Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Dec 1;368(Pt 2):405–413. doi: 10.1042/BJ20021090

The role of myosin in vesicle transport during bovine chromaffin cell secretion.

Patricia Neco 1, Anabel Gil 1, María Del Mar Francés 1, Salvador Viniegra 1, Luis M Gutiérrez 1
PMCID: PMC1223018  PMID: 12225290

Abstract

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.

Full Text

The Full Text of this article is available as a PDF (321.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almazan G., Aunis D., García A. G., Montiel C., Nicolás G. P., Sánchez-García P. Effects of collagenase on the release of [3H]-noradrenaline from bovine cultured adrenal chromaffin cells. Br J Pharmacol. 1984 Apr;81(4):599–610. doi: 10.1111/j.1476-5381.1984.tb16124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aunis D., Guerold B., Bader M. F., Cieselski-Treska J. Immunocytochemical and biochemical demonstration of contractile proteins in chromaffin cells in culture. Neuroscience. 1980;5(12):2261–2277. doi: 10.1016/0306-4522(80)90142-6. [DOI] [PubMed] [Google Scholar]
  3. Burke N. V., Han W., Li D., Takimoto K., Watkins S. C., Levitan E. S. Neuronal peptide release is limited by secretory granule mobility. Neuron. 1997 Nov;19(5):1095–1102. doi: 10.1016/s0896-6273(00)80400-6. [DOI] [PubMed] [Google Scholar]
  4. Chasserot-Golaz S., Hubert P., Thiersé D., Dirrig S., Vlahos C. J., Aunis D., Bader M. F. Possible involvement of phosphatidylinositol 3-kinase in regulated exocytosis: studies in chromaffin cells with inhibitor LY294002. J Neurochem. 1998 Jun;70(6):2347–2356. doi: 10.1046/j.1471-4159.1998.70062347.x. [DOI] [PubMed] [Google Scholar]
  5. Cheek T. R., Burgoyne R. D. Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells. FEBS Lett. 1986 Oct 20;207(1):110–114. doi: 10.1016/0014-5793(86)80022-9. [DOI] [PubMed] [Google Scholar]
  6. Criado M., Gil A., Viniegra S., Gutiérrez L. M. A single amino acid near the C terminus of the synaptosomeassociated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells. Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7256–7261. doi: 10.1073/pnas.96.13.7256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Côté A., Doucet J. P., Trifaró J. M. Phosphorylation and dephosphorylation of chromaffin cell proteins in response to stimulation. Neuroscience. 1986 Oct;19(2):629–645. doi: 10.1016/0306-4522(86)90286-1. [DOI] [PubMed] [Google Scholar]
  8. Espreafico E. M., Cheney R. E., Matteoli M., Nascimento A. A., De Camilli P. V., Larson R. E., Mooseker M. S. Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. J Cell Biol. 1992 Dec;119(6):1541–1557. doi: 10.1083/jcb.119.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gil A., Rueda J., Viniegra S., Gutiérrez L. M. The F-actin cytoskeleton modulates slow secretory components rather than readily releasable vesicle pools in bovine chromaffin cells. Neuroscience. 2000;98(3):605–614. doi: 10.1016/s0306-4522(00)00132-9. [DOI] [PubMed] [Google Scholar]
  10. Gil A., Viniegra S., Gutiérrez L. M. Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells. Eur J Neurosci. 1998 Nov;10(11):3369–3378. doi: 10.1046/j.1460-9568.1998.00341.x. [DOI] [PubMed] [Google Scholar]
  11. Gutierrez L. M., Ballesta J. J., Hidalgo M. J., Gandia L., García A. G., Reig J. A. A two-dimensional electrophoresis study of phosphorylation and dephosphorylation of chromaffin cell proteins in response to a secretory stimulus. J Neurochem. 1988 Oct;51(4):1023–1030. doi: 10.1111/j.1471-4159.1988.tb03063.x. [DOI] [PubMed] [Google Scholar]
  12. Gutierrez L. M., Hidalgo M. J., Palmero M., Ballesta J. J., Reig J. A., Garcia A. G., Viniegra S. Phosphorylation of myosin light chain from adrenomedullary chromaffin cells in culture. Biochem J. 1989 Dec 1;264(2):589–596. doi: 10.1042/bj2640589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gutiérrez L. M., Cànaves J. M., Ferrer-Montiel A. V., Reig J. A., Montal M., Viniegra S. A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca(2+)-dependent exocytosis in chromaffin cells. FEBS Lett. 1995 Sep 18;372(1):39–43. doi: 10.1016/0014-5793(95)00944-5. [DOI] [PubMed] [Google Scholar]
  14. Herrmann C., Wray J., Travers F., Barman T. Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor. Biochemistry. 1992 Dec 8;31(48):12227–12232. doi: 10.1021/bi00163a036. [DOI] [PubMed] [Google Scholar]
  15. Johns L. M., Levitan E. S., Shelden E. A., Holz R. W., Axelrod D. Restriction of secretory granule motion near the plasma membrane of chromaffin cells. J Cell Biol. 2001 Apr 2;153(1):177–190. doi: 10.1083/jcb.153.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson R. G., Jr Accumulation of biological amines into chromaffin granules: a model for hormone and neurotransmitter transport. Physiol Rev. 1988 Jan;68(1):232–307. doi: 10.1152/physrev.1988.68.1.232. [DOI] [PubMed] [Google Scholar]
  17. Kitani S., Teshima R., Morita Y., Ito K., Matsuda Y., Nonomura Y. Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors. Biochem Biophys Res Commun. 1992 Feb 28;183(1):48–54. doi: 10.1016/0006-291x(92)91607-r. [DOI] [PubMed] [Google Scholar]
  18. Kumakura K., Sasaki K., Sakurai T., Ohara-Imaizumi M., Misonou H., Nakamura S., Matsuda Y., Nonomura Y. Essential role of myosin light chain kinase in the mechanism for MgATP-dependent priming of exocytosis in adrenal chromaffin cells. J Neurosci. 1994 Dec;14(12):7695–7703. doi: 10.1523/JNEUROSCI.14-12-07695.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lang T., Wacker I., Wunderlich I., Rohrbach A., Giese G., Soldati T., Almers W. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells. Biophys J. 2000 Jun;78(6):2863–2877. doi: 10.1016/S0006-3495(00)76828-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lazarides E. Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells. J Cell Biol. 1976 Feb;68(2):202–219. doi: 10.1083/jcb.68.2.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Llinás R., McGuinness T. L., Leonard C. S., Sugimori M., Greengard P. Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse. Proc Natl Acad Sci U S A. 1985 May;82(9):3035–3039. doi: 10.1073/pnas.82.9.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lukas T. J., Mirzoeva S., Slomczynska U., Watterson D. M. Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge. J Med Chem. 1999 Mar 11;42(5):910–919. doi: 10.1021/jm980573a. [DOI] [PubMed] [Google Scholar]
  23. Mochida S., Kobayashi H., Matsuda Y., Yuda Y., Muramoto K., Nonomura Y. Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture. Neuron. 1994 Nov;13(5):1131–1142. doi: 10.1016/0896-6273(94)90051-5. [DOI] [PubMed] [Google Scholar]
  24. Molyneaux B. J., Langford G. M. Characterization of antibodies to the head and tail domains of squid brain myosin V. Biol Bull. 1997 Oct;193(2):222–223. doi: 10.1086/BBLv193n2p222. [DOI] [PubMed] [Google Scholar]
  25. Nakanishi A., Yoshizumi M., Hamano S., Morita K., Oka M. Myosin light-chain kinase inhibitor, 1-(5-chlornaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibits catecholamine secretion from adrenal chromaffin cells by inhibiting Ca2+ uptake into the cells. Biochem Pharmacol. 1989 Aug 15;38(16):2615–2619. doi: 10.1016/0006-2952(89)90546-7. [DOI] [PubMed] [Google Scholar]
  26. Nakanishi S., Kakita S., Takahashi I., Kawahara K., Tsukuda E., Sano T., Yamada K., Yoshida M., Kase H., Matsuda Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem. 1992 Feb 5;267(4):2157–2163. [PubMed] [Google Scholar]
  27. Ohara-Imaizumi M., Sakurai T., Nakamura S., Nakanishi S., Matsuda Y., Muramatsu S., Nonomura Y., Kumakura K. Inhibition of Ca(2+)-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells. Biochem Biophys Res Commun. 1992 Jun 30;185(3):1016–1021. doi: 10.1016/0006-291x(92)91728-9. [DOI] [PubMed] [Google Scholar]
  28. Oheim M., Stühmer W. Tracking chromaffin granules on their way through the actin cortex. Eur Biophys J. 2000;29(2):67–89. doi: 10.1007/s002490050253. [DOI] [PubMed] [Google Scholar]
  29. Perrin D., Aunis D. Reorganization of alpha-fodrin induced by stimulation in secretory cells. Nature. 1985 Jun 13;315(6020):589–592. doi: 10.1038/315589a0. [DOI] [PubMed] [Google Scholar]
  30. Reig J. A., Viniegra S., Ballesta J. J., Palmero M., Guitierrez L. M. Naphthalenesulfonamide derivatives ML9 and W7 inhibit catecholamine secretion in intact and permeabilized chromaffin cells. Neurochem Res. 1993 Mar;18(3):317–323. doi: 10.1007/BF00969089. [DOI] [PubMed] [Google Scholar]
  31. Ryan T. A. Inhibitors of myosin light chain kinase block synaptic vesicle pool mobilization during action potential firing. J Neurosci. 1999 Feb 15;19(4):1317–1323. doi: 10.1523/JNEUROSCI.19-04-01317.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steyer J. A., Almers W. Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophys J. 1999 Apr;76(4):2262–2271. doi: 10.1016/S0006-3495(99)77382-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sullivan R., Burnham M., Török K., Koffer A. Calmodulin regulates the disassembly of cortical F-actin in mast cells but is not required for secretion. Cell Calcium. 2000 Jul;28(1):33–46. doi: 10.1054/ceca.2000.0127. [DOI] [PubMed] [Google Scholar]
  34. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  35. Torgerson R. R., McNiven M. A. Agonist-induced changes in cell shape during regulated secretion in rat pancreatic acini. J Cell Physiol. 2000 Mar;182(3):438–447. doi: 10.1002/(SICI)1097-4652(200003)182:3<438::AID-JCP15>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  36. Trifaró J. M., Kenigsberg R. L., Côté A., Lee R. W., Hikita T. Adrenal paraneurone contractile proteins and stimulus-secretion coupling. Can J Physiol Pharmacol. 1984 Apr;62(4):493–501. doi: 10.1139/y84-079. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia adjunct for figure 3(A)
Download video file (396.6KB, mov)
Multimedia adjunct for figure 3(C)
Download video file (394.1KB, mov)

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES