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Effect of hypoxia on phosphatidylcholine biosynthesis in the isolated hamster heart.

ABSTRACT: In hamster heart, the majority of the phosphatidylcholine is synthesized via the CDP-choline pathway, and the rate-limiting step of this pathway is catalysed by CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15). We have shown previously [Choy (1982) J. Biol. Chem. 257, 10928-10933] that, in the myopathic heart, the level of cardiac CTP was diminished during the development of the disease. In order to maintain the level of CDP-choline, and consequently the rate of phosphatidylcholine biosynthesis, cardiac cytidylyltransferase activity was increased. However, it was not clear if the same compensatory mechanism would occur when the cardiac CTP level was decreased rapidly. In this study, hypoxia of the hamster heart was produced by perfusion with buffer saturated with 95% N2. The heart was pulse-labelled with radioactive choline and then chased with non-radioactive choline for various periods under hypoxic conditions. There was a severe decrease in ATP and CTP levels within 60 min of hypoxic perfusion, with a corresponding fall in the rate of phosphatidylcholine biosynthesis. Analysis of the choline-containing metabolites revealed that the lowered ATP level did not affect the phosphorylation of choline to phosphocholine, but the lower CTP level resulted in the decreased conversion of phosphocholine to CDP-choline. Determination of enzyme activities revealed that hypoxic treatment resulted in the enhanced translocation of cytidylyltransferase from the cytosolic to the microsomal form. This enhanced translocation was probably caused by the accumulation of fatty acids in the heart during hypoxia. We postulate that the enhancement of translocation of the cytidylyltransferase to the microsomal form (a more active form) is a mechanism by which the heart can compensate for the decrease in CTP level during hypoxia in order to maintain phosphatidylcholine biosynthesis.

SUBMITTER: Hatch GM

PROVIDER: S-EPMC1131389 | biostudies-other | 1990 May

REPOSITORIES: biostudies-other

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Project description:The pathway for the biosynthesis of new cardiolipin was investigated in the isolated perfused intact rat heart. Isolated rat hearts were perfused in the Langendorff mode for up to 60 min with Krebs-Henseleit buffer containing 0.1 microM [U-14C]glycerol. Analysis of radioactivity incorporated into phospholipids in the organic phase revealed an increase in radioactivity incorporated into phosphatidylglycerol, cardiolipin and other phospholipids with time of perfusion. This was associated with a loss of radioactivity from phosphatidic acid. In contrast, perfusion of hearts for up to 60 min with 0.1 mM [1,(3)-3H]glycerol in the perfusate revealed an increased radioactivity associated with phosphatidic acid as well as cardiolipin, phosphatidylglycerol and other phospholipids. Perfusion of hearts for up to 60 min with [32P]Pi in the perfusate revealed a time-dependent increase in radioactivity associated with all phospholipids. Perfusion of hearts for up to 60 min with 0.1 microM or 0.1 mM glycerol in the perfusate did not affect the concentration of phosphatidic acid, cardiolipin or phosphatidylglycerol. To determine the rate-limiting step of cardiolipin biosynthesis, hearts were pulsed for 5 min with 0.1 microM [1,(3)-3H]glycerol and chased for up to 60 min with 0.1 microM glycerol in the perfusate. Radioactivity was maximum at the start of the chase in phosphatidic acid (and 1,2-diacylglycerol), and was subsequently chased into phosphatidylinositol, phosphatidylglycerol and cardiolipin (and other phospholipids). Significant radioactivity in phosphatidylglycerol phosphate was not detected. Radioactivity in CDP-sn-1,2-diacylglycerol remained constant throughout the chase. The activities of the enzymes of the Kennedy pathway for cardiolipin biosynthesis in the heart were determined. On the basis of continuous-pulse and pulse-chase labelling studies it is postulated that the cardiac polyglycerophospholipids phosphatidylglycerol and cardiolipin are actively synthesized from newly synthesized phosphatidic acid via the Kennedy pathway. In addition, the results suggest that the rate-limiting step of cardiolipin biosynthesis in the intact heart is probably the conversion of phosphatidic acid into CDP-sn-1,2-diacylglycerol.

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Effect of hypoxia on phosphatidylcholine biosynthesis in the isolated hamster heart.

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