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J Physiol. Dec 1993; 472: 665–688.
PMCID: PMC1160506

Exocytosis elicited by action potentials and voltage-clamp calcium currents in individual mouse pancreatic B-cells.

Abstract

1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B-cells. 2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage-dependent Ca2+ currents. The threshold for depolarization-induced release was 0.5 microM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds. 3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations. 4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with > or = four action potentials. 5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B-cell. 6. Inclusion of inhibitors of Ca2+/calmodulin-dependent protein kinase II in the intracellular solution reduced the depolarization-induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery. 7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm. 8. Over short periods, exocytosis may be extremely fast (1 pF/s or 500 granules/s), which is much higher than the rate of endocytosis (18 fF/s or 9 granules/s). Since the latter is in better agreement with the maximum rate of insulin secretion from islets (approximately 2 granules/s), we suggest that membrane retrieval may set an upper limit on the rate of exocytosis during extended periods of secretion.

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  • Adler EM, Augustine GJ, Duffy SN, Charlton MP. Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse. J Neurosci. 1991 Jun;11(6):1496–1507. [PubMed]
  • Ammälä C, Bokvist K, Galt S, Rorsman P. Inhibition of ATP-regulated K(+)-channels by a photoactivatable ATP-analogue in mouse pancreatic beta-cells. Biochim Biophys Acta. 1991 May 17;1092(3):347–349. [PubMed]
  • Ashcroft FM, Rorsman P. Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol. 1989;54(2):87–143. [PubMed]
  • Augustine GJ, Neher E. Calcium requirements for secretion in bovine chromaffin cells. J Physiol. 1992 May;450:247–271. [PMC free article] [PubMed]
  • Bokvist K, Rorsman P, Smith PA. Effects of external tetraethylammonium ions and quinine on delayed rectifying K+ channels in mouse pancreatic beta-cells. J Physiol. 1990 Apr;423:311–325. [PMC free article] [PubMed]
  • Chad JE, Eckert R. Calcium domains associated with individual channels can account for anomalous voltage relations of CA-dependent responses. Biophys J. 1984 May;45(5):993–999. [PMC free article] [PubMed]
  • Colca JR, Brooks CL, Landt M, McDaniel ML. Correlation of Ca2+-and calmodulin-dependent protein kinase activity with secretion of insulin from islets of Langerhans. Biochem J. 1983 Jun 15;212(3):819–827. [PMC free article] [PubMed]
  • Dean PM. Ultrastructural morphometry of the pancreatic -cell. Diabetologia. 1973 Apr;9(2):115–119. [PubMed]
  • Lim NF, Nowycky MC, Bookman RJ. Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals. Nature. 1990 Mar 29;344(6265):449–451. [PubMed]
  • Gillis KD, Misler S. Single cell assay of exocytosis from pancreatic islet B cells. Pflugers Arch. 1992 Jan;420(1):121–123. [PubMed]
  • Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. [PubMed]
  • Harrison DE, Ashcroft SJ. Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans. Biochim Biophys Acta. 1982 Feb 2;714(2):313–319. [PubMed]
  • Harrison DE, Poje M, Rocic B, Ashcroft SJ. Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans. Biochem J. 1986 Jul 1;237(1):191–196. [PMC free article] [PubMed]
  • Henquin JC, Meissner HP. Significance of ionic fluxes and changes in membrane potential for stimulus-secretion coupling in pancreatic B-cells. Experientia. 1984 Oct 15;40(10):1043–1052. [PubMed]
  • Horn R, Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988 Aug;92(2):145–159. [PMC free article] [PubMed]
  • Hutton JC. The insulin secretory granule. Diabetologia. 1989 May;32(5):271–281. [PubMed]
  • Jones PM, Fyles JM, Howell SL. Regulation of insulin secretion by cAMP in rat islets of Langerhans permeabilised by high-voltage discharge. FEBS Lett. 1986 Sep 15;205(2):205–209. [PubMed]
  • Jones PM, Persaud SJ, Howell SL. Time-course of Ca2+-induced insulin secretion from perifused, electrically permeabilised islets of Langerhans: effects of cAMP and a phorbol ester. Biochem Biophys Res Commun. 1989 Aug 15;162(3):998–1003. [PubMed]
  • Jones PM, Persaud SJ, Howell SL. Ca2(+)-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2(+)-induced secretory response is accompanied by loss of Ca2(+)-induced protein phosphorylation. Biochem J. 1992 Aug 1;285(Pt 3):973–978. [PMC free article] [PubMed]
  • Joshi C, Fernandez JM. Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis. Biophys J. 1988 Jun;53(6):885–892. [PMC free article] [PubMed]
  • Li G, Hidaka H, Wollheim CB. Inhibition of voltage-gated Ca2+ channels and insulin secretion in HIT cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca2+ channels. Mol Pharmacol. 1992 Sep;42(3):489–488. [PubMed]
  • Lindau M, Neher E. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch. 1988 Feb;411(2):137–146. [PubMed]
  • Llinás R, Gruner JA, Sugimori M, McGuinness TL, Greengard P. Regulation by synapsin I and Ca(2+)-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse. J Physiol. 1991 May;436:257–282. [PMC free article] [PubMed]
  • Llinás R, Sugimori M, Silver RB. Microdomains of high calcium concentration in a presynaptic terminal. Science. 1992 May 1;256(5057):677–679. [PubMed]
  • Neher E, Augustine GJ. Calcium gradients and buffers in bovine chromaffin cells. J Physiol. 1992 May;450:273–301. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Payne ME, Fong YL, Ono T, Colbran RJ, Kemp BE, Soderling TR, Means AR. Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains. J Biol Chem. 1988 May 25;263(15):7190–7195. [PubMed]
  • Plant TD. Properties and calcium-dependent inactivation of calcium currents in cultured mouse pancreatic B-cells. J Physiol. 1988 Oct;404:731–747. [PMC free article] [PubMed]
  • Pralong WF, Bartley C, Wollheim CB. Single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion. EMBO J. 1990 Jan;9(1):53–60. [PMC free article] [PubMed]
  • Prentki M, Matschinsky FM. Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev. 1987 Oct;67(4):1185–1248. [PubMed]
  • Reetz A, Solimena M, Matteoli M, Folli F, Takei K, De Camilli P. GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J. 1991 May;10(5):1275–1284. [PMC free article] [PubMed]
  • Rorsman P, Ammälä C, Berggren PO, Bokvist K, Larsson O. Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells. EMBO J. 1992 Aug;11(8):2877–2884. [PMC free article] [PubMed]
  • Rorsman P, Ashcroft FM, Trube G. Single Ca channel currents in mouse pancreatic B-cells. Pflugers Arch. 1988 Oct;412(6):597–603. [PubMed]
  • Rorsman P, Trube G. Calcium and delayed potassium currents in mouse pancreatic beta-cells under voltage-clamp conditions. J Physiol. 1986 May;374:531–550. [PMC free article] [PubMed]
  • Rosario LM, Atwater I, Scott AM. Pulsatile insulin release and electrical activity from single ob/ob mouse islets of Langerhans. Adv Exp Med Biol. 1986;211:413–425. [PubMed]
  • Sala F, Hernández-Cruz A. Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties. Biophys J. 1990 Feb;57(2):313–324. [PMC free article] [PubMed]
  • Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B, Valdeolmillos M. Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets. Pflugers Arch. 1991 May;418(4):417–422. [PubMed]
  • Smith PA, Aschroft FM, Fewtrell CM. Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells. J Gen Physiol. 1993 May;101(5):767–797. [PMC free article] [PubMed]
  • Sugden MC, Christie MR, Ashcroft SJ. Presence and possible role of calcium-dependent regulator (calmodulin) in rat islets of Langerhans. FEBS Lett. 1979 Sep 1;105(1):95–100. [PubMed]
  • Theler JM, Mollard P, Guérineau N, Vacher P, Pralong WF, Schlegel W, Wollheim CB. Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP. J Biol Chem. 1992 Sep 5;267(25):18110–18117. [PubMed]
  • Thomas P, Surprenant A, Almers W. Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary. Neuron. 1990 Nov;5(5):723–733. [PubMed]
  • Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, Hidaka H. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 1990 Mar 15;265(8):4315–4320. [PubMed]
  • Trube G, Rorsman P, Ohno-Shosaku T. Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic beta-cells. Pflugers Arch. 1986 Nov;407(5):493–499. [PubMed]
  • Valverde I, Vandermeers A, Anjaneyulu R, Malaisse WJ. Calmodulin activation of adenylate cyclase in pancreatic islets. Science. 1979 Oct 12;206(4415):225–227. [PubMed]
  • Wollheim CB, Ullrich S, Meda P, Vallar L. Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion. Biosci Rep. 1987 May;7(5):443–454. [PubMed]

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