Skip to main content

Advertisement

Log in

Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells

  • Original Research Paper
  • Published:
Inflammation Research Aims and scope Submit manuscript

Abstract

Objective

Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions.

Methods

The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNγ) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-κB) were studied by Western blot.

Results

CARB increased NO synthesis by 57 ± 5%, while a 150 ± 10% increase in NO liberation was triggered by LPS plus IFNγ treatment. CARB added to LPS plus IFNγ potentiated NO synthesis by 227 ± 19%. CARB also upregulated NOS1 protein expression via NF-κB activation. In addition CARB and LPS plus IFNγ stimulated PGE2 synthesis by 72 ± 9 and 42 ± 4%, respectively, while CARB added to LPS plus IFNγ treated cells produced a synergism in PGE2 liberation (130 ± 12%) via COX-2.

Conclusion

Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.

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

Access this article

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
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Stuehr J, Marletta MA. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma. J Immunol. 1987;139:518–25.

    CAS  PubMed  Google Scholar 

  2. Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA. 1993;90:522–6.

    Article  CAS  PubMed  Google Scholar 

  3. O’Brien WJ, Heimann T, Tsao LS, Seet BT, McFadden G, Taylor JL. Regulation of nitric oxide synthase 2 in rabbit corneal cells. Invest Ophthal Vis Sci. 2001;42:713-9.

    Google Scholar 

  4. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406:282–7.

    Article  Google Scholar 

  5. Frey EA, Miller DS, Jahr TG, Sundan A, Bazil V, Espevik T, et al. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med. 1992;176:1665–71.

    Article  CAS  PubMed  Google Scholar 

  6. Jones E, Adcock I, Ahmed B, Punchard N. Modulation of LPS stimulated NF-kappa B mediated nitric oxide production by PKCε and JAK2 in RAW macrophages. J Inflamm. 2007;4:1–9.

    Article  Google Scholar 

  7. Davidson JM. Inflammation: wound repair. New York: Garland Press; 1992.

    Google Scholar 

  8. Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol. 1999;72:209–53.

    Article  CAS  PubMed  Google Scholar 

  9. Buckley CD, Pilling D, Lord JM, Akbar AN, Scheel-Toellner D, Salmon M. Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation. Trends Immunol. 2001;22:199–204.

    Article  CAS  PubMed  Google Scholar 

  10. Buchli R, Ndoye A, Rodriguez JG, Zia S, Webber RJ, Grando SA. Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors. J Cell Biochem. 1999;74:264–77.

    Article  CAS  PubMed  Google Scholar 

  11. Español A, de la Torre E, Sales ME. Parasympathetic modulation of local acute inflammation in murine submandibular glands. Inflammation. 2003;27:97–106.

    Article  PubMed  Google Scholar 

  12. Chen TR. In situ detection of micoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1997;104:255–62.

    Article  Google Scholar 

  13. Granger D, Hidds J, Perfect J, Durack D. Metabolic fate of l-arginine in relation to microbiostatic capability of murine macrophages. Clin Invest. 1990;85:264–73.

    Article  CAS  Google Scholar 

  14. Fiszman G, Cattaneo V, de la Torre E, Español A, Colombo L, Sacerdote de Lustig E, et al. Muscarinic receptors autoantibodies purified from mammary adenocarcinoma bearing mice sera stimulate tumor progression. Int Immunopharma. 2006;6:1323–30.

    Article  CAS  Google Scholar 

  15. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem. 1976;72:248–54.

    Article  CAS  PubMed  Google Scholar 

  16. Español AJ, de la Torre E, Fisman GL, Sales ME. Role of non-neuronal cholinergic system in breast cancer progression. Life Sci. 2007;80:2281–5.

    Article  PubMed  Google Scholar 

  17. Español AJ, Sales ME. Different muscarinic receptors are involved in the proliferation of murine mammary adenocarcinoma cell lines. Int J Mol Med. 2004;13:311–9.

    PubMed  Google Scholar 

  18. Granstrom E, Kindhal H. Radioimmunoassay of prostaglandins and thomboxanes research. New York, Raven Press. 1978;5:119–210.

  19. Goren N, Cuenca J, Martín-Sanz P, Boscá L. Attenuation of NF-kB signaling in rat cardiomyocytes at birth restricts the induction of inflammatory genes. Cardiovascular Res. 2004;64:289–97.

    Article  CAS  Google Scholar 

  20. Bulseco DA, Poluha W, Schonhoff CM, Daou MC, Condon PJ, Ross AH. Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase. J Cell Biochem. 2001;8:193–204.

    Article  Google Scholar 

  21. Noga O, Hanf G, Schäper C, O’Connor A, Kunkel G. The influence of inhalative corticosteroids on circulating nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in allergic asthmatics. Clin Exp Allergy. 2001;31:1906–12.

    Article  CAS  PubMed  Google Scholar 

  22. Eglen RM. Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic function. Auton Autacoid Pharmacol. 2006;26:219–33.

    Article  CAS  PubMed  Google Scholar 

  23. Wessler I, Kilbinger H, Bittinger F, Unger R, Kirkpatrick CJ. The non-neuronal cholinergic system in humans: expression, function and pathophysiology. Life Sci. 2003;72:2055–61.

    Article  CAS  PubMed  Google Scholar 

  24. de la Torre E, Genaro AM, Ribeiro ML, Pagotto R, Pignataro OP, Sales ME. Proliferative actions of muscarinic receptors expressed in macrophages derived from normal and tumor bearing mice. Biochim Biophys Acta. 2008;782:82–9.

    Google Scholar 

  25. Sterin-Borda L, Ganzinelli S, Berra A, Borda E. Novel insight into the mechanisms evolved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels. Neuropharmacology. 2003;45:260–9.

    Article  CAS  PubMed  Google Scholar 

  26. Molina-Holgado E, Khorchid A, Liu H, Almazan G. Regulation of muscarinic receptor function in developing oligodendrocytes by agonist exposure. Br J Pharmacol. 2003;13:47–56.

    Article  Google Scholar 

  27. Carbone DL, Moreno JA, Tjalkens RB. Nuclear factor kappa-B mediates selective induction of neuronal nitric oxide synthase in astrocytes during low-level inflammatory stimulation with MPTP. Brain Res. 2008;1217:1–9.

    Article  CAS  PubMed  Google Scholar 

  28. Guizzetti M, Bordi F, Dieguez-Acuña FJ, Vitalone A, Madia F, Woods JS, et al. Nuclear factor kappaB activation by muscarinic receptors in astroglial cells: effect of ethanol. Neuroscience. 2003;120:941–50.

    Article  CAS  PubMed  Google Scholar 

  29. Pausawasdi N, Ramamoorthy S, Crofford LJ, Askari FK, Todisco A. Regulation and function of COX-2 gene expression in isolated gastric parietal cells. Am J Physiol Gastrointest Liver Physiol. 2002;282:G1069–78.

    Google Scholar 

  30. Fraser CC. G protein-coupled receptor connectivity to NF-kappaB in inflammation and cancer. Int Rev Immunol. 2008;27:320–50.

    Article  CAS  PubMed  Google Scholar 

  31. Dumont I, Peri KG, Hardy P, Hou X, Martinez-Bermudez AK, Molotchnikoff S, et al. PGE2, via EP3 receptors, regulates brain nitric oxide synthase in the perinatal period. Am J Physiol. 1998;275:R1812–21.

    CAS  PubMed  Google Scholar 

  32. Razani-Boroujerdi S, Behl M, Hahn F, Pena-Philippides JC, Hutt J, Sopori ML. Role of muscarinic receptors in the regulation of immune inflammatory response. J Neuroimmunol. 2008;194:82–8.

    Article  Google Scholar 

  33. Salvemini D. Regulation of cyclooxygenase enzymes by nitric oxide. Cell Mol Life Sci. 1997;53:576–82.

    Article  CAS  PubMed  Google Scholar 

  34. Davel L, D′Agostino A, Español A, Jasnis MA, Lauría de Cidre L, de Lustig ES et al. Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration. J Biol Reg Homeost Agent. 2002;16:181–9.

    Google Scholar 

  35. Dolan S, Kelly JG, Huan M, Nolan AM. Transient up-regulation of spinal cyclooxygenase-2 and neuronal nitric oxide synthase following surgical inflammation. Anesthesiology. 2003;98:170–80.

    Article  CAS  PubMed  Google Scholar 

  36. Bonizzi G, Karin M. The two NF-kB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004;25:280–9.

    Article  CAS  PubMed  Google Scholar 

  37. Tran K, Merika M, Thanos D. Distinct functional properties of IkBa and IkBb. Mol Cell Biol. 1997;17:5386–99.

    CAS  PubMed  Google Scholar 

  38. Beg AA, Finco TS, Nantermet PV, Baldwin AS Jr. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IkBa: a mechanism for NF-kB activation. Mol Cell Biol. 1993;13:3301–10.

    CAS  PubMed  Google Scholar 

  39. Gough DJ, Levy DE, Johnstone RW, Clarke CJ. IFNγ signaling—does it mean JAK–STAT? Cytokine Growth Factor Rev 2008;19:383–94.

    Google Scholar 

  40. Boivin MA, Roy PK, Bradley A, Kennedy JC, Rihani T, Ma TY. Mechanism of interferon-gamma-induced increase in T84 intestinal epithelial junction. J Interferon Cytokine Res. 2009;29:45–54.

    Article  CAS  PubMed  Google Scholar 

  41. Schliebs R, Heidel K, Apelt J, Gniezdzinska M, Kirazov L, Szutowicz A. Interaction of interleukin-1β with muscarinic acetylcholine receptor-mediated signaling cascade in cholinergically differentiated SH-SY5Y cells. Brain Res. 2006;1122:78–85.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors want to thank Mrs. María E. Castro and Ana I. Casella for their excellent technical assistance. Alejandro Español, Nora Goren, María L. Ribeiro, and María E. Sales are established investigators from the National Research Council (CONICET). Authors want especially to thank to the University of Buenos Aires for the grant UBACYT MO04, M064 and to the National Agency for the Promotion of Science and Technology (ANPCyT) for the grant PICT 2006-485 that supported this research.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to María Elena Sales.

Additional information

Responsible Editor: M. Katori.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Español, A.J., Goren, N., Ribeiro, M.L. et al. Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells. Inflamm. Res. 59, 227–238 (2010). https://doi.org/10.1007/s00011-009-0097-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-009-0097-4

Keywords

Navigation