Dosis respuesta de diferentes inhibidores para estudios metabólicos en astrocitos con L-lactato en concentraciones perinatales.
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Tovar Franco, J. (2005). Dosis respuesta de diferentes inhibidores para estudios metabólicos en astrocitos con L-lactato en concentraciones perinatales. NOVA, 3(3). https://doi.org/10.22490/24629448.19
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Jairo Alfonso Tovar Franco
Resumen
Los sistemas de transporte como las lanzaderas mitocondrial/citosólicas son importantes para las funciones integrativas del cerebro en desarrollo. Para facilitar su estudio y conocer el papel que estos sistemas de transporte puedan tener en células cerebrales se ha empleado trasadores radiactivos en cultivos primarios. En este trabajo se evaluaron las velocidades de oxidación (respiración) y lipogénesis utilizando lactato que es el principal sustrato cerebral durante la prelactancia en astrocitos en cultivo primario. Se determinó las concentraciones mínimas de inhibidores que interactúan con las principales lanzaderas de manera que no fueran tóxicos para las células, pero que su vez permitieran observar efectos metabólicos. Se utilizó L-[U-14C]- lactato (1 MCi), L-lactato (10.5 mM) en ausencia y presencia de diferentes concentraciones de inhibidores enzimáticos como el dicloroacetato (DCA), un inhibidor de la piruvato deshidrogenasa quinasa y el aminooxiacetato (AOA), un inhibidor de la aspartato aminotransferasa e inhibidores del transporte mitocondrial como el butilmalonato (BM), un inhibidor del transporte de dicarboxilatos y específicamente del L-malato y el 1,2,3-benceno tricarboxilato (BT) un inhibidor del transporte de tricarboxilatos. Los resultados sugieren que a concentraciones de 1 mM de DCA y de 5 mM de AOA, BM y BT son suficientes para evaluar efectos metabólicos en células cerebrales durante el periodo perinatal.
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Referencias
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2. Tabernero A. Regulación del metabolismo del lactato en neuronas y astrocitos en cultivo primario. [Tésis doctoral].
Facultad de Farmacia. Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca. 1993. 186 p.
3. Vicario C. Regulación del metabolismo del lactato en células aisladas de cerebro de rata. [Tésis doctoral]. Universidad de Salamanca. Facultad de Farmacia. Departamento de Bioquímica y Biología Molecular. 1991. 263 p.
4. Vicario C, Medina J. Metabolism of lactate in the rat brain during the early neonatal period. J. Neurochem.
1992;59(1):32-40.
5. Medina JM, Tabernero A, Tovar J, Martin-Barrientos J. Metabolic fuel utilization and pyruvate oxidation during the postnatal period. J.Inher.Metab.Dis. 1996;19:432-442.
6. Arizmendi C, Medina J. Lactate as an oxidizable substrate for rat brain in vitro during the perinatal period. Biochem.J. 1983;214:633-635.
7. Bolaños JP. Efecto del valproato sobre el metabolismo intermediario en cerebro de neonato de rata. [Tésis doctoral].
Facultad de Farmacia. Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca. 1992a. 202 p.
8. Fernández E, Medina, JM. Lactate utilization by the neonatal rat brain in vitro. Biochem.J. 1986;234:489-492.
9. Medina JM, Bolaños JP, Vicario C, Arizmendi, C. Fuel supply to the brain during the early postnatal period. In: Cuezva J, ed. Endocrine and biochemical development of the fetus and neonate. New York: Plenum Press; 1996:175-194.
10. Gladden L. Lactate metabolism: a new paradigm for the third millennium. J. Physiol. 2004;558(1):5-30.
11. Tovar J, Saavedra L, Bryon A. Metabolismo cerebral. En: Niño M y Ferrer, L., ed. Neuroanestesia. Enfoque
perioperatorio en el paciente neurologico. Bogotá D.C. Distribuna Editorial Médica; 2005:33-88.
12. Cohen J, Wilkin G. Neural cell culture. A practical approach. In: Rickwood D, Hames B. Ed. London, England. IRL press at Oxford University Press. 1995.
13. Saneto R, DeVellis J. Neuronal and glial cells: Cell culture of the central nervous system. In: A.J. Turner & H.S. Bachelard, ed. Neurochemistry a Practical Approach. IRL Press. 1987:27- 64.
14. Rose S, Sinha A. Some properties of isolated neuronal cell fractions. J.Neurochem. 1969;16:1319-1328.
15. Kimelberg HK. Primary astrocyte cultures - A key to astrocyte function. Cell.Molec.Neurobiol. 1983;3(1):1-16.
16. Bigmani A, Eng LF, Dahl D, Uyeda, CT. Localization of the glial fibrillary acidic protein in astrocites by inmunofluorescence. Brain Res. 1972;43:429-435.
17. Kimelberg HK, Norenberg MD. Astrocitos. Investigación y Ciencia. 1989;Junio,:18-27.
18. Elliott KAC. The use of brain slices. In: Laftha A, ed. Handbook of neurochemistry. Vol. 2. New York, NY. Plenum Press. 1969:103-115.
19. Gutmann I, Wilhelm A. L-(+)-Lactate. determination with lactate dehydrogenase and NAD. In: Bergmeyer H, ed. Methods of Enzimatic Analysis. Vol. 3. Deerfiel Beach, FL. Academic Press.; 1974:1464-1468.
20. Edmond E, Robbins R, Bergstrom J, Cole R, De Vellis J. Capacity for substrates utilization in oxidative metabolism by neurons, astrocytes and oligodendrocytes from developing brain in primary culture. J.Neurosci.Res. 1987;18(4):551-
561.
21. Sykes J, López-Cardoso M , Van Den Bergh S. Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain. Neurochem.Int. 1986;8:67-75.
22. Folch J, Lees M, Slone GH. A simple method for the isolation and purification of total lipides from animal tissues.
J.Biol.Chem. 1957;226:497-509.
23. Kuroda Y, Toshima K, Watanabe T, Kobashi H, Ito M, Takeda E, Miyao M. Effects of dichloroacetate on pyruvate
metabolism in rat brain in vivo. Pediatric Res. 1984;18:936- 938.
24. Miller AL, Hatch JP, Prihoda TJ. Dichloroacetate increase glucose use and decreases lactate in developing rat brain. Metabolic Brain Disease. 1990;5(4):195-204.
25. Rex-Sheu K-F, Lai JC, Blass JP. Properties and regional distribution of pyruvate dehydrogenase kinase in rat brain.
J.Neurochem. 1984;42:230-236.
26. Katayama Y, Welsh FA. Effects of dichloroacetate on regional energy metabolites and pyruvate dehydrogenase activity during ischemia and reperfusion in gerbil brain. J.Neurochem. 1989;52(6):1817-1822.
27. McAllister A, Allison SP, Randle PJ. Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-
hidroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo. Biochem.J. 1973;134:1067-1081.
28. Abemayor E, Kovachich, G, Haugaard, N. Effects of dichloroacetate on brain pyruvate dehydrogenase.
J.Neurochem. 1984;42(1):38-42.
29. Tomsig JL, Gruenstein E, Dimlich, RV. Inhibition of lactateinduced swelling by dichoroacetate in human astrocytomacells. Brain Res. 1991;568:92-100.
30. Meijer AJ, Van Dam, K. The metabolic significance of anion transport in mitochondria. Biochim.Biophys.Acta.
1974;346:213-244.
31. Flint M, Swartz KJ, Hyman BT, Storey E, Finn SF, Koroshetz W. Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism. J.Neurochem. 1991;57(3):1068-
1073.
32. Palaiologos G, Hertz L, Schousboe A. Role of aspartate aminotransferase and mitochondrial dicarboxylate transport
for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem.Res.
1989;14(4):359-366.
33. Yu A, Schousboe, A, Hertz, L. Metabolic fate of 14C-labeled glutamate in astrocytes in primary culture. J.Neurochem.
1982;39:954-960.
34. Kauppinen RA, Sihra T, Nicholls D. Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve
terminals and prevents the mitochondria from utilizing glycolytic substrates. Biochim.Biophys. Acta. 1987;930:173-
178.
35. McKenna M, Tyson J, Couto R, Stevenson J, Caprio F. The metabolism of malate by cultured rat brain astrocytes.
Neurochem.Res. 1990;15(12):1211-1220.
36. Farinelli S, Nicklas W. Glutamate metabolism in rat cortical astrocyte cultures. J.Neurochem. 1992;58(5):1905-1915.
37. Passarella S, Barile M, Atlante A, Quagliariello E. Oxaloacetate uptake into rat brain mitochondria and
reconstruction of the malate/oxaloacetate shuttle. Biochem.Biophys.Res.Comm. 1984;119:1039-1046.
38. Neale J, Bzdega T, Wroblewska B. N-acetylaspartylglutamate. The most abundant peptide neurotransmitter in the
mammalian central nervous system. J.Neurochem. 2000;75(2):443-452.
39. Chakraborty G, Mekala P, Yahya D, Wu G, Ledeen R. Intraneuronal N-acetylaspartate supplies acetyl groups for
myelin lipids synthesis: evidence for myelin-associated aspartoacylase. J.Neurochem. 2001;78:736-745.
40. Patel T, Clark, J. Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/ cytosolic carbon transport. Biochem.J. 1979;184:539-546.
41. D.Adamo A, Smith J, Phillips I. The piruvate dehydrogenase complex: cloning of the rat somatic E1a subunit and its
coordinate expression with the mRNAs for E1b, E2, and E3 catalytic subunits in developing rat brain. J.Neurochem.
1973;20:1275-1278.
42. D.Adamo A, Yatsu, F. Acetate metabolism in the nervous system. N-acetyl-L-aspartic acid and the biosynthesis of brain lipids. J.Neurochem. 1966;13:961-965.
43. Fitzpatrick S, Cooper A, Duffy T. Use of ß-methylene-D,Laspartate to assess the role of aspartate aminotransferase in cerebral oxidative metabolism. J.Neurochem. 1983;41:1370- 1383.
44. Beal MF, Swartz KJ, Hyman BT, Storey E, Finn SF, Koroshetz W. Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism. J.Neurochem. 1991;57:1068- 1073.
45. Palmieri F, Stipani I, Quagliariello E. Kinetic study of the tricarboxylate carrier in rat liver mitochondria.
Eur.J.Biochem. 1972;26:587-594.
46. Watson JA, Lowenstein, JM. Citrate and the Conversion of Carbohydrate into Fat. Fatty acid synthesis by a combination of cytoplasm and mitochondria. J.Biol.Chem. 1970;245(22):5993-6002.
47. Calvin J, Tubbs P. Mitochondrial transport processes and oxidation of NADH by Hypotonically-treated boar spermatozoa. Europ J Biochem. 1978;89:315-320.
48. Coleman J, Palmer JM. The oxidation of malate by isolated plant mitochondria. Eur. J. Biochem. 1972;26:499-509.
49. Robinson BH, Willians GR, Halperin ML, Leznoff CC. The sensitivity of the exchange reactions of tricarboxylate, 2-
oxoglutarate and dicarboxylate transporting system of rat liver mitochondria to inhibition by 2-pentylmalonate, piodobenzylmalonate, and benzene 1, 2, 3-tricarboxylate. Eur.J.Biochem. 1971;20(1):65-71.
50. Patel MS. Citrate transport oand oxidation by isolated rat brain mitochondria. Brain Res. 1975;98:607-611.
51. Stucki J. Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria. Eur J
Biochem. 1977;78(1):183-187.
52. Stipani I, Palmieri, F. Purification of the active mitochondrial tricarboxylate carrier by hydroxylapatite chromatography. FEBS. 1983;161:269-274.
53. Patel MS. CO2-Fixing enzymes. In: Boulton AA, Baker GB, Butterworth RF, eds. Neuromethods Carbohydrates and Energy Metabolism. Vol. 11. Clifton, N.J.: Humana Press.; 1989:309-341.
54. Kaufman E, Driscoll B. Evidence for cooperativity between neurons and astroglia in the regulation of CO2 fixation in vitro. Dev.Neurosci. 1993;15:299-305.
-------------------------------------------------------------------------------
DOI: http://dx.doi.org/10.22490/24629448.19
2. Tabernero A. Regulación del metabolismo del lactato en neuronas y astrocitos en cultivo primario. [Tésis doctoral].
Facultad de Farmacia. Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca. 1993. 186 p.
3. Vicario C. Regulación del metabolismo del lactato en células aisladas de cerebro de rata. [Tésis doctoral]. Universidad de Salamanca. Facultad de Farmacia. Departamento de Bioquímica y Biología Molecular. 1991. 263 p.
4. Vicario C, Medina J. Metabolism of lactate in the rat brain during the early neonatal period. J. Neurochem.
1992;59(1):32-40.
5. Medina JM, Tabernero A, Tovar J, Martin-Barrientos J. Metabolic fuel utilization and pyruvate oxidation during the postnatal period. J.Inher.Metab.Dis. 1996;19:432-442.
6. Arizmendi C, Medina J. Lactate as an oxidizable substrate for rat brain in vitro during the perinatal period. Biochem.J. 1983;214:633-635.
7. Bolaños JP. Efecto del valproato sobre el metabolismo intermediario en cerebro de neonato de rata. [Tésis doctoral].
Facultad de Farmacia. Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca. 1992a. 202 p.
8. Fernández E, Medina, JM. Lactate utilization by the neonatal rat brain in vitro. Biochem.J. 1986;234:489-492.
9. Medina JM, Bolaños JP, Vicario C, Arizmendi, C. Fuel supply to the brain during the early postnatal period. In: Cuezva J, ed. Endocrine and biochemical development of the fetus and neonate. New York: Plenum Press; 1996:175-194.
10. Gladden L. Lactate metabolism: a new paradigm for the third millennium. J. Physiol. 2004;558(1):5-30.
11. Tovar J, Saavedra L, Bryon A. Metabolismo cerebral. En: Niño M y Ferrer, L., ed. Neuroanestesia. Enfoque
perioperatorio en el paciente neurologico. Bogotá D.C. Distribuna Editorial Médica; 2005:33-88.
12. Cohen J, Wilkin G. Neural cell culture. A practical approach. In: Rickwood D, Hames B. Ed. London, England. IRL press at Oxford University Press. 1995.
13. Saneto R, DeVellis J. Neuronal and glial cells: Cell culture of the central nervous system. In: A.J. Turner & H.S. Bachelard, ed. Neurochemistry a Practical Approach. IRL Press. 1987:27- 64.
14. Rose S, Sinha A. Some properties of isolated neuronal cell fractions. J.Neurochem. 1969;16:1319-1328.
15. Kimelberg HK. Primary astrocyte cultures - A key to astrocyte function. Cell.Molec.Neurobiol. 1983;3(1):1-16.
16. Bigmani A, Eng LF, Dahl D, Uyeda, CT. Localization of the glial fibrillary acidic protein in astrocites by inmunofluorescence. Brain Res. 1972;43:429-435.
17. Kimelberg HK, Norenberg MD. Astrocitos. Investigación y Ciencia. 1989;Junio,:18-27.
18. Elliott KAC. The use of brain slices. In: Laftha A, ed. Handbook of neurochemistry. Vol. 2. New York, NY. Plenum Press. 1969:103-115.
19. Gutmann I, Wilhelm A. L-(+)-Lactate. determination with lactate dehydrogenase and NAD. In: Bergmeyer H, ed. Methods of Enzimatic Analysis. Vol. 3. Deerfiel Beach, FL. Academic Press.; 1974:1464-1468.
20. Edmond E, Robbins R, Bergstrom J, Cole R, De Vellis J. Capacity for substrates utilization in oxidative metabolism by neurons, astrocytes and oligodendrocytes from developing brain in primary culture. J.Neurosci.Res. 1987;18(4):551-
561.
21. Sykes J, López-Cardoso M , Van Den Bergh S. Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain. Neurochem.Int. 1986;8:67-75.
22. Folch J, Lees M, Slone GH. A simple method for the isolation and purification of total lipides from animal tissues.
J.Biol.Chem. 1957;226:497-509.
23. Kuroda Y, Toshima K, Watanabe T, Kobashi H, Ito M, Takeda E, Miyao M. Effects of dichloroacetate on pyruvate
metabolism in rat brain in vivo. Pediatric Res. 1984;18:936- 938.
24. Miller AL, Hatch JP, Prihoda TJ. Dichloroacetate increase glucose use and decreases lactate in developing rat brain. Metabolic Brain Disease. 1990;5(4):195-204.
25. Rex-Sheu K-F, Lai JC, Blass JP. Properties and regional distribution of pyruvate dehydrogenase kinase in rat brain.
J.Neurochem. 1984;42:230-236.
26. Katayama Y, Welsh FA. Effects of dichloroacetate on regional energy metabolites and pyruvate dehydrogenase activity during ischemia and reperfusion in gerbil brain. J.Neurochem. 1989;52(6):1817-1822.
27. McAllister A, Allison SP, Randle PJ. Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-
hidroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo. Biochem.J. 1973;134:1067-1081.
28. Abemayor E, Kovachich, G, Haugaard, N. Effects of dichloroacetate on brain pyruvate dehydrogenase.
J.Neurochem. 1984;42(1):38-42.
29. Tomsig JL, Gruenstein E, Dimlich, RV. Inhibition of lactateinduced swelling by dichoroacetate in human astrocytomacells. Brain Res. 1991;568:92-100.
30. Meijer AJ, Van Dam, K. The metabolic significance of anion transport in mitochondria. Biochim.Biophys.Acta.
1974;346:213-244.
31. Flint M, Swartz KJ, Hyman BT, Storey E, Finn SF, Koroshetz W. Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism. J.Neurochem. 1991;57(3):1068-
1073.
32. Palaiologos G, Hertz L, Schousboe A. Role of aspartate aminotransferase and mitochondrial dicarboxylate transport
for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem.Res.
1989;14(4):359-366.
33. Yu A, Schousboe, A, Hertz, L. Metabolic fate of 14C-labeled glutamate in astrocytes in primary culture. J.Neurochem.
1982;39:954-960.
34. Kauppinen RA, Sihra T, Nicholls D. Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve
terminals and prevents the mitochondria from utilizing glycolytic substrates. Biochim.Biophys. Acta. 1987;930:173-
178.
35. McKenna M, Tyson J, Couto R, Stevenson J, Caprio F. The metabolism of malate by cultured rat brain astrocytes.
Neurochem.Res. 1990;15(12):1211-1220.
36. Farinelli S, Nicklas W. Glutamate metabolism in rat cortical astrocyte cultures. J.Neurochem. 1992;58(5):1905-1915.
37. Passarella S, Barile M, Atlante A, Quagliariello E. Oxaloacetate uptake into rat brain mitochondria and
reconstruction of the malate/oxaloacetate shuttle. Biochem.Biophys.Res.Comm. 1984;119:1039-1046.
38. Neale J, Bzdega T, Wroblewska B. N-acetylaspartylglutamate. The most abundant peptide neurotransmitter in the
mammalian central nervous system. J.Neurochem. 2000;75(2):443-452.
39. Chakraborty G, Mekala P, Yahya D, Wu G, Ledeen R. Intraneuronal N-acetylaspartate supplies acetyl groups for
myelin lipids synthesis: evidence for myelin-associated aspartoacylase. J.Neurochem. 2001;78:736-745.
40. Patel T, Clark, J. Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/ cytosolic carbon transport. Biochem.J. 1979;184:539-546.
41. D.Adamo A, Smith J, Phillips I. The piruvate dehydrogenase complex: cloning of the rat somatic E1a subunit and its
coordinate expression with the mRNAs for E1b, E2, and E3 catalytic subunits in developing rat brain. J.Neurochem.
1973;20:1275-1278.
42. D.Adamo A, Yatsu, F. Acetate metabolism in the nervous system. N-acetyl-L-aspartic acid and the biosynthesis of brain lipids. J.Neurochem. 1966;13:961-965.
43. Fitzpatrick S, Cooper A, Duffy T. Use of ß-methylene-D,Laspartate to assess the role of aspartate aminotransferase in cerebral oxidative metabolism. J.Neurochem. 1983;41:1370- 1383.
44. Beal MF, Swartz KJ, Hyman BT, Storey E, Finn SF, Koroshetz W. Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism. J.Neurochem. 1991;57:1068- 1073.
45. Palmieri F, Stipani I, Quagliariello E. Kinetic study of the tricarboxylate carrier in rat liver mitochondria.
Eur.J.Biochem. 1972;26:587-594.
46. Watson JA, Lowenstein, JM. Citrate and the Conversion of Carbohydrate into Fat. Fatty acid synthesis by a combination of cytoplasm and mitochondria. J.Biol.Chem. 1970;245(22):5993-6002.
47. Calvin J, Tubbs P. Mitochondrial transport processes and oxidation of NADH by Hypotonically-treated boar spermatozoa. Europ J Biochem. 1978;89:315-320.
48. Coleman J, Palmer JM. The oxidation of malate by isolated plant mitochondria. Eur. J. Biochem. 1972;26:499-509.
49. Robinson BH, Willians GR, Halperin ML, Leznoff CC. The sensitivity of the exchange reactions of tricarboxylate, 2-
oxoglutarate and dicarboxylate transporting system of rat liver mitochondria to inhibition by 2-pentylmalonate, piodobenzylmalonate, and benzene 1, 2, 3-tricarboxylate. Eur.J.Biochem. 1971;20(1):65-71.
50. Patel MS. Citrate transport oand oxidation by isolated rat brain mitochondria. Brain Res. 1975;98:607-611.
51. Stucki J. Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria. Eur J
Biochem. 1977;78(1):183-187.
52. Stipani I, Palmieri, F. Purification of the active mitochondrial tricarboxylate carrier by hydroxylapatite chromatography. FEBS. 1983;161:269-274.
53. Patel MS. CO2-Fixing enzymes. In: Boulton AA, Baker GB, Butterworth RF, eds. Neuromethods Carbohydrates and Energy Metabolism. Vol. 11. Clifton, N.J.: Humana Press.; 1989:309-341.
54. Kaufman E, Driscoll B. Evidence for cooperativity between neurons and astroglia in the regulation of CO2 fixation in vitro. Dev.Neurosci. 1993;15:299-305.
-------------------------------------------------------------------------------
DOI: http://dx.doi.org/10.22490/24629448.19
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