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. 1992 Jan;445:515–535. doi: 10.1113/jphysiol.1992.sp018937

Release of intracellular calcium and modulation of membrane currents by caffeine in bull-frog sympathetic neurones.

N V Marrion 1, P R Adams 1
PMCID: PMC1179995  PMID: 1380086

Abstract

1. Calcium release and sequestration were studied in whole-cell voltage-clamped bull-frog sympathetic neurones by image analysis of Fura-2 signals. 2. Application of caffeine (10 mM) to cells voltage clamped at -38 mV caused a rapid increase in intracellular calcium concentration ([Ca2+]i) to a mean value of 352 +/- 33 nM, which activated an outward current. In the continued presence of caffeine the rise in [Ca2+]i slowly declined to a sustained plateau of 196 +/- 20 nM (112 nM above control levels), while the outward current rapidly decayed. Peak calcium release was highest at the edge of the cell. 3. The caffeine-evoked intracellular calcium increase was reduced by two inhibitors of calcium-induced calcium release, ryanodine and procaine. The residual non-suppressible increase in [Ca2+]i may indicate that caffeine can release calcium from two pharmacologically distinct intracellular stores. 4. Inhibition of the caffeine-evoked release of calcium by ryanodine was both concentration and 'use dependent' so that the full inhibitory effect was only observed when caffeine was applied for the second time in the presence of ryanodine. In contrast, the action of procaine did not show any 'use dependence' and unlike ryanodine was fully reversible. 5. The outward current was sensitive to blockers of the large conductance calcium-activated potassium current, Ic. Analysis of variance from this current indicated that it arose at least partly from summation of spontaneous miniature outward currents. 6. The magnitude and duration of calcium release by caffeine was dependent on the resting level of intracellular calcium and the caffeine exposure time. This, together with the pharmacology of the release, suggests that caffeine increases intracellular calcium by sensitizing calcium-induced calcium release. 7. The evoked [Ca2+]i increase was enhanced in amplitude by intracellular application of Ruthenium Red. This effect was mimicked by extracellular application of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) but not by internal application of FCCP or other inhibitors of mitochondrial Ca2+ uptake. This suggests that the evoked increase in [Ca2+]i is predominantly buffered by a Ruthenium Red-sensitive sequestration process which is not mitochondrial.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams P. R., Constanti A., Brown D. A., Clark R. B. Intracellular Ca2+ activates a fast voltage-sensitive K+ current in vertebrate sympathetic neurones. Nature. 1982 Apr 22;296(5859):746–749. doi: 10.1038/296746a0. [DOI] [PubMed] [Google Scholar]
  2. Akaike N., Sadoshima J. Caffeine affects four different ionic currents in the bull-frog sympathetic neurone. J Physiol. 1989 May;412:221–244. doi: 10.1113/jphysiol.1989.sp017612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ambler S. K., Poenie M., Tsien R. Y., Taylor P. Agonist-stimulated oscillations and cycling of intracellular free calcium in individual cultured muscle cells. J Biol Chem. 1988 Feb 5;263(4):1952–1959. [PubMed] [Google Scholar]
  4. Baker P. F., Umbach J. A. Calcium buffering in axons and axoplasm of Loligo. J Physiol. 1987 Feb;383:369–394. doi: 10.1113/jphysiol.1987.sp016414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrett J. N., Magleby K. L., Pallotta B. S. Properties of single calcium-activated potassium channels in cultured rat muscle. J Physiol. 1982 Oct;331:211–230. doi: 10.1113/jphysiol.1982.sp014370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baylor S. M., Hollingworth S., Marshall M. W. Effects of intracellular ruthenium red on excitation-contraction coupling in intact frog skeletal muscle fibres. J Physiol. 1989 Jan;408:617–635. doi: 10.1113/jphysiol.1989.sp017480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blaustein M. P., Ratzlaff R. W., Schweitzer E. S. Calcium buffering in presynaptic nerve terminals. II. Kinetic properties of the nonmitochondrial Ca sequestration mechanism. J Gen Physiol. 1978 Jul;72(1):43–66. doi: 10.1085/jgp.72.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bolton T. B., Lim S. P. Properties of calcium stores and transient outward currents in single smooth muscle cells of rabbit intestine. J Physiol. 1989 Feb;409:385–401. doi: 10.1113/jphysiol.1989.sp017504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bond M., Kitazawa T., Somlyo A. P., Somlyo A. V. Release and recycling of calcium by the sarcoplasmic reticulum in guinea-pig portal vein smooth muscle. J Physiol. 1984 Oct;355:677–695. doi: 10.1113/jphysiol.1984.sp015445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown D. A., Constanti A., Adams P. R. Ca-activated potassium current in vertebrate sympathetic neurons. Cell Calcium. 1983 Dec;4(5-6):407–420. doi: 10.1016/0143-4160(83)90017-9. [DOI] [PubMed] [Google Scholar]
  11. Cheek T. R. Spatial aspects of calcium signalling. J Cell Sci. 1989 Jun;93(Pt 2):211–216. doi: 10.1242/jcs.93.2.211. [DOI] [PubMed] [Google Scholar]
  12. Connor J. A., Wadman W. J., Hockberger P. E., Wong R. K. Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons. Science. 1988 Apr 29;240(4852):649–653. doi: 10.1126/science.2452481. [DOI] [PubMed] [Google Scholar]
  13. Earm Y. E., Noble D. A model of the single atrial cell: relation between calcium current and calcium release. Proc R Soc Lond B Biol Sci. 1990 May 22;240(1297):83–96. doi: 10.1098/rspb.1990.0028. [DOI] [PubMed] [Google Scholar]
  14. Endo M., Tanaka M., Ogawa Y. Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature. 1970 Oct 3;228(5266):34–36. doi: 10.1038/228034a0. [DOI] [PubMed] [Google Scholar]
  15. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983 Jul;245(1):C1–14. doi: 10.1152/ajpcell.1983.245.1.C1. [DOI] [PubMed] [Google Scholar]
  16. Fabiato A. Effects of ryanodine in skinned cardiac cells. Fed Proc. 1985 Dec;44(15):2970–2976. [PubMed] [Google Scholar]
  17. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  18. Fabiato A., Fabiato F. Use of chlorotetracycline fluorescence to demonstrate Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum of skinned cardiac cells. Nature. 1979 Sep 13;281(5727):146–148. doi: 10.1038/281146a0. [DOI] [PubMed] [Google Scholar]
  19. Fleischer S., Ogunbunmi E. M., Dixon M. C., Fleer E. A. Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7256–7259. doi: 10.1073/pnas.82.21.7256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fry C. H., Harding D. P., Miller D. J. Non-mitochondrial calcium ion regulation in rat ventricular myocytes. Proc R Soc Lond B Biol Sci. 1989 Feb 22;236(1282):53–77. doi: 10.1098/rspb.1989.0012. [DOI] [PubMed] [Google Scholar]
  21. Fujimoto S., Yamamoto K., Kuba K., Morita K., Kato E. Calcium localization in the sympathetic ganglion of the bullfrog and effects of caffeine. Brain Res. 1980 Nov 24;202(1):21–32. [PubMed] [Google Scholar]
  22. Gray P. T. The relation of elevation of cytosolic free calcium to activation of membrane conductance in rat parotid acinar cells. Proc R Soc Lond B Biol Sci. 1989 Jun 22;237(1286):99–107. doi: 10.1098/rspb.1989.0039. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Gunter T. E., Wingrove D. E., Banerjee S., Gunter K. K. Mechanisms of mitochondrial calcium transport. Adv Exp Med Biol. 1988;232:1–14. doi: 10.1007/978-1-4757-0007-7_1. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Henkart M., Landis D. M., Reese T. S. Similarity of junctions between plasma membranes and endoplasmic reticulum in muscle and neurons. J Cell Biol. 1976 Aug;70(2 Pt 1):338–347. doi: 10.1083/jcb.70.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hernández-Cruz A., Sala F., Adams P. R. Subcellular calcium transients visualized by confocal microscopy in a voltage-clamped vertebrate neuron. Science. 1990 Feb 16;247(4944):858–862. doi: 10.1126/science.2154851. [DOI] [PubMed] [Google Scholar]
  28. Hwang K. S., Saida K., van Breemen C. Modulation of ryanodine-induced Ca2+ release in amphibian skeletal muscle. Biochem Biophys Res Commun. 1987 Feb 13;142(3):674–679. doi: 10.1016/0006-291x(87)91467-7. [DOI] [PubMed] [Google Scholar]
  29. Jones S. W., Marks T. N. Calcium currents in bullfrog sympathetic neurons. I. Activation kinetics and pharmacology. J Gen Physiol. 1989 Jul;94(1):151–167. doi: 10.1085/jgp.94.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Konishi M., Kurihara S. Effects of caffeine on intracellular calcium concentrations in frog skeletal muscle fibres. J Physiol. 1987 Feb;383:269–283. doi: 10.1113/jphysiol.1987.sp016408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kuffler S. W., Sejnowski T. J. Peptidergic and muscarinic excitation at amphibian sympathetic synapses. J Physiol. 1983 Aug;341:257–278. doi: 10.1113/jphysiol.1983.sp014805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lipscombe D., Madison D. V., Poenie M., Reuter H., Tsien R. W., Tsien R. Y. Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons. Neuron. 1988 Jul;1(5):355–365. doi: 10.1016/0896-6273(88)90185-7. [DOI] [PubMed] [Google Scholar]
  33. Marty A. Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature. 1981 Jun 11;291(5815):497–500. doi: 10.1038/291497a0. [DOI] [PubMed] [Google Scholar]
  34. Marty A. The physiological role of calcium-dependent channels. Trends Neurosci. 1989 Nov;12(11):420–424. doi: 10.1016/0166-2236(89)90090-8. [DOI] [PubMed] [Google Scholar]
  35. Mathers D. A., Barker J. L. Spontaneous voltage and current fluctuations in tissue cultured mouse dorsal root ganglion cells. Brain Res. 1984 Feb 13;293(1):35–47. doi: 10.1016/0006-8993(84)91450-1. [DOI] [PubMed] [Google Scholar]
  36. Miller R. J. Multiple calcium channels and neuronal function. Science. 1987 Jan 2;235(4784):46–52. doi: 10.1126/science.2432656. [DOI] [PubMed] [Google Scholar]
  37. Nachshen D. A. Regulation of cytosolic calcium concentration in presynaptic nerve endings isolated from rat brain. J Physiol. 1985 Jun;363:87–101. doi: 10.1113/jphysiol.1985.sp015697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nagasaki K., Fleischer S. Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium. 1988 Feb;9(1):1–7. doi: 10.1016/0143-4160(88)90032-2. [DOI] [PubMed] [Google Scholar]
  39. Neering I. R., McBurney R. N. Role for microsomal Ca storage in mammalian neurones? Nature. 1984 May 10;309(5964):158–160. doi: 10.1038/309158a0. [DOI] [PubMed] [Google Scholar]
  40. Negulescu P. A., Machen T. E. Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cell. Am J Physiol. 1988 Apr;254(4 Pt 1):C498–C504. doi: 10.1152/ajpcell.1988.254.4.C498. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Pennefather P., Lancaster B., Adams P. R., Nicoll R. A. Two distinct Ca-dependent K currents in bullfrog sympathetic ganglion cells. Proc Natl Acad Sci U S A. 1985 May;82(9):3040–3044. doi: 10.1073/pnas.82.9.3040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pessah I. N., Stambuk R. A., Casida J. E. Ca2+-activated ryanodine binding: mechanisms of sensitivity and intensity modulation by Mg2+, caffeine, and adenine nucleotides. Mol Pharmacol. 1987 Mar;31(3):232–238. [PubMed] [Google Scholar]
  44. Pfaffinger P. J., Leibowitz M. D., Subers E. M., Nathanson N. M., Almers W., Hille B. Agonists that suppress M-current elicit phosphoinositide turnover and Ca2+ transients, but these events do not explain M-current suppression. Neuron. 1988 Aug;1(6):477–484. doi: 10.1016/0896-6273(88)90178-x. [DOI] [PubMed] [Google Scholar]
  45. Rousseau E., Smith J. S., Meissner G. Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am J Physiol. 1987 Sep;253(3 Pt 1):C364–C368. doi: 10.1152/ajpcell.1987.253.3.C364. [DOI] [PubMed] [Google Scholar]
  46. Sadoshima J., Akaike N. Kinetic properties of the caffeine-induced transient outward current in bull-frog sympathetic neurones. J Physiol. 1991 Feb;433:341–355. doi: 10.1113/jphysiol.1991.sp018429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sala F., Hernández-Cruz A. Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties. Biophys J. 1990 Feb;57(2):313–324. doi: 10.1016/S0006-3495(90)82533-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Satin L. S., Adams P. R. Spontaneous miniature outward currents in cultured bullfrog neurons. Brain Res. 1987 Jan 20;401(2):331–339. doi: 10.1016/0006-8993(87)91417-x. [DOI] [PubMed] [Google Scholar]
  49. Smith S. J., MacDermott A. B., Weight F. F. Detection of intracellular Ca2+ transients in sympathetic neurones using arsenazo III. 1983 Jul 28-Aug 3Nature. 304(5924):350–352. doi: 10.1038/304350a0. [DOI] [PubMed] [Google Scholar]
  50. Sutko J. L., Ito K., Kenyon J. L. Ryanodine: a modifier of sarcoplasmic reticulum calcium release in striated muscle. Fed Proc. 1985 Dec;44(15):2984–2988. [PubMed] [Google Scholar]
  51. Thayer S. A., Hirning L. D., Miller R. J. The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro. Mol Pharmacol. 1988 Nov;34(5):664–673. [PubMed] [Google Scholar]
  52. Thayer S. A., Miller R. J. Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro. J Physiol. 1990 Jun;425:85–115. doi: 10.1113/jphysiol.1990.sp018094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Thayer S. A., Perney T. M., Miller R. J. Regulation of calcium homeostasis in sensory neurons by bradykinin. J Neurosci. 1988 Nov;8(11):4089–4097. doi: 10.1523/JNEUROSCI.08-11-04089.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vale M. G., Carvalho A. P. Effects of ruthenium red on Ca2+ uptake and ATPase of sarcoplasmic reticulum of rabbit skeletal muscle. Biochim Biophys Acta. 1973 Oct 19;325(1):29–37. doi: 10.1016/0005-2728(73)90147-3. [DOI] [PubMed] [Google Scholar]
  55. Weber A., Herz R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol. 1968 Nov;52(5):750–759. doi: 10.1085/jgp.52.5.750. [DOI] [PMC free article] [PubMed] [Google Scholar]

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