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Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway

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Summary

  1. 1.

    The effects on catecholamine secretion of activation of protein kinase C and clostridial neurotoxins were examined in digitonin-permeabilized bovine adrenal chromaffin cells.

  2. 2.

    The enhancement by phorbol esters increased only the initial rate of secretion; later rates were unaffected. This enhancement was present over a wide range of Ca2+ concentrations and was elicited at 18 as well as at 27°C.

  3. 3.

    Tetanus toxin inhibited both ATP-dependent and ATP-independent secretion, indicating that the tetanus toxin target is important during the final steps in the pathway.

  4. 4.

    Prior activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl phorbol acetate rendered the primed state more sensitive to inhibition by tetanus toxin. The data indicate that a phosphorylated protein kinase C substrate is either identical to or closely associated with the tetanus toxin target protein at the final steps in the pathway.

  5. 5.

    The interaction between the effect of protein kinase activation and that of tetanus toxin suggests that protein kinase C activation does not stimulate a separate pathway of secretion but, rather, modulates the activity of the ongoing pathway.

  6. 6.

    The enhancement of secretion by protein kinase C is caused, at least in part, by a qualitative change in the characteristics of the primed state. This is indicated by the increased sensitivity of primed secretion to inhibition by tetanus toxin and a threefold increase in sensitivity of primed secretion to Ca2+.

  7. 7.

    Because activation of protein kinase C does not increase the later rates of secretion that are limited by ATP-dependent priming reactions, it is unlikely that enhancement of the maximal rate of secretion by TPA is due to an increased amount of the primed state. Instead, protein kinase C activation may increase the efficacy with which Ca2+ stimulates secretion at all Ca2+ concentrations.

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References

  • Ahnert-Hilger, G., and Weller, U. (1993). Comparison of the intracellular effects of clostridial neurotoxins on exocytosis from streptolysin-O-permeabilized rat pheochromocytoma (PC12) and bovine adrenal chromaffin cells.Neuroscience 53547–552.

    Google Scholar 

  • Ahnert-Hilger, G., Bader, M. F., Bhakdi, S., and Gratzl, M. (1989a). Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: Inhibitory effect of tetanus toxin on catecholamine secretion.J. Neurochem. 521751–1758.

    Google Scholar 

  • Ahnert-Hilger, G., Weller, U., Dauzenroth, M. E., Habermann, E., and Gratzl, M. (1989b). The tetanus toxin light chain inhibits exocytosis.Febs. Lett. 242245–248.

    Google Scholar 

  • Ahnert-Hilger, G., Wegenhorst, U., Stecher, B., Spicher, K., Rosenthal, W., and Gratzl, M. (1992). Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosne 5′-[γ-thio]triphospate and guanosine 5′-[β, γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins.Biochem. J. 284321–326.

    Google Scholar 

  • Ashton, A. C., and Dolly, J. O. (1991). Microtubule-dissociating drugs and A23187 reveal differences in the inhibition of synaptosomal transmitter release by botulinum neurotoxins types A and B.J. Neurochem. 56827–835.

    Google Scholar 

  • Augustine, G. J., Adler, E. M., and Charlton, M. P. (1991). The calcium signal for transmitter secretion from presynaptic nerve terminals.Ann. N.Y. Acad. Sci. 635365–381.

    Google Scholar 

  • Bhattacharyya, S. D., and Sugiyama, H. (1989). Inactivation of botulinum and tetanus toxin by chelators.Infect. Immun. 573053–3057.

    Google Scholar 

  • Bittner, M. A., and Holz, R. W. (1988). Effects of tetanus toxin on catecholamine release from intact and digitonin-permeabilized chromaffin cells.J. Neurochem. 51451–456.

    Google Scholar 

  • Bittner, M. A., and Holz, R. W. (1992a). Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components.J. Biol. Chem. 26716219–16225.

    Google Scholar 

  • Bittner, M. A., and Holz, R. W. (1992b). A temperature-sensitive step in exocytosis.J. Biol. Chem. 26516226–16229.

    Google Scholar 

  • Bittner, M. A., DasGupta, B. R., and Holz, R. W. (1989a). Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells.J. Biol. Chem. 26410354–10360.

    Google Scholar 

  • Bittner, M. A., Habig, W. H., and Holz, R. W. (1989b). Isolated light chain of tetanus toxin inhibits exocytosis: Studies in digitonin-permeabilized cells.J. Neurochem. 53966–968.

    Google Scholar 

  • Bjerrum, J., Schwarzenbach, G., and Sillen, L. G. (1957).Stability Constant of Metal-Ion Complexes, with Solubility Products of Inorganic Substances, Part I: Organic Ligands, Chemical Society, London, p. 422.

    Google Scholar 

  • Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De Camilli, P., Sudhof, T. C., Niemann, H., and Jahn, R. (1993). Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25.Nature 365160–163.

    Google Scholar 

  • Brocklehurst, K. W., and Pollard, H. B. (1985). Enhancement of Ca2+-induced catecholamine release by the phorbol ester TPA in digitonin-permeabilized cultured bovine adrenal chromaffin cells.FEBS Lett. 183107–110.

    Google Scholar 

  • Burgoyne, R. D., Morgan, A., and O'Sullivan, A. J. (1988). A major role for protein kinase C in calcium-activated exocytosis in permeabilised adrenal chromaffin cells.FEBS. Lett. 238151–155.

    Google Scholar 

  • Considine, R. V., Bielicki, J. K., Simpson, L. L., and Sherwin, J. R. (1990). Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells.Toxicon 2813–19.

    Google Scholar 

  • Dreyer, F., and Schmitt, A. (1983). Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature.Pflugers Arch. 399228–234.

    Google Scholar 

  • Dreyer, F., Rosenberg, F., Becker, C., Bigalke, H., and Penner, R. (1987). Differential effects of various secretagogues on quantal transmitter release from mouse motor nerve terminals treated with botulinum A and tetanus toxin.Naunyn Schmiedebergs Arch. Pharmacol. 3351–7.

    Google Scholar 

  • Dunn, L. A., and Holz, R. W. (1983). Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells.J. Biol. Chem. 2584989–4993.

    Google Scholar 

  • Gansel, M., Penner, R., and Dreyer, F. (1987). Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals.Pflugers Arch. 409533–539.

    Google Scholar 

  • Hay, J. C., and Martin, T. F. J. (1992). Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins.J. Cell Biol. 119139–151.

    Google Scholar 

  • Heinemann, C., von Ruden, L., Chow, R. H., and Neher, E. (1993). A two-step model of secretion control in neuroendocrine cells.Pflugers Arch. 424105–112.

    Google Scholar 

  • Holz, R. W., and Bittner, M. A. (1993). The role of protein kinase C in exocytosis. InProtein Kinase C (J. F. Kuo, Ed.), Oxford University Press, New York, pp. 269–289.

    Google Scholar 

  • Holz, R. W., Bittner, M. A., Peppers, S. C., Senter, R. A., and Eberhard, D. A. (1989). MgATP-independent and MgATP-dependent exocytosis. Evidence that MgATP primes adrenal chromaffin cells to undergo exocytosis.J. Biol. Chem. 2645412–5419.

    Google Scholar 

  • Isobe, T., Hiyane, Y., Ichimura, T., Okuyama, T., Takahashi, N., Nakajo, S., and Nakaya, K. (1992). Activation of protein kinase C by the 14-3-3 proteins homologous with Exo1 protein that stimulates calcium-dependent exocytosis.FEBS Lett. 308121–124.

    Google Scholar 

  • Jongeneel, C. V., Bouvier, J., and Bairoch, A. (1989). A unique signature identifies a family of zinc-dependent metallopeptidases.FEBS Lett. 242211–214.

    Google Scholar 

  • Knight, D. E., and Baker, P. F. (1982). Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields.J. Membr. Biol. 68107–140.

    Google Scholar 

  • Knight, D. E., and Baker, P. F. (1983). The phorbol ester TPA increases the affinity of exocytosis for calcium in “leaky” adrenal medullary cells.FEBS Lett. 16098–100.

    Google Scholar 

  • Kurazono, H., Mochida, S., Binz, T., Eisel, U., Quanz, M., Grebenstein, O., Wernars, K., Poulain, B., Tauc, L., and Niemann, H. (1992). Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A.J. Biol. Chem. 26714721–14729.

    Google Scholar 

  • Lazarovici, P., Fujita, K., Contreras, M. L., DiOrio, J. P., and Lelkes, P. I. (1989). Affinity purified tetanus toxin binds to isolated chromaffin granule membranes and inhibits catecholamine release in digitonin-permeabilized chromaffin cells.FEBS Lett. 253121–128.

    Google Scholar 

  • Link, E., Edelman, L., Chou, J. H., Binz, T., Yamasaki, S., Eisel, U., Baumert, M., Sudhof, T. C., Niemann, H., and Jahn, R. (1992). Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis.Biochem. Biophys. Res. Commun. 1891017–1023.

    Google Scholar 

  • Maisey, E. A., Wadsworth, J. D. F., Poulain, B., Shone, C. C., Melling, J., Gibbs, P., Tauc, L., and Dolly, J. O. (1988). Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release.Eur. J. Biochem. 177683–691.

    Google Scholar 

  • Martell, A. E., and Smith, R. M. (1974).Critical Stability Constants, Vol. 1.Amino Acids, Plenum Press, New York, pp. 269–272.

    Google Scholar 

  • Marxen, P., Bartels, F., Ahnert-Hilger, G., and Bigalke, H. (1991). Distinct targets for tetanus and botulinum A neurotoxins within the signal transducing pathway in chromaffin cells.Naunyn Schmiedebergs Arch. Pharmacol. 344387–395.

    Google Scholar 

  • McMahon, H. T., Ushkaryov, Y. A., Edelman, L., Link, E., Binz, T., Niemann, H., Jahn, R., and Sudhof, T. C. (1993). Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein.Nature 364346–349.

    Google Scholar 

  • Mochida, S., Poulain, B., Weller, U., Habermann, E., and Tauc, L. (1989). Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain.FEBS Lett. 25347–51.

    Google Scholar 

  • Morgan, A., and Burgoyne, R. D. (1992). Exol and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells.Nature 355833–836.

    Google Scholar 

  • Neher, E., and Zucker, R. S. (1993). Multiple calcium-dependent processes related to secretion in bovine chromaffin cells.Neuron 1021–30.

    Google Scholar 

  • Nishizaki, T., Walent, J. H., Kowalchyk, J. A., and Martin, T. F. J. (1992). A key role for a 145-kDa cytosolic protein in the stimulation of a Ca2+-dependent secretion by protein kinase C.J. Biol. Chem. 26723972–23981.

    Google Scholar 

  • Penner, R., Neher, E., and Dreyer, F. (1986). Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells.Nature 32476–78.

    Google Scholar 

  • Phillips, J. H. (1982). Dynamic aspects of chromaffin granule structure.Neuroscience 71595–1609.

    Google Scholar 

  • Pocotte, S. L., and Holz, R. W. (1986). Effects of phorbol ester on tyrosine hydroxylase phosphorylation and activation in cultured bovine adrenal chromaffin cells.J. Biol. Chem. 2611873–1877.

    Google Scholar 

  • Pocotte, S. L., Frye, R. A., Senter, R. A., TerBush, D. R., Lee, S. A., and Holz, R. W. (1985). Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.Proc. Natl. Acad. Sci. USA 82930–934.

    Google Scholar 

  • Portzehl, H., Caldwell, P. C., and Reugg, J. C. (1964). The dependence of contraction and relaxation of muscle fibers from the crabMaia squinado on the internal concentration of free calcium ions.Biochim. Biophys. Acta 79581–591.

    Google Scholar 

  • Poulain, B., Tauc, L., Maisey, E. A., Wadsworth, J. D. F., Mohan, P. M., and Dolly, J. O. (1988). Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain.Proc. Natl. Acad. Sci. USA 854090–4094.

    Google Scholar 

  • Sanders, D., and Habermann, E. (1992). Evidence for a link between specific proteolysis and inhibition of [3H]-noradrenaline release by the light chain of tetanus toxin.Naunyn Schmiedebergs Arch. Pharmacol. 346358–361.

    Google Scholar 

  • Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B., and Montecucco, C. (1992a). Tetanus toxin and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin.Nature 359832–835.

    Google Scholar 

  • Schiavo, G., Rossetto, O., Santucci, A., DasGupta, B. R., and Montecucco, C. (1992b). Botulinum neurotoxins are zinc proteins.J. Biol. Chem. 26723479–23483.

    Google Scholar 

  • Schiavo, G., Shone, C. C., Rossetto, O., Alexander, F. C. G., and Montecucco, C. (1993). Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin.J. Biol. Chem. 26811516–11519.

    Google Scholar 

  • Simon, S. M., and Llinas, R. R. (1985). Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release.Biophys. J. 48485–498.

    Google Scholar 

  • Simpson, L. L. (1988). Use of pharmacologic antagonists to deduce commonalities of biologic activity among clostridial neurotoxins.J. Pharmacol. Exp. Ther. 245867–872.

    Google Scholar 

  • TerBush, D. R., and Holz, R. W. (1986). Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells.J. Biol. Chem. 26117099–17106.

    Google Scholar 

  • TerBush, D. R., and Holz, R. W. (1990). Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells: The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporin.J. Biol. Chem. 26521179–21184.

    Google Scholar 

  • TerBush, D. R., Bittner, M. A., and Holz, R. W. (1988). Ca2+ influx causes rapid translocation of protein kinase C to membranes. Studies of the effects of secretagogues in adrenal chromaffin cells.J. Biol. Chem. 26318873–18879.

    Google Scholar 

  • Walent, J. H., Porter, B. W., and Martin, T. F. J. (1992). A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells.Cell 70765–775.

    Google Scholar 

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  1. M6322 Medical Science Building I, Department of Pharmacology, University of Michigan Medical School, 48109-0626, Ann Arbor, Michigan, USA

    Mary A. Bittner & Ronald W. Holz

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  1. Mary A. Bittner
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Bittner, M.A., Holz, R.W. Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway. Cell Mol Neurobiol 13, 649–664 (1993). https://doi.org/10.1007/BF00711564

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