Summary
Preincubation of rat myocardial cells in hypoxic substrate-free Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) resulted in a substantial decline in high energy phosphates (ATP and CP). Thus, 20 and 60 min preincubation produced a 18 and 72% decline in ATP content, whereas the parallel decline in CP content was 51 and 73%. This energy depletion was accompanied by a change in cell morphology from the initial rod-shaped form to rounded up (hyper-contracted) myocytes. In cells preincubated in substrate-free normoxic buffer, both normal morphology and energy homeostasis were maintained. When energy depleted myocytes later were incubated in the presence of phospholipase C (PLC), this resulted in a substantial release of glycerol, amounting to 92 and 137 nmol/106 cells − 2 h in 20 and 60 min energy depleted myocytes, respectively. In addition, PLC caused an increased leakage of lactate dehydrogenase in energy depleted myocytes. Normal cells, on the other hand, were apparently not affected by PLC. These data suggest that PLC selectively attacks energy depleted and/or structurally damaged myocytes. This could well enhance the breakdown of the natural barrier between the extra- and intracellular compartments and thus augment the cellular damage during ischemia. Moreover, energy depleted myocytes appeared exceptionally sensitive to this enzyme, since the levels required to cause glycerol or lactate dehydrogenase release were several orders of magnitude lower than that required to cause membrane permeation in other cell types.
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Chien KR, Pfan RG, Farber JL: Ischemic myocardial cell injury. Prevention by Chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction. Am J Pathol 97: 505–529, 1979
Wolf RA, Gross RW: Identification of a neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipiase A2 in canine myocardium. J Biol Chem 260: 7295–7303, 1985
Roelofsen B: Phospholipases as tools to study the localization of phospholipids in biological membranes. A critical review. J Toxicol Toxin Reviews 1: 87–197, 1982
Kryski Jr A, Kenno KA, Severson DL: Stimulation of lipolysis in rat heart myocytes by isoproterenol. Am J Physiol 248: H208-H216, 1985
Myrnes BJ, Little C: A simple purification sceme yielding crystalline phospholipase C from Bacillus cereus. Acta Chem Scand B 34: 375–377, 1980
Otnaess AB, Little C, Slettin K, Wallin R, Johnsen S, Flengsrud R, Prydz H: Some characteristics of phospholipase C from Bacillus cereus. Eur J Biochem 79: 459–468, 1977
Little C: Phospholipase C from Bacillus cereus. Methods Enzymol 71: 725–730, 1981
Chernick SS: Determination of glycerol in acylglycerols. Methods Enzymol 14: 627–630, 1969
Moss DW: The measurements of enzymes. In: DJ Hearse and de Leiris (eds). Enzymes in Cardiology. John Wiley and Sons 1979, pp 154–198
Sellevold OFM, Jynge P, Aarstad K: High performance liquid chromatography: a rapid isocratic method for determination of creatine compounds and adenine nucleotides in myocardial tissue. J Mol Cell Cardiol 18: 517–527, 1986
Little C, Rumsby MG: Lysis of erythrocytes from stored human blood by phospholipase C (Bacillus cereus). Biochem J 188: 39–46, 1980
Solberg C, Little C, Holme S, Aakre SE: Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-daystored human platelets. Biochem J 222: 389–394, 1984
Gwarsha K, Rumsby MG, Little C: Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations. Neurochem Int 6: 199–206, 1980
Higgins TJC, Bailey PJ, Allsopp D: The influence of ATP depletion on the action of phospholipase C on cardiac myocyte membrane phospholipids. J Mol Cell Cardiol 13:1027–1030, 1981
Ford DA, Gross RW: Identification of endogeneous 1–0alk-1′-enyl-2-acyl-sn-glycerol in myocardium and its effective utilization by choline phosphotransferase. J Biol Chem 263: 2644–2650, 1988
Clark MA, Shorr RGL, Bomalaski JS: Antibodies Prepared to Bacillus cereus phospholipase C crossreact with a phosphatidylcholine preferring phospholipase C in mammalian cells. Biochem Biophys Res Com 140:114–119, 1986
Stam H, Broekhoven-Schokker S, Hulsmann WC: Characterization of mono-, di-, and triacylglycerol lipase activities in the isolated rat heart. Biochim Biophys Acta 875: 76–86, 1986
Dawson RMC, Irvine RF, Bray J, Quinn PJ: Long-chain unsaturated diacylglycerols cause a pertubation in the structure of phospholipid bilayers rendering them susceptible to phospholipase attack. Biochem Biophys Res Com 125: 836–842, 1984
Ikeda V, Arisaka H, Takayasu T, Takeda K, Natsume T, Hosoda S: Protein kinase C activation aggravates hypoxic myocardial injury by stimulating Na+/H+ exhange. J Mol Cell Cardiol 20: 493–500, 1988
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Myrmel, T., Larsen, T.S., Skulberg, A. et al. Phospholipase C-evoked glycerol release in energy depleted rat myocardial cells. Mol Cell Biochem 88, 107–111 (1989). https://doi.org/10.1007/BF00223431
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DOI: https://doi.org/10.1007/BF00223431