Abstract
The pharmacologic agents verapamil, nifedipine, diltiazem, prenylamine, N-oleoylethanolamine, R 24571, trifluoperazine, dibucaine, and quinacrine are examined as potential inhibitors of rat liver mitochondrial phospholipase A2 acting on endogenous phospholipid. Their potency as inhibitors of the enzyme is compared to their activities as inhibitors of phospholipase A2-dependent swelling and ruthenium red-induced Ca2+ release in intact mitochondria. For verapamil, diltiazem, trifluoperazine, dibucaine, and quinacrine, there is complete agreement between the relative potencies as inhibitors of phospholipase A2 and the two other processes. Nifedipine and prenylamine, which are weak inhibitors of phospholipase A2, produce a permeable inner membrane, provided that the mitochondrial have accumulated Ca2+. R 24571, which strongly inhibits the enzyme, disrupts mitochondria by a Ca2+-independent mechanism. N-Oleoylethanolamine, which is an effective inhibitor of swelling, does not inhibit phospholipase A2 or ruthenium red-induced Ca2+ release. The results support a proposed scheme wherein ruthenium red-induced Ca2+ release is viewed as reverse activity of the Ca2+-uptake uniporter occurring subsequent to decline in the proton motive force. The latter effect is proposed to arise from a specific phospholipase A2-dependent increase in inner-membrane H+ conductance of mitochondrial subpopulations. It is further shown that mitochondrial membranes display cyclic oscillations in free fatty acid content which are not dependent on the presence of Ca2+ or on the capacity to generate acylcoenzyme A.
Highlights
Phospholipase Properties of the Mitochondrial Membrane Preparation-We previously reported that polyunsaturated fatty acids [6]and lysophosphatidylethanolamine[33] accumulate in Ca2+-loadedmitochondria treated with N-ethylmaleimide during the period when increased inner-membrane permeability is developing
Samples were taken for the determination of polyunsaturated free fatty acid (FFA) as described under "Experimental Procedures.'' Fatty acid content was calculated on the basis of equivalents of mitochondrial protein from which the washed-membrane preparation was derived. 0, heat-treated preparation. 0,normal preparation
In previous studies on permeability regulation in rat liver mitochondria, we have assumed that changes in the content of polyunsaturated fatty acids and lysophospholipids arise solely from alterations in the relative rates of phospholipid deacylation and reacylation reactions produced by phospholipase Az and the opposing acyltransferase activity, respectively [6,7,33].the bovine serum albumin-sensitive oscillating behavior of the phospholipase progress curves and the rapid reduction in free fatty acid content which occurs upon transferring the previously incubated membranes from ice temperature to 37 “C (Fig. 1) indicate that otheractivities can be involved
Summary
Phospholipase Properties of the Mitochondrial Membrane Preparation-We previously reported that polyunsaturated fatty acids [6]and lysophosphatidylethanolamine[33] accumulate in Ca2+-loadedmitochondria treated with N-ethylmaleimide during the period when increased inner-membrane permeability is developing. Somewhat surprising, property of the washed membrane preparation is illustrated by the single inuerted triangle symbols in panels A and B ofFig. 1 These points represent the amounts of phospholipase Az and AI products, respectively, which were present in the washed membranes after completion of the preparation procedure and storage in the frozen state. The mitochondrialmembrane preparation was assayed for phospholipase A activities as described under "Experimental Procedures." As in Fig. 1,the protein concentration used to calculate the free fatty acid (FFA)content was the equivalent of mitochondrial protein from which the membranes were prepared. N-Oleoylethanolamine is representative of lipid amides which are synthesized in infarcted dog heart [49,50,51] and which antagonize the phospholipase Az-dependentpermeability increase in mitochondria [37] This compound was pounds which effectively prevent the accumulation of polyunsaturated fatty acids (as in Fig. 5A) are classified as strong inhibitors. Bovine serum albumin was not employedto reduce the oscillations in reaction-product curves since, due to the hydrophobic character of the potentialinhibitors being tested (see Fig. 5), binding of the logic agents of different types which share calmodulin-antag- compounds to albumin was anticipated
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