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Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

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Summary

By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water.

Cells rendered ‘leaky’ in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10−8 m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5 m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μm Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions.

Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8 m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5 m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles.

Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca.

Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−>acetate−>Cl−>Br−>SCN−.

Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.

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References

  • Alexander, J. 1937. Colloid Chemistry. p. 143. Dvan Nostrand Co., New York

    Google Scholar 

  • Amy, C.M., Kirshner, N. 1981. Phosphorylation of adrenal medulla cell proteins in conjunction with stimulation of catecholamine secretion.J. Neurochem. 36:847–854

    Google Scholar 

  • Aunis, D., Bouclier, M., Peschelocke, M., Mandel, P. 1977. Properties of membrane bound dopamine-β-hydroxylase in chromaffin granules from bovine adrenal medulla.J. Neurochem. 29:439–447

    Google Scholar 

  • Aunis, D., Serck-Hanssen, G., Helle, K.B. 1978. Dopamine-β-hydroxylase in perfusates of stimulated bovine adrenals: Isolation and characterisation of the released enzyme.Gen. Pharmacol. 9:37

    Google Scholar 

  • Baker, P.F. 1972. Transport and metabolism of calcium ions in nerve.Prog. Biophys. Mol. Biol. 24:177–223

    Google Scholar 

  • Baker, P.F. 1977. Calcium and the control of neurosecretion.In: Biochemistry of Membrane Transport. FEBS Symposium 42. pp 430–441. Springer-Verlag, Berlin

    Google Scholar 

  • Baker, P.F., Hodgkin, A.L., Ridgway, E.B. 1971. Depolarization and calcium entry into squid giant axons.J. Physiol. (London) 218:707–755

    Google Scholar 

  • Baker, P.F., Knight, D.E. 1978a. A high voltage technique for gaining rapid access to the interior of secretory cells.J. Physiol. (London) 284:30P

    Google Scholar 

  • Baker, P.F., Knight, D.E. 1978b. Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes.Nature (London) 276:620–622

    Google Scholar 

  • Baker, P.F., Knight, D.E. 1979a. Calcium-dependent exocytosis has a specific requirement for Mg-ATP.J. Physiol. (London) 295:89P

    Google Scholar 

  • Baker, P.F., Knight, D.E. 1979b. Influence of anions on exocytosis in leaky bovine adrenal medullary cells.J. Physiol. 296:106P

    Google Scholar 

  • Baker, P.F., Knight, D.E. 1981. Calcium control of exocytosis and endocytosis in bovine adrenal medulary cells.Phil. Trans. R. Soc. London B 296:83–103

    Google Scholar 

  • Baker, P.F., Knight, D.E., Whitaker, M.J. 1980. The relation between ionized calcium and cortical granule exocytosis in eggs of the sea urchinEchinus esculentus.Proc. R. Soc. London B 207:149–161

    Google Scholar 

  • Baker, P.F., Rink, T.J. 1975. Catecholamine release from bovine adrenal medulla in response to maintained depolarization.J. Physiol. (London) 253:593–620

    Google Scholar 

  • Banks, P. 1965. Effects of stimulation by carbachol on the metabolism of the bovine adrenal medulla.Biochem. J. 97:555–560

    Google Scholar 

  • Benz, R., Beckers, F., Zimmermann, U. 1979. Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study.J. Membrane Biol. 48:181–204

    Google Scholar 

  • Benz, R., Zimmermann, U. 1980. The resealing process of lipid bilayers after reversible electrical breakdown.Biochim. Biophys. Acta 640:169

    Google Scholar 

  • Bergmeyer, H.-U., Bernt, E., Hess, B. 1965. Lactate dehydrogenase.In: Methods of Enzymatic Analysis. H.U. Bergmeyer, editor. pp. 736–743. Academic Press, New York

    Google Scholar 

  • Blioch, Z.L., Glagoleva, I.M., Liberman, E.A., Nenashev, V.A. 1968. A study of the mechanism of quantal transmitter release at a chemical synapse.J. Physiol. (London) 199:11–35

    Google Scholar 

  • Brandt, B.L., Hagiwara, S., Kidokoro, Y., Miyazaki, S. 1976. Action potentials in the rat chromaffin cell and effects of acetylcholine.J. Physiol. (London) 263:417–439

    Google Scholar 

  • Ceccarelli, B., Hurlbut, W.P. 1980. Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction.J. Cell. Biol. 87:297–303

    Google Scholar 

  • Cole, K.S. 1928. Electric impedance of suspensions of spheres.J. Gen. Physiol. 12:29–36

    Google Scholar 

  • Cole, K.S. 1935. Electrical impedance of Hipponoë eggs.J. Gen. Physiol. 18:877–887

    Google Scholar 

  • Coster, H.G.L., Zimmermann, U. 1975a. The mechanism of electrical breakdown in the membranes ofValonia utricularis.J. Membrane Biol. 22:73–90

    Google Scholar 

  • Coster, H.G.L., Zimmermann, U. 1975b. Dielectric breakdown in the membranes ofValonia utricularis.: The role of energy dissipation.Biochim. Biophys. Acta 382:410–418

    Google Scholar 

  • Crowley, J.N. 1973. Electrical breakdown of bimolecular lipid membranes as an electromechanical instability.Biophys. J. 13:711–724

    Google Scholar 

  • Davis, B., Lazarus, N.R. 1976. Anin vitro system for studying insulin release caused by secretory granule-plasma membrane interaction: Definition of the system.J. Physiol. (London) 256:709–729

    Google Scholar 

  • De Lorenzo, R.J., Freidman, S.D., Yohe, W.B., Maurer, S.C. 1979. Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles.Proc. Natl. Acad. Sci. USA 76:1838–1842

    Google Scholar 

  • Douglas, W.W., Sorimachi, M. 1972. Colchicine inhibits adrenal medullary secretion evoked by acetylcholine without affecting that evoked by potassium.Br. J. Pharmacol. 45:129–132

    Google Scholar 

  • Eimerl, S., Savion, N., Heichal, O., Selinger, Z. 1974. Induction of enzyme secretion in rat pancreatic slices using the ionphore A23187 and calcium.J. Biol. Chem. 249:3991–3993

    Google Scholar 

  • Feinman, R.D., Detwiler, T.C. 1974. Platelet secretion induced by divalent cation ionophore.Nature (London) 249:172–173

    Google Scholar 

  • Fenwick, E.M., Fajdiga, P.B., Howe, N.B.S., Livett, B.G. 1978. Functional and morphological characterization of isolated bovine adrenal medullary cells.J. Cell. Biol. 76:12–30

    Google Scholar 

  • Fried, R.C., Blaustein, M.P. 1978. Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes).J. Cell. Biol. 78:685–700

    Google Scholar 

  • Foreman, J.C., Mongar, J.C., Gomperts, B.D. 1973. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process.Nature (London) 245:249–251

    Google Scholar 

  • Fridovich, I. 1963. Inhibition of acetoacetic decarboxylase by anions.J. Biol. Chem. 283:592–598

    Google Scholar 

  • Gilkey, J.C., Jaffe, L.F., Ridgway, E.B., Reynolds, G.T. 1978. The free calcium wave traverses the activating egg of the medakaOryzias latipes.J. Cell. Biol. 76:438–446

    Google Scholar 

  • Gillespie, J.I. 1979. The effect of repetitive stimulation on the passive electrical properties of the presynaptic terminal of the squid giant synapse.Proc. R. Soc. London B206:293–306

    Google Scholar 

  • Gratzl, M., Dahl, G., Russell, J.T., Thorn, N.A. 1977. Fusion of neurohypophyseal membranesin vitro.Biochim. Biophys. Acta 470:45–47

    Google Scholar 

  • Heuser, J.E., Reese, T.S. 1973. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.J. Cell. Biol. 57:315–344

    Google Scholar 

  • Hillarp, N.A. 1958. The release of catecholamine from the amine containing granules of the adrenal medulla.Acta Physiol. Scand. 43:292–302

    Google Scholar 

  • Hodgkin, A.L., Katz, B. 1949. The effects of calcium on the axoplasm of giant nerve fibres.J. Expt. Biol. 26:292–294

    Google Scholar 

  • Jaffe, L.A., Hagiwara, S., Kado, R.J. 1978. The time course of cortical vesicle fusion in sea urchin eggs observed as membrane capacitance changes.Dev. Biol. 67:243–248

    Google Scholar 

  • Jeltsch, E., Zimmermann, U. 1979. Particles in a homogeneous electric field. A model of the electrical breakdown of living cells in a Coulter counter.Bioelectrochem. Bioenerg. 6:349–384

    Google Scholar 

  • Kanno, T., Cochrane, E.E., Douglas, W.W. 1973. Exocytosis (secretory granule extrusion) induced by injection of calcium into mast cells.Can. J. Physiol. Pharmacol. 51:1001–1004

    Google Scholar 

  • Katz, B., Miledi, R. 1967. A study of synaptic transmission in the absence of nerve impulses.J. Physiol. (London) 192:407–436

    Google Scholar 

  • Kinosita, K., Tsong, T.Y. 1977. Formation and resealing of pores of controlled sizes in human erythrocyte membranes.Nature (London) 268:438–441

    Google Scholar 

  • Kinosita, K., Tsong, T.Y. 1978. Survival of sucrose loaded erythrocytes in the circulation.Nature (London) 272:258–260

    Google Scholar 

  • Knight, D.E. 1981. Rendering cells permeable by exposure to electric fields.In: Techniques in Life Sciences. Vol. 1. pp. 113/1–20. P.F. Baker, editor. Elsevier, Amsterdam (in press)

    Google Scholar 

  • Knight, D.E., Scrutton, M.C. 1980. Direct evidence for a role for Ca2+ in amine storage granule secretion by human platelets.Thrombosis Res. 20:437–446

    Google Scholar 

  • Konings, F., Potter, W. de 1981. Calcium-dependentin vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules as a model for exocytosis.FEBS Lett. 126:103–106

    Google Scholar 

  • Kusano, K., Livengood, D.R., Werman, R. 1967. Correlation of transmitter release with membrane properties of the presynaptic fibre of the squid giant synapse.J. Gen. Physiol. 50:2579–2601

    Google Scholar 

  • Lienhard, G.E., Secemski, I.I. 1973.P 1, P5-Di (adenosine-5′) pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase.J. Biol. Chem. 248:1121–1123

    Google Scholar 

  • Llinas, R., Blinks, T.R., Nicholson, C. 1972. Calcium transient in presynaptic terminal of squid giant synapse. Detection with aequorin.Science 176:1127–1129

    Google Scholar 

  • Llinas, R., Nicholson, C. 1975. Calcium role in depolarization-secretion coupling: An aequorin study in squid giant synapse.Proc. Natl. Acad. Sci. USA 72(1):187–190

    Google Scholar 

  • Mahler, H.R., Cordes, E.H. 1966. Biological Chemistry. p. 644. Harper & Row, London

    Google Scholar 

  • Matthews, E.K. 1970. Calcium and hormone release.In: Calcium and Cellular Function. A.W. Cuthbert, editor. pp. 163–182. Macmillan, London

    Google Scholar 

  • Martell, A., Sillen, L.G. 1964. Stability constants.Spec. Pub. No. 17. The Chemical Society, London

    Google Scholar 

  • Maxwell, J.C. 1892. A treatise on electricity and magnetism. Vol. 1 [3rd ed. (reprinted 1955)], p. 437. Oxford, University Press, London

    Google Scholar 

  • Michell, R.H. 1975. Inositol phospholipids and cell surface receptor function.Biochim. Biophys. Acta 415:81–147

    Google Scholar 

  • Miledi, R. 1973. Transmitter release induced by injection of calcium ions into nerve terminals.Proc. R. Soc. London B183:421–425

    Google Scholar 

  • Neumann, E., Rosenheck, K. 1972. Permeability changes induced by electric impulses in vesicular membranes.J. Membrane Biol. 10:279–290

    Google Scholar 

  • Normann, T.C. 1976. Neurosecretion by exocytosis.Int. Rev. Cytol. 46:1–77

    Google Scholar 

  • Pace, C.S., Tarvin, J.T., Neighbors, A.S. Pirkle, J.A., Greider, M.H. 1980. Use of a high voltage technique to determine the molecular requirements for exocytosis in islet cells.Diabetes 29:911–918

    Google Scholar 

  • Pocock, G. 1979. Parallel measurements of sodium pump activity and catecholamine release in cells isolated from bovine adrenal medulla.J. Physiol. (London) 296:102-103P

    Google Scholar 

  • Pollard, H.B., Pazoles, C.J., Creutz, C.E., Ramu, A., Strott, C.A., Ray, P., Brown, E.M., Aubach, G.D., Tack-Goldman, M., Shulman, N.R. 1977. A role for anion transport in the regulation and release from chromaffin granules and exocytosis from cells.J. Supramolec. Struct. 7:277–285

    Google Scholar 

  • Rubinson, K.A., Baker, P.F. 1979. The flow properties of axoplasm in a defined chemical environment: Influence of anions and calcium.Proc. R. Soc. London B205:323–345

    Google Scholar 

  • Salama, G., Johnson, R.G., Scarpa, A. 1980. Spectrophotometric measurement of transmembrane potential and pH gradients in chromaffin granules.J. Gen. Physiol. 75:109–140

    Google Scholar 

  • Sale, A.J.H., Hamilton, W.A. 1968. Effects of high electric fields in microorganisms: III. Lysis of erythrocytes and protoplasts.Biochim. Biophys. Acta 163:37–43

    Google Scholar 

  • Salisbury, J.C., Condeelis, J.S., Satir, P. 1980. Role of coated vesicles, microfilaments and calmodulin in receptor mediated endocytosis by cultured B lymphoblastoid cells.J. Cell. Biol. 87:132–141

    Google Scholar 

  • Schneider, A.S., Herz, R., Rosenheck, K. 1977. Stimulus secretion coupling in chromaffin cells isolated from bovine adrenal medulla.Proc. Natl. Acad. Sci. USA 74:5036–5040

    Google Scholar 

  • Schwan, H.P. 1957. Electrical properties of tissue and cell suspensions.In: Advances in Biological and Medical Physics. J.H. Lawrence and C.A. Tobias, editors. Vol. V, p. 209 Academic Press, New York

    Google Scholar 

  • Schwan, H.P. 1977. Field interaction with biological matter.Ann. N.Y. Acad. Sci. 303:198–213

    Google Scholar 

  • Steinhardt, R.A., Epel, D. 1974. Activation of sea-urchin eggs by a calcium ionophore.Proc. Natl. Acad. Sci. USA 71:1915–1919

    Google Scholar 

  • Tasaki, I., Singer, I., Takenaka, T. 1965. Effects of internal and external ionic environment on excitability of squid giant axon.J. Gen. Physiol. 48:1095–1123

    Google Scholar 

  • Taylor, D.L., Blinks, J.R., Reynolds, G. 1980. Contractile basis of ameboid movement: VIII. Aequorin luminescence during ameboid movement, endocytosis and capping.J. Cell. Biol. 86:599–607

    Google Scholar 

  • Viveros, O.H., Arqueros, L., Kirshner, N. 1968. Release of catecholamine and dopamine-β-oxidase from the adrenal medulla.Life Sci. 7:609–618

    Google Scholar 

  • Viveros, O.H., Diliberto, E., Axelrod, J. 1977.In: Synapses. G.A. Cottrell and P.N.R. Usherwood, editors. pp. 368–369. Blackie & Son, London

    Google Scholar 

  • Von Euler, V.S., Floding, I. 1961. Improved technique for the fluorimetric estimation of catecholamines.Acta Physiol. Scand. 51:348–356

    Google Scholar 

  • Weiss, B., Levin, R.M. 1978. Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents.Adv. Cyclic Nucleotide Res. 9:285–303

    Google Scholar 

  • Zimmerberg, J., Cohen, F.S., Finkelstein, A. 1980. Fusion of phospholipid vesicles with plasma phospholipid bilayer membranes.J. Gen. Physiol. 75:241–270

    Google Scholar 

  • Zimmermann, U., Pilwat, G., Riemann, F. 1974. Dielectric break-down of cell membranes.Biophys. J. 14:881–899

    Google Scholar 

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  1. Department of Physiology, King's College, WC2R 2LS, London, England

    D. E. Knight & P. F. Baker

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Knight, D.E., Baker, P.F. Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J. Membrain Biol. 68, 107–140 (1982). https://doi.org/10.1007/BF01872259

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