏在37℃下用充入O₂或N₂的底物缺乏培养基（O₂培养基、N₂培养基）进行灌注，并收集流出液用于肌酸磷酸激酶分析。用戊二醛进行组织固定以用于光镜和电镜观察。实验包括以下内容：a）在KCN存在下用O₂培养基或N₂培养基持续灌注；b）分别用N₂培养基灌注45或60分钟，随后用O₂培养基灌注15或180分钟；c）用N₂培养基灌注45分钟，在再用O₂培养基加KCN复氧前15分钟加入KCN；（4）用N₂培养基加KCN灌注60分钟，随后用O₂培养基加KCN灌注180分钟；d）作为不可逆损伤的对照，用无钙O₂培养基灌注21分钟，随后用2.5 mM钙的O₂培养基灌注（“钙反常”）。观察到以下结果：a）与用N₂培养基加KCN灌注的心脏相比，用O₂培养基加KCN灌注的心脏中CPK的初始释放大约在30分钟后出现。b）在缺氧45或60分钟后复氧时，心脏出现氧诱导的CPK释放的突然峰值。大多数不可逆损伤的细胞大量肿胀，有肌膜缺陷和收缩带。同一心脏中可逆损伤的细胞类似于正常心肌。描述了一类先前未被认识的第三种细胞群体。这些细胞的特征是有收缩带，但未肿胀，肌膜完整，含有正常和受损的线粒体，线粒体内有钙积聚颗粒。无法确定这些细胞是可逆损伤还是处于不可逆损伤的早期阶段。c）在缺氧45分钟后的心脏复氧前15分钟加入KCN可抑制氧诱导的CPK释放的突然峰值，但不抑制缓慢持续释放。这些心脏中有少量至中等数量的细胞含有收缩带。d）60分钟后，KCN完全抑制氧诱导的CPK释放和收缩带形成。e）向无钙心脏中加入钙会导致大量CPK释放和收缩带形成。结论是：从用KCN抑制的氧合心脏中CPK释放的开始比从缺氧心脏中大约需要30分钟更长时间；KCN可抑制不可逆损伤的大鼠心肌细胞中氧诱导的CPK释放和收缩带；钙反常中发生的心肌细胞突然挛缩可导致大量CPK释放；收缩带出现在未肿胀的细胞中，因此收缩带可独立于大量细胞肿胀或膜破裂而出现。推测可能存在不可逆心肌损伤的两个阶段：a）收缩控制丧失和b）线粒体及膜完整性的逐渐丧失。
