Cell signalling pathways regulating nitric oxide (NO) production in 'Lymnaea stagnalis' haemocytes

Wright, Bernice (2004) Cell signalling pathways regulating nitric oxide (NO) production in 'Lymnaea stagnalis' haemocytes. (MSc(R) thesis), Kingston University, .


Haemocytes are the primary cells which carry out the cellular immune response in molluscs. They are macrophage-like cells that remove antigens by endocytosis, phagocytosis and encapsulation. Reactive oxygen/nitrogen intermediates are used in haemocyte-mediated mechanisms that allow a recognised organism to be killed. This study aims to describe the production of nitric oxide (NO) in ‘Lymnaea stagnalis’ haemocytes in response to challenge by various compounds and to identify the signalling pathways that regulate NO production in those cells. When haemocytes were challenged with Phorbol Myristate Acetate (PMA) (10 [mu]M), laminarin (10 mg/ml) and yeast particles (zymosan A) (10 [mu]g/ml), NO production was increased (8 times, 4 times and 2.2 times respectively) more than the NO produced by their controls, following a 60 min stimulation. This is in accordance with previous work done on other mollusc species which have shown that NO is produced following exposure to PMA, laminarin and zymosan A, as well as when challenged with bacteria and bacterial lipopolysaccharide. Further characterisation of NO production was carried out by observing the effects of the NOS inhibitors L-nitro arginine methyl ester (L-NAME) and L-nitro monomethyl arginine (L-NMMA) on NO production. L-NAME (10 mM) and L-NMMA (10 mM) partially inhibited PMA (by 90% and 100% respectively) and laminarin-induced (by 106% and 113% respectively) NO production, providing further evidence that a NOS-like protein is involved in NO production in ‘L. stagnalis’ haemocytes. Given that the mitogen-activated protein kinase (MAPK) and the protein kinase C (PKC) pathways play a key role in NO production in mammalian macrophages, the role of these signalling pathways in NOS activation in ‘L. stagnalis’ haemocytes was investigated. Inhibitors of these pathways significantly reduced NO production in haemocytes challenged with PMA or laminarin, with greatest effect seen with the MAPK pathway inhibitor, U0126. These results demonstrate for the first time a key role for MAPK and PKC pathways in NO production by molluscan haemocytes.

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