Forsdahl K, Larsen T S
Department of Medical Physiology, University of Tromsø, Norway.
J Mol Cell Cardiol. 1995 Mar;27(3):893-900. doi: 10.1016/0022-2828(95)90039-x.
In the present study, we investigated possible mechanisms behind exogenous phospholipase C-induced glycerol production in irreversibly damaged myocytes. Rat ventricular myocytes were preincubated for 60 min in substrate-free Krebs-Henseleit bicarbonate buffer equilibrated with 95% N2-5% CO2 (37 degrees C, pH = 7.4), resulting in exhaustion of cellular high energy phosphates and loss of rod-shaped morphology. At the end of the preincubation period, the incubation vials were divided into two groups; one receiving 10 mU/ml phospholipase C (PC-PLC), whereas the other received an equivalent volume of buffer (control incubations). Incubation was then continued for another 60 min under 95% air-5% CO2 atmosphere. Samples for measurement of metabolite levels were taken immediately after cell isolation, at the end of the preincubation period and at the end of the normoxic incubation period. During the 60 min incubation period following reoxygenation, glycerol output was markedly higher from PC-PLC treated than from control myocytes. However, the elevated glycerol output from these cells was not accompanied by a simultaneous rise in glycerol-3-phosphate, nor was it inhibited by inclusion of pyruvate in the incubation buffer. On the other hand, glycerol output from PC-PLC treated myocytes was effectively inhibited by a diacylglycerol lipase inhibitor (U-57908, The Upjohn Company). Analysis of cellular lipids revealed a 22% reduction of phospholipid in PC-PLC treated myocytes (P < 0.02), while the content of triacylglycerol, diacylglycerol and unesterified fatty acids increased by 76, 261 and 103%, respectively (P < 0.02). No significant changes were observed for these parameters in control myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
在本研究中，我们探究了外源性磷脂酶C诱导不可逆损伤心肌细胞产生甘油的可能机制。将大鼠心室肌细胞在无底物的Krebs-Henseleit碳酸氢盐缓冲液中预孵育60分钟，该缓冲液用95% N₂-5% CO₂平衡（37℃，pH = 7.4），导致细胞内高能磷酸盐耗竭且杆状形态丧失。在预孵育期结束时，将孵育瓶分为两组；一组加入10 mU/ml磷脂酶C（PC-PLC），而另一组加入等量体积的缓冲液（对照孵育）。然后在95%空气-5% CO₂气氛下继续孵育60分钟。在细胞分离后、预孵育期结束时和常氧孵育期结束时立即采集用于测量代谢物水平的样本。在复氧后的60分钟孵育期内，PC-PLC处理的心肌细胞甘油产量明显高于对照心肌细胞。然而，这些细胞中甘油产量的升高并未伴随3-磷酸甘油同时升高，也未被孵育缓冲液中加入丙酮酸所抑制。另一方面，PC-PLC处理的心肌细胞的甘油产量被二酰基甘油脂肪酶抑制剂（U-57908，Upjohn公司）有效抑制。细胞脂质分析显示，PC-PLC处理的心肌细胞中磷脂减少了22%（P < 0.02），而三酰基甘油、二酰基甘油和未酯化脂肪酸的含量分别增加了76%、261%和103%（P < 0.02）。对照心肌细胞中这些参数未观察到显著变化。（摘要截断于250字）
