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Phorbol ester binding sites in human brain: Characterization, regional distribution, age-correlation, and alterations in Parkinson’s disease

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Abstract

We have characterized and localized phorbol ester binding sites in human autopsied brains, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu). When the tissue was homogenized in the absence of Ca2+ chelator (10 mM EGTA/2 mM EDTA), Scatchard analysis of the specific [3H]PDBu bindings to both particulate and soluble fractions yielded a single class of high-affinity binding site (K d = 7.1 and 7.4 nM:B max = 45.4 and 3.1 pmol/mg protein, respectively). The particulate fraction retained the majority of [3H]PDBu binding (98% of total binding activity), while the soluble fraction was almost devoid of binding activity (2%). In the presence of Ca2+ chelator, more of the activity was found in the soluble fraction (30%). The binding of [3H]PDBu was potently inhibited by active phorbol esters and related diterpenes withK i of nanomolar concentration but not by inactive ones. Diolein (OAG), a synthetic diacylglycerol, and polymixin B, an inhibitor of protein kinase C (PKC), inhibited the binding moderately (K i = 5.8 and 1.3 µM, respectively). H-7, an inhibitor of PKC and cyclic nucleotides-dependent kinase, did not compete with [3H]PDBu for the binding sites (K i > 100,000 nM). The regional distribution of specific [3H]PDBu binding in the human brain was rather uneven and resembled that of [3H]PDBu autoradiograms and PKC-like immunoreactivities in the rat brain. The binding capacities were generally in the order: rhinencephalon > basal ganglia > cerebral cortex > diencephalon > cerebellum > mesencephalon. Age-related loss of binding sites was observed in the prefrontal cortex of the subjects 33–81 years of age. In Parkinson’s disease, the phorbol ester binding showed a significant reduction in the substantia nigra, caudate putamen, and pallidum, whereas it was unchanged in the prefrontal cortex and caudate nucleus of schizophrenics, when compared with the relevant controls.

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References

  • Ashendel, C.L., Staller, J.M., Boutwell, R.K. (1983) Identification of a calcium- and phospholipid-dependent phorbol ester binding activity in the soluble fraction of mouse tissues. Biochem. Biophys. Res. Commun. 111:340–345

    Article  PubMed  CAS  Google Scholar 

  • Baraban, J.M., Snyder, S.H., Alger, B.E. (1985) Protein kinase C regulates ionic conductance in hippocampal pyramidal neurons: Electrophysiological effects of phorbol esters. Proc. Natl. Acad. Sci. U.S.A. 82:2538–2542

    Article  PubMed  CAS  Google Scholar 

  • Blumberg, P.M. (1980) In vitro studies on the mode of action of the phorbol esters, potent tumor promoters: part 1. C.R.C. Crit. Rev. Toxicol. 8:153–234

    Article  CAS  Google Scholar 

  • Bruns, R.F., Lawson-Wendling, K., Pugsley, T.A. (1983) A rapid filtration assay for soluble receptors using polyethyleneimine-treated filters. Anal. Biochem. 132:74–81

    Article  PubMed  CAS  Google Scholar 

  • Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., Nishizuka, Y. (1982) Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor promoting phorbol esters. J. Biol. Chem. 257:7847–7851

    PubMed  CAS  Google Scholar 

  • Coussens, L., Parker, P.J., Rhee, L., Yang-Feng, T.L., Chen, E., Waterfield, M.D., Francke, U., Ullrich, A. (1986) Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233:859–866

    Article  PubMed  CAS  Google Scholar 

  • Delclos, K.B., Nagle, D.S., Blumberg, P.M. (1980) Specific binding of phorbol ester tumor promoters to mouse skin. Cell 19:1025–1032

    Article  PubMed  CAS  Google Scholar 

  • Diamond, L., O’Brien, T.G., Baird, W.M. (1980) Tumor promoters and the mechanism of tumor promotion. Adv. Cancer Res. 32:1–74

    PubMed  CAS  Google Scholar 

  • Driedger, P.E., Blumberg, P.M. (1980) Specific binding of phorbol ester tumor promoters. Proc. Natl. Acad. Sci. U.S.A. 77:567–571

    Article  PubMed  CAS  Google Scholar 

  • Dunn, J.A., Blumberg, P.M. (1983) Specific binding of [20-3H]12-deoxyphorbol 13-isobutyrate to phorbol ester receptor subclasses in mouse skin particulate preparations. Cancer Res. 43:4632–4637

    PubMed  CAS  Google Scholar 

  • Dunphy, W.G., Delclos, K.B., Blumberg, P.M. (1980) Characterization of specific binding of [3H]phorbol 12,13-dibutyrate and [3H]phorbol 12-myristate 13-acetate to mouse brain. Cancer Res. 40:3635–3641

    PubMed  CAS  Google Scholar 

  • Hammond, C., Paupardin-Tritsch, D., Nairn, A.C., Greengard, P., Gershenfeld, H.M. (1987) Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C. Nature 325:809–811

    Article  PubMed  CAS  Google Scholar 

  • Hecker, E. (1978) Structure-activity relationships in diterpene esters irritant and cocarcinogenic to mouse skin. In Carcinogenesis, Vol. 2. T.J. Slaga, A. Sivak, R.K. Boutwell (eds). Raven Press, New York, pp. 11–48

    Google Scholar 

  • Hidaka, H., Inagaki, M., Kawamoto, S., Sasaki, Y. (1984) Isoquinoline-sulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036–5041

    Article  PubMed  CAS  Google Scholar 

  • Huang, K-P., Huang, F.L. (1986) Immunochemical characterization of rat brain protein kinase C. J. Biol. Chem. 261:14781–14787

    PubMed  CAS  Google Scholar 

  • Huang, K.-P., Nakabayashi, H., Huang, F.L. (1986) Isozymic forms of rat brain Ca2+-activated and phospholipid-dependent protein kinase. Proc. Natl. Acad. Sci. U.S.A. 83:8535–8539

    Article  PubMed  CAS  Google Scholar 

  • Jaken, S., Kiley, S. (1987) Purification and characterization of three types of protein kinase C from rabbit brain cytosol. Proc. Natl. Acad. Sci. U.S.A. 84:4418–4422

    Article  PubMed  CAS  Google Scholar 

  • Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., Nishizuka, Y. (1982) Calcium-activated, phospholipid-dependent protein kinase from rat brain: Subcellular distribution, purification, and properties. J. Biol. Chem. 257:13341–13348

    PubMed  CAS  Google Scholar 

  • Kikkawa, U., Takai, Y., Tanaka, Y., Miyake, R., Nishizuka, Y. (1983) Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J. Biol. Chem. 258:11442–11445

    PubMed  CAS  Google Scholar 

  • Kikkawa, U., Ono, Y., Ogita, K., Fujii, T., Asaoka, Y., Sekiguchi, K., Kosaka, Y., Igarashi, K., Nishizuka, Y. (1987) Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain. FEBS Lett. 217:227–231

    Article  PubMed  CAS  Google Scholar 

  • Kishimoto, A., Kajikawa, N., Shiota, M., Nishizuka, Y. (1983) Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease. J. Biol. Chem. 258:1156–1164

    PubMed  CAS  Google Scholar 

  • Knopf, J.L., Lee, M.-H., Sultzman, L.A., Kritz, R.W., Loomis, C.R., Hewick, R.M., Bell, R.M. (1986) Cloning and expression of multiple protein kinase C cDNAs. Cell 46:491–502

    Article  PubMed  CAS  Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. chem. 193:265–275

    PubMed  CAS  Google Scholar 

  • Makowske, M., Birnbaum, M.J., Ballester, R., Rosen, O.M. (1986) A cDNA encoding protein kinase C identifies two species of mRNA in brain and GH3 cells. J. Biol. Chem. 261:13389–13392

    PubMed  CAS  Google Scholar 

  • Malenka, R.C., Madison, D.V., Nicoll, R.A. (1986) Potentiation of synaptic transmission in the hippocampus. Nature 321:175–177

    Article  PubMed  CAS  Google Scholar 

  • Mazzei, G.J., Katoh, N. Kuo, J.F. (1982) Polymixin B is a more selective inhibitor for phospholipid-sensitive Ca2+-dependent protein kinase than for calmodulinsensitive Ca2+-dependent protein kinase. Biochem. Biophys. Res. Commun. 109:1129–1133

    Article  PubMed  CAS  Google Scholar 

  • McGeer, P.L., McGeer, E.G., Suzuki, J., Colman, C.E., Nagai, T. (1984) Aging, Alzheimer’s disease and the cholinergic system of the basal forebrain. Neurology 34:741–745

    PubMed  CAS  Google Scholar 

  • Michell, R.H. (1983) Polyphosphoinositide breakdown as the initiating reaction in receptor-stimulated inositol phospholipid metabolism. Life Sci. 32:2083–2085

    Article  PubMed  CAS  Google Scholar 

  • Murphy, K.M.M., Gould, R.J., Oster-Granite, M.L., Gearhart, J.D., Snyder, S.H. (1983) Phorbol ester receptors: Autoradiographic identification in the developing rat. Science 222:1036–1038

    Article  PubMed  CAS  Google Scholar 

  • Nagle, D.S., Jaken, S., Castagna, M., Blumberg, P.M. (1981) Variation with embryonic development and regional localization of specific [3H]phorbol 12,13-dibutyrate binding to brain. Cancer Res. 41:89–93

    PubMed  CAS  Google Scholar 

  • Nichols, R.A., Haycock, J.W., Wang, J.K.T., Greengard, P. (1987) Phorbol ester enhancement of neurotransmitter release from rat brain synaptosomes. J. Neurochem. 48:615–621

    Article  PubMed  CAS  Google Scholar 

  • Niedel, J.E., Kuhn, L.J., Vanderbark, G.R. (1983) Phorbol diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 80:36–40

    Article  PubMed  CAS  Google Scholar 

  • Nishino, N., Noguchi-Kuno, S.A., Sugiyama, T., Tanaka, C. (1986) [3H]Nitrendipine binding sites are decreased in the substantia nigra and striatum of the brain from patients with Parkinson’s disease. Brain Res. 377:186–189

    Article  PubMed  CAS  Google Scholar 

  • Nishizuka, Y. (1986) Studies and perspective of protein kinase C. Science 233:305–312

    Article  PubMed  CAS  Google Scholar 

  • Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Inagaki, M., Yokokura, H., Sakoh, T., Hidaka, H. (1987) Tissue-specific expression of three distinct types of rabbit protein kinase C. Nature 325:161–166

    Article  PubMed  CAS  Google Scholar 

  • Ono, Y., Kurokawa, T., Fujii, T., Kawahara, K., Igarashi, K., Kikkawa, U., Ogita, K., Nishizuka, Y. (1986) Two types of complementary DNAs of rat brain protein kinase C: Heterogeneity determined by alternative splicing. FEBS Lett. 206:347–352

    Article  PubMed  CAS  Google Scholar 

  • Ono, Y., Kikkawa, U., Ogita, K., Kurokawa, T., Asaoka, Y., Sekiguchi, K., Ase, K., Igarashi, K. Nishizuka, Y. (1987) Expression and properties of two types of protein kinase C determined by alternative splicing from a single gene. Science 236:1116–1120

    Article  PubMed  CAS  Google Scholar 

  • Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Cheng, E., Stabel, S., Waterfield, M.D., Ullrich, A. (1986) The complete primary structure of protein kinase C: The major phorbol ester receptor. Science 233:853–859

    Article  PubMed  CAS  Google Scholar 

  • Pozzan, T., Gatti, G., Dozio, N., Vicentini, L.M., Meldolesi, J. (1984) Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: A role for protein kinase C activation? J. Cell Biol. 99:628–638

    Article  PubMed  CAS  Google Scholar 

  • Saitoh, T., Dobkins, K.R. (1986) Protein kinase C in human brain and its inhibition by calmodulin. Brain Res. 379:196–199

    Article  PubMed  CAS  Google Scholar 

  • Saitoh, N., Kikkawa, U., Nishizuka, Y., Tanaka, C. (1988) Distribution of protein kinase C-like immunoreactive neurons in rat brain. J. Neurosci. 8:369–382

    Google Scholar 

  • Shapira, R., Silbergerg, S.D., Ginsberg, S., Rahamimoff, R. (1987) Activation of protein kinase C augments evoked transmitter release. Nature 325:58–60

    Article  PubMed  CAS  Google Scholar 

  • Shearman, M.S., Naor, Z., Kikkawa, U., Nishizuka Y. (1987) Differential expression of multiple protein kinase C subspecies in rat central nervous tissue. Biochem. Biophys. Res. Commun. 147:911–919

    Article  PubMed  CAS  Google Scholar 

  • Shoyab, M., Todaro, G.J., Tallman, J.F. (1982) Chlorpromazine and related antipsychotic tricyclic compounds competitively inhibit the interaction between tumor-promoting phorbol esters and their specific receptors. Cancer Lett. 16:171–177

    Article  PubMed  CAS  Google Scholar 

  • Shuntoh, H., Tanaka, C. (1986) Activation of protein kinase C potentiates norepinephrine release from sinus node. Am J. Physiol. 251:C833-C840

    PubMed  CAS  Google Scholar 

  • Tanaka, C., Fujiwara, H., Fujii, Y. (1986) Acetylcholine release from guinea pig caudate slices evoked by phorbol ester and calcium. FEBS Lett. 195:129–134

    Article  PubMed  CAS  Google Scholar 

  • Walaas, S.I., Nairn, A.C., Greengard, P. (1983) Regional distribution of calcium- and cyclic adenosine 3′:5′-monophosphate-regulated protein phosphorylation systems in mammalian brain: II Soluble systems. J. Neurosci. 3:302–311

    PubMed  CAS  Google Scholar 

  • Worley, P.F., Baraban, J.M., Snyder, S.H. (1986) Heterogeneous localization of protein kinase C in rat brain: Autoradiographic analysis of phorbol ester receptor binding. J. Neurosci. 6:199–207

    PubMed  CAS  Google Scholar 

  • Wrenn, R.W., Katoh, N., Schatzman, R.C., Kuo, J.F. (1981) Inhibition by phenothiazine antipsychotic drugs of calcium-dependent phosphorylation of cerebral cortex proteins regulated by phospholipid or calmodulin. Life Sci. 29:725–733

    Article  PubMed  CAS  Google Scholar 

  • Zurgil, N., Zisapel, N. (1985) Phorbol ester and calcium act synergistically to enhance neurotransmitter release by brain neurons in culture. FEBS Lett. 185:257–261

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology, Kobe University School of Medicine, Chuo-ku, 650, Kobe, Japan

    Naoki Nishino, Noboru Kitamura, Takashi Nakai, Takeshi Hashimoto & Chikako Tanaka

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  1. Naoki Nishino
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  2. Noboru Kitamura
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  3. Takashi Nakai
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  4. Takeshi Hashimoto
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This work was supported by a Grant-in-Aid for Special Project Research of Selected Intractable Neurological Disorders from the Ministry of Education, Science and Culture, Japan.

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Nishino, N., Kitamura, N., Nakai, T. et al. Phorbol ester binding sites in human brain: Characterization, regional distribution, age-correlation, and alterations in Parkinson’s disease. J Mol Neurosci 1, 19–26 (1989). https://doi.org/10.1007/BF02896852

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