Myocardial ischemia is accompanied by accelerated phospholipid catabolism resulting in alterations in the chemical constituents of the sarcolemmal membrane which precipitate electrophysiologic dysfunction and myocytic cell death. We have previously demonstrated that plasmalogens are the predominant phospholipid constituents of myocardial sarcolemma and that myocardium contains a novel class of calcium-independent phospholipases A2 which selectively hydrolyze plasmalogen substrate. During the current grant interval we have demonstrated that: 1) sarcolemmal phospholipids are selectively hydrolyzed during myocyte injury; 2) the activity of membrane associated plasmalogen selective phospholipase A2 increases over 9-fold during 15min of myocardial ischemia; and 3) ATP activates calcium- independent plasmalogen selective phospholipase A2 activity in ischemic, but not in control myocardial cytosol. Collectively, these results demonstrate that profound alterations in phospholipid metabolism selectively occur in the electrically active membrane of myocyte which are likely mediated by calcium-independent plasmalogen selective phospholipase A2. Accordingly, the overall goal of the proposed research is the identification of the biochemical mechanisms which result in the activation of these novel phospholipase during myocardial ischemia. Purification of the polypeptide catalyzing membrane associated phospholipase A2 will identify salient physical, structural and kinetic characteristics of this important enzyme. Factors responsible for the induction of membrane associated phospholipase A2 activity during ischemia will be identified and purified. The potential significance of the direct association of cytosolic calcium-independent phospholipase A2 with phosphofructokinase will be delineated and the relationship of this complex to ATP induced activation of cytosolic phospholipase A2 during ischemia will be examined. Cytosolic and membrane bound calcium-independent phospholipase activities will be cloned and their primary sequences will be determined. Critical differences in the stereoelectronic conformation of plasmalogen and diacyl phospholipids will be delineated to assess their role as determinants of the kinetic specificities of these phospholipases A2. Through utilization of this multidisciplinary approach the detailed biochemical mechanisms responsible for the activation of these novel phospholipases will be identified. Collectively, the proposed research will identify critical structural and kinetic characteristics of these novel enzymic targets whose pharmacologic manipulation can potentially prevent lethal ventricular dysrhythmias and attenuate cell death during myocardial ischemia.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL034839-09
Application #
2217674
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1985-09-30
Project End
1995-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
9
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Ramanadham, S; Wolf, M J; Ma, Z et al. (1996) Evidence for association of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from pancreatic islets and HIT insulinoma cells with a phosphofructokinase-like protein. Biochemistry 35:5464-71
Han, X; Gubitosi-Klug, R A; Collins, B J et al. (1996) Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced Biochemistry 35:5822-32
Gross, R W (1995) Myocardial phospholipase A2. J Lipid Mediat Cell Signal 12:131-7
Ramanadham, S; Wolf, M J; Jett, P A et al. (1994) Characterization of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor. Biochemistry 33:7442-52
Ramanadham, S; Bohrer, A; Mueller, M et al. (1993) Mass spectrometric identification and quantitation of arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species. Biochemistry 32:5339-51
Turk, J; Gross, R W; Ramanadham, S (1993) Amplification of insulin secretion by lipid messengers. Diabetes 42:367-74
Gross, R W; Ramanadham, S; Kruszka, K K et al. (1993) Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells. Biochemistry 32:327-36
Ramanadham, S; Gross, R W; Han, X et al. (1993) Inhibition of arachidonate release by secretagogue-stimulated pancreatic islets suppresses both insulin secretion and the rise in beta-cell cytosolic calcium ion concentration. Biochemistry 32:337-46
Ramanadham, S; Bohrer, A; Gross, R W et al. (1993) Mass spectrometric characterization of arachidonate-containing plasmalogens in human pancreatic islets and in rat islet beta-cells and subcellular membranes. Biochemistry 32:13499-509
Ford, D A; Gross, R W (1992) Metabolism of ether-linked diglycerides in brain and myocardium. Methods Enzymol 209:413-23

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