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Calbindin-D28k and calretinin in the rat posterior pituitary; Light and electron microscopic localization and upregulation with dehydration

  • Published:
Journal of Neurocytology

Abstract

Ca2+ binding proteins (CaBPs), calbindin-D28k (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca2+ in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals.

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References

  • AIRAKSINEN, M. S., EILERS, J., GARASCHUK, O., THOENEN, H., KONNERTH, A. & MEYER, M. (1997a) Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutant of the calbindin-D28k gene. Proceedings of The National Academy of Sciences (USA) 94, 1488–1493.

    Google Scholar 

  • AIRAKSINEN, M. S., THOENEN, H. & MEYER, M. (1997b) Vulnerability of midbrain dopaminergic neurons in calbindin-D28k-deficient mice: Lack of evidence for a neuroprotective role of endogenous calbindin in MPTP-treated and weaver mice. European Journal of Neuroscience 9, 120–127.

    PubMed  Google Scholar 

  • AKERFELDT, K. S., COYNE, A. N., WILK, R. R., THULIN, E. & LINSE, S. (1996) Ca2+ binding stoichiometry of calbindin D28k as assessed by spectroscopic analyses of synthetic peptide fragments. Biochemistry 35, 3662–3669.

    PubMed  Google Scholar 

  • ALBRITTON, N. L., MEYER, T. & STRYERS, L. (1992) Range of messenger action of calcium ion and inositol 1,4,5–triphosphate. Science 258, 1812–1815.

    Google Scholar 

  • ARAI, R., JACOBOWITZ, D. W. & DEMURA, S. (1993a) Immunohistochemical localization of calretinin-, calbindin-D28k-and parvalbumin-containing cells in the hypothalamic paraventricular and supraoptic nuclei of the rat. Brain Reseach 618, 323–327.

    Google Scholar 

  • ARAI, R., JACOWOWITZ, D. W. & DEMURA, S. (1993b) Colocalization of Calbindin-D28k with vasopression in hypothalamic cells of the rat: A double-labeling immunofluorescence study. Brain Research 632, 342–345.

    PubMed  Google Scholar 

  • ARAI, R., JACOWOWITZ, D. W. & NAGATSU, I. (1995) Up-regulation of calretinin in the supraoptic nucleus of the rat after chronic salt loading. Brain Research 673, 339–343.

    PubMed  Google Scholar 

  • ARAI, R., JACOWOWITZ, D. W. & NAGATSU, I. (1996) Calretinin is differentially localized in magnocellular oxytocin neurons of the rat hypothalamus. A double-labeling immunofluorescence study. Brain Research 735, 154–158.

    PubMed  Google Scholar 

  • ARMSTRONG, W. E., SMITH, B. N. & TIAN, B. N. (1994) Electrophysiological characteristics of immunochemically identified rat oxytocin and vasopressin neurones in vitro. Journal of Physiology 475, 115–128.

    PubMed  Google Scholar 

  • BAIMBRIDGE, K. G., CELIO, M. R. & ROGERS, J. H. (1992) Calcium-binding proteins n the nervous system. Trends in Neuroscience 15, 303–308.

    PubMed  Google Scholar 

  • BALMENT, R. J., BRIMBLE, M. J. & FORSLING, M. L. (1980) Release of oxytocin induced by salt loading and its influence on renal excretion in the male rat. Journal of Physiology 308, 439–449.

    PubMed  Google Scholar 

  • BEN-BARAK, Y., RUSSEL, J.-T., WHITNALL, M. H., OZATO, K. & GAINER, H. (1985) Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterisation of monoclonal antibodies. Journal of Neuroscience 5, 81–97.

    PubMed  Google Scholar 

  • BERDAL, A., NANCI, A., SMITH, C.-E., AHLUWALIA, J. P., THOASSET, M., CUISINIER-GLEIZES, P. & MATHIEU, H. (1991) Differential expression of calbindin-D 28kDa in rat incisor ameloblasts throughout enamel development. Anatomical Record 230, 149–163.

    PubMed  Google Scholar 

  • BICKNELL, R. J. (1993) Calcium signaling in neurosecretory terminals and pituicytes. Annals of New York Academy of Science 689, 177–182.

    Google Scholar 

  • CELIO, M. R. (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475.

    PubMed  Google Scholar 

  • CHARD, P. S., BLEAKMAN, D., CHRISTAKOS, S., FULLMER, C. S. & MILLER, R. J. (1993) Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. Journal of Physiology 472, 341–357.

    PubMed  Google Scholar 

  • CHARD, P. S., JORDAN, J., MARCUCCILLI, C. J., MILLER, R. J., LEIDEN, J. M., ROOS, R. P. & GHHADGE, G. D. (1995) Regulation of excitatory transmission at hippocampal synapses by calbindin D28k. Proceedings of The National Academy of Science (USA) 92, 5144–5148.

    Google Scholar 

  • CHEUNG, W. T., RICHARDS, D. E. & ROGERS, J. H. (1993) Calcium binding by chick calretinin and rat calbindin D28k synthesised in bacteria. European Journal of Biochemistry 215, 401–410.

    PubMed  Google Scholar 

  • CIMINI, V., ISAACS, K. R. & JACOBOWITZ, D. M. (1997) Calretinin in the rat pituitary: Colocaliation with thyroid-stimulating hormone. Neuroendocrinology 65, 179–188.

    PubMed  Google Scholar 

  • COBBET, P., SMITHSON, K. G. & HATTON, G. I. (1986) Immunoreactivity to vasopressin-but not oxytocin-associated neurophysin antiserum in phasic neurons of rat paraventricular nucleus. Brain Research 362, 7–16.

    PubMed  Google Scholar 

  • DIFIGLIA, M., CHRISTAKOS, S. & ARONIN, N. (1989) Ultrastructural localization of immunoreactive calbindin-D28k in the rat and monkey basal gabglia, including subcellular distribution with colloidal gold labeling. Journal of Comparative Neurology 279, 653–665.

    PubMed  Google Scholar 

  • FELDMAN, S. C. & CHRISTAKOS, S. (1983) Vitamin D-dependent calcium binding protein in rat brain: Biochemical and immunocytochemical characterization. Endocrinology 112, 290–302.

    PubMed  Google Scholar 

  • GEPPERT, M., GODA, Y., HAMMER, R. E., LI, C., ROSAHL, T. W., STEVENS, C. F. & SUDHOF, T. C. (1994) Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse. Cell 9, 717–727.

    Google Scholar 

  • GOTZOS, V., SCHWALLER, B., HETZEL, N., BUSTOS-CASTILLO, M. & CELIO, M. R. (1992) Expression of 16 MIYATA, NAKAI, KIYOHARA, and HATTON the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle. Experimental Cell Research 202, 292–302.

    PubMed  Google Scholar 

  • HATTON, G. I. (1990) Emerging concepts of structure-function dynamics in adult brain: The hypothalamo-neurohypophysial system. Progress in Neurobiology 34, 437–504.

    PubMed  Google Scholar 

  • HATTON, G. I. (1997) Function-related plasticity in hypothalamus. Annual Review of Neuroscience 20, 375–397.

    PubMed  Google Scholar 

  • HATTON, G. I. (1999) Astroglial modulation of neurotransmitter/peptide release from the neurohypophysis status. Journal of Chemical Neuroanatomy 16, 203–222.

    PubMed  Google Scholar 

  • HEIZMANN, C. W. & BRAUN, K. (1992) Changes in Ca2+-binding proteins in human neurodegenerative disorders. Trends in Neuroscience 15, 259–264.

    Google Scholar 

  • HSU, S.-F. & JACKSON, M. B. (1991) Rapid exocytosis and endocytosis in nerve terminals of the rat posterior pituitary. Journal of Physiology 494, 539–553.

    Google Scholar 

  • HUBBARD, M. J. & MCHUGH, N. J. (1995) Calbindin 28kDa and calbindin30kDa (calretinin) are substantially localized in the particulate fraction of rat brain. FEBS Letters 374, 333–337.

    PubMed  Google Scholar 

  • ISSA, N. P. & HUDSPETH, A. J. (1996) The entry and clearance of at individual presynaptic active zones of hair cells from the bullfrog's sacculus. Proceedings of National Academy of Science (USA) 93, 9527–9532.

    Google Scholar 

  • JANDE, S. S., MALER, L. & LAWSON, D. E. M. (1981) Immunohistochemical mapping of Vitamin D-dependent calcium-binding protein in brain. Nature 294, 765–767.

    PubMed  Google Scholar 

  • JONES, C. W. & PICKERING, B. T. (1969) Comparison of the effect of water deprivation and sodium chloride imbition on the hormone content of the neurohypophysis of the rat. Journal of Physiology 203, 449–485.

    PubMed  Google Scholar 

  • KAWASAKI, H., NAKAYAMA, S. & KRETSINGER, R. H. (1998) Classification and evolution of EF-hand proteins. Biometals 11, 277–295.

    PubMed  Google Scholar 

  • KRAMER, R. H. & ZUCKER, R. S. (1985) Calcium-dependent inward current in Aplysia bursting pacemaker neurones. Journal of Physiology 362, 107–130.

    PubMed  Google Scholar 

  • KLAPSTEIN, G. J., VIETLA, S., LIEBERMAN, D. N., GRAY, P. A., AIRAKSINEN, M. S., THOENEN, H., MEYER, M. & MODY, I. (1998) Calbindin-D28k fails to protect hippocampal neurons against ischemia in spite of its cytoplasmic calcium buffering properties: Evidence from calbindin-D28k knockout mice. Neuroscience 85, 361–373.

    PubMed  Google Scholar 

  • LEATHERS, V., LINSE, S., FORSEN, S. & NORMAN, A. W. (1990) Calbindin-D28K, a 1,25–dihydroxyvitamin D3–induced calcium protein, binds five or six calcium with high affinity. Journal of Biological Chemistry 265, 9838–9841.

    PubMed  Google Scholar 

  • LEHMAN, I. R. & KAGUNI, L. S. (1989) DNA polymerase α. Journal of Biological Chemistry 264, 4265–4268.

    PubMed  Google Scholar 

  • LI, Z., DECAVEL, C. & HATTON, G. I. (1995) Calbindin-D28k: Role in determining intrinsically generated firing patterns in rat supraoptic neurones. Journal of Physiology 488, 601–608.

    PubMed  Google Scholar 

  • LI, Z. & HATTON, G. I. (1996) Oscillatory bursting of phasically firing rat supraoptic neurones in low-Ca2+medium: Na+ influx, cytosolic Ca2+ and gap junctions. Journal of Physiology 496, 379–394.

    PubMed  Google Scholar 

  • LI, Z. & HATTON, G. I. (1997) Ca2+ release from internal stores: Role in generating depolarizing after-potentials in rat supraoptic neurones. Journal of Physiology 498, 339–350.

    PubMed  Google Scholar 

  • LUKAS, W. & JONES, W. A. (1994) Cortical neurons containing calretinin are selectively resistant to calcium overload and excitotoxicity in vitro. Neuroscience 61, 307–316.

    PubMed  Google Scholar 

  • LYSAKOWSKI, A., FIGUERAS, H., PRICE, S. D. & PENG, Y. Y. (1999) Dense-cored vesicles, smooth endoplasmic reticulum, and mitochondria are closely associated with non-specialized parts of plasma membrane of nerve terminals: Implications for exocytosis and calcium buffering by intraterminal organelles. Journal of Comparative Neurology 403, 378–390.

    PubMed  Google Scholar 

  • MATSUNAGA, W., MIYATA, S., HASHIMOTO, Y., LIN, S.-H., NAKASHIMA, T., KIYOHARA, T. & MATSUMOTO, T. (1999) Microtubule-associated protein-2 in the hypothalamo-neurohypophysial system: Low-molecular-weight microtubuleassociated protein-2 in pituitary astrocytes. Neuroscience 88, 1289–1297.

    PubMed  Google Scholar 

  • MIYATA, S., ARSHAD, K. & HATTON, G. I. (1998) Colocalization of calretinin and calbindin-D28k with oxytocin and vasopressin in rat supraoptic nucleus neurons: A quantitative study. Brain Research 785, 178–182.

    PubMed  Google Scholar 

  • MIYATA, S., MATSUSHIMA, O. & HATTON, G. I. (1997) Taurine in rat posterior pituitary: Localization in astrocytes and selective release by hypoosmotic stimulation. Journal of Comparative Neurology 381, 513–523.

    PubMed  Google Scholar 

  • MOLINARI, S., BATTINI, R., FERRARI, S., POZZI, L., KILLCROSS, A. S., ROBBINS, T. W., JOUVENCEAU, A., BILLARD, J.-M., DUTAR, P., LAMOUR, Y., BAKER, W. A., COX, H. & EMSON, P. C. (1996) Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28k expression. Proceedings of The National Academy of Science (USA) 93, 8028–8033.

    Google Scholar 

  • NAGASAWA, J., DOUGLAS, W. W. & SCHULZ, R. A. (1971) Micropinocytotic origin of coated and smooth microvesicles ("synaptic vesicles") in neurosecretory terminals of posterior pituitary glands demonstrated by incorporation of horseradish peroxidase. Nature 232, 341–342.

    PubMed  Google Scholar 

  • NAVONE, F., DI GIOIA, G., JAHN, R., BROWNING, M., GREENGARD, P. & DE CAMILLI, P. (1989) Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals. Journal of Cell Biology 109, 3425–3433.

    PubMed  Google Scholar 

  • NAVONE, F., JAHN, R., DI GIOIA, G., STUKENBROK, H., GREENGARD, OP. & DE CAMILLI, P. (1986) Protein p38: An integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. Journal of Cell Biology 103, 2511–2527.

    PubMed  Google Scholar 

  • NEHER, E. & ZUCKER, R. S. (1993) Multiple calcium-dependent processes related to secretion in bovine chromaffin cells. Neuron 10, 21–30.

    Article  PubMed  Google Scholar 

  • NEMERE, I., OPPERMAN, L. A., ROSS, F. P. & NORMAN, A. W. (1992) Noncytoplasmic and filamentous appearance of Calbindin-D28k and tubulin in double, indirect immunofluorescent staining of embryonic chick tissue. Molecular & Cellular Endocrinology 86, 83–91.

    Google Scholar 

  • PERSECHINI, A., MONCRIEF, A. D. & KRETSINGER, R. H. (1989) The EF-hand family of calcium-modulated proteins. Trends in Neuroscience 12, 462–467.

    Google Scholar 

  • PEVSNER, J., HSU, S., BRAUN, J. E., CALAKOS, N., TING, A. E., BENNETT, M. K. & SCHELLER, R. H. (1994) Specificity and regulation of a synaptic vesicle docking complex. Neuron 13, 353–361.

    PubMed  Google Scholar 

  • REN, K. & RUDA, M. K. (1994) A comparative study of the calcium-binding proteins calbindin-D28k, calretinin, calmodulin and paralbumin in the rat spinal cord. Brain Research 19, 163–179.

    Google Scholar 

  • ROBERTS, W. M. (1994) Localization of calcium signals by a mobile calcium buffer in frog saccular hair cells. Journal of Neuroscience 14, 3246–3262.

    PubMed  Google Scholar 

  • ROBERTS, W. M., JACOBS, R. A. & HUDSPETH, A. J. (1990) Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. Journal of Neuroscience 10, 3664–3684.

    PubMed  Google Scholar 

  • SANCHES, F., ALONSO, J. R., AREVALO, R., CARRETERO, J., VAZQUEZ, R. & AIJON, J. (1992) Calbindin D-28k-and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat. Brain Research Bulletin 28, 39–46.

    PubMed  Google Scholar 

  • SCHURMANS, S., SCHIFFMANN, S. N., GURDEN, H., LEMAIRE, M., LIPP, H. P., SCHWAM, V., POCHET, R., IMPERATO, A., BOHME, G. A. & PARMENTIER, M. (1997) Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice. Proceedings of The National Academy of Science (USA) 94, 10415–10420.

    Google Scholar 

  • SCHWALLER, B., DURUSSEL, I., JERMANN, D., HERRMANN, B. & COX, J. A. (1997) Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin. Journal of Biological Chemistry 272, 29663–29671.

    PubMed  Google Scholar 

  • SMITH, S. J. & AUGUSTINE, G. J. (1988) Calcium ions, active zones and synaptic transmitter release. Trends in Neuroscience 11, 458–464.

    Google Scholar 

  • STEVENS, J. & ROGERS, J. H. (1997) Chick calretinin: Purification, composition, and metal binding activity of native and recombinant forms. Protein Experiment and Purification 9, 171–181.

    Google Scholar 

  • TWEEDLE, C. D. (1983) Ultrastructural manifestations of increased hormone release in the neurohypophysis. Progress in Brain Research 60, 259–272.

    PubMed  Google Scholar 

  • VERHAGE, M., MCMAHON, H. T., GHIJSEN, W. E. J. M., BOOMSMA, F., SCHOLTEN, G., WIEGANT, V. M. & NICHOLLS, D. G. (1991) Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals. Neuron 6, 517–524.

    PubMed  Google Scholar 

  • VOISIN, D. L., FENELON, V. S. & HERBISON, A. E. (1996) Calbindin-D28k mRNA expression in magnocellular hypothalamic neurons of female rats during parturition, lactation and following dehydration. Molecular Brain Research 42, 279–286.

    PubMed  Google Scholar 

  • VON GERSDORFF, H. & MATTHEWS, G. (1994) Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735–739.

    Article  PubMed  Google Scholar 

  • WINSKY, L. & KUZNICKI, J. (1995) Distribution of calretinin, calbindinD28k, and parvalbumin in subcellular fractions of rat cerebellum: Effect of calcium. Journal of Neurochemistry 65, 381–388.

    PubMed  Google Scholar 

  • WINSKY, L., NAKATA, H., MARTIN, B. M. & JACOBOWITZ, D. M. (1989) Isolation, partial amino acid sequence, and immunohistochemical localization of a brain-specific calcium-binding protein. Proceedings of The National Academy of Science (USA) 86, 139–143.

    Google Scholar 

  • ZHOU, Z. & NEHER, E. (1993) Mobile and immobile calcium buffers in bovine adrenal chromaffin cells. Journal of Physiology 469, 245–273.

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606–8585, Japan

    Seiji Miyata, Sadamu Nakai & Toshikazu Kiyohara

  2. Department of Neuroscience, University of California, Riverside, CA, 92521, USA

    Glenn I. Hatton

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  1. Seiji Miyata
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  2. Sadamu Nakai
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  3. Toshikazu Kiyohara
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  4. Glenn I. Hatton
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Miyata, S., Nakai, S., Kiyohara, T. et al. Calbindin-D28k and calretinin in the rat posterior pituitary; Light and electron microscopic localization and upregulation with dehydration. J Neurocytol 29, 5–17 (2000). https://doi.org/10.1023/A:1007180328597

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