Abstract
Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K+, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloridetreated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K+- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment.
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References
Bilinski M, Plattner H, Matt H (1981) Secretory protein de-condensation as a distinct Ca2+-mediated event during the final steps of exocytosis in Paramecium cells. J Cell Biol 88:179–188
Breckenridge LJ, Almers W (1987) Currents through the fusion pore that forms during exocytosis of a secretory vesicle. Nature 328:814–817
Caulfield JP, Lewis RA, Hein A, Austen KF (1980) Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge. J Cell Biol 85: 299–311
Chandler DE (1988) Exocytosis and endocytosis: membrane fusion events captured in rapidly frozen cells. Curr Top Membr Trans 32:169–202
Cohen FS, Zimmerberg J, Finkelstein A (1980) Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane. J Gen Physiol 75:251–270
Cullis PR, deKruijff B, Verkleij AJ, Hope MJ (1986) Lipid polymorphism and membrane fusion. Biochem Soc Trans 14: 242–245
Curran MJ, Brodwick MS, Edwards C (1984) Direct visualization of exocytosis in mast cells. Biophys J 45:170a
Fernandez JM, Neher E, Gomperts BD (1984) Capacitance measurements reveal stepwise fusion events in degranulating mast cells. Nature 312:453–455
Finkelstein A, Zimmerberg J, Cohen FS (1986) Osmotic swelling of vesicles. Ann Rev Physiol 48:163–174
Green DPL (1982) The course of the acrosome reaction in guineapig sperm. J Cell Sci 84:161–171
Green DPL (1982) Granule swelling and membrane fusion in exocytosis. J Cell Sci 88: 547–549
Hampton RY, Holz RW (1983) Effects of changes inxosmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis. J Cell Biol 96:1082–1088
Helle K, Serck-Hansen G (1975) The adrenal medulla: a model for studies of hormonal and neuronal storage and releese mechanisms. Mol Cell Biochem 6:127–146
Hermans MP, Henquin JC (1986) Is there a role for osmotic events in the exocytotic release of insulin? Endocrinology 119:105–111
Holz RW (1986) The role of osmotic forces in exocytosis from adrenal chromaffin cells. Ann Rev Physiol 48:175–189
Holz RW, Senter RA, Sharp RR (1983) Evidence that the H+ electrochemical gradient across membranes of chromaffin granules is not involved in exocytosis. J Biol Chem 258 7506–7513
Knight DE, Baker PF (1982) Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J Membr Biol 68:107–140
Knight DE Baker PF (1985) The chromaffin granule proton pump and calcium-dependent exocytosis in bovine adrenal medullary cells. J Membr Biol 83:147–156
Kuijpers GAJ, Rosario LM, Ornberg RL (1989) Role of intracellular pH in secretion from adrenal medulla chromaffin cells. J Biol Chem 264: 698–705
Kwok R, Evans E (1981) Thermoelasticity of large lecithin bilayer membrane vesicles. Biophys J 35:637–652
Ladona MG, Bader MF, Aunis D (1987) Influence of hypertonic solutions on catecholamine release from intact and permeabilized cultured chromaffin cells. Biochim Biophys Acta 927:18–25
Loeb J (1919) Artificial parthenogenesis and fertilization, Chicago University Press Chicago pp 209–210
Lucy JA, Ahkong QH (1986) An osmotic model for the fusion of biological membranes FEBS Lett 199:1–11
Miller C, Racker E (1976) Ca2+-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J Membr Biol 30:283–300
Morgenstern E, Neumann K, Patscheke H (1987) The exocytosis of human blood palatelets. A fast freezing and freeze-substitution analysis. Eur J Cell Biol 43: 273–282
Ornberg RL, Reese TS (1981) Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes. J Cell Biol 90:40–54
Ornberg RL, Duong LT, Pollard HB (1986) Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells. Cell Tissue Res 245:547–553
Plattner H (1989) Regulation of membrane fusion during exocytosis. Intern Rev Cytol 119197–286
Pollard HB, Pazoles CJ, Creutz CE, Zinder O (1979) The chromaffin granule and possible mechanisms of exocytosis. International Rev Cytol 58:159–197
Pollard HB, Pazoles CE, Creutz CE Scott JH, Zinder OH, Hotchkiss A (1984) An osmotic mechnism for exocytosis from dissociated chromaffin cells. J Biol Chem 259:1114–1121
Pollard HB, Burns AL Rojas E, (1988) A molecular basis for synexin-driven, calcium dependent membrane fusion. J Exp Biol 139:267–286
Pollard HB, Rojas E, Pastor RW, Rojas EM, Guy HR, Burns AL (1991) Synexin: molecular mechanism of calcium-dependent membrane fusion and voltage-dependent calcium channel activity. Evidence in support of the “Hydrophobic Bridge Hypothesis” for exocytotic membrane fusion. Ann NY Acad Sci 635: 328–351
Rosario LM, Stutzin A, Cragoe EJ, Pollard HB (1991) Modulation of intracellular pH by secretagogues and the An+/H+ antiporter in cultured bovine chromaffin cells. Neuroscience 41:269–276
Satir B, Schooley C, Satir P (1973) Membrane fusion in a model system. Mucocyst secretion in Tetrahymena. J Cell Biol 56:153–176
Schmauder-Chock EA, Chock SP (1987) Mechanism of secretory granule exocytosis: can granule enlargement precede pore formation? Histochemical J 19:413–418
Stanley ER, Ehrenstein G (1985) A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles? Life Sci 37: 1985–1995
Talmon Y, Burns JL, Chestnut MH, Siegel DP (1990) Time-resolved cryotransmission electron microscopy. J Electron Micro Tech 14:6–12
Winkler H, Carmichel SW (1982) The chromaffin granule. In: Poisner AM, Trifaro JM (eds) The secretory granule. Elsevier, Amsterdam pp 3–79
Zimmerberg J, Whitaker M (1985) Irreversible swelling of secretory granules during exocytosis caused by calcium. Nature 315:581–584
Zimmerberg J, Curran M, Cohen FS, Brodwick M (1987) Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc Natl Acad Sci USA 84: 1585–1589
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Ornberg, R.L., Furuya, S., Goping, G. et al. Granule swelling in stimulated bovine adrenal chromaffin cells: Regulation by internal granule pH. Cell Tissue Res 279, 85–92 (1995). https://doi.org/10.1007/BF00300694
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DOI: https://doi.org/10.1007/BF00300694