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

Abstract

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.

Phospholipid-mediated signaling in diseased myocardium

“…Activation of PLC activity has been evidenced in I-R, however, this was observed in a microsomal fraction that was isolated from whole ventricular tissue [76]. Other investigators have reported a decrease in PLC activity in ischemia and an increase in reperfusion [79]; however, this was conducted in a total membrane preparation from whole ventricular tissue. Similarly, although a recent study has reported that myocardial ischemia induces an increase in PLC activity, measurement of the activity was conducted in left ventricular total homogenate [77].…”

“…While the degradation of cardiac membrane inositol phospholipids has been associated with cardiac dysfunction due to I-R [74][75][76][77][78][79], in view of the role of PLC products in cardiomyocyte Ca 2+ -homeostasis, it is possible that activation of PLC may contribute to the occurrence of intracellular Ca 2+ -overload and I-R injury. A small number of studies have reported alterations in inositol phospholipid metabolism in I-R due to PLC.…”

A Rodent Model of Alcoholic Heart Muscle Disease and Its Evaluation by Echocardiography

“…Isolated Langendorff perfusion experiments were performed as previously described Schwertz and Halverson, 1992). Hearts were perfused retrogradely (constant flow 12 ml/min, mean coronary perfusion pressure 100 mmHg) with an oxygenated Krebs-Ringer solution (in mmol: NaCl 100, KCL 4.74, CaCl 2 1.115, KH 2 PO 4 1.18, MgSO 4 1.18, glucose 11.5, pyruvate 24.6, fumarate 26.9, NaHCO 3 25) maintained at 37°C, pH 7.4 and paced at a rate of 300 beats per minute (Grass Instruments, Quincy, MA).…”

A Study on Dilazep: I. Mechanism of Anti-ischemic Action of Dilazep Is Not Coronary Vasodilation but Decreased Cardiac Mechanical Function in the Isolated, Working Rat Heart

“…There is circumstantial evidence that myocardial phospholipases A2 and C mainly contribute to the post-ischemic reperfusion injury (31,32). Because the optimum pH range for the myocardial phospholipase A2 is neutral to alkaline (33), the enzyme would be activated during reperfusion to a greater extent than during ischemia.…”