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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

View larger version (11K): [in this window] [in a new window]   Fig. 1. The effects of laminarin, PMA and zymosan on PKC phosphorylation. Lymnaea stagnalis haemocytes were challenged with (A) laminarin (10 mg ml-1), (B) PMA (10 µmol l-1) or (C) zymosan (10 µg ml-1) for 0-30 min and their effects on PKC phosphorylation determined by western blotting. Equal amounts of protein were loaded in each lane and membranes were probed with anti-phospho PKC (pan), anti-phospho PKC, or anti-phospho PKCßII antibodies as indicated in the figure. Anti-actin antibodies were used to confirm equal loading of proteins. Relative PKC phosphorylation levels (shown in the graph in A) detected with anti-phospho PKC (pan) antibodies were determined by image analysis. Values are means ± s.d. from four independent experiments. *P0.05 and ***P0.001 when compared to control values (time 0).

View larger version (22K): [in this window] [in a new window]   Fig. 2. GF103209X attenuates PKC, MEK and ERK 1/2 phosphorylation in laminarin-challenged haemocytes. Haemocyte monolayers were incubated with various concentrations of GF103209X (0.001-10 µmol l-1), a competitive inhibitor of the ATP-binding site of PKC, or vehicle (0.1% DMSO), prior to challenge with laminarin (10 mg ml-1) for 10 min. Protein extracts from haemocytes were prepared and western blotting was performed using polyclonal phospho-specific antibodies either to (A) PKC, (B) MEK or (C) ERK. Anti-actin antibodies were used to confirm equal loading of proteins. Relative PKC, MEK and ERK phosphorylation levels were determined by image analysis of blots from four independent experiments. Values are mean ± s.d. *P0.05, **P0.01, ***P0.001 when compared with phosphorylation levels observed in stimulated, uninhibited cells.

View larger version (18K): [in this window] [in a new window]   Fig. 3. The PKC inhibitor, calphostin C reduces MEK and ERK 1/2 phosphorylation in laminarin-challenged haemocytes. Haemocyte monolayers were incubated with calphostin C (0.001-10 µmol l-1), a PKC inhibitor that interacts with the cysteine-rich zinc finger structure of the PKC regulatory domain, or vehicle (0.1% DMSO), prior challenge with laminarin (10 mg ml-1) for 10 min. Protein extracts were subjected to SDS-PAGE followed by western blotting using polyclonal phospho-specific antibodies to (A) MEK and to (B) ERK. Anti-actin antibodies were used to confirm equal loading of proteins. Immunoblots are representative of two independent experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Distribution and levels of phosphorylated PKC in L. stagnalis haemocytes investigated by immunocytochemistry. Phosphorylated PKC in (A) untreated haemocytes, (B) haemocytes challenged with laminarin (10 mg ml-1) for 10 min, and (C) haemocytes incubated with GF109203X (10 µmol l-1) for 30 min prior challenge with laminarin (10 mg ml-1) for 10 min. Rhodamine phalloidin (red) stains F-actin and fluorescein (green) shows the phosphorylated PKC detected with the anti-phospho PKC (pan) antibody. Haemocytes were observed with a Leica laser scanning confocal microscope. Results are representative of three independent experiments. Bar, 20 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 5. PKC phosphorylation is PI-3-K-independent but phospholipase C-dependent in laminarin-stimulated haemocytes. Haemocyte monolayers were pre-treated for 30 min with the phospholipase C inhibitors U-73122 and ET-18-OCH3 or the PI-3-K inhibitor LY294002 at similar range of concentrations (0.001-10 µmol l-1), or vehicle prior to the addition of laminarin (10 mg ml-1) for 10 min. Phosphorylated PKC was detected by immunoblotting with the anti-phospho PKC (pan) antibody. (A) For U-73122 inhibition studies, immunoblots are representative of four independent experiments. In the case of (B) ET-18-OCH3 and (C) LY294002 inhibition studies, immunoblots are representative of three independent experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 6. Stimulation of H2O2 production in L. stagnalis haemocytes by laminarin. The generation of H2O2 was investigated in (A) haemocytes stimulated with different doses of laminarin (1-10 mg ml-1) for 30 min, and (B) in haemocytes stimulated with laminarin (10 mg ml-1) over 30 min. Values are relative fluorescence (mean ± s.d.) of two independent experiments, each done in triplicate. *P0.05, **P0.01 and ***P0.001 when compared to control (unstimulated) values.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Inhibition of laminarin-mediated H2O2 generation in haemocytes. The production of H2O2 was investigated in haemocytes pre-incubated with the PKC inhibitors (A) GF109203X (0.01-10 µmol l-1) and (B) Gö 6976 (0.01-10 µmol l-1), or (C) the NADPH oxidase inhibitor apocynin (10-500 µmol l-1), for 30 min prior to stimulation with laminarin (10 mg ml-1). Values are means ± s.d. of two independent experiments each performed in triplicate. *P0.05, **P0.01 and ***P0.001 when compared to H2O2 production in stimulated haemocytes not exposed to either inhibitor (shown as 100%, broken line).