Skip to main content
SpringerLink
Log in

4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Recently, we have shown that stimulation of [3H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K+, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 μM to 5 mM. 4-AP (100 μM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K+ for 15 seconds. The effects of both 4-AP or K+ depolarization on B-50 phosphorylation were abolished at low extracellular Ca2+ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K+ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca2+-dependent transmitter release evoked by 4-AP.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Allgaier C., Von Kügelgen O., Hertting G. (1986). Enhancement of noradrenaline release by 12-O-tetradecanoyl phorbol-13-acetate, an activator of protein kinase C. Eur. J. Pharmacol. 129:389–392

    Article  PubMed  CAS  Google Scholar 

  • Augustine, G.J., Charlton M.P., Smith S.J. (1987). Calcium action in synaptic transmitter release. Annu. Rev. Neurosci. 10:633–693

    Article  PubMed  CAS  Google Scholar 

  • Agoston D., Hargittai P., Nagy A. (1983). Effects of 4-aminopyridine in calcium movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex. J. Neurochem. 41:745–751

    Article  PubMed  CAS  Google Scholar 

  • Basi G.S., Jacobson R.D., Virág I., Schilling, J., Skene J.H.P. (1987). Primary structure and transcriptional regulation of GAP43, a protein associated with nerve-growth. Cell 49:785–791

    Article  PubMed  CAS  Google Scholar 

  • Benowitz L.I., Routtenberg A. (1987). A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity. Trends Neurosci. 10:527–532

    Article  CAS  Google Scholar 

  • Bradford M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem. 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Buckle, P.J., Haas H.L. (1982). Enhancement of synaptic transmission by 4-aminopyridine in hippocampal slices of the rat. J. Physiol. 326:109–122

    PubMed  CAS  Google Scholar 

  • Cimler B.M., Giebelhaus D.H., Wakim B.T., Storm D.R., Moon R.T. (1987). Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein. J. Biol. Chem. 262:12158–12163

    PubMed  CAS  Google Scholar 

  • Colbran R.J., Schworer C.M., Hashimoto Y., Fong Y.-L., Rich, D.P., Smith M.K., Soderling T.R. (1989). Calcium/calmodulin-dependent protein kinase II. Biochem J. 258:313–325

    PubMed  CAS  Google Scholar 

  • Cotman C.W., Haycock J.W., White F.F. (1976). Stimulus-secretion processes in brain: Analysis of noradrenaline and GABA release. J. Physiol. London 254:475–505

    PubMed  CAS  Google Scholar 

  • De Graan P.N.E., Heemskerk F.M.J., Dekker L.V., Melchers B.P.C., Gianotti C., Schrama L.H. (1988). Phorbol esters induce long- and short-term enhancement of B-50/GAP-43 phosphorylation in rat hippocampal slices. Neurosci. Res. Commun. 3:175–182

    Google Scholar 

  • De Graan P.N.E., Dekker, L.V., Oestreicher, A.B., Van der Voorn L., Gispen W.H. (1989). Determination of changes in the phosphorylation state of the neuron-specific protein kinase C substrate B-50 (GAP43) by quantitative immunoprecipitation. J. Neurochem. 52:17–23

    Article  PubMed  Google Scholar 

  • Dekker L.V., De Graan P.N.E., Versteeg D.H.G., Oestreicher A.B., Gispen W.H. (1989). Phosphorylation of B-50 (GAP43) is correlated with neurotransmitter release in rat hippocampal slices. J. Neurochem. 52:24–30

    Article  PubMed  CAS  Google Scholar 

  • Dekker L.V., De Graan P.N.E., De Wit M., Hens J.J.H., Gispen W.H. (1990). Depolarization-induced phosphorylation of the protein kinase C substrate B-50 (GAP43) in rat cortical synaptosomes. J. Neurochem. 54:in press

  • Diaz-Guerra M.J.M., Sánchez-Prieto J., Bosca L., Pocock J., Barrie A., Nicholls D. (1988). Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release. Biochim. Biophys. Acta 970:157–165

    Article  PubMed  CAS  Google Scholar 

  • Doležal V., Tuçek S. (1983). The effects of 4-aminopyridine and tetrodotoxin on the release of acetylcholine from rat striatal slices. Naunyn-Schiedeberg's Arch. Pharmacol. 323:90–95

    Article  Google Scholar 

  • Dunkley P.R., Robinson P.J. (1986). Depolarisation-dependent protein phosphorylation in synaptosomes: Mechanisms and significance. Prog. Brain Res. 69:273–293

    Article  PubMed  CAS  Google Scholar 

  • Dunkley P.R., Heath J.W., Harrison S.M., Jarvie P.E., Glenfield P.J., Rostas A.P. (1988). A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1-fraction: Homogeneity and morphology of subcellular fractions. Brain Res. 441:59–71

    Article  PubMed  CAS  Google Scholar 

  • Foldes F.F., Ludvig N., Nagashima H., Vizi E.S. (1988). The influence of aminopyridines on Ca2+-dependent evoked release of acetylcholine from rat cortex slices. Neurochem. Res. 13:761–764

    Article  PubMed  CAS  Google Scholar 

  • Gibson G.E., Manger T. (1988). Changes in cytosolic free calcium with 1,2,3,4-tetrahydro-5-aminoacridine, 4-aminopyridine and 3,4-diaminopyridine. Biochem. Pharmacol. 37:4191–4196

    Article  PubMed  CAS  Google Scholar 

  • Gispen W.H., Leunissen J.L.M., Oestreicher A.B., Verkleij A.J., Zwiers H. (1985). Presynaptic localization of B-50 phosphoprotein: The (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism. Brain Res. 328:381–385

    Article  PubMed  CAS  Google Scholar 

  • Gispen W.H., De Graan P.N.E., Chan S.Y., Routtenberg A. (1986). Comparison between the neural acidic proteins B-50 and F1. Prog. Brain Res. 69:383–386

    PubMed  CAS  Google Scholar 

  • Heemskerk F.M.J., Schrama L.H., De Graan P.N.E., Ghijsen W.E.J.M., Lopes da Silva F.H., Gispen W.H. (1989a). 4-Aminopyridine increases B-50 (GAP-43) phosphorylation and calcium levels in rat brain synaptosomes. Am. Neurosci. Abstr. 189. 15

    Google Scholar 

  • Heemskerk F.M.J., Schrama L.H., Ghijsen W.E.J.M., De Graan P.N.E., Lopes da Silva F.H., Gispen W.H. (1989b). Increased B-50/GAP-43 phosphorylation and Ca2+ levels in synaptosomes by 4-aminopyridine. J. Neurochem. Suppl. 52:106B

    Google Scholar 

  • Heemskerk F.M.J., Schrama L.H., Gianotti C., Spierenburg H., Versteeg D.H.G., De Graan P.N.E., Gispen W.H. (1990). 4-Aminopyrdine stimulates B-50/GAP-43 phosphorylation and [3H]-noradrenaline release in rat hippocampal slices. J. Neurochem. 54: in press

  • Hertting G., Allgaier C. (1988). Participation of protein kinase C and regulatory G proteins in modulation of the evoked noradrenaline release in brain. Cell. Mol. Neurobiol. 8:105–114

    Article  PubMed  CAS  Google Scholar 

  • Kaczmarek L.K. (1987). The role of PKC in regulation of ion channels and neurotransmitter release. Trends Neurosci. 10:30–34.

    Article  CAS  Google Scholar 

  • Llinás R., McGuiness T.L., Leonard C.S., Sugimori M., Greengard P. (1985). Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release in the squid giant synapse. Proc. Natl. Acad. Sci. U.S.A. 82:3035–3039

    Article  PubMed  Google Scholar 

  • Löffelholz K., Weide W. (1982). Aminopyridines and the release of acetylcholine. Trends Pharmacol. Sci. 4: 147–149

    Article  Google Scholar 

  • Lundh H., Thesleff S. (1977). The mode of action of 4-aminopyridine and guanidine on transmitter release from motor nerve terminals. Eur. J. Pharmacol. 42: 411–412

    Article  PubMed  CAS  Google Scholar 

  • Nachshen D.A. (1985). Regulation of cytosolic calcium concentration in presynaptic nerve endings isolated from rat brain. J. Physiol. 363:87–101

    PubMed  CAS  Google Scholar 

  • Nachshen D.A., Blaustein M.P. (1980). Some properties of potassium-stimulated calcium influx in presynaptic nerve endings. J. Gen. Physiol. 76:709–728

    Article  PubMed  CAS  Google Scholar 

  • Nicholls D.G., Tibbs G., Barrie A.P. (1989). Cytosolic free calcium in synaptosomes and its coupling to glutamate exocytosis. J. Neurochem. Suppl. 52:47D

    Google Scholar 

  • Nielander H.B., Schrama L.H., Van Rozen A.J., Kasperaitis M., Oestreicher A.B., De Graan P.N.E., Gispen W.H., Schotman P. (1987). Primary structure of the neuron-specific phosphoprotein B-50 is identical to growth-associated protein GAP-43. Neurosci. Res. Commun. 1:163–172

    CAS  Google Scholar 

  • Peterson C., Gibson G.E. (1983). Aging and 3,4-diamino-pyridine alter synaptosomal calcium uptake. J. Biol. Chem. 258:11482–11486

    PubMed  CAS  Google Scholar 

  • Peterson C., Gibson G.E. (1985). Synaptosomal calcium metabolism during hypoxia and 3,4-diaminopyridine treatment. J. Neurochem. 42:248–253

    Article  Google Scholar 

  • Rodnight R., Perrett C. (1986). Protein phosphorylation and synaptic transmission: Receptor mediated modulation of protein kinase C in a rat brain fraction enriched in synaptosomes. J. Physiol. (Paris) 81:340–348

    CAS  Google Scholar 

  • Rogawski M.A. (1985). The A-current: How ubiquitous a feature of excitable cells is it? Trends Neurosci. 5: 214–219

    Article  Google Scholar 

  • Rogawski M.A., Barker J.L. (1983). Effects of 4-amino-pyridine on calcium action potentials and calcium current under voltage clamp in spinal neurons. Brain Res. 280:180–185

    Article  PubMed  CAS  Google Scholar 

  • Rutecki P.A., Lebeda F.J., Johnston D. (1987). 4-Amino-pyridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition. J. Neurophysiol. 57:1922–1924

    Google Scholar 

  • Schrama L.H., Edwards P.M., Schotman P. (1984). Modulation of protein synthesis in a cell-free system derived from rat brain by corticotropin (ACTH), magnesium, and spermine. J. Neurosci. Res. 11:67–77

    Article  PubMed  CAS  Google Scholar 

  • Segal M., Rogawski M.A., Barker J.L. (1984). A transient potassium conductance regulates the excitability of cultured hippocampal and spinal neurons. J. Neurosci. 4:604–609

    PubMed  CAS  Google Scholar 

  • Skene J.H.P. (1989). Axonal growth-associated proteins. Annu. Rev. Neurosci. 12:127–156

    Article  PubMed  CAS  Google Scholar 

  • Sörensen R.G., Kleine L.P., Mahler H.R. (1981). Presynaptic localization of phosphoprotein B-50. Brain Res. Bull. 7:57–61

    Article  PubMed  Google Scholar 

  • Szente M., Baranyi A. (1987). Mechanism of aminopyridine-induced ictal seizure activity in the cat neocortex. Brain Res. 413:368–373

    Article  PubMed  CAS  Google Scholar 

  • Tapia R., Sitges M. (1982). Effect of 4-aminopyridine on transmitter release in synaptosomes. Brain Res. 250: 291–299

    Article  PubMed  CAS  Google Scholar 

  • Tapia R., Sitges M., Morales E. (1985). Mechanism of the calcium-dependent stimulation of transmitter release by 4-aminopyridine in synaptosomes. Brain Res. 361:373–382

    Article  PubMed  CAS  Google Scholar 

  • Tibbs G.R., Dolly J.O., Nicholls D.G. (1989). Dendrotoxin, 4-aminopyridine and β-bungarotoxin act at common loci but by two distinct mechanisms to induce Ca2+-dependent release of glutamate from guinea-pig cerebrocortical synaptosomes. J. Neurochem. 52:201–206

    Article  PubMed  CAS  Google Scholar 

  • Thesleff S. (1980). Aminopyridines and synaptic transmission. Neuroscience 5:1413–1419

    Article  PubMed  CAS  Google Scholar 

  • Van Lookeren Campagne M., Oestreicher A.B., Van Bergen en Henegouwen P.M.P., Gispen W.H. (1989). Ultrastructural immunocytochemical localization of B-50/GAP43, a protein kinase C substrate, in isolated presynaptic nerve terminals and neuronal growth cones. J. Neurocytol. 18:479–489

    Article  Google Scholar 

  • Versteeg D.H.G., Florijn W.J. (1986). Phorbol 12,13-dibutyrate enhances electrically stimulated neuromessenger release from rat dorsal hippocampal slices in vitro. Life Sci. 40:1237–1243

    Article  Google Scholar 

  • Versteeg D.H.G., Ulenkate H.J.L.M. (1987). Basal and electrically stimulated release of [3H]noradrenaline and [3H]dopamine from rat amygdala slices in vitro: Effects of 4-phorbol 12,13-dibutyrate, 4α-12,13-didecanoate and polymyxin B. Brain Res. 416:343–348

    Article  PubMed  CAS  Google Scholar 

  • Voskuyl R.A., Albus H. (1985). Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine. Brain Res. 342:54–66

    Article  PubMed  CAS  Google Scholar 

  • Wang J.K.T., Walaas S.I., Greengard P. (1988). Protein phosphorylation in nerve terminals: Comparison of calcium/calmodulin-dependent and calcium/diacyl-glycerol-dependent systems. J. Neurosci. 8:281–288

    PubMed  CAS  Google Scholar 

  • Wu W.C.-S., Walaas S.I., Nairn A.C., Greengard P. (1982). Calcium/phospholipid regulates phosphorylation of a Mr “87 k” substrate protein in rat brain synaptosomes. Proc. Natl. Acad. Sci. U.S.A. 79:5249–5253

    Article  PubMed  CAS  Google Scholar 

  • Zwiers H., Schotman P., Gispen W.H. (1980). Purification and some characteristics of an ACTH-sensitive protein kinase and its substrate protein in rat brain membranes. J. Neurochem. 34:1689–1699

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Molecular Neurobiology, Institute for Molecular Biology and Medical Biotechnology, Rudolf Magnus Institute, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands

    F. M. J. Heemskerk, L. H. Schrama, P. N. E. De Graan & W. H. Gispen

  2. Laboratory of Physiological Chemistry, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands

    F. M. J. Heemskerk, L. H. Schrama, P. N. E. De Graan & W. H. Gispen

Authors
  1. F. M. J. Heemskerk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. H. Schrama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. N. E. De Graan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. W. H. Gispen
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This research was supported by CLEO-TNO grant A66 of the Dutch Epilepsy Foundation.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Heemskerk, F.M.J., Schrama, L.H., De Graan, P.N.E. et al. 4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes. J Mol Neurosci 2, 11–17 (1990). https://doi.org/10.1007/BF02896921

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02896921

Keywords

Search

Navigation