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. 1991 Mar 1;97(3):473–497. doi: 10.1085/jgp.97.3.473

Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs. Direct evidence from changes in membrane capacitance

PMCID: PMC2216487  PMID: 2037838

Abstract

The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2(+)-containing solutions. The resting Cm of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 microF/cm2. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 microns in diameter). High Ca2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm.

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Selected References

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  1. Ben-Jonathan N. Dopamine: a prolactin-inhibiting hormone. Endocr Rev. 1985 Fall;6(4):564–589. doi: 10.1210/edrv-6-4-564. [DOI] [PubMed] [Google Scholar]
  2. Benham C. D. Voltage-gated and agonist-mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp. J Physiol. 1989 Aug;415:143–158. doi: 10.1113/jphysiol.1989.sp017716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bittner M. A., Holz R. W., Neubig R. R. Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells. J Biol Chem. 1986 Aug 5;261(22):10182–10188. [PubMed] [Google Scholar]
  4. Breckenridge L. J., Almers W. Currents through the fusion pore that forms during exocytosis of a secretory vesicle. 1987 Aug 27-Sep 2Nature. 328(6133):814–817. doi: 10.1038/328814a0. [DOI] [PubMed] [Google Scholar]
  5. Breckenridge L. J., Almers W. Final steps in exocytosis observed in a cell with giant secretory granules. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1945–1949. doi: 10.1073/pnas.84.7.1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheek T. R., Burgoyne R. D. Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells. FEBS Lett. 1986 Oct 20;207(1):110–114. doi: 10.1016/0014-5793(86)80022-9. [DOI] [PubMed] [Google Scholar]
  7. Clapham D. E., Neher E. Trifluoperazine reduces inward ionic currents and secretion by separate mechanisms in bovine chromaffin cells. J Physiol. 1984 Aug;353:541–564. doi: 10.1113/jphysiol.1984.sp015350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cobbett P., Ingram C. D., Mason W. T. Sodium and potassium currents involved in action potential propagation in normal bovine lactotrophs. J Physiol. 1987 Nov;392:273–299. doi: 10.1113/jphysiol.1987.sp016780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cobbett P., Ingram C. D., Mason W. T. Voltage-activated currents through calcium channels in normal bovine lactotrophs. Neuroscience. 1987 Nov;23(2):661–677. doi: 10.1016/0306-4522(87)90084-4. [DOI] [PubMed] [Google Scholar]
  10. Douglas W. W. Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Br J Pharmacol. 1968 Nov;34(3):451–474. doi: 10.1111/j.1476-5381.1968.tb08474.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fernandez J. M., Neher E., Gomperts B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. 1984 Nov 29-Dec 5Nature. 312(5993):453–455. doi: 10.1038/312453a0. [DOI] [PubMed] [Google Scholar]
  12. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  13. Hagiwara S., Byerly L. Calcium channel. Annu Rev Neurosci. 1981;4:69–125. doi: 10.1146/annurev.ne.04.030181.000441. [DOI] [PubMed] [Google Scholar]
  14. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  15. Ingram C. D., Bicknell R. J., Mason W. T. Intracellular recordings from bovine anterior pituitary cells: modulation of spontaneous activity by regulators of prolactin secretion. Endocrinology. 1986 Dec;119(6):2508–2518. doi: 10.1210/endo-119-6-2508. [DOI] [PubMed] [Google Scholar]
  16. Ingram C. D., Keefe P. D., Wooding F. B., Bicknell R. J. Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique. Cell Tissue Res. 1988 Jun;252(3):655–659. doi: 10.1007/BF00216653. [DOI] [PubMed] [Google Scholar]
  17. Joshi C., Fernandez J. M. Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis. Biophys J. 1988 Jun;53(6):885–892. doi: 10.1016/S0006-3495(88)83169-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Keeton T. K., Campbell W. B. The pharmacologic alteration of renin release. Pharmacol Rev. 1980 Jun;32(2):81–227. [PubMed] [Google Scholar]
  19. Kidokoro Y., Miyazaki S., Ozawa S. Acetylcholine-induced membrane depolarization and potential fluctuations in the rat adrenal chromaffin cell. J Physiol. 1982 Mar;324:203–220. doi: 10.1113/jphysiol.1982.sp014107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kidokoro Y. Spontaneous calcium action potentials in a clonal pituitary cell line and their relationship to prolactin secretion. Nature. 1975 Dec 25;258(5537):741–742. doi: 10.1038/258741a0. [DOI] [PubMed] [Google Scholar]
  21. Knight D. E., Baker P. F. Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J Membr Biol. 1982;68(2):107–140. doi: 10.1007/BF01872259. [DOI] [PubMed] [Google Scholar]
  22. Knight D. E., Baker P. F. Guanine nucleotides and Ca-dependent exocytosis. Studies on two adrenal cell preparations. FEBS Lett. 1985 Sep 23;189(2):345–349. doi: 10.1016/0014-5793(85)81053-x. [DOI] [PubMed] [Google Scholar]
  23. Lindau M., Neher E. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch. 1988 Feb;411(2):137–146. doi: 10.1007/BF00582306. [DOI] [PubMed] [Google Scholar]
  24. Lingle C. J., Sombati S., Freeman M. E. Membrane currents in identified lactotrophs of rat anterior pituitary. J Neurosci. 1986 Oct;6(10):2995–3005. doi: 10.1523/JNEUROSCI.06-10-02995.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maruyama Y. Ca2+-induced excess capacitance fluctuation studied by phase-sensitive detection method in exocrine pancreatic acinar cells. Pflugers Arch. 1986 Nov;407(5):561–563. doi: 10.1007/BF00657517. [DOI] [PubMed] [Google Scholar]
  26. Mason W. T., Ingram C. D. Techniques for studying the role of electrical activity in control of secretion by normal anterior pituitary cells. Methods Enzymol. 1986;124:207–242. doi: 10.1016/0076-6879(86)24017-3. [DOI] [PubMed] [Google Scholar]
  27. Mason W. T., Rawlings S. R., Cobbett P., Sikdar S. K., Zorec R., Akerman S. N., Benham C. D., Berridge M. J., Cheek T., Moreton R. B. Control of secretion in anterior pituitary cells--linking ion channels, messengers and exocytosis. J Exp Biol. 1988 Sep;139:287–316. doi: 10.1242/jeb.139.1.287. [DOI] [PubMed] [Google Scholar]
  28. Mason W. T., Sikdar S. K., Zorec R., Akerman S., Rawlings S. R., Cheek T., Moreton R., Berridge M. Ion channels, intracellular calcium, and exocytosis: control of hormone secretion in cultured bovine pituitary lactotrophs. Soc Gen Physiol Ser. 1989;44:225–238. [PubMed] [Google Scholar]
  29. Mason W. T., Waring D. W. Electrophysiological recordings from gonadotrophs. Evidence for Ca2+ channels mediated by gonadotrophin-releasing hormone. Neuroendocrinology. 1985 Sep;41(3):258–268. doi: 10.1159/000124186. [DOI] [PubMed] [Google Scholar]
  30. Mason W. T., Waring D. W. Patch clamp recordings of single ion channel activation by gonadotrophin-releasing hormone in ovine pituitary gonadotrophs. Neuroendocrinology. 1986;43(2):205–219. doi: 10.1159/000124529. [DOI] [PubMed] [Google Scholar]
  31. McBurney R. N., Neering I. R. The measurement of changes in intracellular free calcium during action potentials in mammalian neurones. J Neurosci Methods. 1985 Mar;13(1):65–76. doi: 10.1016/0165-0270(85)90044-5. [DOI] [PubMed] [Google Scholar]
  32. Miller D. J., Smith G. L. EGTA purity and the buffering of calcium ions in physiological solutions. Am J Physiol. 1984 Jan;246(1 Pt 1):C160–C166. doi: 10.1152/ajpcell.1984.246.1.C160. [DOI] [PubMed] [Google Scholar]
  33. 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. doi: 10.1073/pnas.79.21.6712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Neher E. The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum. J Physiol. 1988 Jan;395:193–214. doi: 10.1113/jphysiol.1988.sp016914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nüsse O., Lindau M. The dynamics of exocytosis in human neutrophils. J Cell Biol. 1988 Dec;107(6 Pt 1):2117–2123. doi: 10.1083/jcb.107.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. O'Sullivan A. J., Cheek T. R., Moreton R. B., Berridge M. J., Burgoyne R. D. Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2. EMBO J. 1989 Feb;8(2):401–411. doi: 10.1002/j.1460-2075.1989.tb03391.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ozawa S., Sand O. Action potentials in non-tumor cells from the anterior pituitary gland. Experientia. 1978 Apr 15;34(4):542–544. doi: 10.1007/BF01935981. [DOI] [PubMed] [Google Scholar]
  38. Ozawa S., Sand O. Electrophysiology of excitable endocrine cells. Physiol Rev. 1986 Oct;66(4):887–952. doi: 10.1152/physrev.1986.66.4.887. [DOI] [PubMed] [Google Scholar]
  39. Penner R., Neher E., Dreyer F. Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells. Nature. 1986 Nov 6;324(6092):76–78. doi: 10.1038/324076a0. [DOI] [PubMed] [Google Scholar]
  40. Penner R., Neher E. The patch-clamp technique in the study of secretion. Trends Neurosci. 1989 Apr;12(4):159–163. doi: 10.1016/0166-2236(89)90059-3. [DOI] [PubMed] [Google Scholar]
  41. Penner R., Neher E. The role of calcium in stimulus-secretion coupling in excitable and non-excitable cells. J Exp Biol. 1988 Sep;139:329–345. doi: 10.1242/jeb.139.1.329. [DOI] [PubMed] [Google Scholar]
  42. Penner R., Pusch M., Neher E. Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling. Biosci Rep. 1987 Apr;7(4):313–321. doi: 10.1007/BF01121453. [DOI] [PubMed] [Google Scholar]
  43. Pusch M., Neher E. Rates of diffusional exchange between small cells and a measuring patch pipette. Pflugers Arch. 1988 Feb;411(2):204–211. doi: 10.1007/BF00582316. [DOI] [PubMed] [Google Scholar]
  44. Rink T. J., Knight D. E. Stimulus-secretion coupling: a perspective highlighting the contributions of Peter Baker. J Exp Biol. 1988 Sep;139:1–30. [PubMed] [Google Scholar]
  45. Ronning S. A., Martin T. F. Characterization of phorbol ester- and diacylglycerol-stimulated secretion in permeable GH3 pituitary cells. Interaction with Ca2+. J Biol Chem. 1986 Jun 15;261(17):7840–7845. [PubMed] [Google Scholar]
  46. Schlegel W., Winiger B. P., Mollard P., Vacher P., Wuarin F., Zahnd G. R., Wollheim C. B., Dufy B. Oscillations of cytosolic Ca2+ in pituitary cells due to action potentials. Nature. 1987 Oct 22;329(6141):719–721. doi: 10.1038/329719a0. [DOI] [PubMed] [Google Scholar]
  47. Sikdar S. K., Zorec R., Brown D., Mason W. T. Dual effects of G-protein activation on Ca-dependent exocytosis in bovine lactotrophs. FEBS Lett. 1989 Aug 14;253(1-2):88–92. doi: 10.1016/0014-5793(89)80936-6. [DOI] [PubMed] [Google Scholar]
  48. Taraskevich P. S., Douglas W. W. Action potentials occur in cells of the normal anterior pituitary gland and are stimulated by the hypophysiotropic peptide thyrotropin-releasing hormone. Proc Natl Acad Sci U S A. 1977 Sep;74(9):4064–4067. doi: 10.1073/pnas.74.9.4064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Taraskevich P. S., Douglas W. W. Electrical activity in adenohypophyseal cells and effects of hypophyseotropic substances. Fed Proc. 1984 Jun;43(9):2373–2378. [PubMed] [Google Scholar]
  50. Tomiko S. A., Taraskevich P. S., Douglas W. W. Effects of veratridine, tetrodotoxin and other drugs that alter electrical behaviour on secretion of melanocyte-stimulating hormone from melanotrophs of the pituitary pars intermedia. Neuroscience. 1984 Aug;12(4):1223–1228. doi: 10.1016/0306-4522(84)90016-2. [DOI] [PubMed] [Google Scholar]
  51. Vale W., Rivier C., Brown M. Regulatory peptides of the hypothalamus. Annu Rev Physiol. 1977;39:473–527. doi: 10.1146/annurev.ph.39.030177.002353. [DOI] [PubMed] [Google Scholar]
  52. Zimmerberg J., Curran M., Cohen F. S., Brodwick M. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1585–1589. doi: 10.1073/pnas.84.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]

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