Abstract
We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.
Full Text
The Full Text of this article is available as a PDF (779.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Breckenridge L. J., Almers W. Final steps in exocytosis observed in a cell with giant secretory granules. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1945–1949. doi: 10.1073/pnas.84.7.1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brooks J. C., Treml S. Catecholamine secretion by chemically skinned cultured chromaffin cells. J Neurochem. 1983 Feb;40(2):468–473. doi: 10.1111/j.1471-4159.1983.tb11306.x. [DOI] [PubMed] [Google Scholar]
- Busa W. B., Ferguson J. E., Joseph S. K., Williamson J. R., Nuccitelli R. Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores. J Cell Biol. 1985 Aug;101(2):677–682. doi: 10.1083/jcb.101.2.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Busa W. B., Nuccitelli R. An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis. J Cell Biol. 1985 Apr;100(4):1325–1329. doi: 10.1083/jcb.100.4.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Charbonneau M., Grey R. D. The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laevis. Dev Biol. 1984 Mar;102(1):90–97. doi: 10.1016/0012-1606(84)90177-5. [DOI] [PubMed] [Google Scholar]
- Dunn L. A., Holz R. W. Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4989–4993. [PubMed] [Google Scholar]
- Elinson R. P. Fertilization in amphibians: the ancestry of the block to polyspermy. Int Rev Cytol. 1986;101:59–100. doi: 10.1016/s0074-7696(08)60246-6. [DOI] [PubMed] [Google Scholar]
- Grey R. D., Wolf D. P., Hedrick J. L. Formation and structure of the fertilization envelope in Xenopus laevis. Dev Biol. 1974 Jan;36(1):44–61. doi: 10.1016/0012-1606(74)90189-4. [DOI] [PubMed] [Google Scholar]
- Hollinger T. G., Dumont J. N., Wallace R. A. Calcium-induced dehiscence of cortical granules in Xenopus laevis oocytes. J Exp Zool. 1979 Oct;210(1):107–115. doi: 10.1002/jez.1402100111. [DOI] [PubMed] [Google Scholar]
- Holz R. W., Senter R. A. Effects of osmolality and ionic strength on secretion from adrenal chromaffin cells permeabilized with digitonin. J Neurochem. 1986 Jun;46(6):1835–1842. doi: 10.1111/j.1471-4159.1986.tb08502.x. [DOI] [PubMed] [Google Scholar]
- Holz R. W. The role of osmotic forces in exocytosis from adrenal chromaffin cells. Annu Rev Physiol. 1986;48:175–189. doi: 10.1146/annurev.ph.48.030186.001135. [DOI] [PubMed] [Google Scholar]
- Kilpatrick D. L., Ledbetter F. H., Carson K. A., Kirshner A. G., Slepetis R., Kirshner N. Stability of bovine adrenal medulla cells in culture. J Neurochem. 1980 Sep;35(3):679–692. doi: 10.1111/j.1471-4159.1980.tb03707.x. [DOI] [PubMed] [Google Scholar]
- Kline D., Simoncini L., Mandel G., Maue R. A., Kado R. T., Jaffe L. A. Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs. Science. 1988 Jul 22;241(4864):464–467. doi: 10.1126/science.3134693. [DOI] [PubMed] [Google Scholar]
- Knight D. E., Baker P. F. Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J Membr Biol. 1982;68(2):107–140. doi: 10.1007/BF01872259. [DOI] [PubMed] [Google Scholar]
- Kopell W. N., Westhead E. W. Osmotic pressures of solutions of ATP and catecholamines relating to storage in chromaffin granules. J Biol Chem. 1982 May 25;257(10):5707–5710. [PubMed] [Google Scholar]
- Kubota H. Y., Yoshimoto Y., Yoneda M., Hiramoto Y. Free calcium wave upon activation in Xenopus eggs. Dev Biol. 1987 Jan;119(1):129–136. doi: 10.1016/0012-1606(87)90214-4. [DOI] [PubMed] [Google Scholar]
- Lew P. D., Monod A., Waldvogel F. A., Dewald B., Baggiolini M., Pozzan T. Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils. J Cell Biol. 1986 Jun;102(6):2197–2204. doi: 10.1083/jcb.102.6.2197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neher E., Marty A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6712–6716. doi: 10.1073/pnas.79.21.6712. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Njus D., Kelley P. M., Harnadek G. J. Bioenergetics of secretory vesicles. Biochim Biophys Acta. 1986;853(3-4):237–265. doi: 10.1016/0304-4173(87)90003-6. [DOI] [PubMed] [Google Scholar]
- Patzak A., Böck G., Fischer-Colbrie R., Schauenstein K., Schmidt W., Lingg G., Winkler H. Exocytotic exposure and retrieval of membrane antigens of chromaffin granules: quantitative evaluation of immunofluorescence on the surface of chromaffin cells. J Cell Biol. 1984 May;98(5):1817–1824. doi: 10.1083/jcb.98.5.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Phillips J. H., Burridge K., Wilson S. P., Kirshner N. Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells. J Cell Biol. 1983 Dec;97(6):1906–1917. doi: 10.1083/jcb.97.6.1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson D. W., Wilcox C. A., Flynn G. C., Chen E., Kuang W. J., Henzel W. J., Block M. R., Ullrich A., Rothman J. E. A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature. 1989 Jun 1;339(6223):355–359. doi: 10.1038/339355a0. [DOI] [PubMed] [Google Scholar]
- Wilson S. P., Kirshner N. Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4994–5000. [PubMed] [Google Scholar]
- Winkler H. The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience. 1976;1(2):65–80. doi: 10.1016/0306-4522(76)90001-4. [DOI] [PubMed] [Google Scholar]
- Winkler H., Westhead E. The molecular organization of adrenal chromaffin granules. Neuroscience. 1980;5(11):1803–1823. doi: 10.1016/0306-4522(80)90031-7. [DOI] [PubMed] [Google Scholar]
- Zimmerberg J., Curran M., Cohen F. S., Brodwick M. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1585–1589. doi: 10.1073/pnas.84.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]