β-1, 3-glucan modulates PKC signalling inLymnaea stagnalisdefence cells: a role for PKC in H2O2 production and downstream ERK activation

Abstract

SUMMARY 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.

Retinoic acid affects calcium signaling in adult molluscan neurons

“…RA has previously been shown to affect protein synthesis Chen 2008), PKA signaling (Kholodenko et al 2007) and PLC activation (Liou et al 2005). However, despite our use of pharmacological inhibitors that 1) are capable of blocking other effects of RA (Farrar et al 2009;Liou et al 2005) and 2) are known effective blockers in Lymnaea (Dobson et al 2006;Farrar et al 2009;Hamakawa et al 1999;Lacchini et al 2006;Marra et al 2013), there was no evidence to support a role for these particular pathways in the effects of atRA on neuronal firing. We did, however, find that acute exposure to atRA resulted in a significant decline in [Ca 2ϩ ] i as early as 15 min after application.…”

“…The final bath concentration of the PKA inhibitors (PKAi), Rp-adenosine 3=,5=-cyclic monophosphorothioate (Rp-cAMPs) was 10 M and for H-89 was 5 M (Marra et al 2013). The final bath concentration of the PLC inhibitor (PLCi), U-73122, was 20 M (Lacchini et al 2006). The vehicle controls for the above inhibitors used 0.1% EtOH in the bath, and atRA was applied in the presence of EtOH.…”

Regulation of hydrogen peroxide release in circulating hemocytes of the planorbid snail Biomphalaria glabrata

“…In contrast, PKC does not appear to be significantly involved in BSA-gal-stimulated H 2 O 2 release in B. glabrata. This was unexpected given the involvement of PKC in both PMA-and laminarininduced H 2 O 2 release by L. stagnalis hemocytes [20] as well as in vertebrate cells as described previously [21]. In addition, the importance of PKC in a variety of molluscan immune-related functions has become increasingly apparent in recent years [23,24].…”

“…The PKC inhibitor Bis I significantly decreased H 2 O 2 production in response to PMA, which is not surprising as PMA activates DAG-dependent PKCs. PKC regulation of H 2 O 2 release was likewise demonstrated in the snail Lymnaea stagnalis, when hemocytes were stimulated with either PMA or laminarin [20]. A role for PKC in vertebrate immune cell H 2 O 2 production is well documented and it has been shown that in bovine leucocytes, NADPH oxidasedependent superoxide production and specifically p47 phosphorylation are PKC dependent [21].…”

Avian Schistosomes and Outbreaks of Cercarial Dermatitis

“…It has been suggested that parasite excretory-secretory products participate in the modulation of hemocyte activities (149). Disruption of hemocyte signaling pathways, such as protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways, may also influence hemocyte activities (150)(151)(152). For example, carbohydrates known to be present on larval surfaces of T. szidati and T. regenti, such as D-galactose and L-fucose (153)(154)(155)(156), affected hemocyte PKC and ERK signaling in L. stagnalis, which suggests an immunosuppressive role (157,158).…”