Skip to main content
SpringerLink
Log in

Tight mitochondrial control of calcium and exocytotic signals in chromaffin cells at embryonic life

  • Signaling and cell physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca2+ and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca2+ buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca2+ buffering during cell activation. We found that the clearance of cytosolic Ca2+ transients ([Ca2+]c) elicited by high K+ was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca2+]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca2+ buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K+-elicited [Ca2+]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K+-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca2+ and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

[Ca2+]c :

Cytosolic Ca2+ concentrations

[Ca2+]m :

Mitochondrial Ca2+ concentrations

CCs:

Chromaffin cells

VACCs:

Voltage-activated calcium channels

EM:

Electron microscopy

References

  1. Adams MB, Simonetta G, McMillen IC (1996) The non-neurogenic catecholamine response of the fetal adrenal to hypoxia is dependent on activation of voltage sensitive Ca2+ channels. Brain Res Dev Brain Res 94:182–189

    Article  CAS  PubMed  Google Scholar 

  2. Aggett PJ, Fenwick PK, Kirk H (1982) The effect of amphotericin B on the permeability of lipid bilayers to divalent trace metals. Biochim Biophys Acta 684:291–294

    Article  CAS  PubMed  Google Scholar 

  3. Augustine GJ, Neher E (1992) Calcium requirements for secretion in bovine chromaffin cells. J Physiol 450:247–271

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Babcock DF, Herrington J, Goodwin PC, Park YB, Hille B (1997) Mitochondrial participation in the intracellular Ca2+ network. J Cell Biol 136:833–844

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y et al (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476:341–345

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Bernardi P (1999) Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79:1127–1155

    CAS  PubMed  Google Scholar 

  7. Bournaud R, Hidalgo J, Yu H, Jaimovich E, Shimahara T (2001) Low threshold T-type calcium current in rat embryonic chromaffin cells. J Physiol 537:35–44

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Carafoli E (1979) The calcium cycle of mitochondria. FEBS Lett 104:1–5

    Article  CAS  PubMed  Google Scholar 

  9. Caricati-Neto A, Padin JF, Silva-Junior ED, Fernandez-Morales JC, de Diego AM, Jurkiewicz A et al (2013) Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37 degrees C. Physiol Rep 1:e00182

    Article  PubMed Central  PubMed  Google Scholar 

  10. Chalmers S, Nicholls DG (2003) The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria. J Biol Chem 278:19062–19070

    Article  CAS  PubMed  Google Scholar 

  11. Coll KE, Joseph SK, Corkey BE, Williamson JR (1982) Determination of the matrix free Ca2+ concentration and kinetics of Ca2+ efflux in liver and heart mitochondria. J Biol Chem 257:8696–8704

    CAS  PubMed  Google Scholar 

  12. Comline RS, Silver M (1966) Development of activity in the adrenal medulla of the foetus and new-born animal. Br Med Bull 22:16–20

    CAS  PubMed  Google Scholar 

  13. Crompton M, Heid I (1978) The cycling of calcium, sodium, and protons across the inner membrane of cardiac mitochondria. Eur J Biochem 91:599–608

    Article  CAS  PubMed  Google Scholar 

  14. Csordás G, Thomas AP, Hajnóczky G (1999) Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria. EMBO J 18:96–108

    Article  PubMed Central  PubMed  Google Scholar 

  15. Cuchillo-Ibanez I, Lejen T, Albillos A, Rose SD, Olivares R, Villarroya M et al (2004) Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and secretion in bovine chromaffin cells. J Physiol 560:63–76

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476:336–340

    Article  PubMed Central  PubMed  Google Scholar 

  17. Duchen MR (1999) Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol 516:1–17

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Duchen MR (2000) Mitochondria and calcium: from cell signalling to cell death. J Physiol 529:57–68

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Feldberg W, Minz B, Tsudzimura H (1934) The mechanism of the nervous discharge of adrenaline. J Physiol 81:286–304

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Fernández-Morales JC, Cortés-Gil L, García AG, de Diego AM (2009) Differences in the quantal release of catecholamines in chromaffin cells of rat embryos and their mothers. Am J Physiol Cell Physiol 297:C407–C418

    Article  PubMed  Google Scholar 

  21. Fernández-Morales JC, Padin JF, Arranz-Tagarro JA, Vestring S, Garcia AG, de Diego AM (2014) Hypoxia-elicited catecholamine release is controlled by L-type as well as N/PQ types of calcium channels in rat embryo chromaffin cells. Am J Physiol Cell Physiol 307:C455–C465

    Article  PubMed  Google Scholar 

  22. Garcia AG, Garcia-De-Diego AM, Gandia L, Borges R, Garcia-Sancho J (2006) Calcium signaling and exocytosis in adrenal chromaffin cells. Physiol Rev 86:1093–1131

    Article  CAS  PubMed  Google Scholar 

  23. Garcia-Fernandez M, Mejias R, Lopez-Barneo J (2007) Developmental changes of chromaffin cell secretory response to hypoxia studied in thin adrenal slices. Pflugers Arch 454:93–100

    Article  CAS  PubMed  Google Scholar 

  24. Giovannucci DR, Hlubek MD, Stuenkel EL (1999) Mitochondria regulate the Ca(2+)-exocytosis relationship of bovine adrenal chromaffin cells. J Neurosci 19:9261–9270

    CAS  PubMed  Google Scholar 

  25. Gunter TE, Pfeiffer DR (1990) Mechanisms by which mitochondria transport calcium. Am J Physiol 258:C755–C786

    CAS  PubMed  Google Scholar 

  26. Hernandez-Guijo JM, Maneu-Flores VE, Ruiz-Nuno A, Villarroya M, Garcia AG, Gandia L (2001) Calcium-dependent inhibition of L, N, and P/Q Ca2+ channels in chromaffin cells: role of mitochondria. J Neurosci 21:2553–2560

    CAS  PubMed  Google Scholar 

  27. Herrington J, Park YB, Babcock DF, Hille B (1996) Dominant role of mitochondria in clearance of large Ca2+ loads from rat adrenal chromaffin cells. Neuron 16:219–228

    Article  CAS  PubMed  Google Scholar 

  28. Horikawa Y, Goel A, Somlyo AP, Somlyo AV (1998) Mitochondrial calcium in relaxed and tetanized myocardium. Biophys J 74:1579–1590

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Levitsky KL, Lopez-Barneo J (2009) Developmental change of T-type Ca2+ channel expression and its role in rat chromaffin cell responsiveness to acute hypoxia. J Physiol 587:1917–1929

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Machado DJ, Montesinos MS, Borges R (2008) Good practices in single-cell amperometry. Methods Mol Biol 440:297–313

    Article  CAS  PubMed  Google Scholar 

  31. Mahapatra S, Calorio C, Vandael DH, Marcantoni A, Carabelli V, Carbone E (2012) Calcium channel types contributing to chromaffin cell excitability, exocytosis and endocytosis. Cell Calcium 51:321–330

    Article  CAS  PubMed  Google Scholar 

  32. Miranda-Ferreira R, de Pascual R, Caricati-Neto A, Gandia L, Jurkiewicz A, Garcia AG (2009) Role of the endoplasmic reticulum and mitochondria on quantal catecholamine release from chromaffin cells of control and hypertensive rats. J Pharmacol Exp Ther 329:231–240

    Article  CAS  PubMed  Google Scholar 

  33. Mojet MH, Mills E, Duchen MR (1997) Hypoxia-induced catecholamine secretion in isolated newborn rat adrenal chromaffin cells is mimicked by inhibition of mitochondrial respiration. J Physiol 504:175–189

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Montero M, Alonso MT, Albillos A, Cuchillo-Ibanez I, Olivares R, Villalobos C et al (2002) Effect of inositol 1,4,5-trisphosphate receptor stimulation on mitochondrial [Ca2+] and secretion in chromaffin cells. Biochem J 365:451–459

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Montero M, Alonso MT, Carnicero E, Cuchillo-Ibáñez I, Albillos A, García AG, García-Sancho J, Alvarez J (2000) Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion. Nat Cell Biol 2:57–61

    Article  CAS  PubMed  Google Scholar 

  36. Neher E (1998) Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20:389–399

    Article  CAS  PubMed  Google Scholar 

  37. Park YB, Herrington J, Babcock DF, Hille B (1996) Ca2+ clearance mechanisms in isolated rat adrenal chromaffin cells. J Physiol 492:329–346

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Reynafarje B, Lehninger AL (1977) Electric charge stoichiometry of calcium translocation in mitochondria. Biochem Biophys Res Commun 77:1273–1279

    Article  CAS  PubMed  Google Scholar 

  39. Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469–477

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Seidler FJ, Slotkin TA (1985) Adrenomedullary function in the neonatal rat: responses to acute hypoxia. J Physiol 358:1–16

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Seidler FJ, Slotkin TA (1986) Non-neurogenic adrenal catecholamine release in the neonatal rat: exocytosis or diffusion? Brain Res 393:274–277

    Article  CAS  PubMed  Google Scholar 

  42. Seidler FJ, Slotkin TA (1986) Ontogeny of adrenomedullary responses to hypoxia and hypoglycemia: role of splanchnic innervation. Brain Res Bull 16:11–14

    Article  CAS  PubMed  Google Scholar 

  43. Sorensen JB, Nagy G, Varoqueaux F, Nehring RB, Brose N, Wilson MC et al (2003) Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23. Cell 114:75–86

    Article  CAS  PubMed  Google Scholar 

  44. Takeuchi Y, Mochizuki-Oda N, Yamada H, Kurokawa K, Watanabe Y (2001) Nonneurogenic hypoxia sensitivity in rat adrenal slices. Biochem Biophys Res Commun 289:51–56

    Article  CAS  PubMed  Google Scholar 

  45. Thompson RJ, Jackson A, Nurse CA (1997) Developmental loss of hypoxic chemosensitivity in rat adrenomedullary chromaffin cells. J Physiol 498:503–510

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Uceda G, García AG, Guantes JM, Michelena P, Montiel C (1995) Effects of Ca2+ channel antagonist subtypes on mitochondrial Ca2+ transport. Eur J Pharmacol 289:73–80

    Article  CAS  PubMed  Google Scholar 

  47. Villalobos C, Nunez L, Montero M, Garcia AG, Alonso MT, Chamero P et al (2002) Redistribution of Ca2+ among cytosol and organelle during stimulation of bovine chromaffin cells. FASEB J 16:343–353

    Article  CAS  PubMed  Google Scholar 

  48. Villanueva J, Viniegra S, Gimenez-Molina Y, García-Martinez V, Expósito-Romero G, del Mar Frances M, García-Sancho J, Gutiérrez LM (2014) The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells. J Cell Sci 127:5105–5114

    Article  CAS  PubMed  Google Scholar 

  49. Wang GJ, Thayer SA (2002) NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture. J Neurophysiol 87:740–749

    CAS  PubMed  Google Scholar 

  50. Warashina A (2006) Mode of mitochondrial Ca2+ clearance and its influence on secretory responses in stimulated chromaffin cells. Cell Calcium 39:35–46

    Article  CAS  PubMed  Google Scholar 

  51. Werth JL, Thayer SA (1994) Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons. J Neurosci 14:348–356

    CAS  PubMed  Google Scholar 

  52. White RJ, Reynolds IJ (1997) Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones. J Physiol 498:31–47

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Wightman RM, Jankowski JA, Kennedy RT, Kawagoe KT, Schroeder TJ, Leszczyszyn DJ et al (1991) Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc Natl Acad Sci U S A 88:10754–10758

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. Xu T, Naraghi M, Kang H, Neher E (1997) Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. Biophys J 73:532–545

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We want to express our gratitude to Professor Luis Santamaría-Solís (Department of Anatomy, Histology, and Neuroscience, School of Medicine, University Autónoma of Madrid) for helping us with the data analysis of EM imaging. This work was supported by the following grants to AGG: (1) SAF 2010–21795 and (2) SAF 2013–44108, Ministerio de Economía y Competitividad, Spain; (3) CABICYC, UAM/Bioibérica, Spain; and (4) the continued support of Fundación Teófilo Hernando, Madrid, Spain.

Authors’ contributions

Antonio G. García, Antonio-Miguel G. de Diego, Juan-Fernando Padín and José-Carlos Fernández-Morales participated in the research design; Stefan Vestring, José-Carlos Fernández-Morales, Antonio-Miguel G. de Diego, Juan-Fernando Padín, Diego Castro-Musial and Iago Méndez-López conducted the experiments; José-Carlos Fernández-Morales, Stefan Vestring, Juan-Fernando Padín and Iago Méndez-López performed the data analysis; and Antonio G. García and Juan-Fernando Padín wrote or contributed to the writing of the manuscript.

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Instituto Teófilo Hernando, Universidad Autónoma de Madrid, 28029, Madrid, Spain

    Stefan Vestring, José C. Fernández-Morales, Iago Méndez-López, Diego C. Musial, Antonio-Miguel G. de Diego, J. Fernando Padín & Antonio G. García

  2. Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Avda. Arzobispo Morcillo, 4, 28029, Madrid, Spain

    José C. Fernández-Morales, Iago Méndez-López, Diego C. Musial, Antonio-Miguel G. de Diego, J. Fernando Padín & Antonio G. García

  3. Servicio de Farmacología Clínica, Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, UAM, Madrid, Spain

    Antonio G. García

  4. Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

    Stefan Vestring

  5. Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil

    Diego C. Musial

Authors
  1. Stefan Vestring
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. José C. Fernández-Morales
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Iago Méndez-López
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diego C. Musial
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Antonio-Miguel G. de Diego
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. Fernando Padín
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Antonio G. García
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Antonio G. García.

Additional information

Stefan Vestring and José C. Fernández-Morales are equal junior contributors, and Fernando Padín and Antonio G. García are equal senior contributors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vestring, S., Fernández-Morales, J.C., Méndez-López, I. et al. Tight mitochondrial control of calcium and exocytotic signals in chromaffin cells at embryonic life. Pflugers Arch - Eur J Physiol 467, 2589–2601 (2015). https://doi.org/10.1007/s00424-015-1724-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-015-1724-2

Keywords

Search

Navigation