Analytical and Bioanalytical Chemistry

, Volume 394, Issue 5, pp 1497–1504 | Cite as

Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7

  • Michaela Pekarova
  • Jana Kralova
  • Lukas Kubala
  • Milan Ciz
  • Antonin Lojek
  • Cenek Gregor
  • Jan Hrbac
Original Paper


In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium.


Nitric oxide Macrophages RAW 264.7 Nitric oxide sensor Nitrites iNOS expression 



This study was conducted under the research plans AVOZ50040507, AVOZ50040702, and 1M6198959201, supported from the Grant Agency of the Czech Republic (project no. 524/05/P135), the Grant Agency of the AS CR (no. 1QS 500040507), and MEYS (no. OC08058).

Supplementary material

216_2009_2813_MOESM1_ESM.pdf (205 kb)
ESM 1 (PDF 204 kb)


  1. 1.
    Napoli CN, Nigris F, Williams-Ignarro S, Pignalosa O, Sica V, Ignarro LJ (2006) Nitric Oxide 15:265–27CrossRefGoogle Scholar
  2. 2.
    Rawlingson A (2003) Burns 29:631–640CrossRefGoogle Scholar
  3. 3.
    Lancaster JR (1997) Nitric Oxide 1:18–30CrossRefGoogle Scholar
  4. 4.
    Moncada S, Palme RM, Higgs EA (1991) Pharmacol Rev 43:109–142Google Scholar
  5. 5.
    Halliwell B, Gutteridge JMC (2001) Free radicals in biology and medicine. Oxford University Press, EnglandGoogle Scholar
  6. 6.
    Mayer B, Hemmens B (1997) Trends Biochem Sci 22:477–481CrossRefGoogle Scholar
  7. 7.
    Noda T, Amano F (1997) J Biochem 121:38–46Google Scholar
  8. 8.
    Van Miert ASJPAM (2002) Vet Res Commun 26:111–126CrossRefGoogle Scholar
  9. 9.
    Yao D, Vlessidis AG, Evmiridis NP (2004) Microchim Acta 147:1–20CrossRefGoogle Scholar
  10. 10.
    Josji MS, Ponthier JL, Lancaster JR Jr (1999) Free Radic Biol Med 27:1357–1366CrossRefGoogle Scholar
  11. 11.
    Archer S (1993) FASEB J 7:349–360Google Scholar
  12. 12.
    Bryan NS, Grisham MB (2007) Free Radic Biol Med 43:645–657CrossRefGoogle Scholar
  13. 13.
    Aoki T (1990) Biomed Chromatogr 4:128–130CrossRefGoogle Scholar
  14. 14.
    Kleschyov AL, Wenzel P, Munzel TJ, Chromatogr B (2007) Analyt Technol Biomed Life Sci 851:12–20CrossRefGoogle Scholar
  15. 15.
    Xia Y, Zweier JL (1997) Proc Natl Acad Sci U S A 64:12705–12710CrossRefGoogle Scholar
  16. 16.
    Joshi MS, Lancaster JR Jr, Xiaoping L, Ferguson B Jr (2001) Nitric Oxide 5:561–56CrossRefGoogle Scholar
  17. 17.
    Boo YC, Tressel SL, Jo H (2007) Nitric Oxide 16:306–312CrossRefGoogle Scholar
  18. 18.
    Amatore Ch, Arbault S, Bouton C, Drapier JC, Ghandour H, Koh ACW (2008) Chembiochem 9:1472–1480CrossRefGoogle Scholar
  19. 19.
    Amatore Ch, Arbault S, Chen Y, Crozatier C, Tapsoba I (2007) Lab Chip 7:233–238CrossRefGoogle Scholar
  20. 20.
    Amatore Ch, Arbault S, Bouton C, Coffi K, Drapier JC, Ghandour H, Tong Y (2006) Chembiochem 7:653–661CrossRefGoogle Scholar
  21. 21.
    Amatore Ch, Arbault S, Bouret Y, Cauli B, Guille M, Rancillac A, Rossier J (2006) Chembiochem 7:181–187CrossRefGoogle Scholar
  22. 22.
    Malinski T, Taha Z (1992) Nature 358:676–678CrossRefGoogle Scholar
  23. 23.
    Fabre B, Burlet S, Cespuglio R, Bidan G (1997) J Electroanal Chem 426:75–83CrossRefGoogle Scholar
  24. 24.
    Villeneuve N, Bedioui F, Voituriez K, Avaro S, Vilaine JP (1998) J Pharmacol Toxicol Meth 40:95–100CrossRefGoogle Scholar
  25. 25.
    Macherzynski M, Bilecki W, Gorecki J, Przewlocki R, Golas J (2000) Electroanalysis 12(13):1046–1050CrossRefGoogle Scholar
  26. 26.
    Dalbasti T, Cagli S, Kilinc E, Oktar N, Ozsoz M (2002) Nitric Oxide 7:301–305CrossRefGoogle Scholar
  27. 27.
    Carvalho FA, Martins-Silva J, Saldanha C (2004) Biosens Bioelect 20:505–508CrossRefGoogle Scholar
  28. 28.
    Ferreira NR, Ledo A, Frade JG, Gerhardt GA, Laranjinha J, Barbosa RM (2005) Anal Chim Acta 535:1–7CrossRefGoogle Scholar
  29. 29.
    Brovkovych V, Stolarczyk E, Oman J, Tomboulian P, Malinsky T (1999) Pharm. Biomed Anal 19:135–143CrossRefGoogle Scholar
  30. 30.
    Chang SC, Pereira-Rodrigues N, Henderson JR, Cole A, Bedioui F, McNeil CJ (2005) Biosens Bioelect 21:917–922CrossRefGoogle Scholar
  31. 31.
    Isik S, Castillo J, Blochl A, Csoregi E, Schuhmann W (2007) Bioelectrochemistry 70:173–179CrossRefGoogle Scholar
  32. 32.
    Wang Y, Hu S (2006) Biosens Bioelect 22:10–17CrossRefGoogle Scholar
  33. 33.
    Kono T, Saito M, Kinoshita Y, Satoh I, Shinbori C, Satoh K (2007) Mol Cell Biochem 286:139–145CrossRefGoogle Scholar
  34. 34.
    Herrero F, Morales D, Baamonde C, Salas E, Berrazueta JR, Casanova D (2006) Transplant Proc 38:2600–2602CrossRefGoogle Scholar
  35. 35.
    Yomura Y, Shoji Y, Asai D, Murakami E, Ueno S, Nakashima H (2007) Life Sciences 80:1449–1457CrossRefGoogle Scholar
  36. 36.
    Cserey A, Gratzl M (2001) Anal Chem 73:3965–397CrossRefGoogle Scholar
  37. 37.
    Kamei K, Mie M, Yanagida Y, Aizawa M, Kobatake E (2004) Sens Actuators, B, Chem 99:106–112CrossRefGoogle Scholar
  38. 38.
    Hrbac J, Gregor C, Machova M, Kralova J, Bystron T, Ciz M, Lojek A (2007) Bioelectrochemistry 71:46–53CrossRefGoogle Scholar
  39. 39.
    Hrbac J, Vostalova J, Kralova J, Ciz M, Lojek A (2006) Electrochemical Society Transactions 3(10):109–115Google Scholar
  40. 40.
    Friedemann MN, Robinson SW, Gerhardt G (1996) Anal Chem 68:2621CrossRefGoogle Scholar
  41. 41.
    Arias-Negrete S, Jimenez-Romero LA, Solis-Martinez MO, Ramirez-Emiliano J, Avila EE, Cuellar-Mata P (2004) Anal Biochem 328:14–21CrossRefGoogle Scholar
  42. 42.
    Ciz M, Pavelkova M, Gallova L, Kralova J, Kubala L, Lojek A (2008) Physiol Res 57:393–402Google Scholar
  43. 43.
    Nakane M, Klinghofer V, Kuk JE, Donnelly JL, Budzik GP, Pollock JS, Basha F (1995) Mol Pharmacol 47:831–834Google Scholar
  44. 44.
    Boer R, Ulrich WR, Klein T, Mirau B, Haas S, Baur I (2000) Mol Pharmacol 58:1026–1034Google Scholar
  45. 45.
    Southan GJ, Szabo c (1996) Biochem Pharmacol 51:383–394CrossRefGoogle Scholar
  46. 46.
    Stuehr DJ, Marletta MA (1985) Proc Natl Acad Sci U S A 82:7738–7742CrossRefGoogle Scholar
  47. 47.
    Weber TJ, Smallwood HS, Kathmann LE, Markillie LM, Squier TC, Thrall BD (2006) Am J Physiol Cell Physiol 290:1512–1520CrossRefGoogle Scholar
  48. 48.
    Nussler AK, Billiar TR, Liu ZZ, Moris SM (1994) J Biol Chem 269:1257–1261Google Scholar
  49. 49.
    Chiou WF, Chen ChF, Lin JJ (2000) Br J Pharmacol 129:1553–1560CrossRefGoogle Scholar
  50. 50.
    Kleinert H, Pautz A, Punker K, Schwarz PM (2004) Eur J Pharmacol 500:255–266CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Michaela Pekarova
    • 1
  • Jana Kralova
    • 1
  • Lukas Kubala
    • 1
  • Milan Ciz
    • 1
  • Antonin Lojek
    • 1
  • Cenek Gregor
    • 2
  • Jan Hrbac
    • 2
  1. 1.Institute of BiophysicsAcademy of Sciences of the Czech RepublicBrnoCzech Republic
  2. 2.Department of Physical Chemistry, Faculty of SciencePalacky UniversityOlomoucCzech Republic

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