Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption.
A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.
Sarri
Elisabet
E
Cardiology Service, Institut de Recerca Hospital Universitari Vall d'Hebron, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain.
Garcia-Dorado
David
D
Abellan
Arancha
A
Soler-Soler
Jordi
J
D000138:Acidosis / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase / Q000378:metabolism*; D005947:Glucose / Q000172:deficiency* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009336:Necrosis / Q000378:metabolism; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D012508:Sarcolemma / Q000378:metabolism*; D013997:Time Factors
D010713:Phosphatidylcholines; D007770:L-Lactate Dehydrogenase; D019916:Choline-Phosphate Cytidylyltransferase; D005947:Glucose; D002794:Choline