Summary
In the present study we have investigated whether enzyme histochemical parameters can be applied to detect early ischemic damage in rat heart after ischemia without restoration of the blood flow. Ischemia was induced by incubating heart fragments for 0, 10, 20, 30, 60, 120 and 240 min at 37°C. The activity and localization of the following enzymes was studied in unfixed cryostat sections using quantitative histochemical methods: lactate dehydrogenase, creatine kinase, succinate dehydrogenase, phosphofructokinase, acid phosphatase, 5′-nucleotidase and glycogen phosphorylase. Moreover, the ultrastructure of the tissue was studied with special attention to the appearance of flocculent densities in mitochondria, which can be seen as a sign of irreversible cell damage. It was shown that glycogen phosphorylase activity in rat heart decreased after short periods (30 min) of in vitro ischemia, whereas all other enzymes studied were not decreased up to 240 min, with the exception of lactate dehydrogenase and phosphofructokinase activities which were diminished only at 240 and 120 min of ischemia, respectively. Some reaction product was found after incubating for 5′-nucleotidase activity in the absence of substrate, indicating the presence of endogenous substrate(s). This endogenous substrate disappeared from the myocytes after 20 min of ischemia. It is assumed that AMP and/or other phosphate-containing compounds play an essential role in the activation of glycogen phosphorylase. Significant reduction of glycogen phosphorylase activity is correlated with the irreversible stage of damage of myocytes as judged from the ultrastructure.
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References
Decker RS, Wildenthal K (1980) Role of lysosomes and latent hydrolytic enzymes in ischemic damage and repair of the heart. In: Wildenthal K (ed) Degradative processes in heart and skeletal muscle. North Holland Biomedical Press, Amsterdam, pp. 389–418
De Leiris J, Hearse DJ (1986) Myocardial enzyme leakage as an indicator of cellular injury: principles and application. In: Dhalla NS (ed) Methods in studying cardiac membranes. CRC Press, Boca Raton, Florida, vol. 1:253–277
Derias NW, Adams CWM (1982) Macroscopic enzyme histochemistry in myocardial infarction: artefactual nature of the creatine phosphokinase reaction. J Clin Pathol 35:407–409
Ewen LM, Griffiths J (1971) Patterns of enzyme activity following myocardial infarction and ischemia. Am J Clin Path 56:614–622
Frederiks WM, Marx F (1987) Changes in cytoplasmic and mitochondrial enzymes in rat liver after ischemia followed by reperfusion. Exp Mol Pathol 47:291–299
Frederiks WM, Marx F (1988) A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol. Histochem J 20:207–214
Frederiks WM, Marx F (1989) Changes in acid phosphatase activity in rat liver after ischemia. Histochemistry 93:161–166
Frederiks WM, Marx F (1990) The effect of ischemia on glycogen phosphorylase activity in rat liver: a quantitative histochemical study. Anal Cell Pathol 2:347–355
Frederiks WM, Marx F, Myagkaya GL (1988) A quantitative histochemical study of 5′-nucleotidase activity in rat liver after ischemia. J Pathol 154:277–286
Frederiks WM, Marx F, Van Noorden CJF (1987) Quantitative histochemical assessment of heterogeneity of glycogen phosphorylase activity in liver parenchyma from fasted rats using the semipermeable membrane technique and the PAS-reaction. Histochem J 19:150–156
Frederiks WM, Marx F, Van Noorden CJF (1988) Quantitative histochemistry of creatine kinase in rat myocardium and skeletal muscle. Histochem J 20:624–628
Frederiks WM, Marx WM, Van Noorden CJF (1991) Homogeneous distribution of phosphofructokinase in the rat liver acinus. A quantitative histochemical study. Hepatology 14:634–639
Frederiks WM, Myagkaya GL, Bosch KS, Fronik GM, Van Veen H, Vogels IMC, James J (1983) The value of enzyme leakage for the prediction of necrosis in liver ischemia. Histochemistry 78:459–472
Fukuhara I, Kawashima T, Kubota I, Mitsunami K, Motomura M, Bito K, Kinoshita M, Kawakita S (1987) Changes of calcium ATPase and cytochrome oxidase activity of myocardial cells under early and late ischemia in comparison with ultrastructural changes. Jpn Circ J 51:403–410
Hiltunen JK, Saukko P, Hirvonen J (1985) Correlations between enzyme histochemical reactions and respective enzyme activities in global ischaemic rat hearts. Br J Exp Path 66:743–752
Ishiharajima S, Aida T, Nakagawa R, Kameyama K, Sugano K, Ogura T, Asano G (1986) Early membrane damage during ischemia in rat heart. Exp Mol Pathol 44:1–6
James J, Frederiks WM, Van Noorden CJF, Tas J (1986) Detection of metabolic changes in heopatocytes by quantitative cytochemistry. Histochemistry 24:308–316
Jennings RB, Reimer KA (1981) Lethal myocardial ischemic injury. Am J Pathol 102:241–255
Klein HH, Puschmann S, Schaper J, Schaper W (1981) The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction. Virchows Arch A (Pathol Anat) 393:287–297
Meijer AEFH (1968) Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase. I. The use of glucosyl acceptor dextran. Histochemie 12:244–252
Michaelidis B, Story KB (1990) Phosphofructokinase from the anterior byssus retractor muscle of mytilus edulis: modification of the enzyme in anoxia and by endogenous protein kinases. Int J Biochem 22:759–765
Myagkaya GL, Van Veen H, James J (1985) Quantitative analysis of mitochondrial flocculent densities in rat hepatocytes during normothermic and hypothermic ischemia in vitro. Virchows Arch B (Cell Pathol) 49:61–72
Passonneau JV, Lowry OH (1964) The role of phosphofructokinase in metabolic regulation. In: Weber G (ed) Advances in Enzyme Regulation. Pergamon Press, London, vol 2, pp 265–274
Penttila A, Ahonen A (1976) Electron microscopical and enzyme histochemical changes in the rat myocardium during prolonged autolysis. Beitr Path Bd 157:126–141
Sandritter HW, Jestadt R (1958) Triphenyltetrazoliumchlorid (TTC) als Reduktionsindikator zur makroskopischen Diagnose des frischen Herzinfarktes. Verh Dtsch Ges Pathol 41:165–170
Siegel RJ, Edwalds G, Rej R, Fishbein MC (1984) Distribution of cytosolic and mitochondrial aspartate aminotransferase in normal, ischemic, and necrotic myocardium. Lab Invest 51:648–654
Schaper J, Mulch J, Winkler B, Schaper W (1979) Ultrastructural, functional and biochemical criteria for estimation of reversibility of ischemic injury: A study on the effects of global ischemia on the isolated dog heart. J Mol Cell Cardiol 11:521–541
Trump BF, Berezesky IK, Osornio-Vargas AG (1982) Cell death and the disease process. The role of calcium. In: Bowen ID, Lockshin RA (eds) Cell Death in Biology and Pathology, Chapman and Hall, London, pp 209–242
Will-Shahab L, Krause EG, Bartel S, Schulze W, Kuttner I (1985) Reversible inhibition of adenylate cyclase activity in the ischemic myocardium. J Cardiovasc Pharmacol 7:S23-S58
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Frederiks, W.M., Schellens, J.P.M., Marx, F. et al. Histochemical detection of glycogen phosphorylase activity as parameter for early ischemic damage in rat heart. Basic Res Cardiol 88, 130–140 (1993). https://doi.org/10.1007/BF00798261
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DOI: https://doi.org/10.1007/BF00798261