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Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study

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Journal of Neurocytology

Abstract

Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.

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References

  • Altschuler, R. A., Wenthold, R. J., Schwartz, A. M., Haser, W. G., Curthoys, N. P., Parakkal, M. H. & Fex, J. (1984) Immunocytochemical localization of glutaminase-like immunoreactivity in the auditory nerve. Brain Research 291, 173–178.

    Google Scholar 

  • Benjamin, A. M. & Quastel, J. H. (1972) Locations of amino acids in brain slices from the rat. Tetrodotoxinsensitive release of amino acids. Biochemical Journal 128, 631–646.

    Google Scholar 

  • Blackstad, T. W., Karag ¬ ulle, T. & Ottersen, O. P. (1990) MORFOREL, a computer program for twodimensional analysis of micrographs of biological specimens, with emphasis on immunogold preparations. Computers in Biology and Medicine 20, 15–34.

    Google Scholar 

  • Chaudhery, F. A., Lehre, K. P., Van lookern campagne, M., Ottersen, O. P., Danbolt, N. C. & Storm-mathisen, J. (1995) Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 15, 711–720.

    Google Scholar 

  • Dõaldin, C., Caicedo, A., Ruel, J., Renard, N., Pujol, R. & Puel, J. L. (1998) Antisense oligonucleotides to the GluR2 AMPA receptor subunit modify excitatory synaptic transmission in vivo. Molecular Brain Research 55, 151–164.

    Google Scholar 

  • Dallos, P. & Evans, B. N. (1995) High-frequency motility of outer hair cells and the cochlear amplifier. Science 267, 2006–2009.

    Google Scholar 

  • Danbolt, N. C. (1994) The high affinity uptake system for excitatory amino acids in the brain. Progress in Neurobiology 44, 377–396.

    Google Scholar 

  • Erecinska, M. & Silver, I. A. (1990) Metabolism and role of glutamate in mammalian brain. Progress in Neurobiology 35, 245–296.

    Google Scholar 

  • Ericson, A. C., Blomqvist, A., Craig, A. D., Ottersen, O. P. & Broman, J. (1995) Evidence for glutamate as neurotransmitter in trigemino-and spinothalamic tract terminals in the nucleus submedius of cats. European Journal of Neuroscience 7, 305–317.

    Google Scholar 

  • Eybalin, M. (1993) Neurotransmitters and neuromodulators of the mammalian cochlea. Physiological Reviews 73, 309–373.

    Google Scholar 

  • Fex, J., Kachar, B., Rubio, J. A., Parakkal, M. H. & Altschuler, R. A. (1985) Glutaminase-like immunoreactivity in the organ of Corti of guinea pig. Hearing Research 17, 101–113.

    Google Scholar 

  • Fonnum, F. (1984) Glutamate: a neurotransmitter in mammalian brain. Journal of Neurochemistry 42, 1–11.

    Google Scholar 

  • Friedmann, I. & Ballantyne, J. (1984) Ultrastructural Atlas of the Inner Ear. London: Butterworths.

    Google Scholar 

  • Furness, D. N. & Lehre, K. P. (1997) Immunocytochemical localization of a high-affinity glutamateaspartate transporter, GLAST, in the rat and guinea-pig cochlea. European Journal of Neuroscience 9, 1961–1969.

    Google Scholar 

  • Gundersen, V., Shupliakov, O., Brodin, L., Ottersen, O. P. & Storm-mathisen, J. (1995) Quantification of excitatory amino acid uptake at intact glutamatergic synapses by immunocytochemistry of exogenous D-aspartate. Journal of Neuroscience 15, 4417–4428.

    Google Scholar 

  • Haser, W. G., Shapiro, R. A. & Curthoys, N. P. (1985) Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney. Biochemical Journal 229, 399–408.

    Google Scholar 

  • Hollmann, M. & Heinemann, S. (1994) Cloned glutamate receptors. Annual Reviews of Neuroscience 17, 31–108.

    Google Scholar 

  • Kaneko, T., Itoh, K., Shigemoto, R. & Mizuno, N. (1989) Glutaminase-like immunoreactivity in the lower brainstem and cerebellum of the adult rat. Neuroscience 32, 79–98.

    Google Scholar 

  • Kaneko, T., Kang, Y. & Mizuno, N. (1995) Glutaminase-positive and glutaminase-negative pyramidal cells in layer VI of the primary motor and somatosensory cortices: a combined analysis by intracellular staining and immunocytochemistry in the rat. Journal of Neuroscience 15, 8362–8377.

    Google Scholar 

  • Kaneko, T. & Mizuno, N. (1994) Glutamate-synthesizing enzymes in GABAergic neurons of the neocortex: a double immunofluorescence study in the rat. Neuroscience 61, 839–849.

    Google Scholar 

  • Kaneko, T., Urade, Y., Watanabe, Y. & Mizuno, N. (1987) Production, characterization, and immunohistochemical application of monoclonal antibodies to glutaminase purified from rat brain. Journal of Neuroscience 7, 302–309.

    Google Scholar 

  • Kataoka, Y. & Ohmori, H. (1994) Activation of glutamate receptors in response to membrane depolarization of hair cells isolated from chick cochlea. Journal of Physiology (Lond.) 477, 403–414.

    Google Scholar 

  • Kataoka, Y. & Ohmori, H. (1996) Of known neurotransmitters, glutamate is the most likely to be released from chick cochlear hair cells. Journal of Neurophysiology 76, 1870–1879.

    Google Scholar 

  • Kvamme, E. (1988) Glutamine and Glutamate in Mammals. Vol.I. Boca Raton, Florida: CRC Press.

    Google Scholar 

  • Kvamme, E. (1998) Synthesis of glutamate and its regulation. Progress in Brain Research 116, 73–85.

    Google Scholar 

  • Kvamme, E., Svenneby, G. & Torgner, I. A. (1988) Glutaminases. In Glutamine and Glutamate in Mammals (edited by Kvamme, E.) pp. 53–67. Boca Raton, Florida: CRC Press.

    Google Scholar 

  • Laake, J. H., Slyngstad, T. A., Haug, F. M. & Ottersen, O. P. (1995) Glutamine from glial cells is essential for the maintenance of the nerve terminal pool of glutamate: immunogold evidence from hippocampal slice cultures. Journal of Neurochemistry 65, 871–881.

    Google Scholar 

  • Laake, J. H., Takumi, Y., Eidet, J., Torgner, I. A., Roberg, B., Kvamme, E. & Ottersen, O. P. (1999) Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum. Neuroscience 88, 1137–1151.

    Google Scholar 

  • Mart´inez-hern ´ andez, A., Bell, K. P. & Norenberg, M. D. (1977) Glutamine synthetase: glial localization in brain. Science 195, 1356–1358.

    Google Scholar 

  • Matsubara, A., Laake, J. H., Davanger, S., Usami, S. & Ottersen, O. P. (1996) Organization of AMPAreceptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti. Journal of Neuroscience 16, 4457–4467.

    Google Scholar 

  • Matsubara, A., Takumi, Y., Nakagawa, T., Usami, S., Shinkawa, H. & Ottersen, O. P. (1999) Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs. Brain Research 819, 58–64.

    Google Scholar 

  • Moráan, J. & Rivera-gaxiola, M. (1992) Effect of potassium and N-methyl-D-aspartate on the aspartate aminotransferase activity in cultured cerebellar granule cells. Journal of Neuroscience Research 33, 239–247.

    Google Scholar 

  • Nimmo, G. A. & Tipton, K. P. (1979) The distribution of soluble and membrane-bound forms of glutaminase in pig brain. Journal of Neurochemistry 33, 1083–1084.

    Google Scholar 

  • Nobili, R., Mammano, F. & Ashmore, J. (1998) How well do we understand the cochlea? Trends in Neurosciences 21, 159–167.

    Google Scholar 

  • Norenberg, M. D. & Mart´inez-hern ´ andez, A. (1979) Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Research 161, 303–310.

    Google Scholar 

  • Ottersen, O. P. (1989) Quantitative electron microscopic immunocytochemistry of amino acids. Anatomy and Embryology 180, 1–15.

    Google Scholar 

  • Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H. & Usami, S. (1998) Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear. Progress in Neurobiology 54, 127–148.

    Google Scholar 

  • Ottersen, O. P., Zhang, N. & Walberg, F. (1992) Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 46, 519–53

    Google Scholar 

  • Palay, S. L. & Chan-palay, V. (1974) Cerebellar Cortex: Cytology and Organization. New York: Springer.

    Google Scholar 

  • Puel, J. L. (1995) Chemical synaptic transmission in the cochlea. Progress in Neurobiology 47, 449–476.

    Google Scholar 

  • Puel, J. L., Ruel, J., Gervais dõaldin, C. & Pujol, R. (1998) Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. Neuroreport 9, 2109–2114.

    Google Scholar 

  • Pujol, R., Rebillard, G., Puel, J. L., Lenoir, M., Eybalin, M. & Recasens, M. (1990) Glutamate neurotoxicity in the cochlea: a possible consequence of ischaemic or anoxic conditions occurring in ageing. Acta Otolaryngologica Suppl. (Stockh.) 476, 32–36.

    Google Scholar 

  • Roberg, B., Torgner, I. A. & Kuamme, E. (1997) Soluble and membrane bound forms of phosphate activated glutaminase with different kinetic characteristics in pig and rat kidney mitochondria. In Nutritional and Acid-Base Aspects of Amino Acid Metabolism. Contributions to Nephrology, Vol. 121 (edited by O. Donovan, D. J., Doo, H. N., Schoolwerth, G. C., Tizianello, G. & Walls, L.), pp. 11–18. Basel: Karser.

    Google Scholar 

  • Salganicoff, L. & Derobertis, E. (1965) Subcellular distribution of the enzymes of the glutamic acid, glutamic and °-aminobutyric acid cycles in rat brain. Journal of Neurochemistry 12, 287–309.

    Google Scholar 

  • Shank, R. P. & Aprison, M. H. (1981) Present status and significance of the glutamine cycle in neural tissues. Life Sciences 28, 837–842.

    Google Scholar 

  • Shank, R. P., Bennett, G. S., Freytag, S. O. & Campbell, G. L. (1985) Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Research 329, 364–367.

    Google Scholar 

  • Shapiro, R. A., Rarrell, L., Srinivasan, M. & Curthoys, N. P. (1991) Isolation, characterization, and in vitro expression of a cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase. Journal of Biological Chemistry 266, 18792–18796.

    Google Scholar 

  • Takumi, Y., Matsubara, A., Danbolt, N. C., Laake, J. H., Storm-mathisen, J., Usami, S., Shinkawa, H. & Ottersen, O. P. (1997) Discrete cellular and subcellular localization of glutamine synthetase and the glutamate transporter GLAST in the rat vestibular end organ. Neuroscience 79, 1137–1144.

    Google Scholar 

  • Takumi, Y., Nagelhus, E. A., Eidet, J., Matsubara, A., Usami, S., Shinkawa, H., Nielsen, S. & Ottersen, O. P. (1998) Select types of supporting cell in the inner ear express aquaporin-4 water channel protein. European Journal of Neuroscience 10, 3584–3595.

    Google Scholar 

  • Takumi, Y., Ram´irez-le´on, V., Laake, P., Rinvik, E. & Ottersen, O. P. (1999) Different modes of expression ofAMPAandNMDAreceptors in hippocampal synapses. Nature Neuroscience 2, 618–624.

    Google Scholar 

  • Usami, S., Osen, K. K., Zhang, N. & Ottersen, O. P. (1992) Distribution of glutamate-like and glutaminelike immunoreactivities in the rat organ of Corti: a light microscopic and semiquantitative electron microscopic analysis with a note on the localization of aspartate. Experimental Brain Research 91, 1–11.

    Google Scholar 

  • Usami, S. & Ottersen, O. P. (1995) Differential cellular distribution of glutamate and glutamine in the rat vestibular endorgans: an immunocytochemical study. Brain Research 676, 285–292.

    Google Scholar 

  • Van den berg, C. J., Matheson, D. F., Ronda, G., Reijnierse, G. L. A., Blokhuis, G. G. D., Kroon, M. C., Clarke, D. D. & Garfinkel, D. (1975) A model of glutamate metabolism in brain: a biochemical analysis of a heterogeneous structure. In Metabolic Compartmentation and Neurotransmission. Relation to Brain Structure and Function (edited by Berl, S., Clarke, D.D. & Schneider, D.) pp. 515–543. New York: Plenum Press.

    Google Scholar 

  • Veruki, M. L. & W¬assle, H. (1996) Immunohistochemistry localization of dopamine D1 receptors in rat retina. European Journal of Neuroscience 8, 2286–2297.

    Google Scholar 

  • Wenthold, R. J. (1981) Glutamate and aspartate as neurotransmitters for the auditory nerve. In Glutamate as a Neurotransmitter (edited by Di Chiara, G. & Gessa, G. L.) pp. 69–78. New York: Raven Press.

    Google Scholar 

  • Wenthold, R. J. & Gulley, R. L. (1977) Aspartic acid and glutamic acid levels in the cochlear nucleus after auditory nerve lesion. Brain Research 138, 111–123.

    Google Scholar 

  • Wenthold, R. J., Skaggs, K. K. & Altschuler, R. A. (1986) Immunocytochemical localization of aspartate aminotransferase and glutaminase immunoreactivities in the cerebellum. Brain Research 363, 371–375.

    Google Scholar 

  • Westergaard, N., Fosmark, H. & Schousboe, A. (1991) Metabolism and release of glutamate in cerebellar granule cells cocultured with astrocytes from cerebellum or cerebral cortex. Journal of Neurochemistry 56, 59–66.

    Google Scholar 

  • Witt, C. M., Hu, H. Y., Brownell, W. E. & Bertrand, D. (1994) Physiologically silent sodium channels in mammalian outer hair cells. Journal of Neurophysiology 72, 1037–1040.

    Google Scholar 

  • Yu, A. C., Drejer, J., Hertz, L. & Schousboe, A. (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. Journal of Neurochemistry 41, 1484–1487.

    Google Scholar 

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Authors and Affiliations

  1. Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, 0317, Oslo, Norway

    Yutaka Takumi, Jon H. Laake, Vania Ramírez-León & Ole P. Ottersen

  2. Department of Otorhinolaryngology, Hirosaki University School of Medicine, Hirosaki, 036-8562, Japan

    Yutaka Takumi, Atsushi Matsubara & Shin-ichi Usami

  3. Neurochemical Laboratory, Institute of Basic Medical Sciences, University of Oslo, 0317, Oslo, Norway

    Bjørg Roberg, Ingeborg Torgner & Elling Kvamme

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  1. Yutaka Takumi
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  2. Atsushi Matsubara
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  3. Jon H. Laake
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  4. Vania Ramírez-León
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  7. Elling Kvamme
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  9. Ole P. Ottersen
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Takumi, Y., Matsubara, A., Laake, J.H. et al. Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study. J Neurocytol 28, 223–237 (1999). https://doi.org/10.1023/A:1007076007642

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