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First published online December 1, 2006
Journal of Experimental Biology 209, 4829-4840 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02561

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ß-1, 3-glucan modulates PKC signalling in Lymnaea stagnalis defence cells: a role for PKC in H₂O₂ production and downstream ERK activation

Audrey H. Lacchini, Angela J. Davies, David Mackintosh and Anthony J. Walker^*

School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey, KT1 2EE, UK

^* Author for correspondence (e-mail: t.walker{at}kingston.ac.uk)

Accepted 25 September 2006

Haemocytes from the gastropod snail Lymnaea stagnalis (Linnaeus) were used as a model to characterize protein kinase C (PKC) signalling events in molluscan defence cells. Challenge of freshly collected haemocytes with the ß-1, 3-glucan laminarin resulted in a transient increase in the phosphorylation of haemocyte PKC, with maximal phosphorylation (represented by a 3.5-fold increase) occurring at 10 min; this effect was blocked by the PKC inhibitor, GF109203X. Moreover, extracellular signal-regulated kinase (ERK) was found to be a downstream target of molluscan PKC, operating via a MAPK/ERK kinase (MEK)-dependent mechanism. Pharmacological inhibition of PKC phosphorylation by U-73122 and ET-18-OCH₃ suggested that laminarin-dependent PKC signalling was modulated via phospholipase C (PLC); however, a role for phosphatidylinositol-3-kinase (PI-3-K) is unlikely since the PI-3-K inhibitor LY294002 was without effect. Generation of H₂O₂ by haemocytes in response to laminarin was also investigated. H₂O₂ output increased in a dose- and time-dependent manner, with 10 mg ml^-1 laminarin eliciting a 9.5-fold increase in H₂O₂ production after 30 min. H₂O₂ production was significantly attenuated by the PKC inhibitors, GF109203X and Gö 6976, and by the NADPH-oxidase inhibitor, apocynin. In conclusion, these data further our understanding of PKC signalling events in molluscan haemocytes and for the first time define a role for PKC in H₂O₂ production by these defence cells. Given that H₂O₂ is an important anti-pathogen molecule, and that haemocytes play a crucial role in the elimination of invading organisms, PKC signalling in these cells is likely to be crucial to the molluscan innate defence response.

Key words: haemocyte, mollusc defence, PKC, ERK, hydrogen peroxide, reactive oxygen species