Related Experiment Videos
Effect of hypoxia on phosphatidylcholine biosynthesis in the isolated hamster heart
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Insights
Hypoxia in hamster hearts rapidly decreases energy and CDP-choline levels, slowing phosphatidylcholine synthesis. However, the enzyme cytidylyltransferase shifts to a more active form to compensate, maintaining essential lipid production.
Area of Science:
- Biochemistry
- Cardiovascular Physiology
- Cellular Metabolism
Background:
- Phosphatidylcholine is crucial for heart cell membranes, synthesized mainly via the CDP-choline pathway.
- CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyzes the rate-limiting step in this pathway.
- Previous studies showed diminished cardiac CTP in myopathy, with increased cytidylyltransferase activity to compensate.
Purpose of the Study:
- To investigate if cardiac cytidylyltransferase activity increases to compensate for rapidly decreased CTP levels during hypoxia.
- To understand the mechanism of phosphatidylcholine biosynthesis regulation under acute hypoxic stress.
Main Methods:
- Hamster hearts were perfused with a hypoxic buffer (95% N2).
- Hearts were pulse-labeled with radioactive choline and chased with non-radioactive choline.
- Choline metabolites, ATP, CTP levels, and enzyme activities were analyzed.
Main Results:
- Hypoxia caused rapid decreases in ATP and CTP levels within 60 minutes.
- Phosphatidylcholine biosynthesis rate declined, primarily due to reduced conversion of phosphocholine to CDP-choline.
- Hypoxic treatment enhanced the translocation of cytidylyltransferase from the cytosol to the microsomal fraction.
Conclusions:
- The heart compensates for hypoxia-induced decreases in CTP by enhancing cytidylyltransferase translocation to the more active microsomal form.
- This enzyme shift aims to maintain phosphatidylcholine biosynthesis despite reduced substrate availability.
- Fatty acid accumulation during hypoxia may trigger this compensatory enzyme translocation.
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.