Changes in phosphoinositide-specific phospholipase C and phospholipase A2 activity in ischemic and reperfused rat heart

Summary 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 postischemic 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, alpha₁-adrenergic, beta-adrenergic, and adenosine receptor blockers did not alter ischemiaelicited 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.Supported by NIH grant NR 02203