Schwertz D W, Halverson J
Department of Pharmacology and Medical Surgical Nsg, University of Illinois, Chicago.
Basic Res Cardiol. 1992 Mar-Apr;87(2):113-27. doi: 10.1007/BF00801959.
Phospholipid metabolism is altered during ischemia and post-ischemic reperfusion. Past studies demonstrating elevated myocardial free fatty acid and lysophospholipid content infer accelerated phospholipid degradation involving phospholipase A activity. Recently, ischemic and post-ischemic reperfusion (reperfusion) have been shown to affect levels of phosphoinositide (PPI) degradation products. Considering the role of PPI turnover in regulation of cellular calcium homeostasis, our laboratory and others have suggested that alteration in the metabolism of the inositol phospholipids could play a role in the development of ischemia-induced calcium overload injury. Using an isolated rat heart model (Langendorff perfusion), this study examines the effect of global ischemia and reperfusion on ventricular phosphoinositide-specific phospholipase C (PLC) activity and PLA2 activity. The primary purpose was to determine if ischemia and reperfusion-induced changes in PLC activity could explain previously observed changes in PPI degradation products, and whether PLC and PLA2 activities were similarly or differentially altered by ischemia and reperfusion. PLC and PLA2 activities were measured in cytosolic and total membrane fractions from control (perfused), ischemic (5, 10, 30, and 60 min), and post-ischemic reperfused ventricular tissue. Phospholipase activity was determined under optimal in vitro conditions using exogenous radiolabeled substrates. Alterations in membrane-associated PPI-PLC activity correlated with reported ischemia and reperfusion-induced changes in ventricular content of PPI metabolites. Membrane PLC activity increased slightly at 5 min of ischemia, decreased significantly at 10 min of ischemia, and continued to decrease with longer duration of ischemia (73% of control after 60 min). Cytosolic PPI-PLC activity was decreased at 5 min, and then significantly increased by longer durations of ischemia, while cytosolic PLA2 activity was reduced at all time points. Pretreatment with muscarinic, alpha 1-adrenergic, beta-adrenergic, and adenosine receptor blockers did not alter ischemia-elicited changes in PLC activity. Reperfusion caused a 140% to 200% rise in the activities of all phospholipases in all fractions after 40 min of ischemia, but not after 10 min of ischemia. Results suggest 1) ischemia and reperfusion-elicited alterations in membrane-associated PPI-PLC activity can explain previously observed changes in phosphoinositide turnover metabolites, 2) cytosolic and membrane-associated PPI-PLC and PLA2 activities are not uniformly affected by ischemia, 3) reperfusion following ischemia of sufficient duration initiates uniform activation of PIP2-PLC and PLA2, and 4) because ischemia and reperfusion-induced changes in phospholipase activity can be detected under optimal in vitro assay conditions (removed from the in vivo ischemic microenvironment), it is likely that the enzymes themselves have been altered.
在缺血及缺血后再灌注过程中，磷脂代谢会发生改变。过去的研究表明，心肌游离脂肪酸和溶血磷脂含量升高，这意味着涉及磷脂酶A活性的磷脂降解加速。最近，已证实缺血及缺血后再灌注会影响磷酸肌醇（PPI）降解产物的水平。考虑到PPI周转在调节细胞钙稳态中的作用，我们实验室及其他研究团队认为，肌醇磷脂代谢的改变可能在缺血诱导的钙超载损伤的发展过程中发挥作用。本研究采用离体大鼠心脏模型（Langendorff灌注），检测全心缺血及再灌注对心室磷酸肌醇特异性磷脂酶C（PLC）活性和磷脂酶A2（PLA2）活性的影响。主要目的是确定缺血及再灌注诱导的PLC活性变化是否能够解释先前观察到的PPI降解产物的变化，以及PLC和PLA2活性在缺血及再灌注过程中是发生相似还是不同的改变。在对照（灌注）、缺血（5、10、30和60分钟）及缺血后再灌注的心室组织的胞质和总膜组分中测量PLC和PLA2活性。使用外源性放射性标记底物在最佳体外条件下测定磷脂酶活性。膜相关PPI-PLC活性的改变与报道的缺血及再灌注诱导的心室PPI代谢物含量变化相关。缺血5分钟时膜PLC活性略有增加，缺血10分钟时显著降低，且随着缺血时间延长持续降低（60分钟后为对照的73%）。胞质PPI-PLC活性在5分钟时降低，随后随着缺血时间延长显著增加，而胞质PLA2活性在所有时间点均降低。用毒蕈碱、α1肾上腺素能、β肾上腺素能和腺苷受体阻滞剂预处理并未改变缺血引起的PLC活性变化。缺血40分钟后再灌注导致所有组分中所有磷脂酶的活性升高140%至200%，但缺血10分钟后再灌注则未出现这种情况。结果表明：1）缺血及再灌注引起的膜相关PPI-PLC活性改变能够解释先前观察到的磷酸肌醇周转代谢物的变化；2）胞质和膜相关的PPI-PLC及PLA₂活性受缺血影响并不一致；3）足够长时间缺血后的再灌注会引发PIP₂-PLC和PLA₂的一致激活；4）由于在最佳体外测定条件下（脱离体内缺血微环境）能够检测到缺血及再灌注诱导的磷脂酶活性变化，因此很可能酶本身已发生改变。
