Skip to main content
SpringerLink
Log in

Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The carotid body is an arterial chemoreceptor organ that senses arterial pO2 and pH. Previous studies have indicated that both reactive oxygen species (ROS) and nitric oxide (NO) are important potential mediators that may be involved in the response of the carotid body to hypoxia. However, whether their production by the chemosensitive elements of the carotid body is indeed oxygen-dependent is currently unclear. Thus, we have investigated their production under normoxic (20% O2) and hypoxic (1% O2) conditions in slice preparations of the rat carotid body by using fluorescent indicators and confocal microscopy. NO-synthesizing enzymes were identified by immunohistochemistry and histochemistry, and the subcellular localization of the NO-sensitive indicator diaminofluorescein was determined by a photoconversion technique and electron microscopy. Glomus cells of the carotid body responded to hypoxia by increases in both ROS and NO production. The hypoxia-induced increase in NO generation required (to a large extent, but not completely) extracellular calcium. Glomus cells were immunoreactive to endothelial NO synthase but not to the neuronal or inducible isoforms. Ultrastructurally, the NO-sensitive indicator was observed in mitochondrial membranes after exposure to hypoxia. The data show that glomus cells respond to exposure to hypoxia by the enhanced production of both ROS and NO. NO production by glomus cells is probably mediated by endothelial NO synthase, which is activated by calcium influx. The presence of NO indicator in mitochondria suggests the hypoxic regulation of mitochondrial function via NO in glomus cells.

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

Similar content being viewed by others

References

  • Acker H, Dufau E, Huber J, Sylvester D (1989) Indications to an NADPH oxidase as a possible pO2 sensor in the rat carotid body. FEBS Lett 256:75–78

    Article  PubMed  CAS  Google Scholar 

  • Acker H, Bolling B, Delpiano MA, Dufau E, Gorlach A, Holtermann G (1992) The meaning of H2O2 generation in carotid body cells for PO2 chemoreception. J Auton Nerv Syst 41:41–51

    Article  PubMed  CAS  Google Scholar 

  • Beltran B, Mathur A, Duchen MR, Erusalimsky JD, Moncada S (2000) The effect of nitric oxide on cell respiration: a key to understanding its role in cell survival or death. Proc Natl Acad Sci USA 97:14602–14607

    Article  PubMed  CAS  Google Scholar 

  • Bloch W, Mehlhorn U, Krahwinkel A, Reiner M, Dittrich M, Schmidt A, Addicks K (2001) Ischemia increases detectable endothelial nitric oxide synthase in rat and human myocardium. Nitric Oxide 5:317–333

    Article  PubMed  CAS  Google Scholar 

  • Buckler KJ (1997) A novel oxygen-sensitive potassium current in rat carotid body type I cells. J Physiol (Lond) 498:649–662

    CAS  Google Scholar 

  • Buckler KJ, Vaughan-Jones RD (1998) Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells. J Physiol (Lond) 513:819–833

    Article  CAS  Google Scholar 

  • Cathcart R, Schwiers E, Ames BN (1983) Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal Biochem 134:111–116

    Article  PubMed  CAS  Google Scholar 

  • Chandel NS, Schumacker PT (2000) Cellular oxygen sensing by mitochondria: old questions, new insight. J Appl Physiol 88:1880–1889

    Article  PubMed  CAS  Google Scholar 

  • Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95:11715–11720

    Article  PubMed  CAS  Google Scholar 

  • Chugh DK, Katayama M, Mokashi A, Bebout DE, Ray DK, Lahiri S (1994) Nitric oxide-related inhibition of carotid chemosensory nerve activity in the cat. Respir Physiol 97:147–156

    Article  PubMed  CAS  Google Scholar 

  • Cross AR, Henderson L, Jones OT, Delpiano MA, Hentschel J, Acker H (1990) Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body. Biochem J 272:743–747

    PubMed  CAS  Google Scholar 

  • Duranteau J, Chandel NS, Kulisz A, Shao Z, Schumacker PT (1998) Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem 273:11619–11624

    Article  PubMed  CAS  Google Scholar 

  • Fung ML, Ye JS, Fung PC (2001) Acute hypoxia elevates nitric oxide generation in rat carotid body in vitro. Pflügers Arch 442:903–909

    Article  PubMed  CAS  Google Scholar 

  • Gonzalez C, Sanz-Alfayate G, Agapito MT, Gomez-Nino A, Rocher A, Obeso A (2002) Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia. Respir Physiol Neurobiol 132:17–41

    Article  PubMed  CAS  Google Scholar 

  • Grimes PA, Mokashi A, Stone RA, Lahiri S (1995) Nitric oxide synthase in autonomic innervation of the cat carotid body. J Auton Nerv Syst 54:80–86

    Article  PubMed  CAS  Google Scholar 

  • He L, Chen J, Dinger B, Sanders K, Sundar K, Hoidal J, Fidone S (2002) Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice. Am J Physiol Cell Physiol 282:C27–C33

    PubMed  CAS  Google Scholar 

  • Hempel SL, Buettner GR, O’Malley YQ, Wessels DA, Flaherty DM (1999) Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic Biol Med 27:146–159

    Article  PubMed  CAS  Google Scholar 

  • Henrich M, Hoffmann K, König P, Gruss M, Fischbach T, Gödecke A, Hempelmann G, Kummer W (2002) Sensory neurons respond to hypoxia with NO production associated with mitochondria. Mol Cell Neurosci 20:307–322

    Article  PubMed  CAS  Google Scholar 

  • Höhler B, Mayer B, Kummer W (1994) Nitric oxide synthase in the rat carotid body and carotid sinus. Cell Tissue Res 276:559–564

    Article  PubMed  Google Scholar 

  • Höhler B, Lange B, Holzapfel B, Goldenberg A, Hänze J, Sell A, Testan H, Möller W, Kummer W (1999) Hypoxic upregulation of tyrosine hydroxylase gene expression is paralleled, but not induced, by increased generation of reactive oxygen species in PC12 cells. FEBS Lett 457:53–56

    Article  PubMed  Google Scholar 

  • Iturriaga R, Mosqueira M, Villanueva S (2000a) Effects of nitric oxide gas on cat carotid body chemosensory response to hypoxia. Brain Res 855:282–286

    Article  PubMed  CAS  Google Scholar 

  • Iturriaga R, Villanueva S, Mosqueira M (2000b) Dual effects of nitric oxide on cat carotid body chemoreception. J Appl Physiol 89:1005–1012

    PubMed  CAS  Google Scholar 

  • Jourd’heuil D (2002) Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide. Free Rad Biol Med 33:676–684

    Article  PubMed  CAS  Google Scholar 

  • Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70:2446–2453

    Article  PubMed  CAS  Google Scholar 

  • König P, Dedio J, Müller-Esterl W, Kummer W (2002) Distribution of the novel eNOS-interacting protein NOSIP in the liver, pancreas, and gastrointestinal tract of the rat. Gastroenterology 123:314–324

    Article  PubMed  CAS  Google Scholar 

  • Kourie JI (1998) Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol 275:C1–C24

    PubMed  CAS  Google Scholar 

  • Kowaltowski AJ, Castilho RF, Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress. FEBS Lett 495:12–15

    Article  PubMed  CAS  Google Scholar 

  • Kroll SL, Czyzyk-Krzeska MF (1998) Role of H2O2 and heme-containing O2 sensors in hypoxic regulation of tyrosine hydroxylase gene expression. Am J Physiol 274:C167–C174

    PubMed  CAS  Google Scholar 

  • Kummer W, Acker H (1997) Cytochrome b558 and hydrogen peroxide production in small intensely fluorescent cells of sympathetic ganglia. Histochem Cell Biol 107:151–158

    Article  PubMed  CAS  Google Scholar 

  • Kummer W, Höhler B, Goldenberg A, Lange B (2000) Subcellular localization and function of B-type cytochromes in carotid body and other paraganglionic cells. Adv Exp Med Biol 475:371–375

    Article  PubMed  CAS  Google Scholar 

  • Lahiri S, Buerk DG, Chugh D, Osanai S, Mokashi A (1995) Reciprocal photolabile O2 consumption and chemoreceptor excitation by carbon monoxide in the cat carotid body: evidence for cytochrome a3 as the primary O2 sensor. Brain Res 684:194–200

    Article  PubMed  CAS  Google Scholar 

  • Lahiri S, Osanai S, Buerk DG, Mokashi A, Chugh DK (1996) Thapsigargin enhances carotid body chemosensory discharge in response to hypoxia in zero [Ca2+]e: evidence for intracellular Ca2+ release. Brain Res 709:141–144

    Article  PubMed  CAS  Google Scholar 

  • Lahiri S, Ehleben W, Acker H (1999) Chemoreceptor discharges and cytochrome redox changes of the rat carotid body: role of heme ligands. Proc Natl Acad Sci USA 96:9427–9432

    Article  PubMed  CAS  Google Scholar 

  • Lahiri S, Rozanov C, Roy A, Storey B, Buerk DG (2001) Regulation of oxygen sensing in peripheral arterial chemoreceptors. Int J Biochem Cell Biol 33:755–774

    Article  PubMed  CAS  Google Scholar 

  • Lopez-Barneo J (1996) Oxygen-sensing by ion channels and the regulation of cellular functions. Trends Neurosci 19:435–440

    PubMed  CAS  Google Scholar 

  • Mills E, Jobsis FF (1972) Mitochondrial respiratory chain of carotid body and chemoreceptor response to changes in oxygen tension. J Neurophysiol 35:405–428

    PubMed  CAS  Google Scholar 

  • Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, Bracale R, Valerio A, Francolini M, Moncada S, Carruba MO (2003) Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science 299:896–899

    Article  PubMed  CAS  Google Scholar 

  • Obeso A, Gomez-Nino A, Gonzalez C (1999) NADPH oxidase inhibition does not interfere with low PO2 transduction in rat and rabbit CB chemoreceptor cells. Am J Physiol 276:C593–C601

    PubMed  CAS  Google Scholar 

  • Paddenberg R, Ishaq B, Goldenberg A, Faulhammer P, Rose F, Weissmann N, Braun-Dullaeus RC, Kummer W (2003) Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature. Am J Physiol Lung Cell Mol Physiol 284:L710–L719

    PubMed  CAS  Google Scholar 

  • Prabhakar NR (1999) NO and CO as second messengers in oxygen sensing in the carotid body. Respir Physiol 115:161–168

    Article  PubMed  CAS  Google Scholar 

  • Prabhakar NR, Kumar GK, Chang CH, Agani FH, Haxhiu MA (1993) Nitric oxide in the sensory function of the carotid body. Brain Res 625:16–22

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez J, Specian V, Maloney R, Jourd'heuil D, Feelisch M (2005) Performance of diamino fluorophores for the localization of sources and targets of nitric oxide. Free Radic Biol Med 38:356–368

    Article  PubMed  CAS  Google Scholar 

  • Sarkela TM, Berthiaume J, Elfering S, Gybina AA, Giulivi C (2001) The modulation of oxygen radical production by nitric oxide in mitochondria. J Biol Chem 276:6945–6949

    Article  PubMed  CAS  Google Scholar 

  • Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD (1999) Cell transformation by the superoxide-generating oxidase Mox1. Nature 401:79–82

    Article  PubMed  CAS  Google Scholar 

  • Sugimoto K, Fujii S, Takemasa T, Yamashita K (2000) Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5-diaminofluorescein diacetate. Histochem Cell Biol 113:341–347

    PubMed  CAS  Google Scholar 

  • Tanaka K, Chiba T (1994) Nitric oxide synthase containing neurons in the carotid body and sinus of the guinea pig. Microsc Res Tech 29:90–93

    Article  PubMed  CAS  Google Scholar 

  • Valdes V, Mosqueira M, Rey S, Del Rio R, Iturriaga R (2003) Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms. Am J Physiol Lung Cell Mol Physiol 284:L57–L68

    PubMed  CAS  Google Scholar 

  • Wang ZZ, Bredt DS, Fidone SJ, Stensaas LJ (1993) Neurons synthesizing nitric oxide innervate the mammalian carotid body. J Comp Neurol 336:419–432

    Article  PubMed  CAS  Google Scholar 

  • Wang ZZ, Stensaas LJ, Bredt DS, Dinger B, Fidone SJ (1994) Localization and actions of nitric oxide in the cat carotid body. Neuroscience 60:275–286

    Article  PubMed  CAS  Google Scholar 

  • Wang ZZ, Stensaas LJ, Dinger BG, Fidone SJ (1995) Nitric oxide mediates chemoreceptor inhibition in the cat carotid body. Neuroscience 65:217–229

    Article  PubMed  CAS  Google Scholar 

  • Wilson DF, Mokashi A, Chugh D, Vinogradov S, Osanai S, Lahiri S (1994) The primary oxygen sensor of the cat carotid body is cytochrome a3 of the mitochondrial respiratory chain. FEBS Lett 351:370–374

    Article  PubMed  CAS  Google Scholar 

  • Wink DA, Mitchell JB (1998) Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25:434–456

    Article  PubMed  CAS  Google Scholar 

  • Wyatt CN, Wright C, Bee D, Peers C (1995) O2-sensitive K+ currents in carotid body chemoreceptor cells from normoxic and chronically hypoxic rats and their roles in hypoxic chemotransduction. Proc Natl Acad Sci USA 92:295–299

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto Y, Henrich M, Snipes RL, Kummer W (2003) Altered production of nitric oxide and reactive oxygen species in rat nodose ganglion neurons during acute hypoxia. Brain Res 961:1–9

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Mrs. T. Papadakis, Mr. M. Bodenbenner, Mr. G. Kripp, and Ms. K. Michael for skillful technical assistance.

Author information

Authors and Affiliations

  1. Institute of Anatomy and Cell Biology, Justus Liebig University, Aulweg 123, 35385, Giessen, Germany

    Yoshio Yamamoto, Peter König, Michael Henrich & Wolfgang Kummer

  2. Department of Anesthesiology, Intensive Care, Pain Therapy, Justus Liebig University, Rudolf-Buchheim-Strasse 7, 35385, Germany

    Michael Henrich

  3. Disease Group, Cardiovascular Diseases, Aventis Pharma Deutschland, Frankfurt, Germany

    Jürgen Dedio

  4. Laboratory of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Morioka Iwate, 020-8550, Japan

    Yoshio Yamamoto

Authors
  1. Yoshio Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter König
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Henrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jürgen Dedio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Kummer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoshio Yamamoto.

Additional information

This study was supported by the DFG (SFB 547, project C1; W.K.), Grant-in-Aid (15780185) from the JSPS, Japan (Y.Y.), and by Young Scientists grants from the Faculty of Medicine of the Justus Liebig University, Giessen, Germany (M.H. and P.K.).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yamamoto, Y., König, P., Henrich, M. et al. Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body. Cell Tissue Res 325, 3–11 (2006). https://doi.org/10.1007/s00441-006-0178-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-006-0178-4

Keywords

Search

Navigation