Summary
Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca2+ and Mg2+ (t1/2=1 min) and much slower in the presence of Ca2+ or Mg2+ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca2+ and Mg2+, and by equivalent interaction of ionophore with membranes of whole and sonicated cells.
A23187 (10 μm) increased amylase release 200% in the presence of extracellular Ca2+ and Mg2+. In the absence of Ca2+ (but in the presence of Mg2+) A23187 did not increase amylase release. A23187 (10 μm) also produced Ca2+-dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 μm) increased amylase release 140% without increasing LDH release or permeability to trypan blue.
Similar content being viewed by others
References
Amsterdam, A., Jamieson, J.D. 1972. Structural and functional characterization of isolated pancreatic exocrine cells.Proc. Nat. Acad. Sci. USA 69:3028
Amsterdam, A., Jamieson, J.D. 1974. Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells.J. Cell Biol. 63:1037
Ashby, J.P., Speake, R.N. 1975. Insulin and glucagon secretion from isolated islets of langerhans. Effects of calcium ionophores.Biochem. J. 150:89
Binet, A., Volfin, P. 1975. Effect of the A23187 ionophore on mitochondrial Mg2+ and Ca2+.FEBS Lett. 49:400
Case, R.M., Clausen, T. 1973. The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas.J. Physiol. (London) 235:75
Case, G.D., Vanderkooi, J.M., Scarpa, A. 1974. Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187.Arch. Biochem. Biophys. 162:174
Caswell, A.H., Pressman, B.C. 1972. Kinetics of transport of divalent cations across sarcoplasmic reticulum vesicles induced by ionophores.Biochem. Biophys. Res. Commun. 49:292
Chandler, D.E. 1976. Stimulus-secretion coupling in the exocrine pancreas: Use of the fluorescent probe chlorotetracycline to detect divalent cation redistribution.Fed. Proc. 35:603 (Abstr.)
Chandler, D.E., Williams, J.A. 1974. Pancreatic acinar cells: Effects of lanthanum ions on amylase release and calcium ion fluxes.J. Physiol. (London) 243:831
Charles, M.A., Lawecki, J., Pictet, R., Grodsky, G.M. 1975. Insulin secretion. Interrelationships of glucose, cyclic adenosine 3′∶5′-monophosphate and calcium.J. Biol. Chem. 250:6134
Cochrane, D.E., Douglas, W.W. 1974. Calcium induced extrusion of secretory granules exposed to 48/80 or the ionophores A23187 and X-537A.Proc. Nat. Acad. Sci. USA 71:408
Cochrane, D.E., Douglas, W.W., Mouri, T., Nakazato, Y. 1975. Calcium and stimulus-secretion coupling in the adrenal medulla: Contrasting stimulating effects of the ionophores X-537A and A23187 on catecholamine output.J. Physiol. (London) 252:363
Foreman, J.C., Mongar, J.L., Gomperts, B.D. 1973. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process.Nature (London) 245:249
Garcia, A.G., Kirpekar, S.M., Prat, J.C. 1975. A calcium ionophore stimulating the secretion of catecholamines from the cat adrenal.J. Physiol. (London) 244:253
Gardner, J.D., Conlon, T.P., Klaeveman, H.L., Adams, T.D., Ondetti, M.A. 1975. Action of cholecystokinin and cholingergic agents on calcium transport in isolated pancreatic acinar cells.J. Clin. Invest. 56:366
Hainaut, K., Desmedt, J.E. 1974. Calcium ionophore A23187 potentiates twitch and intracellular calcium release in single muscle fibers.Nature (London) 252:407
Heinz, E., Hoffman, J.F. 1965. Phosphate incorporation and Na, K-ATPase activity in human red blood cell ghosts.J. Cell Comp. Physiol. 65:31
Karl, R.C., Zawalich, W.S., Ferrendelli, J.A., Matschinsky, F.M. 1975. The role of Ca2+ and cyclic adenosine 3′∶5′-monophosphate in insulin release induced in vitro by the divalent cation ionophore A23187.J. Biol. Chem. 250:4575
Kondo, S., Schulz, I. 1976. Calcium ion uptake in isolated pancreas cells induced by secretagogues.Biochim. Biophys. Acta. 419:76
Murray, J.J., Reed, P.W., Fay, F.S. 1975. Contraction of isolated smooth muscle cells by ionophore A23187.Proc. Nat. Acad. Sci. USA 72:4459
Petris, S. de 1975. Concanavalin A receptors, immunoglobulins, and θ-antigen of the lymphocyte surface.J. Cell Biol. 65:123
Pfeiffer, D.R., Reed, P.W., Lardy, H.A. 1974. Ultraviolet and fluorescent spectral properties of the divalent cation ionophore A23187 and its metal ion complexes.Biochemistry 13:4007
Poulsen, J.H., Williams, J.A. 1976. Pancreatic acinar cell depolarization and amylase release evoked by Ca2+-ionophore A23187 and bethanechol.Fed. Proc. 35:603 (Abstr.)
Pressman, B.C. 1972. Carboxylic ionophores as mobile carriers for divalent ions.In: The Role of Membranes in Metabolic Regulation M.A. Mehlman and R.W. Hanson, editors. p. 149. Academic Press, New York
Reed, P.W., Lardy, H.A. 1972a. Antibiotic A23187 as a probe for the study of calcium and magnesium in biological systems.In: The Role of Membrane in Metabolic Regulation, A.M. Mehlman and R.W. Hanson, editors. p. 111. Academic Press, New York
Reed, P.W., Lardy, H.A. 1972b. A23187: A divalent cation ionophore.J. Biol. Chem. 247:6970
Rinderknecht, H., Wilding, P., Haverback, B.J. 1967. A new method for the determination of α-amylase.Experientia 23:805
Robberecht, P., Christophe, J. 1971. Secretion of hydrolases by perfused fragments of rat pancreas: Effects of calcium.Am. J. Physiol. 220:911
Russell, J.T., Hansen, E.L., Thorn, N.A. 1974. Calcium and stimulus-secretion coupling in the neurohypophysis. III. Ca2+ ionophore (A23187)-induced release of vasopressin from isolated rat neurohypophysis.Acta Endocrinol. (Copenhagen) 77:443
Scarpa, A., Baldassare, J., Inesi, G. 1972. The effect of calcium ionophores on fragmented sarcoplasmic reticulum.J. Gen. Physiol. 60:735
Schreurs, V.V.A.M., Swarts, H.G.P., dePont, J.J.H.M., Bonting, S.L. 1976. Role of calcium in exocrine pancreatic secretion. II. Comparison of the effects of carbachol and the ionophore A23187 on enzyme secretion and calcium movements in rabbit pancreas.Biochim. Biophys. Acta 419:320
Schroeder, T.E., Strickland, D.L. 1974. Ionophore A23187, calcium and contractility in frog eggs.Exp. Cell Res. 83:139
Selinger, Z., Eimerl, S., Savion, N., Schramm, M. 1974. A Ca++ ionophore (A23187) simulating hormone and neurotransmitter action in the rat parotid and pancreas glands.In: Secretory Mechanisms of Exocrine Glands, N.A. Thorn and O.H. Petersen, editors. p. 68. Munksgaard, Copenhagen
Sordahl, L.A. 1975. Effects of magnesium, ruthenium red and the antibiotic ionophore A23187 on initial rates of calcium uptake and release by heart mitochondria.Arch. Biochem. Biophys. 167:104
Steinhardt, R.A., Epel, D. 1974. Activation of sea-urchin eggs by a calcium ionophore.Proc. Nat. Acad. Sci. USA 71:1915
Thoa, N.B., Costa, N.L., Moss, J., Kopin, I.J. 1974. Mechanism of release of norepinephrine from peripheral adrenergic neurones by the calcium ionophore X-537A and A23187.Life Sci. 14:1705
Unanue, E.R., Perkins, W.D., Karnovsky, M.J. 1972. Ligand induced movement of lymphocyte membrane macromolecules. I. Analysis by immunofluorescence and ultrastructural radioautography.J. Exp. Med. 136:885
Wacker, W.E.C., Ulmer, D.D., Vallee, B.L. 1956. Metalloenzymes and myocardial infarction. II. Malic and latic dehydrogenase activities and zinc concentrations in serum.N. Engl. J. Med. 255:449
Williams, J.A., Cary, P.C., Moffat, B. 1976. Effects of ions on amylase release by dissociated pancreatic acinar cells.Am. J. Physiol. 231:1562
Williams, J.A., Chandler, D. 1975. Ca++ and pancreatic amylase release.Am. J. Physiol. 228:1729
Williams, J.A., Lee, M. 1974. Pancreatic acinar cells: Use of a Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling.Biochem. Biophys. Res. Commun. 60:542
Wollheim, C.B., Blondel, B., Trueheart, P.A., Renold, A.E., Sharp, G.W.G. 1975. Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.J. Biol. Chem. 250:1354
Wong, D.T., Wilkinson, J.R., Hamill, R.L., Horng, J.-S. 1973. Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria.Arch. Biochem. Biophys. 156:578
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chandler, D.E., Williams, J.A. Intracellular uptake and α-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells. J. Membrain Biol. 32, 201–230 (1977). https://doi.org/10.1007/BF01905220
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01905220