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A fluorimetric and amperometric study of calcium and secretion in isolated mouse pancreatic β-cells

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  • Molecular and Cellular Physiology
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Abstract

We have examined the temporal relationship between intracellular Ca2+ concentration ([Ca2+]i) and secretion in single intact pancreatic β-cells. Secretion was detected as the release of 5-hydroxytryptamine from pre-loaded β-cells, using amperometry, and changes in [Ca2+]i were monitored by microfluorimetry. Stimulation of β-cells by elevation of the extracellular K+ concentration ([K+]o), acetylcholine or glucose increased [Ca2+]i and, after a delay of 2–7 s, evoked amperometric currents. In the presence of glucose, we observed oscillations in [Ca2+]i which were associated with oscillations in the amplitude and frequency of amperometric currents: however, the temporal correlation was not exact, suggesting that there is a significant latency between the increase in average [Ca2+]i and exocytosis. Both the amplitude and frequency of the amperometric currents elicited by 50 mM KCl declined with successive stimulation, but were restored by agents which elevate intracellular adenosine 3'∶5′:cyclic monophosphate (cAMP). This suggests that β-cells may possess a readily releasable pool of granules which is replenished by cAMP. The variable amplitude of the amperometric currents is discussed in terms of a model in which several secretory granules fuse simultaneously with the plasma membrane.

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References

  1. Ämmälä C, Ashcroft FM, Rorsman P (1993) Calcium-independent potentiation of insulin secretion by cyclic AMP in single β-cells. Nature 363:356–358

    Google Scholar 

  2. Ämmälä C, Eliasson L, Bokvist K, Larsson O, Ashcroft FM, Rorsman P (1993) Exocytosis elicited by action potentials and voltage-clamp calcium currents in individual mouse pancreatic β-cells. J Physiol 472:665–688

    Google Scholar 

  3. Armstrong-James M, Millar J (1979) Carbon-fibre microelectrodes. J Neurosci Methods 1:279–287

    Google Scholar 

  4. Ashcroft FM, Rorsman P (1989) Electrophysiology of the pancreatic β-cell. Prog Biophys Mol Biol 54:87–144

    Google Scholar 

  5. Bokvist K, Eliasson L, Ämmälä C, Renström E, Rorsman P (1995) Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic β-cells. EMBO J 14:50–57

    Google Scholar 

  6. Chow RH, Ruden L, Neher E (1992) Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356:60–63

    Google Scholar 

  7. Chow RH, Klingauf J, Neher E (1994) Time course of Ca2+ concentration triggering exocytosis in neuroendocrine cells. Proc Natl Acad Sci USA 91:12765–12769

    Google Scholar 

  8. Colquhoun D, Sigworth FJ (1985) In: Sakmann B, Neher E (eds) Single channel recording. Plenum, New York, pp 191–263

    Google Scholar 

  9. Cregell L (1968) The occurrence of biogenic amines in the mammalian endocrine pancreas. Acta Physiol Scand Suppl 314:1–46

    Google Scholar 

  10. Dean (1973) Ultrastructural morphometry of the pancreatic β-cell. Diabetologia 9:115–119

    Google Scholar 

  11. Duchen MR, Smith PA, Ashcroft FM (1993) Substrate dependent changes in mitochondrial function, [Ca2+]i and membrane channels in pancreatic β-cells. Biochem J 294:35–42

    Google Scholar 

  12. Ekholm R, Ericson LE, Lundquist I (1971) Monoamines in the pancreatic islets of the mouse. Diabetologia 7:339–348

    Google Scholar 

  13. Ericson LE, Håkanson R, Lundquist I (1977) Accumulation of dopamine in mouse pancreatic β-cells following injection of L-DOPA. Localisation to secretory granules and inhibition of insulin secretion. Diabetologia 13:117–124

    Google Scholar 

  14. Falck B, Hellman B (1963) Evidence for the presence of biogenic amines in pancreatic islets. Experientia 19:139–140

    Google Scholar 

  15. Gagliardino JJ, Nierle C, Pfeifer EF (1974) The effect of serotonin on in vitro insulin secretion and biosynthesis in mice. Diabetologia 10:411–414

    Google Scholar 

  16. Gillis KD, Misler S (1993) Enhancers of cytosolic cAMP augment depolarisation-induced exocytosis from pancreatic β-cells: evidence for effects distal to Ca2+ entry. Pflügers Arch 424: 195–197

    Google Scholar 

  17. Gilon P, Shepherd RM, Henquin J-C (1993) Oscillations of secretion driven by oscillations of cytoplasmic Ca2+ as evidenced in single pancreatic islets. J Biol Chem 268:22265–22268

    Google Scholar 

  18. Gylfe E (1978) Association between 5-hydroxytryptamine release and insulin secretion. J Endocrinol 78:239–248

    Google Scholar 

  19. Gylfe E (1980) Effects of metabolic inhibitors on the efflux of 5-hydroxytryptamine from pancreatic β-cells. Acta Physiol Scand 109:155–161

    Google Scholar 

  20. Glyfe E, Hellman N, Sehlin J, Täljedal I-B (1973) Amino acid conversion into 5-hydroxytryptamine in pancreatic β-cells. Endocrinology 93:932–937

    Google Scholar 

  21. Hellman B, Lernmark Å, Sehlin J, Täljedal I-B (1972) Transport and storage of 5-hydroxytryptamine in pancreatic β-cells Biochem Pharmacol 21:695–706

    Google Scholar 

  22. Hughes SJ, Chalk JG, Ashcroft SJH (1990) The role of cytosolic free Ca2+ and protein kinase C in acetylcholine-induced insulin release in the clonal β-cell line HIT T15. Biochem J 267: 227–232

    Google Scholar 

  23. Hutton JC, Peshavaria M, Tooke NE (1983) 5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma. Biochem J 210:803–810

    Google Scholar 

  24. Jones PM, Fyles JM, Howell SL (1986) Regulation of insulin secretion by cAMP in rat islets of Langerhans permeabilised by high voltage discharge. FEBS Lett 205:205–209

    Google Scholar 

  25. Jones PM, Persaud SJ, Howell SL (1989) Time course of Ca2+induced secretion from perifused electrically permeabilised islets of Langerhans: effects of cAMP and a phorbol ester. Biochem Biophys Res Commun 162:998–1003

    Google Scholar 

  26. Lernmark A (1971) The significance of 5-hydroxytryptamine for insulin secretion in the mouse. Horm Metab Res 3:305–309

    Google Scholar 

  27. Neher E, Marty A (1982) Discrete changes of cell capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci USA 79: 6712–6716

    Google Scholar 

  28. Persaud SJ, Jones PM, Howell SL (1990) Glucose-stimulated insulin secretion is not dependent on activation of protein kinase A. Biochem Biophys Res Commun 173:833–839

    Google Scholar 

  29. Persaud SJ, Jones PM, Howell SL (1992) The role of protein kinase C in insulin secretion. In: Flatt PR (ed) Nutrient regulation of insulin secretion. Portland Press, London, pp 247–271

    Google Scholar 

  30. Pipeleers DG, Schuit FC, In't Veld PA, Maes E, Hooghe-Peters EL, Van de Winkel M, Gepts W (1985) Interplay of nutrients and hormones in the regulation of insulin release. Endocrinology 117:824–833

    Google Scholar 

  31. Robitaille R, Garcia ML, Kaczorowski GJ, Charlton MP (1993) Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11:645–655

    Google Scholar 

  32. Rorsman P, Ashcroft FM, Trube G (1988) Single Ca channel currents in mouse pancreatic β-cells. Pflügers Arch 412: 597–603

    Google Scholar 

  33. Santos RM, Rosario LM, Nadel A, Garcia-Sancho J, Soria B, Valdeolmillos M (1991) Widespread synchronous oscillations due to bursting electrical activity in single pancreatic islets. Pflügers Arch 418: 417–422

    Google Scholar 

  34. Schroeder TJ, Janowski JA, Kagoe KT, Wightman RM (1992) Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis. J Gen Physiol 101:767–797

    Google Scholar 

  35. Smith PA, Duchen MR, Ashcroft FM (1993) Voltammetric monitoring of 5-HT secretion from isolated mouse pancreatic β-cells. J Physiol (Lond) 475:157P

    Google Scholar 

  36. Smith PA, Ashcroft FM, Fewtrell C (1993) Permeation and gating properties of the L-type calcium channel in mouse pancreatic β-cells. J Gen Physiol 101:767–797

    Google Scholar 

  37. Smith PA, Proks P, Ashcroft FM (1994) Exocytosis monitored by simultaneous measurement of cell capacitance and 5-HT release in isolated mouse pancreatic β-cells. J Physiol (Lond) 480:123P

    Google Scholar 

  38. Tatham PE, Duchen MR, Millar J (1991) Monitoring exocytosis from single mast cells by fast voltammetry. Pflügers Arch 419:409–414

    Google Scholar 

  39. Tomita Y, Inooka G, Shimada H, Maruyama Y (1994) Ca2+-dependent unidirectional vesicular release detected with a carbon fibre electrode in rat pancreatic acinar cells. Pflügers Arch 428:69–75

    Google Scholar 

  40. Valdeolmillos M, Santos RM, Contreras D, Soria B, Rosario LM (1989) Glucose-induced oscillations of intracellular Ca2+ concentration matching bursting electrical activity in single mouse islets of Langerhans. FEBS Lett 259:19–23

    Google Scholar 

  41. Wightman RM, Jankowski JA, Kennedy RT, Kawagoe KT, Shroeder TJ, Leszczyszyn, DJ, Near JA, Diliberto EJ, Viveros OH (1991) Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc Natl Acad Sci USA 88:10754–10758

    Google Scholar 

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Authors and Affiliations

  1. University Laboratory of Physiology, Parks Road, OX1 3PT, Oxford, UK

    Paul A. Smith & Frances M. Ashcroft

  2. Department of Physiology, University College London, Gower Street, London, UK

    Michael R. Duchen

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  1. Paul A. Smith
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  2. Michael R. Duchen
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  3. Frances M. Ashcroft
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Smith, P.A., Duchen, M.R. & Ashcroft, F.M. A fluorimetric and amperometric study of calcium and secretion in isolated mouse pancreatic β-cells. Pflugers Arch. 430, 808–818 (1995). https://doi.org/10.1007/BF00386180

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  • DOI: https://doi.org/10.1007/BF00386180

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