...rat nervous tissues by using isozyme‐specific antibodies...

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Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme‐specific antibodies - Pfeiffer‐Guglielmi - 2003 - Journal of Neurochemistry - Wiley Online Library First published: 28 February 2003 https://doi.org/10.1046/j.1471-4159.2003.01644.xCitations: 115 Address correspondence and reprint requests to Dr Bernd Hamprecht, Physiologisch-Chemisches Institut, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany. E-mail: bernd.hamprecht@uni-tuebingen.de Dedicated to Dr Monique Sensenbrenner, Strasbourg, on the occasion of her 70th birthday. Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Isozyme-specific antibodies were raised against peptides from the low-homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double-labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co-localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system. Abbreviations used BSA bovine serum albumin CFA complete Freund\'s adjuvant DRG dorsal root ganglion GP glycogen phosphorylase IFA incomplete Freund\'s adjuvant KLH keyhole limpet haemocyanin Me5 mesencephalic trigeminal nucleus 5 PBS phosphate-buffered saline PMSF phenylmethylsulphonylfluoride TRITC tetramethylrhodamine isothiocyanate Glycogen is the main energy reserve in nervous tissues where it is found predominantly in astrocytes. It has been proposed to be an emergency fuel store during physiological or pathological stress such as hypoglycaemia and cerebral ischaemia (Swanson etal. 1989; Swanson and Choi 1993), but there is some evidence for a role of glycogen in normal metabolism also (Choi etal. 1999; Shulman etal. 2001). The response of glycogen metabolism to functional conditions is exemplified by the influence of various factors, including noradrenaline, vasoactive intestinal peptide (Hamprecht etal. 1993), cyclic AMP and adenosine (Sorg and Magistretti 1991, 1992; Cardineaux and Magistretti 1996), insulin and insulin-like growth factor 1 (Dringen and Hamprecht 1992; Hamai etal. 1999), K+ and Ca2+ (Cambray-Deakin etal. 1988; Hof etal. 1988), glutamate (Swanson etal. 1990; Hamai etal. 1999), arachidonic acid (Sorg etal. 1995), peroxides (Rahman etal. 2000) and cell volume changes (Dombro etal. 2000). Glycogen phosphorylase (GP) is the key enzyme in glycogen degradation. The homodimeric enzyme exists as three isoforms named according to the tissues they predominate in, LL (liver), MM (skeletal muscle) and BB (brain). The GP isoforms differ in their responses to activation by phosphorylation and allosteric control (Fletterick etal. 1986; Crerar etal. 1995), and can thus meet the energy requirements of different tissues or cells. With the exception of liver and skeletal muscle, adult rat tissues have been shown to contain messenger RNAs for more than one isozyme and express a typical isozyme pattern (Richter etal. 1983; David and Crerar 1986) which can be demonstrated by polyacrylamide gel electrophoresis under non-denaturing conditions followed by activity staining for GP. In rat nervous tissues this method has revealed the presence of isozymes BB and MM (Richter etal. 1983; David and Crerar 1986; Pfeiffer-Guglielmi etal. 2000). Owing to poor resolution and low sensitivity this electrophoretic method does not lend itself to the clarification of the exact regional and cellular distribution pattern of GP isozymes in nervous tissues. Such clarification might shed light on the local functions of glycogen. To amend this situation, the generation of isozyme-specific antibodies was chosen as an appropriate way of investigating isozyme patterns in tissue homogenates as well as in tissue slices and cell cultures at a cellular level. Studies with such antibodies undertaken so far have been restricted to only one isoform or non-nervous tissues (Kato etal. 1989; Ignacio etal. 1990; Nihira etal. 1995; Rabitzsch etal. 1995; Krause etal. 1996; Mair 1998; Uno etal. 1998). Therefore, we generated in rabbits and guinea pigs isozyme-specific antibodies against peptides derived from the isozyme-specific amino acid sequences of the rat BB and MM isozymes. These antibodies allowed the mapping of specific isozymes by means of immunocytochemical double-labelling experiments on frozen sections of rat nervous tissues. For coupling of peptides to keyhole limpet haemocyanin (KLH) or bovine serum albumin (BSA), the Imject® Maleimide Activated Immunogen Conjugate Kit (Pierce, Rockford, IL, USA) was used. Microtitre plates (Maxisorp®) were from Nunc (Wiesbaden, Germany). Phenylmethylsulfonylfluoride (PMSF), leupeptin, pepstatin and aprotinin were from Sigma (Deisenhofen, Germany). Goat anti-rabbit–alkaline phosphatase conjugate was from Zymed Laboratories (San Francisco, CA, USA; purchased from Zytomed, Berlin, Germany). All other secondary antibody conjugates were from Jackson ImmunoResearch (West Grove, PA, USA; purchased from Dianova, Hamburg, Germany) or Molecular Probes Europe (Leiden, The Netherlands). Vectashield® mounting medium was obtained from Vector Laboratories (Burlingame, CA, USA; purchased from Alexis, Gruenberg, Germany). Wistar rats were used for all experiments and purchased from Charles River Laboratories (Sulzfeld, Germany). Owing to the high degree of homology of GP isozymes (87%) the choice of the peptides for raising isozyme-specific antisera was restricted. Comparison of the amino acid sequences of the three rat isozymes (accession no. s 37 300, s 34 624, s 22 338; Protein Information Resource, PIR) revealed that only the carboxy-terminal regions were suited. Peptides for immunization should have a minimal length of 12–15 amino acids. Therefore, the brain-specific sequence 826–842 GVEPSDLQIPPPNLPKD and the muscle-specific sequence 826–841 GLEPSRQRLPAPDEKI were chosen for synthesis of the peptides. As the high sequence homology between the latter and the rabbit muscle isozyme suggested the possibility of poor antigenicity in rabbits, a third peptide with the muscle-specific sequence 774–786 CQDKVSELYKNPR was synthesized. An additional cysteinyl residue was coupled to the amino terminal amino acids of the brain-specific sequence 826–842 GVEPSDLQIPPPNLPKD and the muscle-specific sequence 826–841 GLEPSRQRLPAPDEKI. These cysteinyl peptides were designated BB peptide and MM1 peptide, respectively. The muscle-specific sequence 774–786 CQDKVSELYKNPR was designated MM2 peptide. Comparison of the sequences with several protein data bases using the BLASTP program (Altschul etal. 1997) revealed no sequence identities with relevant proteins. Synthetic peptides were prepared by multiple solid-phase peptide synthesis on a robotic system (Syro MultiSynTech, Bochum, Germany) using fluorenylmethoxycarbonyl/t-butyl (Fmoc/tBu) chemistry and 2-chlorotrityl resin (Senn Chemicals, Dielsdorf, Switzerland) (Jung and Beck-Sickinger 1992; Fleckenstein etal. 1999). For coupling, 10 equivalents of di-isopropylcarbodiimide, 5 equivalents of 1-hydroxybenzotriazole and 5 equivalents of protected amino acids were used. Peptides were cleaved off the resin and side chains were deprotected with a solution of 2.5% ethanedithiol, 2.5% water and 2.5% tri-isopropylsilane in trifluoroacetic acid for 3 h. Peptides were precipitated by adding ice-cold diethylether, washed three times with diethylether and lyophilized from tert-butylalcohol/water (4 : 1). The identity of the peptides was confirmed by electrospray mass spectrometry and their purity was analysed by reversed phase HPLC. Peptides BB, MM1 and MM2 were coupled to KLH according to the manufacturer\'s protocol. The degree of conjugation was determined using Ellman\'s reagent (Ellman 1959). Peptides coupled accordingly to BSA were used in ELISAs for determining antibody titres. Immunizations were carried out commercially by Charles River Laboratories (Kisslegg, Germany). One rabbit and one guinea pig were immunized with BB, MM1 or MM2 peptide–KLH conjugates. Rabbits were immunized subcutaneously with KLH conjugate corresponding to 400 µg peptide in a volume of 1 mL containing 0.5 mL complete Freund\'s adjuvant (CFA). Three booster injections were applied with the same amount of antigen using incomplete Freund\'s adjuvant (IFA). Guinea pigs were immunized subcutaneously with KLH conjugate corresponding to 100 µg peptide in a volume of 0.5 mL containing 0.25 mL CFA. Two booster injections were applied with the same amount of antigen in IFA. Immune sera were collected on day 70 after the first injection and stored in aliquots over liquid nitrogen. Microtitre plates were coated overnight at 4°C with 100 ng per 100 µL per well of the peptide–BSA conjugate dissolved in 50 mm sodium carbonate buffer, pH 9.6. The wells were washed twice with phosphate-buffered saline (PBS) containing 0.05% (w/v) Tween 20, and blocked with the same buffer plus 1% (w/v) skimmed milk powder at room temperature (23°C) for 30 min. The immune sera were diluted serially in the washing buffer containing 0.6% (w/v) skimmed milk powder. These solutions were applied in duplicate to the wells (100 µL/well), and the reaction proceeded at room temperature for 3 h. The wells were washed as described above, and peroxidase-conjugated goat anti-rabbit IgG or goat anti-guinea pig IgG in PBS/0.05% (w/v) Tween 20 at a dilution of 1 : 50 000 was added (100 µL/well). The plate was incubated at room temperature for 1 h. After washing as described above, the substrate solution [4 mmo-phenylenediamine/0.03% (v/v) H2O2 in 50 mm Na2HPO4 and 25 mm citric acid, pH 5.0] was added (100 µL/well) and the plate stored in the dark for 30 min. The peroxidase reaction was stopped by addition of 50 µL 0.25 m H2SO4 per well, and absorbance was measured in an ELISA reader at 492 nm. Pre-immune sera were used as negative controls. Cross-reactivity towards the other peptides was monitored by using microtitre plates coated with the BSA conjugates of the other peptides. Tissues were homogenized as described previously (Pfeiffer-Guglielmi etal. 2000) except that the homogenization buffer contained protease inhibitors (100 µm PMSF, 1 µm leupeptin, 1 µm pepstatin and 0.3 µm aprotinin). Pure GP BB isozyme from rat brain was prepared as reported (Mayer etal. 1992). The protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 5% stacking gel and a 10% separating gel. Proteins were transferred to a nitrocellulose membrane (100 V constant voltage for 1 h in precooled buffer). The membrane was blocked at room temperature for 30 min by incubation in PBS containing 0.05% (w/v) Tween 20 and 5% (w/v) skimmed milk powder. Subsequently, the membrane was incubated with antiserum (diluted 1 : 1000 in PBS/0.05% Tween 20) at room temperature for 2 h. After a washing step in PBS/0.5% Tween 20, the membrane was incubated with goat anti-rabbit IgG–alkaline phosphatase conjugate (diluted 1 : 2000) or goat anti-guinea pig IgG–alkaline phosphatase conjugate (diluted 1 : 10 000) at room temperature for 1 h. The membrane was washed and the blot developed for 5–10 min in 0.2 m Tris/HCl buffer, pH 9.5, with 10 mm MgCl2, containing 33 mg nitroblue tetrazolium chloride and 16 mg 5-bromo-4-chloro-3-indolylphosphate in 100 mL. The reaction was stopped with PBS/20 mm EDTA. Pre-immune sera and antisera pre-absorbed with the antigens were used as negative controls. For pre-absorption of antisera, peptides were absorbed on to nitrocellulose discs and the discs were incubated with the antisera as described by Ignacio etal. (1990). Tissues were fixed with 4% paraformaldehyde/PBS, either by transcardial perfusion or by immersion at 4°C for 24 h, and subsequently cryoprotected in 30% (w/v) sucrose/PBS at 4°C for 48 h. Tissues were frozen and stored in liquid nitrogen. Frozen sections of 10 µm were cut with a cryostat (Microm, Walldorf, Germany), wrapped in aluminium foil and stored at − 20°C for up to 1 week. After thawing and equilibrating with PBS, sections were used for immunofluorescence studies. For immunocytochemical studies antisera were diluted 1 : 500–1 : 2000 in PBS/0.1% (w/v) Triton X-100/10% (v/v) goat normal serum. For double-labelling experiments, primary antibodies were mixed and incubated together, as were secondary antibodies at the following incubation step. Tissues or cells were incubated with antisera at room temperature for 2 h. After washing in PBS/0.1% Triton X-100 for 10 min, secondary antibodies [goat anti-rabbit IgG/tetramethylrhodamine isothiocyanate (TRITC) and goat anti-guinea pig IgG/FITC, both diluted 1 : 100; goat anti-rabbit IgG/Alexa Fluor 488 and goat anti-guinea pig IgG/Alexa Fluor 568, both diluted 1 : 1000] were applied at room temperature for 1 h. After washing for 10 min, the sections were mounted. Negative controls were performed by substituting immune sera with pre-immune sera or antisera pre-absorbed with antigens. Secondary antibodies were tested for non-specific staining by omitting the primary antibodies. Cross-reactivity of secondary antibodies in double-labelling experiments was tested by combining rabbit antisera with anti-guinea pig secondary antibodies, and vice versa. The isozyme specificity of antisera was tested by using sections of rat skeletal muscle. For immunofluorescence double-labelling experiments, two combinations of antisera (rabbit anti-BB isozyme/guinea pig anti-MM isozyme or guinea pig anti-BB isozyme/rabbit anti-MM isozyme) were applied. The animals immunized (three rabbits and three guinea pigs) developed antibody titres of 1 : 14 000–1 : 79 000 as measured by ELISA. Pre-immune sera did not react. Cross-reactivities were negligible for the antisera raised against BB peptide and MM1 peptide but reached nearly 50% with the antisera raised against MM2 peptide. Therefore, the latter antisera were not investigated further. Antisera were tested for isozyme specificity by western blotting. Antisera raised against the BB peptide detected a single band of 97 kDa in rat brain homogenate, but not in skeletal muscle homogenate (Figs 1a and e). Antisera raised against the MM1 peptide detected a single band of 97 kDa in homogenates of both rat brain and skeletal muscle (Figs 1b and f). Anti-BB sera reacted with GP isozyme BB purified from rat brain (Figs 1a and e). Pre-immune sera or antisera pre-absorbed with the antigens generated no signals (Figs 1c and g, and d and h, respectively). Antisera did not cross-react with supernatants of rat liver homogenates (not shown). Western blots demonstrating isozyme specificity. Blots contained supernatants of homogenates from rat brain (lanes b; 5 mU of activity applied) or skeletal muscle (lanes sk; 5 mU applied), and purified rat brain BB isozyme (lanes BB; 1 mU applied). (a–d) Sera from rabbits, (e–h) sera from guinea pigs, (a and e) antisera raised against BB peptide and (b and f) antisera raised against MM1 peptide. (c and g) Negative controls in which pre-immune sera were used instead of antisera and (d and h) negative controls in which antisera were pre-absorbed with antigen. Immunofluorescence staining experiments revealed the presence of the GP isozymes BB and MM in astrocytes throughout the brain and spinal cord. Double-labelling experiments showed perfect co-localization of both isozymes. In the cerebellum (Fig. 2), Bergmann glial cells and fibres, astrocytes of the granular layer, and fibrous astrocytes of the cerebellar white matter displayed immunoreactivity for both isozymes (Figs 2a–d). Pre-immune sera or sera pre-absorbed with antigens generated no signal; only some autofluorescence of Purkinje cells was visible (Figs 2e and h). In the hippocampus, protoplasmic astrocytes were positive for BB and MM isoforms (Figs 3a and b). This applied also to cortical astrocytes (Figs 3c and d) and capillary endfeet (Figs 3c and d). In the spinal cord, astrocytes showed immunoreactivity for either isozyme (Figs 4a–f). The astrocytic nature of the GP-immunoreactive structures in brain and spinal cord was verified by double-labelling in combination with antibodies against glial fibrillary acidic protein (not shown). Immunocytochemical double-labelling of rat cerebellum with rabbit antiserum against GP isozyme BB (a and c) and guinea pig antiserum against GP isozyme MM (b, d). Secondary antibodies were conjugated to TRITC (a), FITC (b), Alexa Fluor 568 (c) or Alexa Fluor 488 (d). Short arrows indicate Bergmann glial cells in (a) and (b) and white matter astrocytes in (c) and (d). Long arrows indicate granular layer astrocytes in (a) and (b), showing immunoreactivity for both isozymes. Purkinje cells [black arrowheads in (a) and (b)] are not immunoreactive. (e and f) Negative control to (a) and (b); pre-immune sera were applied instead of antisera. (g and h) Negative controls to (a) and (b); antisera pre-absorbed with antigens were applied instead of antisera. Note the autofluorescence of Purkinje cells in (e)– (h). The bar in (a) corresponds to 50 µm in (a)–(h). (a and b) Immunocytochemical double-labelling of rat hippocampus with guinea pig antiserum against GP isozyme BB (a) and rabbit antiserum against GP isozyme MM (b). Secondary antibodies were conjugated to FITC (a) or TRITC (b). Arrows point to astrocytes showing immunoreactivity for both isozymes. Neurones (arrowheads) are not immunoreactive. (c and d) Immunocytochemical double-labelling of rat cerebral cortex with rabbit antiserum against GP isozyme BB (c) and guinea pig antiserum against GP isozyme MM (d). Secondary antibodies were conjugated to Alexa Fluor 488 (c) or Alexa Fluor 568 (d). Small arrows point to astrocytes showing immunoreactivity for both isozymes. The large arrow marks a capillary with astrocytic endfeet. (e and f) Immunocytochemical double-labelling of rat third ventricle ependyma with guinea pig antiserum against GP isozyme BB (e) and rabbit antiserum against GP isozyme MM (f). Secondary antibodies were conjugated to FITC (e) or TRITC (f). Only the BB isozyme is expressed in ependymal cells. Subependymal glial fibres show immunoreactivity for both isozymes (arrow). The bar in (a) corresponds to 50 µm in (a)–(f). Immunocytochemical double-labelling of rat spinal nerve and spinal cord with guinea pig antiserum against GP isozyme BB (a) and rabbit antiserum against GP isozyme MM (b) as well as with rabbit antiserum against GP isozyme BB (c and e) and guinea pig antiserum against GP isozyme MM (d and f). Secondary antibodies were conjugated to Alexa Fluor 488 (anti-rabbit IgG) or Alexa Fluor 568 (anti-guinea pig IgG). (a and b) Cross-section of the dorsal root of a spinal nerve; axons of nerve fibers (cut perpendicularly) stain for the BB isozyme only (a). (c and d) Dorsal column; ascending nerve fibre bundles (cut perpendicularly) stain for the BB isozyme only (arrowheads), whereas astrocytes and their fibres stain for both isozymes (arrows). (e and f) Ventral horn; large motoneurones express the BB isozyme only (arrows). The bar in (a) corresponds to 50 µm in (a)– (f). In contrast to astrocytes, ependymal cells lining the ventricles and the central canal of the spinal cord were only positive for the BB isozyme (Figs 3e and f). Cells of the plexus choroideus were not immunoreactive (not shown). Most neurones were lacking GP immunoreactivity. This is exemplified by Purkinje cells and granular cells of the cerebellum (Figs 2a–d), hippocampal pyramidal cells (Figs 3a and b) and cortical neurones (Figs 3c and d). Primary sensory neurones of the mesencephalic trigeminal nucleus 5 (Me5) (Figs 5a and b) expressed the BB isozyme only. This applied also to neurones of the dorsal root ganglia (DRG), including their fibres (Figs 5c and d). Me5 as well as DRG neurones showed varying levels of BB immunoreactivity, ranging from none to very high intensities. The weak signal seen in DRG neurones stained for the MM isozyme (Fig. 5d) is non-specific as proven by negative controls with pre-immume sera and pre-absorbed antisera (not shown). Spinal nerves showed immunoreactivity exclusively for the BB isozyme (Figs 4a and b) and neuronal fibre bundles of all parts of the spinal cord were positive solely for the BB isozyme (Figs 4c and d). Some large motoneurones of the ventral horn of the spinal cord displayed immunoreactivity for BB but lacked that for MM (Figs 4e and f). In addition, rare neuronal nuclei of the brain stem, probably including cochlear nuclei, were found to express only the BB isozyme of GP (not shown). (a and b) Immunocytochemical double-labelling of Me5 neurones in the rat brain stem with rabbit antiserum against GP isozyme BB (a) and guinea pig antiserum against GP isozyme MM (b). Secondary antibodies were conjugated to TRITC (a) or FITC (b). Only the BB isozyme is expressed in neurones (arrows). (c and d) Immunocytochemical double-labelling of DRG neurones with guinea pig antiserum against GP isozyme BB (c) and rabbit antiserum against GP isozyme MM (d). Secondary antibodies were conjugated to FITC (c) or TRITC (d). DRG neurones (arrows) and their axons (cut perpendicularly; arrowheads) express the BB isozyme only. The bar in (a) corresponds to 50 µm in (a)–(d). It has been established by immunocytochemical methods that GP in nervous tissues is mainly localized in astrocytes (Pfeiffer etal. 1990; Reinhart etal. 1990; Richter etal. 1996). In addition, ependymal cells of the ventricles contain GP (Pfeiffer etal. 1990, 1992). Neuronal localization appeared to be restricted to rare neurones of the PNS (Pfeiffer etal. 1995). The monoclonal antibodies used in these studies recognized all three isoforms equally well, so the presence of GP was revealed without identifying the isoform(s) responsible for the positive immunoreaction. Of the three known GP isozymes, the BB and MM isoforms have been shown to be expressed in adult rat brain (Richter etal. 1983; David and Crerar 1986). Electrophoretic analysis of homogenates from various regions of the rat nervous system revealed no difference in the distribution patterns of these isozymes. In addition, in immature brain and cultured astroglial cells, the mRNAs for all three GP isozymes could be detected. These results supported the idea that all cell types with GP could express two or even three isozymes simultaneously (Pfeiffer-Guglielmi etal. 2000). However, the considerable limitations in resolution and sensitivity of the method and its principle inability to elucidate isozyme patterns at a cellular level warranted the introduction of the more powerful method of double-labelling immunocytochemistry. The use of antibodies specific for GP isozymes BB and MM was envisaged to overcome these limitations and achieve a differentiated analysis. Commercially available monoclonal antibodies raised against human GP isozyme BB cross-reacted with the muscle isoform or were not suited for immunocytochemistry (B. Pfeiffer-Guglielmi, unpublished observations). Antibodies raised against isozyme-specific peptide sequences as antigens appeared more promising. Such antibodies have already been raised against peptides from the human BB isozyme sequence and applied successfully in immunohistochemistry (Shimada etal. 1986; Ignacio etal. 1990; Uno etal. 1998). The BB isozyme was localized in astrocytes of the macaque and rat cerebrum and cerebellum (Ignacio etal. 1990). Studies with MM-specific antibodies have been restricted to monkey and human retina (Nihira etal. 1995). Antibodies of value in immunocytochemical mapping of both isozymes in double-labelling experiments must be raised in different species, they should afford an unambiguous signal with low background, and they should not cross-react with the other isoforms. Our immunization protocol resulted in high-titre antisera that fulfilled these criteria without the need for antibody purification. The present immunocytochemical experiments on sections of rat nervous tissues revealed that only astrocytes express both isozymes in perfect co-localization, and throughout the brain and spinal cord. In contrast, ependymal cells and the rare GP-positive neurones express solely the BB isoform. The double-labelling pattern of astrocytes was identical for both combinations of antisera [rabbit antiserum against isozyme BB/guinea pig antiserum against isozyme MM, see Figs 2 and 3(c)and (d); and guinea pig antiserum against isozyme BB/rabbit antiserum against isozyme MM, see Figs 3(a) and (b)]. In neurones, both combinations displayed a signal solely with the antisera against isozyme BB, irrespective of the host (see Fig. 5). This was also the case for ependymal cells (not shown). What is the physiological importance of this particular isozyme distribution pattern? Glycogen metabolism is controlled precisely by several interlocking mechanisms. A key point of this control is GP. The MM and BB isoforms of GP differ in their regulatory properties (Crerar etal. 1995). Detailed kinetic analysis has been carried out primarily with the MM isozyme (Newgard etal. 1989; Johnson 1992). This isoform is strongly activated by phosphorylation (conversion of phosphorylase b to phosphorylase a) as well as by the allosteric activator AMP. Less is known about the kinetic properties of the BB isoform. Investigations with GP isolated from rabbit brain (Lowry etal. 1967) and rabbit heart (Guenard etal. 1977) might in fact have been investigations with a mixture of both isozymes. However, more recent data obtained with chimeric enzymes constructed from rabbit muscle and human brain GP cDNAs expressed in Escherichia coli (Crerar etal. 1995) have confirmed that the BB isoform, in contrast to the MM isoform, needs AMP for full activation even in the a form. In addition, the glycogen affinity of the BB isozyme is lower than that of the MM isozyme but increases in the presence of AMP. This means that the MM isoform is primarily tailored to respond to extracellular control via signals triggering the phosphorylation cascade, whereas the BB isoform is mainly sensitive to the AMP level of the cell and therefore adapted to provide energy for internal benefits. This is in accordance with the prevalence of the BB isoform in tissues sensitive to anoxia or hypoglycaemia such as fetal tissues (Richter etal. 1983) and cardiac myocytes (Kato etal. 1989; Krause etal. 1996; Mair 1998). Several types of cancer tissue have also been reported to contain the isozyme BB (Sato and Weinhouse 1973; Sato etal. 1976; Takashi etal. 1989; Mayer etal. 1992; Shimada etal. 1999; Tashima etal. 2000), in the anoxic environment of which this isoform may have a similar function. In the nervous system, astrocytes are equipped with both isozymes. This suggests a very versatile role of glycogen in this cell type. Astrocytes might respond to external neurotransmitter-borne demands of neighbouring cells as well as to a low energy charge of the cell itself. Astroglial cells in culture have been shown to release lactate generated by the breakdown of glycogen (Dringen etal. 1993a). In brain, such lactate is thought to be taken up by neurones and used as fuel for aerobic generation of energy (Schurr etal. 1988; Dringen etal. 1993b). Under physiological conditions and at metabolite concentrations in situ, this function might be fulfilled by the MM isozyme. In fact, evidence has been presented that CNS axonal function can be supported through the breakdown of astrocytic glycogen (Ransom and Fern 1997). The BB isozyme is probably used to fulfil local demands of energy and of NADPH (Rahman etal. 2000) in the astrocytes. Although most neurones contain no GP at all, certain neurones and their fibres contain high levels of isozyme BB. Most of these neurones form part of somatosensory pathways (neurones of Me5, DRG, fibre bundles in the dorsal horn of the spinal cord and the brain stem). These systems are of special survival value for the organism as a whole because they rapidly transfer information from the body surface to higher centres. GP isozyme BB might therefore enable these neurones to react autonomously when the glucose supply fails to meet their requirements and the energy charge of the cells drops. The ensuing AMP would be the intracellular signal for breaking down glycogen, the reserve fuel. In contrast to astrocytes, ependymal cells express only the BB isozyme of GP. It would therefore appear that these cells, rather than the astrocytes which support neighbouring cells, use their glycogen stores mainly for meeting their own needs. This is in accordance with their postulated barrier and detoxification functions in the adult brain (DelBigio 1995). 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