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β-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, Anthony J. Walker


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 theβ -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.

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