The objective of this proposal is to test the hypothesis that, in the isolated perfused rabbit heart, the intracellular Mg2+ concentrations are important in regulating the arachidonic acid content and overall fatty acid composition of phospholipids. In the isolated rabbit heart, the synthesis of prostaglandins from exogenous arachidonic acid is significantly increased during perfusion with Ca2+ free buffer or during perfusion with 35mM Na+/104 mMLi+ buffer, while the incorporation of labeled arachidonate into tissue phospholipids, especially phosphatidyl choline and phosphatidyl ethanolamine, is significantly reduced. Both of these perfusion conditions significantly reduce the tissue Mg2+ content. The differences in prostaglandin output in response to exogenous arachidonic acid are linearly correlated with the tissue Mg2+ content, and cannot be accounted for by differences in the Km or Vmax of cyclooxygenase, changes in the permeability of the heart to arachidonate or changes in the profile of prostaglandins synthesized. Experiments are proposed to: (1) determine whether the activities of fatty acyl CoA synthetase and/or lysophosphatidyl choline acyl transferase (enzymes which are important in the incorporation of arachidonic and other fatty acids into phospholipids) are changes during Mg2+ depletion; (2) study more extensively the effects of Mg2+ depletion on eicosanoids synthesis; (3) study the effects of Mg2+ depletion on the incorporation of arachidonic acid into tissue lipids; and (4) determine whether anoxia/hypoxia induced Mg2+ loss might mediate anoxic tissue injury. Magnesium depletion will be induced by changing the ionic compositions of the perfusion buffer, by adding ionophore, isoproterenol or a cAMP analog to the perfusion buffer, or by reducing the oxygen tension of the perfusion buffer. Conversation of arachidonic acid to prostaglandins and the incorporation of radiolabelled arachidonic acid into neutral lipids and phospholipids will be measured. The data will be examined for significant correlations between changes in the disposition of arachidonic acid (i.e., changes in the synthesis of acid into lipids and shifts in the Mg2+ content of the tissue. The activities (Km and Vmax for arachidonic acid) and substrate preference ratios of fatty acyl CoA synthetase and lysophosphatidyl choline acyl transferase will be measured in hearts under conditions known to change the Mg2+ content of the tissue. The direct effect of added Mg2+ on the activities of these enzymes will also be measured.
