Skip to main content
SpringerLink
Log in

Effect of hydrogen peroxide on secretory response, calcium mobilisation and caspase-3 activity in the isolated rat parotid gland

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H2O2) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca2+]i) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H2O2 had little or no effect on amylase secretion compared to basal level. Combining H2O2 with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca2+]i comprising an initial peak followed by a plateau. In a Ca2+-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca2+]i. H2O2 alone evoked a gradual and dose-dependent increase in [Ca2+]i. Combining H2O2 with CCh resulted in a decrease in [Ca2+]i compared to the effect of CCh alone. In a Ca2+-free medium, H2O2 still evoked a small increase in [Ca2+]i, but this response was less compared to the results obtained with H2O2 in normal [Ca2+]0. Combining H2O2 with CCh resulted in only a small transient increase in [Ca2+]i. Following CCh stimulation, H2O2 application resulted in a large increase in [Ca2+]i in normal [Ca2+]0. This effect of H2O2 was partially abolished in a nominally free Calcium medium containing EGTA. H2O2 can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H2O2 can also mobilise Ca2+ from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H2O2 may be due to its activity to induce apoptosis in the parotid gland, since H2O2 can stimulate the activity of caspase-3, a marker of cellular apoptosis.

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

References

  1. Forsberg L, de Faire U, Morgenstern R (2001) Oxidative stress, human genetic variation, and disease. Arch Biochem Biophys 389:84–93

    Article  PubMed  CAS  Google Scholar 

  2. Cross CE, Halliwell B, Borish ET, Pryor WA, Ames BN, Saul RL, McCord JM, Harman D (1987) Oxygen radicals and human disease. Ann Intern Med 107:526–545

    PubMed  CAS  Google Scholar 

  3. Halliwell B, Gutteridge JM (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219:1–14

    PubMed  CAS  Google Scholar 

  4. Holbrook NJ, Ikeyama S (2002) Age-related decline in cellular response to oxidative stress: links to growth factor signaling pathways with common defects. Biochem Pharmacol 64:999–1005

    Article  PubMed  CAS  Google Scholar 

  5. Ryter SW, Kim HP, Hoetzel A et al (2007) Mechanisms of cell death in oxidative stress. Antioxid Redox Signal 9:49–89

    Article  PubMed  CAS  Google Scholar 

  6. Curtin JF, Donovan M, Cotter TG (2002) Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 265:49–72

    Article  PubMed  CAS  Google Scholar 

  7. Favero TG, Zable AC, Abramson JJ (1995) Hydrogen peroxide stimulates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum. J Biol Chem 270:25557–25563

    Article  PubMed  CAS  Google Scholar 

  8. Redondo PC, Salido GM, Pariente JA, Rosado JA (2004) Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets. Biochem Pharmacol 67:1065–1076

    Article  PubMed  CAS  Google Scholar 

  9. Rosado JA, Gonzalez A, Salido GM, Pariente JA (2002) Effects of reactive oxygen species on actin filament polymerisation and amylase secretion in mouse pancreatic acinar cells. Cell Signal 14:547–556

    Article  PubMed  CAS  Google Scholar 

  10. Supinski GS, Callahan LA (2005) Diaphragmatic free radical generation increases in an animal model of heart failure. J Appl Physiol 99:1078–1084

    Article  PubMed  Google Scholar 

  11. Sweiry JH, Shibuya I, Asada N et al (1999) Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas. Biochim Biophys Acta 1454:19–30

    PubMed  CAS  Google Scholar 

  12. Granados MP, Salido GM, Pariente JA et al (2004) Generation of ROS in response to CCK-8 stimulation in mouse pancreatic acinar cells. Mitochondrion 3:285–296

    Article  PubMed  CAS  Google Scholar 

  13. Rosado JA, Redondo PC, Salido GM et al (2004) Hydrogen peroxide generation induces pp60src activation in human platelets: evidence for the involvement of this pathway in store-mediated calcium entry. J Biol Chem 279:1665–1675

    Article  PubMed  CAS  Google Scholar 

  14. Wang X, Takeda S, Mochizuki S et al (1999) Mechanisms of hydrogen peroxide-induced increase in intracellular calcium in cardiomyocytes. J Cardiovasc Pharmacol Ther 4:41–48

    Article  PubMed  CAS  Google Scholar 

  15. Korzets A, Chagnac A, Weinstein T et al (1999) H2O2 induces DNA repair in mononuclear cells: evidence for association with cytosolic Ca2+ fluxes. J Lab Clin Med 133:362–369

    Article  PubMed  CAS  Google Scholar 

  16. Krippeit-Drews P, Haberland C, Fingerle J et al (1995) Effects of H2O2 on membrane potential and [Ca2+]i of cultured rat arterial smooth muscle cells. Biochem Biophys Res Commun 209:139–145

    Article  PubMed  CAS  Google Scholar 

  17. Moreau VH, Castilho RF, Ferreira ST et al (1998) Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase at submicromolar iron concentrations: evidence for metal-catalyzed oxidation. Free Radic Biol Med 25:554–560

    Article  PubMed  CAS  Google Scholar 

  18. Pariente JA, Camello C, Camello PJ et al (2001) Release of calcium from mitochondrial and nonmitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide. J Membr Biol 179:27–35

    Article  PubMed  CAS  Google Scholar 

  19. Weber H, Roesner JP, Nebe B et al (1998) Increased cytosolic Ca2+ amplifies oxygen radical-induced alterations of the ultrastructure and the energy metabolism of isolated rat pancreatic acinar cells. Digestion 59:175–185

    Article  PubMed  CAS  Google Scholar 

  20. Kim MH, Cho HS, Jung M et al (2005) Extra-cellular signal-regulated kinase and AP-1 pathways are involved in reactive oxygen species-induced urokinase plasminogen activator receptor expression in human gastric cancer cells. Int J Oncol 26:1669–1674

    PubMed  CAS  Google Scholar 

  21. Mata A, Marques D, Mesquita MF, Singh J (2002) Effect of extra-cellular magnesium on secretagogue-evoked amylase secretion in the isolated rat parotid gland segments. Mag Res 15:161–165

    CAS  Google Scholar 

  22. Rinderknecht H, Marbach EP (1970) A new automated method for the determination of serum-amylase. Clinica et Chemica Acta 29:107–110

    Article  CAS  Google Scholar 

  23. Michalek R, Templeton D (1987) Description of an automated assay for measurement of alpha-amylase in vitro from rat parotid gland slices. Gen Pharmacol 18:555–558

    PubMed  CAS  Google Scholar 

  24. Baum BJ, Ambudkar IS, Helman J et al (1990) Dispersed salivary gland acinar cell preparations for use in studies of neuroreceptor-coupled secretory events. Methods Enzymol 192:26–37

    Article  PubMed  CAS  Google Scholar 

  25. Bejarano I, Lozano GM, Ortiz A, García JF, Paredes SD, Rodríguez AB, Pariente JA (2008) Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertil Steril (In press). doi:10.1016/j.fertnstert.2007.08.069

  26. Petersen OH (1992) Stimulus-secretion coupling: cytoplasmic Ca2+ signals and control of ion channels in exocrine acinar cells. J Physiol 448:1–51

    PubMed  CAS  Google Scholar 

  27. Schulz I, Streb H, Bayerdorffer E, Thevenod F (1985) Stimulus-secretion coupling in exocrine glands: role of inositol 1,4,5-trisphosphate, calcium and cAMP. Curr Eye Res 4:467–473

    Article  PubMed  CAS  Google Scholar 

  28. Ambudkar IS, Lockwich T, Hiramatsu Y et al (1992) Calcium entry in rat parotid acinar cells. Mol Cell Biochem 114:73–77

    Google Scholar 

  29. Ambudkar IS (2004) Cellular domains that contribute to Ca2+ entry events. Sci STKE 2004:pe32

  30. Baum BJ, Ambudkar IS (1988) Regulation of calcium handling by rat parotid acinar cells. Mol Cell Biochem 82:67–73

    Article  PubMed  CAS  Google Scholar 

  31. Mata AD (2003) Influence of magnesium on salivary gland secretion: physiological and pathophysiological studies. PhD Thesis, University of Central Lancashire, Preston, United Kingdom

  32. Fulda S, Scaffidi C, Susin SA et al (1998) Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J Biol Chem 273:33942–33948

    Article  PubMed  CAS  Google Scholar 

  33. González-Matheos A, Camello PJ, Salido GM et al (2001) Effect of xanthine oxidase-catalyzed reactive oxygen species generation on secretagogue-evoked calcium mobilization in mouse pancreatic acinar cells. Biochem Pharmacol 62(12):1621–1627

    Article  Google Scholar 

  34. Chakraborti T, Das S, Mondal M et al (1999) Oxidant, mitochondria and calcium: an overview. Cell Signal 11:77–85

    Article  PubMed  CAS  Google Scholar 

  35. Jacobson J, Duchen MR (2002) Mitochondrial oxidative stress and cell death in astrocytes-requirement for stored Ca2+ and sustained opening of the permeability transition pore. J Cell Sci 115:1175–1188

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by MEC-DGI Grant BFU2007-60091.

Author information

Authors and Affiliations

  1. Faculdade de Medicina Dentária da Universidade de Lisboa, UICOB - Oral Sciences and Biomedical Research Group, Cidade Universitária, Lisbon, 1649-003, Portugal

    António Mata, Duarte Marques, João Silveira & Joana Marques

  2. Cell Physiology Research Group, Department of Physiology, University of Extremadura, Caceres, Spain

    María A. Martínez-Burgos, José A. Pariente & Gines M. Salido

  3. Oral Biology Research Group, Instituto Superior de Ciências da Saúde Egas Moniz, Monte de Caparica, Portugal

    Maria F. Mesquita

  4. School of Forensic and Investigative Sciences, Faculty of Science and Technology, University of Central Lancashire, Preston, PR1 2HE, Lancashire, UK

    Jaipaul Singh

Authors
  1. António Mata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Duarte Marques
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. María A. Martínez-Burgos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. João Silveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joana Marques
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Maria F. Mesquita
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. José A. Pariente
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gines M. Salido
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jaipaul Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to António Mata or Jaipaul Singh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mata, A., Marques, D., Martínez-Burgos, M.A. et al. Effect of hydrogen peroxide on secretory response, calcium mobilisation and caspase-3 activity in the isolated rat parotid gland. Mol Cell Biochem 319, 23–31 (2008). https://doi.org/10.1007/s11010-008-9873-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-008-9873-7

Keywords

Search

Navigation