Cellular signalling and regulation of nitric oxide synthesis

Bulut, Vedat (1996) Cellular signalling and regulation of nitric oxide synthesis. PhD thesis, University of Glasgow.

Abstract

Murine macrophages synthesise nitric oxide (NO) from L-arginine, and the reaction is catalysed by NO synthases (NOS). Macrophages stimulated by interferon-gamma (IFN-gamma) and low dose of lipopolysaccharide (LPS) or a high concentration of LPS alone can produce large amounts of NO. Using a murine macrophage cell line, J774, we have investigated the signalling mechanisms for NO production. Transient increases in cyclic adenosine monophosphate (cAMP) did not affect the production of NO. However, prolonged elevation of intracellular cAMP levels, using the prostaglandin E2 and phosphodiesterase inhibitors isobutylmethylxanthine or rolipram, was found to inhibit NO production. This inhibition did not occur at the level of gene transcription, but at the post transcriptional level. Interferon-gamma and LPS treatment resulted in protein kinase C (PKC) activation and translocation into the plasma membrane, which correlated with NO production and NOS activity. The activation and the translocation of PKC was inhibited by IL-4, a physiological inhibitor of NO synthesis. IL-4 and the PKC inhibitor, Ro31-8220, inhibited the transcription of inducible NOS (iNOS) gene. IL-4 did not induce an elevation of cAMP levels in macrophages. These data suggest that IL-4 acts by inhibiting the activation of PKC, which is essential in the induction of iNOS gene transcription, but not via the induction of cAMP generation. We also showed that pertussis toxin (Ptx) sensitive-G-proteins are not involved in the induction of iNOS expression in macrophages, and modification of G-proteins by Ptx and cholera toxin (Ctx) did not alter NO production in J774 cells. NO production, stimulated by IFN-gamma and LPS, was not inhibited by Ctx, even although Ctx induced significant levels of intracellular cAMP. Furthermore, since phospholipase A2 (PLA2) inhibitors, such as 4-4-octadecyl-4- oxobenzene butenoic acid, 4-bromophenacyl bromide and dexamethasone, have no effect on NO production by J774 cells, it is suggested that arachidonic acid metabolites and PLA2 are not involved in NO production in macrophages.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Liew, Professor Foo Y., Severn, Dr. A. and Sands, Dr. William A.
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-71734
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 11 Nov 2021 16:00
• Thesis DOI: 10.5525/gla.thesis.71734
• URI: https://theses.gla.ac.uk/id/eprint/71734