The phospholipase D (PL D), which catalyzes the formation of phosphatidic acid (PA), was studied in rat myocardium using 14C-labelled phosphatidylcholine (PC) as an exogenous substrate. Subcellular distribution experiments indicated the presence of PL D in particulate fractions only. Different procedures for the isolation of purified cardiac subcellular organelles showed the presence of PL D in sarcolemma (SL), sarcoplasmic reticulum (SR) and mitochondria with 14-, 11- and 5-fold enrichment when compared to the homogenate value, respectively. The activity of SL PL D was observed over a narrow acid pH range with an optimum at 6.5, and it showed a high specificity for PC while phosphatidylethanolamine and phosphatidylinositol showed a low rate of hydrolysis. Under optimal conditions, PA formation was linear for a 90-min period of incubation and the reaction rate was constant for 10 to 100 μg SL protein in the assay medium. The SR PL D displayed properties similar to those seen with the SL PL D. In membrane fractions PL D was also found to catalyze a transphosphatidylation reaction for the synthesis of phosphatidylglycerol. Assessment of the intramembranal levels of radioactive 1,2-diacylglycerol (DAG) in the absence or presence of KF suggested the presence of an active PA phosphohydrolase activity. This study indicates that a PC-specific PL D activity is localized in different membrane systems of the myocardium and may be associated with PA phosphohydrolase to act in a coordinated manner. The functional significance of PL D-dependent formation of PA in cardiac membranes is discussed.

The mitochondrial oxidation of long-chain fatty acids is a major pathway for energy production in the heart. L-carnitine, a naturally occurring highly polar compound, is essential for transporting long-chain fatty acids across the inner mitochondrial membrane to the site of oxidation, as well as for the export of intramitochondrially produced short-chain acyl esters and for the disposal of unphysiological acyl metabolites (1,2). In fact, myocardial carnitine deficiency has been documented in several heart diseases, both in humans (3,4) and in experimental animal models (5,6). Accordingly, carnitine deficiency can be seen to be associated with an energy deficit arising from the unavailability of fatty acids within the mitochondria. Moreover, the accumulation of long-chain fatty acids and their derivatives, fatty acyl CoAthio esters and fatty acylcarnitine esters due to carnitine deficiency could produce deleterious effects on cardiac structure and function. These metabolites are active detergents and bind to the cell membranes (7), and there is evidence (7,8) that these lipid intermediates alter the functional properties of the myocardial membranes. It has also been suggested that the long-chain acyl derivatives may contribute to the decline of myocardial contractility and may cause intracellular Ca2+ overload (8).

This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-l-methionine (0.055, 10, and 150 μM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.