Abstract
Summary. The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.
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Arbones, L., Picatoste, E., & Garcia, A. (1990) Histamine stimulates glycogen breakdown and increases 45Ca2+ permeability in rat astrocytes in primary cultures. Molecular Pharmacology 37, 921–27.
Bignami, A. & Dahl, D. (1973) Differentiation of astrocytes in the cerebellar cortex and the pyramidal tracts of the newborn rat. An immunofluorescene study with antibodies to a protein specific to astrocytes. Brain Research 49, 393–402.
Bignami, A. & Dahl, D. (1974) Astrocyte-specific protein and neuroglial differentiation. An immunofluorescene study with antibodies to the glial fibrillary acidic protein. Journal of Comparative Neurology 153, 27–38.
Bignami, A. & Dahl, D. (1977) Specificity of the glial fibrillary acidic protein for astroglia. Journal of Histochemistry and Cytochemistry 25, 466–49.
Bologa-Sandru, L., Siegrist, H. P., Z′Graggen, A., Hofmann, K., Wiesmann, U., Dahl, D. & Herschkowitz, N. (1981) Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures. Brain Research 210, 217–29.
Borke, R. C. & Nau, M. E. (1984) Glycogen, its transient occurrence in neurons of the rat CNS during normal postnatal development. Developmental Brain Research 16, 277–84.
BrÑckner, G. & Biesold, D. (1981) Histochemistry of glycogen deposition in perinatal rat brain: importance of radial glial cells. Journal of Neurocytology 10, 749–57.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–54.
Browning, M., Bennett, W. & Lynch, G. (1979a) Phosphorylase kinase phophorylates a brain protein which is influenced by repetitive synaptic activation. Nature 278, 273–5.
Browning, M., Dunwiddie, T., Bennett, W., Gispen, W. & Lynch, G. (1979b) Synaptic phosphoproteins: specific changes after repetitive stimulation of the hippocampal slice. Science 203, 60–2.
Cambray-Deakin, M., Pearce, B., Morrow, C. & Murphy, S. (1988a) Effects of neurotransmitters on astrocyte glycogen stores in vitro. Journal of Neurochemistry 51, 1852–7.
Cambray-Deakin, M., Pearce, B., Morrow, C. & Murphy, S. (1988b) Effects of extracellular potassium on glycogen stores of astrocytes in vitro. Journal of Neurochemistry 51, 1846–51.
Catald, M. B., Fox, D. T. & Hanks, S. K. (1992) Molecular cloning and enzymatic analysis of the rat homolog of ″PhK-γT, ″ an isoform of phosphorylase kinase catalytic subunit. Journal of Biological Chemistry 267, 1455–63.
Cataldo, A. M. & Broadwell, R. D. (1986a) Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions: I. Neurons and glia. Journal of Electron Microscopy Techniques 3, 413–37.
Cataldo, A. M. & Broadwell, R. D. (1986b) Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions. II. Choroid plexus and ependymal epithelia, endothelia and pericytes. Journal of Neurocytology 15, 511–24.
Chamberlain, J. S., Vantuinen, P., Reeves, A. A., Philip, B. A. & Caskey, C. T. (1987) Isolation of cDNA clones for the catalytic γ subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice. Proceedings of the National Academy of Sciences USA 84, 2886–90.
Coopersmith, R. & Leon, M. (1995) Olfactory bulb glycogen metabolism: noradrenergic modulation in the young rat. Brain Research 674, 230–7.
Dahl, D., Rueger, D. C. & Bignami, A. (1981) Vimentin, the 57000 molecular weight protein in fibroblast filaments, is the major cytoskeletal component in immature glia. European Journal of Cell Biology 24, 191–6.
Dennis, M. J. & Gerschenfeld, H. M. (1969) Some physiological properties of identified mammalian neuroglial cells. Journal of Physiology 203, 211–22.
Dringen, R. & Hamprecht, B. (1992) Glucose, insulin, and insulin-like growth factor I regulate the glycogen content of astroglia-rich primary cultures. Journal of Neurochemistry 58, 511–7.
Drummond, G. I. & Bellward, G. (1970) Studies on phosphorylase b kinase from neural tissues. Journal of Neurochemistry 17, 475–82.
Eugenin, E. A., Saez, C. G., Garces, G. & Saez, J. C. (1997) Regulation of glycogen content in rat pineal gland by norepinephrine. Brain Research 760, 34–41.
Ferendelli, J. & McDougal, D. (1971) The effects of audiogenic seizures on regional energy reserves, glycolysis and citric acid cycle flux. Journal of Neurochemistry 17, 1207–20.
Fischer, E. H. & Krebs, E. G. (1962) Muscle phosphorylase b. In: Methods in Enzymology, Vol. 5, (edited by Colowick, S. P. & Kaplan, N. O.) pp. 369–73. New York: Academy Press.
Glowinski, J. & Iversen, L. I. (1966) Regional studies of catecholamines in rat brain. I. The deposition of [3H]-norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. Journal of Neurochemistry 13, 655–69.
Gross, S. R. & Mayer, S. E. (1974) Characterization of the phosphorylase b to a converting activity in skeletal muscle extracts of mice with the phosphorylase b kinase deficiency mutation. Journal of Biological Chemistry 249, 6710–8.
Hallermayer, K. & Hamprecht, B. (1984) Cellular heterogeneity in primary cultures of brain cells revealed by immunocytochemical localization of glutamine synthatase. Brain Research 295, 1–11.
Hallermayer, K., Harmering, C. & Hamprecht, B. (1981) Cellular localization and regulation of glutamine synthatase in primary cultures of brain cells from newborn mice. Journal of Neurochemistry 37, 43–52.
Hamprecht, B. & LÖffler, F. (1985) Primary glial cultures as a model for studying hormone action. In: Methods in Enzymology, Vol. 109, (edited by Birnbaumer, L. & O′Malley, B. W.) pp. 341–5. New York: Academy Press.
Hamprecht, B. (1986) Astroglia cells in culture: receptors and cyclic nucleotides. In: Astrocytes: Biochemistry, Physiology and Pharmacology of Astrocytes, Vol. 2, (edited by Fedorof F,S. & Vernadakis, A.) pp. 77–106. Orlando, FL: Academic Press.
Hanks, S. K. (1989) Messenger ribonucleic acid encoding an apparent isoform of phosphorylase kinase catalytic subunit is abundant in the adult testis. Molecular Endocrinology 3, 110–6.
Hansson, E. (1990) Regional heterogeneity among astrocytes in the central nervous system. Neurochemistry International 16, 237–45.
Harboe, N. & Ingild, A. (1973) Immunization isolation of immunoglobulins, estimation of antibody titre. Scandinavian Journal of Immunology 2, (Suppl. 1). 161–4.
Harik, S. I., Lust, W. D., Jones, S. C., Lauro, K. L., Pundik, S. & Lamanna, J. C. (1995) Brain glucose metabolism in hypobaric hypoxia. Journal of Applied Physiology 79, 136–40.
Harmann, B., Zander, N. F. & Kilimann, M. W. (1991) Isoform diversity of phosphorylase kinase α and β subunits generated by alternative RNA splicing. Journal of Biological Chemistry 266, 15631–7.
Helmreich, E. & Cori, C. F. (1964) The role of the adenylic acid in the activation of phosphorylase. Proceedings of the National Academy of Sciences USA 51, 131–8.
Henn, F. A., Haljamae, H. & Hamberger, A. (1972) Glia cell function: active control of extracellular K+ concentration. Brain Research 43, 437–43.
Hertz, L., Yager, J. Y. & Juurlink, B. H. (1995) Astrocyte survival in the absence of exogenous substrates: comparison of immature and mature cells. International Journal of Developmental Neuroscience 13, 523–7.
Hof, P. R., Celio, M. R. & Magistretti, P. J. (1989) Age-dependent supersensitivity to the glycogenolytic effect of K+ in the cerebral cortex of the spontaneously epileptic quaking mouse mutant. Developmental Brain Research 46, 107–13.
Hof, P. R., Pascale, E. & Magistretti, P. J. (1988) K+ at concentrations reached in the extracellular space during neuronal activity promotes a Ca2+-dependent glycogen hydrolysis in mouse cerebral cortex. Journal of Neuroscience 8, 1922–28.
Horl, W. H., Jennissen, H. P. & Heilmeyer, L. M. G. Jr. (1978) Evidence for the participation of a Ca2+-dependent protein kinase and a protein phosphatase in the regulation of the Ca2+ transport ATPase of the sarcoplasmic reticulum. 1. Effect of inhibitors of the Ca2+-dependent protein kinase and protein phosphatase. Biochemistry 17, 759–72.
Ignacio, P. C., Baldwin, B. A., Vijayan, V. K., Tait, R. C. & Gorin, F. A. (1990) Brain isoenzyme of glycogen phosphorylase: immunohistological localization within the central nervous system. Brain Research 529, 42–9.
Jones, C. T. (1991) Control of glucose metabolism in the perinatal period. Journal of Developmental Physiology 15, 81–9.
Katz, M. S., Dax, E. M. & Gregerman, R. I. (1993) Beta adrenergic regulation of rat liver glycogenolysis during aging. Experimental Gerontology 28, 329–40.
Knull, H. R. & Khandelwal, R. L. (1982) Glycogen metabolizing enzymes in brain. Neurochemical Research 7, 1307–17.
Kohle, S. J. & Vannucci, R. C. (1977) Glycogen metabolism in fetal and postnatal rat brain: influence of birth. Journal of Neurochemistry 28, 441–3.
Kuffler, S. W. & Nicholls, J. G. (1996) The physiology of neuroglial cells. Ergebnisse der Physiologie, Biologischen Chemie und Experimentellen Pharmakologie 57, 1–90.
Lazarov, N. (1996) Fine structure and synaptic organization of the mesencephalic trigeminal nucleus of the cat: a quantitative electron microscopic study. European Journal of Morphology 34, 95–106.
LÖffler, F., Lohmann, S. M., Walckoff, B., Walter, U. & Hamprecht, B. (1986) Immunocytochemical characterization of neuron-rich primary cultures of embryonic rat brain cells by established neuronal and glial markers and by monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein synapsin I. Brain Research 363, 205–21.
Lowry, O. H., Passonneau, J. V., Hasselberger, F. H. & Schulz, D. W. (1964) Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. Journal of Biological Chemistry 239, 18–30.
Magistretti, P. J., Hof, P. R. & Martin, J. L. (1986) Adenosine stimulates glycogenolysis in mouse cerebral cortex: a possible coupling mechanism between neuronal activity and energy metabolism. Journal of Neuroscience 6, 2558–62.
Magistretti, P. J., Morrison, J. H., Shoemaker, W. J., Sapin, V. & Bloom, F. B. (1981) Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism. Proceedings of the National Academy of Sciences USA 78, 6535–9.
Maniscalco, W. M., Wilson, C. M., Gross, I., Gobran, L., Rooney, S. A. & Warsaw, J. B. (1978) Development of glycogen and phospholipid metabolism in fetal and newborn rat lung. Biochimica et Biophysica Acta 530, 333–46.
McCandless, D. W., Feussner, G. K., Lust, W. D. & Passoneau, J. V. (1979) Metabolite levels in brain following experimental seizures: the effects of maximal electroshock and phenytoin in cerebellar layers. Journal of Neurochemistry 32, 743–53.
Nelson, S. R., Schulz, D. W., Passonneau, J. V. & Lowry, O. H. (1968) Control of glycogen levels in brain. Journal of Neurochemistry 15, 1271–9.
O′Dowd, B. S., Barrington, J., Ng, K. T., Hertz, E. & Hertz, L. (1995) Glycogenolytic response of primary chick and mouse cultures of astrocytes to noradrenaline across development. Developmental Brain Research 88, 220–3.
O′Dowd, B. S., Gibbs, M. E., Ng, K. T., Hertz, E. & Hertz, L. (1994) Astrocytic glycogenolysis energizes memory processes in neonate chicks. Developmental Brain Research 78, 137–41.
Orkand, R. K. (1969) Neuroglial–neuronal interactions. In: Basic Mechanisms of the Epilepsies (edited by Jasper, H. H., Ward, A. A. & Pope, A.), pp. 737–46. Boston: Little, Brown and Co.
Orkand, R. K., Nicholls, J. G., & Kuffler, S. W. (1966) Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia. Journal of Neurophysiology 29, 788–806.
Paudel, H. K. (1997) The regulatory Ser262 of microtubule-associated protein Tau is phosphorylated by phosphorylase kinase. Journal of Biological Chemistry 272, 1777–85.
Paudel, H. K., Zwiers, H. & Wang, J. H. (1993) Phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of neuronal tissue-specific proteins B-50 (GAP-43) and neurogranin. Journal of Biological Chemistry 268, 6207–13.
Paxinos, G. & Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd ed. London: Academic Press.
Pellerin, L., Stolz, M., Sorg, O., Martin, J. L., Deschepper, C. F. & Magistretti, P. J. (1997) Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line. Glia 21, 74–83.
Pfeiffer, B., Buse, E., Meyermann, R., Rocha, M. J. A. & Hamprecht, B. (1993) Glycogen phosphorylase activity and immunoreactivity during pre-and postnatal development of rat brain. Histochemistry 100, 265–70.
Pfeiffer, B., Elmer, K., Roggendorf, W., Reinhart, P. H. & Hamprecht, B. (1990) Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices. Histochemistry 94, 73–80.
Pfeiffer, B., Grosche, J., Reichenbach, A. & Hamprecht, B. (1994) Immunocytochemical demonstration of glycogen phosphorylase in Müller (glial) cells of the mammalian retina. Glia 12, 62–7.
Pfeiffer, B., Meyermann, R. & Hamprecht, B. (1992) Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain slices. Histochemistry 97, 405–12.
Phelps, C. H. (1972) Barbiturate-induced glycogen accumulation in brain. An electron microscopy study. Brain Research 39, 225–34.
Poitry-Yamate, C. & Tsacopoulos, M. (1991) Glial (Müller) cells take up and phosphorylate {3H}2-deoxy-D-glucose in a mammalian retina. Neuroscience Letters 122, 241–4.
Priestley, J. V. (1987) Immunocytochemical techniques for the localization of neurochemically characterized nerve pathways. In: Neurochemistry: A Practical Approach (edited by Turner, A. J. & Bachelard, H. S.), pp. 64–111. Oxford, Washington, DC: IRL Press.
Proux, D., Alexandre, Y., Delain, D. & Dreyfus, J. C. (1974) The isozymes of phosphorylase kinase in various mammalian tissues. Biochimie 56, 1559–64.
Psarra, A.-M. G. & Sotiroudis, T. G. (1996) Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes. International Journal of Biochemistry and Cell Biology 28, 29–42.
Quach, T. T., Duchemin, A. M., Rose, C. & Schwartz, J.-C. (1980) {3H} Glycogen hydrolysis elicited by histamine in mouse brain slices: selective involvement of H1 receptors. Molecular Pharmacology 17, 301–8.
Quach, T. T., Duchemin, A. M., Rose, C. & Schwartz, J.-C. (1982) Glycogenolysis induced by serotonin in brain: identification of a new class of receptor. Nature 298, 373–5.
Ransom, B. R. & Fern, R. (1997) Does astrocytic glycogen benefit axon function and survival in CNS white matter during glucose deprivation? Glia 21, 134–41.
Reinhart, P. H., Pfeiffer, B., Spengler, S. & Hamprecht, B. (1990) Purification of glycogen phosphorylase from bovine brain and immunocytochemical examination of rat glial primary cultures using monoclonal antibodies raised against this enzyme. Journal of Neurochemistry 54, 1474–83.
Richter, K., Hamprecht, B. & Scheich, H. (1996) Ultrastractural localization of glycogen phosphorylase predominantly in astrocytes of the gerbil brain. Glia 17, 263–73.
Rosenberg, P. A. & Dichter, M. A. (1987) A small subset of cortical astrocytes in culture accumulates glycogen. International Journal of Developmental Neuroscience 5, 227–35.
Sagar, S. M., Sharp, F. R. & Swanson, R. A. (1987) The regional distribution of glycogen in rat brain fixed by microwave irradiation. Brain Research 417, 172–4.
Saheki, S., Takeda, A. & Shimazu, T. (1985) Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity. Analytical Biochemistry 148, 277–81.
Sorg, O., Pellerin, L., Stolz, M., Beggah, S. & Magistretti, P. J. (1995) Adenosine triphosphate and arachidonic acid stimulate glycogenolysis in primary cultures of mouse cerebral cortical astrocytes. Neuroscience Letters 188, 109–12.
Subbarao, K. V. & Hertz, L. (1990) Effect of adrenergic agonists on glycogenolysis in primary cultures of astrocytes. Brain Research 536, 220–6.
Swanson, R. A. (1992) Physiologic coupling of glial glycogen metabolism to neuronal activity in brain. Canadian Journal Physiology and Pharmacology 70, S138–44.
Swanson, R. A. & Choi, D. W. (1993) Glial glycogen stores affect neuronal survival during glucose deprivation in vitro. Journal of Cerebral Blood Flow and Metabolism 13, 162–9.
Swanson, R. A., Morton, M. M., Sagar, S. M. & Sharp, F. R. (1992) Sensory stimulation induces local cerebral glycogenolysis: demonstration by autoradiography. Neuroscience 51, 451–61.
Swanson, R. A., Shiraishi, K., Morton, M. T. & Sharp, F. R. (1990) Methionine sulfoximine reduces infarct size in rats after middle cerebral artery occlusion. Stroke 21, 322–7.
Taira, T., Kii, R., Sakai, K., Tabuchi, H., Takimoto, S., Nakamura, S., Takahashi, J., Hashimoto, E., Yamamura, H. & Nishizuka, Y. (1982) Comparison of glycogen phosphorylase kinase of various rat tissues. Journal of Biochemistry 91, 883–8.
Trachtenberg, M. C., & Pollen, D. A. (1970) Neuroglia: biophysical properties and physiological functions. Science 167, 1248–52.
Tsacopoulos, M. & Magistretti, P. J. (1996) Metabolic coupling between glia and neurons. Journal of Neuroscience 16, 877–85.
Tsacopoulos, M., EvÊquoz-Nercier, V., Perrottet, P. & Buchner, E. (1988) Honeybee retinal glial cells transform glucose and supply the neurons with metabolic substrate. Proceedings of the National Academy of Sciences USA 85, 8727–31.
Ververken, D., van Veldhoven, P., Proost, C., Carton, H. & Dewulf, H. (1982) On the role of calcium ions in the regulation of glycogenolysis in mouse brain cortical slices. Journal of Neurochemistry 38, 1286–95.
Wagner, S. R. 4th & Lanier, M. L. (1994) Metabolism of glucose, glycogen, and high-energy phosphates during complete cerebral ischemia. A comparison of normoglycemic, chronically hyperglycemic diabetic, and acutely hyperglycemic nondiabetic rats. Anesthesiology 81, 1516–26.
Yager, J. Y., Kala, G., Hertz, L. & Juurlink, B. H. (1994) Correlation between content of high-energy phosphates and hypoxic ischemia damage in immature and mature astrocytes. Developmental Brain Research 82, 62–8.
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Psarra, AM.G., Pfeiffer, B., Giannakopoulou, M. et al. Immunocytochemical localization of glycogen phosphorylase kinase in rat brain sections and in glial and neuronal primary cultures. J Neurocytol 27, 779–790 (1998). https://doi.org/10.1023/A:1006970429961
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DOI: https://doi.org/10.1023/A:1006970429961