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. 1994 Aug;14(4):315–340. doi: 10.1007/BF02088714

Concomitant protein phosphorylation and endogenous acetylcholine release induced by nicotine: Dependency on neuronal nicotinic receptors and desensitization

Enrique L M Ochoa 1,✉, Sean M O'Shea 1
PMCID: PMC11566932  PMID: 7788641

Abstract

1. Nicotine stimulated two Ca2+-dependent processes in rat frontal cortex synaptosomes: the phosphorylation of an 80-kDa protein band and the release of endogenous ACh.3 Both effects were mediated by neuronal nAChRs and coincided with depolarization of the synaptosomal plasma membrane induced by the drug. Changes in the state of phosphorylation of the 80-kDa band (presumed to contain synapsin I) were correlated with changes in the release of ACh as follows, from 2 to 4.

2. Blockade of predominant, nerve terminal P-type Ca2+ channels with ω-agatoxin-IVA, did not prevent nicotine from stimulating ACh release. In contrast, exposure to the toxin partially inhibited the release promoted by the depolarizing agent veratridine and attenuated protein phosphorylation induced by either nicotine or veratridine. Taken together, these data suggest that, upon nicotine stimulation, Ca2+ enters nerve terminals through two distinct pathways. The first, via Ca2+ channels, is necessary (but not sufficient) for both nicotine-induced phosphorylation and ACh release. The second, both necessary and sufficient for nicotine-induced phosphorylation and release, is the neuronal nAChR itself.

3. Preincubation of the synaptosomes with a subeffective concentration of nicotine inactivated both nicotine-induced ACh liberation and phosphorylation. This shows that diminished release is associated to decreased phosphorylation of the 80-kDa protein band, most likely as a consequence of nicotine-promoted nAChR desensitization.

4. Augmented ACh release and phosphorylation of the 80-kDa protein band were achieved by using the protein phosphatase inhibitor okadaic acid. However, okadaic acid did not summate with either nicotine or veratridine to increase ACh release further. This is probably because okadaic acid, as in other neurons, increases intracellular Ca2+ (Cholewinskiet al., 1993), thus promoting desensitization of ACh release.

Key words: nicotine dependence, nicotine-induced desensitization, protein phosphorylation, synapsins

Abbreviations used

DCFscein

2′,7′-dichlorofluorescein

CDFDA

2′,7′-dichlorofluorescin diacetate

DCFscin

2′,7′-dichlorofluorescin

Ach

acetylcholine

AChE

acetylcholinesterase

α-BuTx

α-bungarotoxin

Ca2+-free HBS

calcium-free HEPES buffered saline

Cho

choline oxidase

κ-BuTx

κ-bungarotoxin

nAChRs

nicotinic acetylcholine receptors

OKA

okadaic acid

ω-ATX

ω-agatoxin IVA

PEG

polyethylene glycol

PP

protein phosphatase

Em

synaptosomal membrane potential

TPP+

tetra-[14C]phenylphosphonium ion

TTX

tetrodotoxin

DTT

dithiothreitol

SDS

sodium dodecyl sulfate

PAGE

polyacrylamide gel electrophoresis

References

  1. Abdul, G. M., Kravitz, E. A., Meiri, H., and Rahamimoff, R. (1991). Protein phosphatase inhibitor okadaic acid enhances transmitter release at neuromuscular junctions.Proc. Natl. Acad. Sci. USA88:1803–1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams, D. J., Takeda, K., and Umbach, J. A. (1985). Inhibitors of calcium buffering depress evoked transmitter release at the squid giant synapse.J. Physiol. (Lond.)369:145–159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benowitz, N. (1990). Pharmacokinetic considerations in understanding nicotine dependence. In,The Biology of Nicotine Dependence, Ciba Foundation Symposium 152, Wiley, Chichester, pp. 186–209. [DOI] [PubMed] [Google Scholar]
  4. Cholewinski, A., Burgess, G. M., and Bevan, S. (1993). The role of calcium in capsaicin-induced desensitization in rat cultured dorsal root ganglion neurons.Neuroscience55:1015–1023. [DOI] [PubMed] [Google Scholar]
  5. Cohen, P., Holmes, C. F. B., and Tsukitani, Y. (1990). Okadaic acid—A new probe for the study of cellular regulation.Trends Biochem. Sci.15:98–102. [DOI] [PubMed] [Google Scholar]
  6. Drapeau, P. (1988). Long-term storage of functional, isolated nerve endings by slow freezing and rapid thawing.J. Neurosci. Meth.24:111–115. [DOI] [PubMed] [Google Scholar]
  7. Enriquez, J. A., Sanchez-Prieto, J., Muino Blanco, M. T., Hernandez-Yago, J., and Lopez-Perez, M. J. (1990). Rat brain synaptosomes prepared by phase partition.J. Neurochem.55:1841–1849. [DOI] [PubMed] [Google Scholar]
  8. Firestone, J. A., and Browning, M. D. (1992). Synapsin II phosphorylation and catecholamine release in bovine adrenal chromaffin cells: Additive effects of histamine and nicotine.J. Neurochem.58:441–447. [DOI] [PubMed] [Google Scholar]
  9. Flores, C. M., Rogers, S. W., Pabreza, L. A., Wolfe, B. B., and Kellar, K. J. (1992). A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits and is up-regulated by chronic nicotine treatment.Mol. Pharmacol.41:31–37. [PubMed] [Google Scholar]
  10. Grady, S. R., Marks, M. J., and Collins, A. C. (1994). Desensitization of nicotine-stimulated [3H]dopamine release from mouse striatal synaptosomes.J. Neurochem.62:1390–1398. [DOI] [PubMed] [Google Scholar]
  11. Greengard, P., Valtorta, F., Czernik, A. J., and Benfenati, F. (1993). Synaptic versicle phosphoproteins and regulation of synaptic function.Science259:780–785. [DOI] [PubMed] [Google Scholar]
  12. Haycock, J. W., Greengard, P., and Browning, M. D. (1988). Cholinergic regulation of protein III phosphorylation in bovine adrenal chromaffin cells.J. Neurosci.8:3233–3239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hillard, C. J., and Graf, W. K. (1991). Studies of the effect of nicotine on synaptosomal calcium accumulation using FURA-2.Nida Res. Monogr.105:331–332. [PubMed] [Google Scholar]
  14. Hillard, C. J., and Pounds, J. J. (1991). [3H]-Tetraphenylphosphonium accululation in cerebral cortical synaptosomes as a measure of nicotine-induced changes in membrane potential.J. Pharmacol. Exp. Ther.259:1118–1123. [PubMed] [Google Scholar]
  15. Israel, M., Meunier, F. M., Morel, N., and Lesbats, B. (1987). Calcium-induced desensitization of acetylcholine release from synaptosomes or proteoliposomes equipped with mediatophore, a presynaptic membrane protein.J. Neurochem.49:975–982. [DOI] [PubMed] [Google Scholar]
  16. Jessell, T. M., and Kandel, E. R. (1993). Synaptic transmission: A bidirectional and self-modifiable form of cell-cell communication.Cell 1–30. 72 Suppl. [DOI] [PubMed]
  17. Johnston, M. V., McKinney, M., and Coyle, J. T. (1979). Evidence for a cholinergic projection to neocortex from neurons in basal forebrain.Proc. Natl. Acad. Sci. USA76:5392–5396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Katz, B., and Miledi, R. (1969). Tetrodotoxin-resistant electric activity in pre-synaptic terminals.J. Physiol. (Lond.)203:459–487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krueger, B. K., Forn, J., and Greengard, P. (1977). Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.J. Biol. Chem.252:2764–2773. [PubMed] [Google Scholar]
  20. Llinas, R., Gruner, J. A., Sugimori, M., McGuinness, T. L., and Greengard, P. (1991). Regulation by synapsin I and Ca(2+)-calmodulin-dependent protein kinase II of transmitter release in squid giant synapse.J. Physiol. (Lond.)436:257–282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Llinas, R., Sugimori, M., and Silver, R. B. (1992). Microdomains of high calcium concentration in a presynaptic terminal.Science256:677–679. [DOI] [PubMed] [Google Scholar]
  22. Marks, M. J., and Collins, A. C. (1993). Desensitization of nicotine-induced86Rb+ efflux.Soc. Neurosci. Abstr.19:289. [Google Scholar]
  23. Marks, M. J., Burchs, J. B., and Collins, A. C. (1983). Effects of chronic nicotine infusion on tolerance development and cholinergic receptors.J. Pharmacol. Exp. Ther.226:806–816. [PubMed] [Google Scholar]
  24. Marks, M. J., Pauly, J. R., Gross, S. D., Deneris, E. S., Hermans-Borgmeyer, I., Heinemann, S. F., and Collins, A. C. (1992). Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment.J. Neurosci.12:2765–2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mintz, I. M., Venema, V. J., Swinderek, K. M., Lee, T. D., Bean, B. P., and Adams, M. E. (1992). P-type calcium channels blocked by the spider toxin omega-Aga-IVA.Nature355:827–829. [DOI] [PubMed] [Google Scholar]
  26. Mulle, C., Choquet, D., Korn, H., and Changeux, J. P. (1992). Calcium influx through nicotinic receptor in rat central neurons: Its relevance to cellular regulation.Neuron8:135–143. [DOI] [PubMed] [Google Scholar]
  27. Nairn, A. C., and Shenolikar, S. (1992). The role of protein phosphatases in synaptic transmission, plasticity, and neuronal development.Curr. Opin. Neurobiol.2:296–301. [DOI] [PubMed] [Google Scholar]
  28. Nichols, R. A., Chilcote, T. J., Czernik, A. J., and Greengard, P. (1992). Synapsin-I regulates glutamate release from rat brain synaptosomes.J. Neurochem.58:783–785. [DOI] [PubMed] [Google Scholar]
  29. Ochoa, E. L., Li, L., and McNamee, M. G. (1990). Desensitization of central cholinergic mechanisms and neuroadaptation to nicotine.Mol. Neurobiol.4:251–287. [DOI] [PubMed] [Google Scholar]
  30. Ochoa, E. L. M., Wennberg, R. P., An, Y., Tandon, T., Takashima, T., Nguyen, T., and Chui, A. (1993). Interactions of bilirubin with isolated presynaptic nerve terminals: Functional effects on the uptake and release of neurotransmitters.Cell. Mol. Neurobiol.13:69–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Shea, S. M., and Ochoa, E. L. M. (1993). Nicotine-induced synapsin I phosphorylation and endogenous acetylcholine release in cholinergic nerve endings.Soc. Neurosci. Abstr.19:902. [Google Scholar]
  32. Parfitt, K. D., Hoffer, B. J., and Browning, M. D. (1991). Norepinephrine and isoproterenol increase the phosphorylation of synapsin I and synapsin II in dentate slices of young but not aged Fisher 344 rats.Proc. Natl. Acad. Sci. USA88:2361–2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parfitt, K. D., Doze, V. A., Madison, D. V., and Browning, M. D. (1992). Isoproterenol increases the phosphorylation of the synapsins and increases synaptic transmission in dentate gyrus, but not in area CA1, of the hippocampus.Hippocampus2:59–64. [DOI] [PubMed] [Google Scholar]
  34. Patrick, J., Sequela, P., Vernino, S., Amador, M., Luetje, C., and Dani, J. A. (1993). Functional diversity of neuronal nicotinic acetylcholine receptors.Progr. Brain Res.98:113–120. [DOI] [PubMed] [Google Scholar]
  35. Protti, D. A., and Uchitel, O. D. (1993). Transmitter release and presynaptic Ca2+ currents blocked by the spider toxin omega-AGA-IVA.Neuroreport5:333–336. [DOI] [PubMed] [Google Scholar]
  36. Rapier, C., Lunt, G. G., and Wonnacott, S. (1988). Stereoselective nicotine-induced release of dopamine from striatal synaptosomes: Concentration dependence and repetitive stimulation.J. Neurochem.50:1123–1130. [DOI] [PubMed] [Google Scholar]
  37. Robinson, P. J. (1991). The role of protein kinase C and its neuronal substrates dephosphine, B-50, and MARCKS in neurotransmitter release.Mol. Neurobiol.5:87–130. [DOI] [PubMed] [Google Scholar]
  38. Rowell, P. P. (1987). Current concepts on the effects of nicotine on neurotransmitter release in the central nervous system. InTobacco Smoking and Nicotine: A Neurobiological Approach (W. R. Martin, G. R. Van Loon, E. T. Iwamoto, and L. Davis, Eds.), Plenum Press, New York and London, pp. 191–208. [Google Scholar]
  39. Rowell, P. P., and Hillebrand, J. A. (1993). Desensitization of nicotine-stimulated [3H]-dopamine release from superfused rat striatal synaptosomes.Soc. Neurosci. Abstr.19:289. [Google Scholar]
  40. Russell, M. A. H., Jarvis, M., Iyer, R., and Feyerabend, C. (1980). Relation of nicotine yield of cigarettes to blood nicotine concentrations in smokers.Br. Med. J.280:972–975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanderson, E. M., Drasdo, A. L., McCrea, K., and Wonnacott, S. (1993). Upregulation of nicotinic receptors following continuous infusion of nicotine is brain-region-specific.Brain Res.617:349–352. [DOI] [PubMed] [Google Scholar]
  42. Sargent, P. B. (1993). The diversity of neuronal nicotinic acetylcholine receptors.Annu. Rev. Neurosci.16:403–443. [DOI] [PubMed] [Google Scholar]
  43. Schwartz, R. D., and Kellar, K. J. (1985). In vivo regulation of [3H] acetylcholine recognition sites in brain by nicotinic cholinergic drugs.J. Neurochem.45:427–433. [DOI] [PubMed] [Google Scholar]
  44. Seguela, P., Wadiche, J., Dineley, M. K., Dani, J. A., and Patrick, J. W. (1993). Molecular cloning, functional properties, and distribution of rat brain alpha 7: A nicotinic cation channel highly permeable to calcium.J. Neurosci.13:596–604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sihra, T. S., Bogonez, E., and Nicholls, D. G. (1992). Localized Ca2+ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release.J. Biol. Chem.267:1983–1989. [PubMed] [Google Scholar]
  46. Tandon, T., and Ochoa, E. L. M. (1992). Calcium and nicotine-induced desensitization of endogenous acetylcholine release from mammalian brain cholinergic nerve endings.Soc. Neurosci. Abstr.18:634. [Google Scholar]
  47. Uchitel, O. D., Protti, D. A., Sanchez, V., Cherksey, B. D., Sugimori, M., and Llinas, R. (1992). P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.Proc. Natl. Acad. Sci. USA89:3330–3333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vernino, S., Amador, M., Luetje, C. W., Patrick, J., and Dani, J. A. (1992). Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.Neuron8:127–134. [DOI] [PubMed] [Google Scholar]
  49. Vidal, C., and Changeux, J. P. (1993). Nicotinic and muscarinic modulations of excitatory synaptic transmission in the rat prefrontal cortex in vitro.Neuroscience56:23–32. [DOI] [PubMed] [Google Scholar]
  50. Wonnacott, S., Irons, J., Rapier, C., Thorne, B., and Lunt, G. G. (1989). Presynatpic modulation of transmitter release by nicotinic receptors. InProgr. Brain Res. (A. Nordberg, K. Fuxe, B. Holmstedt, and A. Sundwall, Eds.), Elsevier, Amsterdam, pp. 157–163. [DOI] [PubMed] [Google Scholar]
  51. Zhang, Z. W., Vijayaraghavan, S., and Berg, D. K. (1994). Neuronal acetylcholine receptors that bind alpha-bungarotoxin with high affinity function as ligand-gated ion channels.Neuron12:167–177. [DOI] [PubMed] [Google Scholar]
  52. Zoccarato, F., Deana, R., Cavallini, L., and Alexandre, A., (1989). Generation of hydrogen peroxide by cerebral-cortex synaptosomes.Eur. J. Biochem.180:473–478. [DOI] [PubMed] [Google Scholar]
  53. Zucker, R. S., and Haydon, P. G. (1988). Membrane potential has no direct role in evoking neurotransmitter release.Nature335:360–362. [DOI] [PubMed] [Google Scholar]

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