regulation of nitric oxide production in snail (lymnaea stagnalis) defence cells: a role for pkc and...

Biol. Cell (2006) 98, 265–278 (Printed in Great Britain) doi:10.1042/BC20050066 Research article

Regulation of nitric oxide productionin snail (Lymnaea stagnalis) defencecells: a role for PKC and ERKsignalling pathwaysBernice Wright, Audrey H. Lacchini, Angela J. Davies and Anthony J. Walker1

School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, U.K.

Background information. Nitric oxide (NO) is an important molecule in innate immune responses. In molluscs NO isproduced by mobile defence cells called haemocytes; however, the molecular mechanisms that regulate NO pro-duction in these cells is poorly understood. The present study focused on the role of cell signalling pathways inNO production by primary haemocytes from the snail Lymnaea stagnalis.

Results. When haemocytes were challenged with PMA (10 µM) or the β-1,3-glucan laminarin (10 mg/ml), an 8-foldand 4-fold increase in NO production were observed after 60 min respectively. Moreover, the NOS (NO synthase)inhibitors L-NAME (NG-nitro-L-arginine methyl ester) and L-NMMA (NG-monomethyl-L-arginine) were found to blocklaminarin- and PMA-induced NO synthesis. Treatment of haemocytes with PMA or laminarin also increased thephosphorylation (activation) status of PKC (protein kinase C). When haemocytes were preincubated with PKCinhibitors (calphostin C or GF109203X) or inhibitors of the ERK (extracellular-signal-regulated kinase) pathway(PD98059 or U0126) prior to challenge, significant reductions in PKC and ERK phosphorylation and NO productionwere observed following exposure to laminarin or PMA. The greatest effect on NO production was seen withGF109203X and U0126, with PMA-induced NO production inhibited by 94% and 87% and laminarin-induced NOproduction by 50% and 91% respectively.

Conclusions. These data suggest that ERK and PKC comprise part of the signalling machinery that regulates NOSactivation and subsequent production of NO in molluscan haemocytes. To our knowledge, this is the first reportthat shows a role for these signalling proteins in the generation of NO in invertebrate defence cells.

IntroductionThe innate immune system represents an ancient firstline of defence against a variety of invading organ-isms (Hoffmann et al., 1999). In molluscs, innate

1To whom correspondence should be addressed ([email protected]).Key words: extracellular-signal-regulated kinase (ERK), haemocyte, molluscdefence, nitric oxide, protein kinase C (PKC).Abbreviations used: DAF-FM diacetate, 4-amino-5-methylamino-2′7′-difluorescein diacetate; ERK, extracellular-signal-regulated kinase;IFN-γ, interferon-γ; LPS, lipopolysaccharide; MAPK, mitogen-activatedprotein kinase; MEK, MAPK/ERK kinase; D-NAME, NG-nitro-D-arginine methylester; D-NMMA, NG-monomethyl-D-arginine; L-NAME, NG-nitro-L-argininemethyl ester; L-NMMA, NG-monomethyl-L-arginine; NOS, NO synthase;eNOS, endothelial NOS; iNOS, inducible NOS; nNOS, neuronal NOS;Lym-nNOS, nNOS from Lymnaea stagnalis; PKC, protein kinase C; SSS,sterile snail saline.

immunity relies on humoral and cellular defencemechanisms, with cellular defence being co-ordi-nated by circulating haemocytes that functionallyresemble mammalian macrophages (van der Knaapet al., 1993). Molluscan haemocytes can recognize andsubsequently eliminate, or sequester, invading patho-gens through processes that include phagocytosis, en-capsulation and the production of lysosomal enzymesand bacteriostatic substances (van der Knaap et al.,1993; Nunez et al., 1994; Yoshino and Vasta, 1996).In addition, molluscan haemocytes can produce reac-tive oxygen and nitrogen intermediates; these cyto-toxic molecules are thought to play an importantrole in the destruction of micro-organisms and para-sites (Dikkeboom et al., 1988; Adema et al., 1994;

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B. Wright and others

Conte and Ottaviani, 1995; Hahn et al., 2000, 2001;Zelck et al., 2005). In mammals, NO also possessesanti-microbial activity and can act as a protectiveagent against various parasites, including the proto-zoans Trypanosoma cruzi (Pinge-Filho et al., 2005) andLeishmania major (Blos et al., 2003), and the helminthSchistosoma mansoni ( James et al., 1998).

The short-lived reactive nitrogen intermediate,nitric oxide (NO), is generated by NOS (nitric oxidesynthase) isoforms which oxidize the guanadino ni-trogen of L-arginine, a reaction which results in theproduction of equal amounts of L-citrulline and NO(Nathan and Xie, 1994). In mammals, three types ofNOS are known to exist with the Ca2+-depend-ent nNOS (neuronal NOS) and eNOS (endothelialNOS) (also called NOS I and NOS III respectively)isoforms being constitutively expressed, and the ex-pression of the iNOS (inducible NOS) (NOS II) iso-form being up-regulated in various cell types, in-cluding macrophages, following infection (Griffithand Stuehr, 1995). NOS isoforms have also beenidentified in molluscs; mRNA encoding an nNOS(Lym-nNOS) has been cloned and sequenced from thecentral nervous system of the gastropod snail Lymnaeastagnalis and is expressed in key modulatory neuronsof the feeding network (Korneev et al., 1998). More-over, suppression of Lym-nNOS mRNA levels inthis snail reduces its feeding behaviour (Korneevet al., 2002). A Ca2+/calmodulin-dependent NOSfrom the central nervous system of the marine mol-lusc Aplysia californica has also recently been char-acterized at the biochemical level (Bodnarova et al.,2005).

In mammalian cells, activation of nNOS or eNOSand the expression of iNOS are regulated by sig-nal transduction pathways. For example, eNOS andiNOS are expressed in rat adipocytes, and activ-ation of the eNOS isoform by insulin is depend-ent on ERK1/2 (extracellular-signal-regulated kinase1/2) (Ribiere et al., 2002). PKC (protein kinase C)also plays a role in the regulation of eNOS by phos-phorylating the enzyme on the calmodulin-bindingdomain, such that phosphorylation by PKC neg-atively regulates eNOS activity (Matsubara et al.,2003). In contrast with eNOS and nNOS, regulationof iNOS is generally believed to occur at the level ofexpression (Kleinert et al., 2004). Various signallingmodules, including ERK1/2, PKC, p38 MAPK (p38mitogen-activated protein kinase) and JNK (c-Jun

N-terminal kinase) pathways, up-regulate the pro-duction of iNOS in macrophages through the down-stream activation of transcription factors such as NF-κB (nuclear factor κB) (Chen and Wang, 1999; Chanand Riches, 2001; He and Kogut, 2003; Chio et al.,2004). The generation of NO (or its stable end pro-ducts) in response to immunological challenge hasbeen described in haemocytes from a few molluscspecies, including the bivalves Mytilus galloprovin-cialis (Arumugam et al., 2000; Gourdon et al., 2001;Tafalla et al., 2002; Novas et al., 2004), Ruditapesdecussatus (Tafalla et al., 2003) and Crassostrea gigas(Nakayama and Maruyama, 1998), and the gastropodViviparus ater (Conte and Ottaviani, 1995). However,only one report has investigated the cell signallingmechanisms that regulate NO production in mol-luscs, and this suggested a role for PKA (proteinkinase A) in the regulation of NO synthesis inM. galloprovincialis haemocytes in response to inter-leukin-2 (Novas et al., 2004).

Recently, we have reported the presence of ERK-and PKC-like proteins in haemocytes of L. stagnalis,an intermediate host to various trematode parasites,including the avian schistosome Trichobilharzia ocel-lata (Walker and Plows, 2003; Plows et al., 2004). Inaddition, we have shown that the activities of theseproteins are modulated following challenge with bac-terial LPS (lipopolysaccharide), and that ERK andPKC are involved in the phagocytic response of thesecells (Walker and Plows, 2003; Plows et al., 2004). Inthe present study, we report for the first time that NOproduction in L. stagnalis haemocytes is increased inresponse to challenge by the β-1,3-glucan laminarin(from Laminaria digitata) or PMA. Whereas PMA is aphorbol ester which can activate PKC, β-1,3-glucansare well-known PAMP (pathogen-associated molecu-lar pattern) molecules which are biologically activeand elicit various innate immune reactions in mam-mals and invertebrates. Importantly, in the present re-port we also show that laminarin- or PMA-dependentNO synthesis is, at least in part, under the controlof ERK and PKC signal transduction pathways inL. stagnalis haemocytes.

ResultsNO production in stimulated haemocytesUsing the NO probe DAF-FM diacetate (4-amino-5-methylamino-2′7′-difluorescein diacetate) to

266 C© Portland Press 2006 | www.biolcell.org

PKC and ERK regulate NO output in snail haemocytes Research article

determine cellular NO production, the various com-pounds tested showed different abilities to stimulateNO synthesis in L. stagnalis haemocytes. Challengeof haemocytes with PMA resulted in a significantincrease in NO production with time (P � 0.001;Figure 1A). The greatest increase in fluorescencewas observed with haemocytes treated with 10 µMPMA, and the change in fluorescence over 60 minwas approx. 8-fold greater than that of unchallenged(control) haemocytes (Figure 1A). Lower concen-trations of PMA (0.01–1 µM) also significantlyincreased NO production after 60 min (P � 0.001),although the lowest dose (0.001 µM) did not have aneffect.

The β-1,3-glucan laminarin also significantlystimulated NO production in haemocytes in a time-dependent manner (P � 0.001; Figure 1B). Whenused at 10 mg/ml, fluorescence levels increased toapprox. 4-fold greater than that of the control haemo-cytes after 60 min. Whereas 5 mg/ml laminarin alsoincreased haemocyte NO production significantlyover 60 min (P � 0.001; Figure 1B), the lowest dosetested (1 mg/ml) was ineffective.

In contrast with PMA and laminarin, zymosanA only significantly increased NO production atthe highest dose used (10 µg/ml), with the changein fluorescence after 60 min being 2.2-fold greaterthan that of control cells (P � 0.001; Figure 1C).The lower doses of zymosan A were also withouteffect after longer incubation (up to 6 h; data notshown). Bacterial LPS, used as an immune modulatorin vertebrates [often with IFN-γ (interferon-γ)] andinvertebrates, did not significantly increase NO pro-duction in haemocytes at any dose tested (0.01–10 µg/ml), even after 6 h challenge (data not shown).Furthermore, no increase was observed when LPS wasdelivered to haemocytes in combination with IFN-γ.Finally, the effect of insulin on NO production inhaemocytes was investigated, but this hormone didnot stimulate NO synthesis at any of the concentra-tions used (data not shown).

Various NOS inhibitors, which block the activationof mammalian NOS isoforms, inhibited the produc-tion of NO in L. stagnalis haemocytes following chal-lenge with PMA (10 µM) and laminarin (10 mg/ml).When haemocytes were preincubated with 10 mML-NAME (NG-nitro-L-arginine methyl ester) prior tochallenge, the NO production in response to PMAwas significantly reduced by 89% (P � 0.001; Fig-

ure 2A). Although a lower dose of L-NAME (1 mM)was without effect, 1 mM and 10 mM L-NMMA (NG-monomethyl-L-arginine) completely blocked PMA-induced NO production, resulting in levels of NOproduction similar to those seen in non-stimulatedcells (P � 0.01; Figure 2A). The inactive analoguesof these inhibitors [D-NAME (NG-nitro-D-argininemethyl ester) and D-NMMA (NG-monomethyl-D-arginine) respectively] did not inhibit NO produc-tion in response to PMA challenge (Figure 2A). Whenhaemocytes were challenged with laminarin in thepresence of 10 mM L-NAME, the stimulatory effectsof laminarin on NO synthesis were significantly at-tenuated (P � 0.001; Figure 2B); moreover, a similareffect was also seen when cells were preincubatedwith either 1 mM or 10 mM L-NMMA (P � 0.001;Figure 2B). Laminarin-induced activation of haemo-cyte NOS was sustained in the presence of 1 mM or10 mM D-NMMA, but it was inhibited by 10 mMD-NAME (Figure 2B). Also, L-NMMA sometimesincreased baseline fluorescence in control cells com-pared with those lacking the inhibitor; however, thisincrease was not always significant (data not shown).Finally the effect of 1400W, an inhibitor of mam-malian iNOS, on NO production was tested, but nosignificant inhibition was observed. At 10 µM and100 µM, this inhibitor increased NO production by13% and 28% in PMA-treated haemocytes and re-duced it by 7% and 2% in laminarin-treated haemo-cytes respectively.

PKC and ERK phosphorylationTo evaluate the effects of PMA and laminarin onhaemocyte PKC and ERK phosphorylation, haemo-cytes were challenged for various times and the cellextracts processed for Western blotting with anti-(phospho-p44/42 MAPK) and anti-phospho-PKC(pan) antibodies, which detect the active forms ofthese kinases in L. stagnalis (Walker and Plows, 2003;Plows et al., 2004, 2005). Both 1 µM PMA and10 mg/ml laminarin induced a transient increase inPKC phosphorylation over time, with maximal ac-tivation occurring after 10 min (Figure 3); analysis ofimmunoblots revealed the increases in PKC phos-phorylation levels after 10 min to be approx. 2–3-fold greater than the control (0 min). Althoughexposure to PMA appeared to result in a slight in-crease in p44 ERK phosphorylation, this was notapparent following laminarin stimulation (Figure 3).



Figure 1 NO production in L. stagnalis haemocytes incubated with various concentrations of (A) PMA, (B) laminarin or(C) zymosan A for 60 minNO was detected using the fluorescent probe DAF-FM diacetate as described in the Materials and methods section. The relative

fluorescence values are shown (means +− S.E.M.; n = 6) and represent the fluorescence signal as a proportion of that obtained

at 0 min (- - - -). *P < 0.05 and ***P < 0.001 (ANOVA) when compared with the control values at the various time points.



Figure 2 Effect of NOS inhibition on NO production in L. stagnalis haemocytesVarious concentrations of NOS inhibitors (L-NAME or L-NMMA) or their corresponding inactive analogues (D-NAME or D-NMMA),

or vehicle (for control cells lacking inhibitors/inactive analogues), were preincubated with haemocytes for 30 min prior to stim-

ulation with (A) 10 µM PMA or (B) 10 mg/ml laminarin for 60 min. NO production was detected using the fluorescent probe

DAF-FM diacetate. The relative fluorescence values are shown [means +− S.E.M.; n = 6 or n = 3 for L-NAME in (B)] and represent

the fluorescence signal as a proportion of that obtained in stimulated cells treated with vehicle alone (100%, - - - -). **P < 0.01

and ***P < 0.001 (ANOVA) when compared with control cells.

Consistently, however, although the strength of theimmunoreactive signal was low, an increase inthe phosphorylation of the lower band (p42 ERK)was always observed after 10 min.

Effect of PKC and ERK inhibition on NOproduction and PKC/ERK phosphorylationThe PKC inhibitors calphostin C and GF109203X,which have previously been validated for use in



Figure 3 PKC and ERK phosphorylation in haemocytesfollowing challenge and inhibition of signallingHaemocytes were challenged with (A) 1 µM PMA or (B)

10 mg/ml laminarin for various times in the presence or ab-

sence of the PKC or MEK inhibitors GF109203X (10 µM)

and U0126 (10 µM) respectively. Equal amounts of haemo-

cyte proteins were loaded on to each lane and membranes

were probed with either the anti-phospho-PKC (pan) or

anti-(phospho-p44/42 MAPK) antibodies. An anti-actin anti-

body was used to confirm equal loading. Blots are represent-

ative of at least two independent experiments.

L. stagnalis haemocytes (Walker and Plows 2003;Plows et al., 2004), significantly inhibited NO pro-duction in PMA-stimulated haemocytes over 60 min(P � 0.001; Figure 4A). These inhibitors (10 µM)reduced NO production by 82% and 94% respect-ively (P � 0.001; Figure 4A). Moreover, the effectof GF109203X appeared to be dose-responsive, withincreasing concentrations of the inhibitor reducingmean NO levels (Figure 4A). GF109203X acts asa competitive inhibitor of the ATP-binding site ofPKC and inhibits PKC autophosphorylation at asite (Ser660 in PKC βII) recognized by the anti-phospho-PKC (pan) antibody. The effect of this in-

hibitor on PKC phosphorylation was therefore as-sessed and, when used at 10 µM, was found to be ex-tremely effective at inhibiting PKC phosphorylation(activation) following challenge by PMA (Figure 3A).When challenged with laminarin, the inhibitory ef-fect of 10 µM calphostin C on NO production wasless marked than that seen following PMA challenge,since only a 51% inhibition of laminarin-inducedNO production was observed (P � 0.05; Figure 4B).A similar degree of inhibition, 50% (P � 0.05; Fig-ure 4B), was also observed following treatment with10 µM GF109203X; this inhibitor was also foundto be an effective inhibitor of PKC phosphorylationfollowing laminarin challenge (Figure 3B).

MEK (MAPK/ERK kinase) is the upstream ac-tivator of ERK1/2, and we have previously shownthat the MEK inhibitors PD98059 and U0126 blockERK1/2 phosphorylation (activation) in L. stagnalishaemocytes challenged with LPS (Plows et al., 2004).When tested in the present study, U0126 was foundto be a very effective inhibitor of ERK phosphoryl-ation (activation) following challenge of haemocyteswith either PMA or laminarin (Figure 3). U0126 alsoproved an effective inhibitor of NO production fol-lowing challenge with these compounds, with 1 µMand 10 µM U0126 significantly inhibiting NO pro-duction by 35% and 87% (for PMA), and 55% and91% (for laminarin) respectively (P � 0.001; Fig-ure 5). Furthermore, ANOVA revealed that the ef-fect of this inhibitor on NO production was dose-dependent (P � 0.001). The effects of PD98059on NO production following challenge were lessmarked. When used at 100 µM, PD98059 signifi-cantly inhibited NO production by 48% inlaminarin-treated haemocytes (P � 0.001; Fig-ure 5B); although 28% inhibition was achieved inPMA-stimulated haemocytes, the effect was not sig-nificantly different from control values (Figure 5A).

Visualization of NO within haemocytesMicroscopy revealed that the intracellular NO-de-rived fluorescence in control (unchallenged) haemo-cytes was low and scattered in distinct patches(Figure 6A), whereas the fluorescence in laminarin-stimulated haemocytes was intense and spreadthrough the entire cell (Figure 6B). Haemocytestreated with calphostin C (10 µm; Figure 6C) orGF109203X (10 µM; Figure 6D) prior to challengewith laminarin displayed lower NO levels; however,



Figure 4 Effect of PKC pathway inhibition on NO production in L. stagnalis haemocytesVarious concentrations of the PKC inhibitors calphostin C or GF109203X, or vehicle (for control cells not incubated in the

inhibitors), were preincubated with haemocytes for 30 min prior to stimulation with (A) 10 µM PMA or (B) 10 mg/ml laminarin for

60 min. NO production was detected using the fluorescent probe DAF-FM diacetate. The relative fluorescence values are shown

(means +− S.E.M.; n = 6) and represent the fluorescence signal as a proportion of that obtained in stimulated cells treated with

vehicle alone (100%, - - - -). *P < 0.05 and ***P < 0.001 (ANOVA) when compared with control cells.

NO was still detected in most parts of the cell.Although NO-derived intracellular fluorescence re-mained relatively high when cells were pre-treatedwith PD98059 (100 µM) prior to challenge withlaminarin (Figure 6E), U0126 appeared to reduce

the signal to basal (control) levels (Figure 6F). Res-ults obtained with or without inhibitors and PMAchallenge (data not shown) were broadly similarto those described following stimulation withlaminarin.



Figure 5 Effect of ERK pathway inhibition on NO production in L. stagnalis haemocytesVarious concentrations of the MEK inhibitors PD98059 or U0126, or vehicle (for control cells not incubated in the inhibitors),

were preincubated with haemocytes for 30 min prior to stimulation with (A) 10 µM PMA or (B) 10 mg/ml laminarin for 60 min.

NO production was detected using the fluorescent probe DAF-FM diacetate. The relative fluorescence values are shown

(means +− S.E.M.; n = 6) and represent the fluorescence signal as a proportion of that obtained in stimulated cells treated with

vehicle alone (100%, - - - -). **P < 0.01 and ***P < 0.001 (ANOVA) when compared with control cells.

DiscussionAlthough the cellular mechanisms that regulate NOSactivity and subsequent NO production in vert-ebrate cells have been well studied, they have re-

ceived little attention in invertebrates. In the presentstudy, we report that PMA and laminarin stim-ulated NO production in L. stagnalis haemocytesand showed that NO synthesis is, at least in part,



Figure 6 NO production in L. stagnalis haemocytesobserved by fluorescence microscopyCells were either (A) unchallenged or (B) challenged with

10 mg/ml laminarin for 60 min. Haemocytes were also treated

with the PKC inhibitors calphostin C (10 µM) (C) or GF109203X

(10 µM) (D), with the MEK inhibitors PD98059 (100 µM) (E) or

U0126 (10 µM) (F), or with vehicle alone, for 30 min prior to

challenge with laminarin for 60 min. After challenge, haemo-

cytes were fixed and processed for fluorescence microscopy,

as decribed in the Materials and methods section.

regulated through PKC and ERK signalling path-ways. This report is therefore the first to show arole for both PKC and ERK signal transductionpathways in NO production in invertebrate defencecells.

The NO probe DAF-FM diacetate was employedto determine NO levels in control and treated haemo-cytes with time. This probe has been used in many celltypes including neuroblastoma-derived Neuro2A,cholinergic SN56, COS-1 and PC12 cells (Lopez-Figueroa et al., 2001; Arundine et al., 2003). Whenchallenged with PMA, laminarin or zymosan A, NOproduction in L. stagnalis haemocytes significantly in-creased, with PMA and laminarin having the greatest

effect. Research carried out elsewhere has shown thatthese substances elicit NO production in haemo-cytes from other molluscs, particularly bivalves. Forexample, when M. galloprovincialis haemocytes werestimulated with PMA, Tafalla et al. (2002) reporteda significant increase in NO production within 2 h,whereas Arumugam et al. (2000) reported a 10-foldincrease after 60 min, this being similar to the8-fold increase seen in the present study. PMA alsostimulated NO production in C. gigas with max-imal output occurring after 15 min (Nakayama andMaruyama, 1998). M. galloprovincialis haemocytesalso produce NO when challenged with laminarin,with the increase in NO synthesis after 60 min (3.5-fold) (Arumugam et al., 2000) being similar to thatreported in the present study (4-fold). Also, similarto the present study, Tafalla et al. (2003) found a lim-ited, but significant, stimulatory effect of zymosanA on NO production in haemocytes of R. decussatus.LPS is often used in vertebrate systems to stimulateimmune cells, and has been used widely in studieswith macrophages to elicit the production of NO(for example, see Chen and Wang, 1999; Chan andRiches, 2001). The effects of LPS on NO productionin molluscan haemocytes are, however, controversial.Although LPS did not stimulate NO production inL. stagnalis haemocytes (either alone or in combin-ation with IFN-γ) in the present study, it has beenshown to do so in haemocytes of V. ater (Conte andOttaviani,1995) and R.decussatus (Tafalla et al., 2003),but not in M. galloprovincialis (Novas et al., 2004). Inconclusion, the NO responses of L. stagnalis haemo-cytes to challenge by the compounds tested appearto be broadly similar to those of M. galloprovincialishaemocytes.

Either PMA- or laminarin-induced NO produc-tion in haemocytes was blocked by the NOS inhibi-tors L-NAME or L-NMMA. Moreover, with the ex-ception of 10 mM D-NAME in laminarin-stimulatedhaemocytes, the inactive analogues did not inhibitNOS activity. Although various workers have usedthese or similar inhibitors to limit NOS activation inmolluscan haemocytes (Conte and Ottaviani, 1995;Arumugam et al., 2000; Gourdon et al., 2001;Tafalla et al., 2002, 2003), the use of inactive ana-logues as controls has been seldom reported. The ac-tions of various NOS inhibitors can vary between spe-cies (Nakane et al., 1995) and, similar to the presentstudy, the mammalian iNOS inhibitor 1400W did



not inhibit NO production in an insect (Lymantriadispar) cell line (Ottaviani et al., 2001). The unex-pected effect of D-NAME on laminarin-stimulatedNO production requires further investigation, butsubtle differences in NOS between invertebrates andvertebrates may be responsible for this finding.

The anti-phospho-PKC (pan) antibody recognizesPKC isoforms (α, βI, βII, ε, η; and δ) only whenautophosphorylated at a residue homologous to Ser660

of human PKCβII; this antibody has been validatedfor use in L. stagnalis and only recognizes active PKCin this snail (Walker and Plows, 2003). Challengeof haemocytes with either PMA or laminarin in-duced a 2–3-fold increase in PKC phosphorylationthat was maximal at 10 min and could be blockedby preincubation of cells with the PKC inhibitorGF109203X. The degree of activation of this enzymefollowing PMA or laminarin treatment is similar tothat reported by us for haemocytes challenged withbacterial LPS (Walker and Plows, 2003). Althoughwe were able to detect PKC activation following chal-lenge by PMA and laminarin, it should be noted thatadditional PKC-like proteins not recognized by theanti-phospho-PKC (pan) antibody may exist and beoperational in haemocytes, and may play a role intransmitting signals following exposure to these com-pounds. The anti-(phospho-p44/42 MAPK) antibodywas used in the present study to assess ERK activationfollowing challenge, and although no large changesin overall ERK activation were detected, a small, butconsistent, increase in phosphorylation was observedin the p42 ERK isoform after 10 min. In the presentstudy and previous work (Plows et al., 2004, 2005),we consistently observed high basal phosphorylationof p44 ERK and presume that this phenomenon isa consequence of cell extraction and working withprimary haemocytes; lengthy equilibration periodsdo not alleviate this problem and other workers havereported similar findings in primary haemocytes fromother mollusc species (Canesi et al., 2002). In thiscontext, constitutively activated ERK may drive cer-tain signalling events and functional responses andstudies exploring the effects of ERK inhibition onthe cells may be more revealing.

Although PMA has been shown to stimulate NOproduction in molluscan haemocytes (Nakayama andMaruyama, 1998; Arumugam et al., 2000; Tafallaet al., 2002), thus implying a role for PKC in NOSactivation, this phenomenon had not been tested with

the use of pathway inhibitors prior to the presentstudy. We have recently demonstrated the presenceof PKC in L. stagnalis haemocytes, and have shownthat the PKC inhibitor GF109203X is able to blockthe phosphorylation and activation of this enzyme ina dose-dependent manner (Walker and Plows, 2003).When used here, GF109203X significantly inhibi-ted both PMA- and laminarin-induced PKC phos-phorylation (activation) and NO production, with10 µM GF109203X reducing NO synthesis by 90%and 47% respectively. The PKC inhibitor calphostinC also attenuated NO production in haemocytes to asimilar extent. Moreover, the MEK inhibitor U0126inhibited ERK phosphorylation (activation) and NOproduction in haemocytes, strongly implying a rolefor the downstream target of MEK, ERK, in NOS ac-tivation; indeed, U0126 was found to be a very potentinhibitor of laminarin-dependent NO production. Fi-nally, the MEK inhibitor PD98059 also blocked NOproduction. We have also recently shown that U0126and PD98059 inhibit the phosphorylation and ac-tivation of L. stagnalis haemocyte ERK in responseto LPS in a dose-dependent manner (Plows et al.,2004). That the MEK1/2 inhibitor U0126 was moreeffective than the MEK1 inhibitor PD98059 at in-hibiting NO production following PMA or laminarinchallenge, suggests that both MEK1 and MEK2 iso-forms are important mediators of downstream NOSactivation in haemocytes. Furthermore, given the dif-ferential effects of the PKC and MEK inhibitors onNO production following laminarin stimulation, itappears that PKC-dependent or -independent path-ways may lead to ERK-mediated activation of NOSfollowing challenge by this compound. Although thedata of the present study show a role for PKC andERK in NO production following challenge, cross-talk between these and other kinases that may beinvolved in the regulation of NO output in haemo-cytes is likely to occur. Furthermore, the signal-basedregulation of NO production is certain to be depen-dent on the nature of the immune challenge and thenumber of different NOS isoforms present in haemo-cytes.

PKC and ERK signal the up-regulation of NO pro-duction in mammalian macrophages following im-mune challenge, but this is a consequence of increasediNOS expression after a period of many hours (Chenand Wang, 1999; Chan and Riches 2001; He andKogut, 2003; Chio et al., 2004). In the present study,



the relatively rapid increase in NO production follow-ing challenge suggests that regulation via de novo pro-tein synthesis is not necessary for L. stagnalis haemo-cytes to mount an NO response. It is possible thatL. stagnalis haemocytes express an iNOS-like enzyme,particularly given that Novas et al. (2004) detected aprotein of a similar molecular mass to mammalianiNOS in M. galloprovincialis haemocyte extracts,both before and after stimulation with interleukin-2,usingananti-(mouse iNOS) antibody.However, usinga similar antibody, we were unable to successfullydetect this protein in L. stagnalis haemocytes (A.J.Walker and B. Wright, unpublished data). Our res-ults do, however, show that NOS seems to be regu-lated in these cells at a level independent of proteinexpression. Such regulatory behaviour is more akin tothe constitutively expressed mammalian eNOS andnNOS isoforms, although PKC is generally believedto negatively regulate eNOS activity (Matsubaraet al., 2003). NO has been studied widely in molluscsin the context of neurobiology and has been shown toplay a role in the modulation of neurotransmitter re-lease, neurosecretion and behavioural activities, suchas feeding (Stefano and Ottaviani, 2002). In L. stag-nalis, nNOS (Lym-nNOS) has been identified in keymodulatory neurons of the feeding network (Korneevet al., 1998), and in A. californica (Bodnarova et al.,2005) a NOS isoform that shares enzymatic char-acteristics with constitutive mammalian and insectnNOS has recently been characterized at the bio-chemical level. Clearly, it is possible that L. stag-nalis haemocyte NOS may resemble L. stagnalis andA. californica nNOS. Although there are no reportsconcerning the signal-based regulation of nNOS-likeproteins in molluscs, a role for ERK signalling in theinduction of nNOS has been demonstrated in mam-malian cells (Schonhoff et al., 2001). Studies into thesignalling mechanisms that regulate NOS expressionand activity in lower organisms are lacking; perhapsL. stagnalis will emerge as a useful model for definingthese events in invertebrate systems.

In conclusion, this study significantly advances ourknowledge of the signal based-regulation of the in-vertebrate defence response. The activation of PKCin response to PMA and laminarin challenge, and theeffects of the inhibitors on both PKC and ERK activ-ation (phosphorylation) and stimulant-induced NOsynthesis are consistent with PKC and ERK playinga role in NOS activation and subsequent NO pro-

duction in L. stagnalis haemocytes. L. stagnalis is anintermediate host to trematode parasites, and we haverecently reported that carbohydrates that mimicschistosome surface-coat components down-regulateERK and PKC signalling in haemocytes of this snail(Plows et al., 2005). Given the findings of the presentstudy and that NO is considered an important anti-parasite molecule, we are currently exploring the poss-ibility that intra-molluscan stages of parasites switchoff host NO defence responses by down-regulatingthe activities of haemocyte ERK and PKC signallingpathways. Moreover, NO is generally considered tobe an ancestral defence molecule (Franchini et al.,1995), and our results suggest that the intracellularcontrols that govern the activity of vertebrate NOSisoforms may have developed from cell signalling-based regulatory mechanisms that originally evolvedin invertebrates.

Materials and methodsReagentsU0126 and the anti-(phospho-p44/42 MAPK) (ERK1/2) andanti-phospho-PKC (pan) antibodies were obtained from NewEngland Biolabs (Hitchin, Herts., U.K.). Recombinant humanIFN-γ, stripping buffer and enhanced chemiluminescence (ECL)reagent were from Perbio Science (Tattenhall, Cheshire, U.K.),and Hybond nitrocellulose membrane was from AmershamBiosciences (Amersham, Bucks., U.K.). 1400W, L-NMMA,L-NAME, D-NMMA, calphostin C (from Cladosporium cla-dosporioides) and GF109203X were purchased from Calbiochem(Nottingham, U.K.). D-NAME was obtained from Alexis Bio-chemicals (Nottingham, U.K.) and PD98059 was from Promega(Southampton, U.K.). Both Vectashield and DAF-FM diacetatewere purchased from Molecular Probes (Europe BV, Leiden, TheNetherlands). Zymosan A (from Saccharomyces cerevisiae), PMA,laminarin (from Laminaria digitata), insulin (from bovine pan-creas), Escherichia coli LPS (serotype 0111:B4), DMSO, anti-actin antibody and all other chemicals were from Sigma–Aldrich(Poole, Dorset, U.K.).

SnailsLaboratory cultures of L. stagnalis were reared from eggs laid bysnails that were commercially obtained from Blades Biologicals(Cowden, Edenbridge, Kent, U.K.). Juveniles that emerged fromegg masses were kept at room temperature until they reached ashell length of 20–30 mm. They were then transferred into hold-ing tanks within a programmable incubator (Sanyo) set at 12 hlight/12 h dark cycles at 20◦C. All tanks contained continuouslyaerated tap water that had been filtered through a Brimak/carbonfiltration unit (Silverline Ltd, Winkleigh, Devon, U.K.). Snailswere fed fresh washed lettuce ad libitum.

Haemolymph extraction and haemocyte treatmentsL. stagnalis were washed in distilled water; haemolymph, extrac-ted from several snails by head–foot retraction (Sminia, 1972),was pooled and placed on ice in SSS (sterile snail saline; 1:2,



SSS/haemolymph) (Adema et al., 1994). Haemocyte monolay-ers were then prepared by allowing extracted cells to adhere toindividual wells (200 µl of diluted haemolymph per well) of a96-well culture plate (Nunc) for 30 min at 22◦C. The monolayerswere washed twice with 250 µl of SSS to remove dead and non-adherent cells. After washing, the haemocytes were incubated in200 µl of SSS containing the fluorescent NO indicator, DAF-FMdiacetate (5 µM) for 1 h in the dark (Nakatsubo et al., 1998). Thecells were subsequently washed in 250 µl of SSS prior to beingincubated in 200 µl of SSS for 30 min to de-esterify the intracel-lular DAF-FM diacetate. Haemocytes were then challenged withLPS (0.001–10 µg/ml), zymosan A (0.001–10 µg/ml), lamin-arin (1–10 mg/ml), insulin (25–500 nM), PMA (0.001–10 µM),IFN-γ (0.1–10 ng/ml) or vehicle to establish their effects on NOproduction. In addition, haemocytes were challenged with LPS(1 µg/ml) and IFN-γ (0.1–10 ng/ml) together. Where applic-able, the concentration ranges chosen here were similar to thoseemployed by other workers studying NO production in mol-luscs. Control wells for background fluorescence included SSS orhaemocytes alone, and haemocytes with SSS and each stimulant.All incubations and washes were performed at 22◦C. Followingchallenge, the fluorescence signal was measured every 5 min for1 h, using a Fluorstar Optima microplate reader (BMG Labtech,Aylesbury, Bucks., U.K.), calibrated for excitation at 485 nmand emission at 520 nm. In experiments where haemocytes werechallenged for periods of up to 6 h, the fluorescence signal wasmeasured every 15 min.

For inhibition assays, inhibitors were added to haemocytemonolayers at various concentrations at the beginning of thede-esterification stage (i.e. 30 min prior to challenge). Forinhibition of NOS, L-NMMA (1 and 10 mM), L-NAME (1and 10 mM) or 1400W (5 µM–1 mM) were used (Conte andOttaviani, 1995; Garvey et al., 1997; Tafalla et al., 2002, 2003);the effects of the inactive isomers, D-NAME (1 and 10 mM) andD-NMMA (1 and 10 mM) for L-NAME and L-NMMA respect-ively, were also tested. Controls were carried out with or withoutthe inhibitor/inactive analogue for all treatments. Calphostin C(0.01–10 µM) or GF109203X (0.01–10 µM) were used to in-hibit PKC (Walker and Plows, 2003), whereas PD98059 (0.01–100 µM) or U0126 (0.001–10 µM) were used to inhibit theERK pathway in haemocytes (Plows et al., 2004); controls weretreated with the vehicle for each inhibitor and, as we have re-ported previously, the inhibitors do not appear to be toxic toL. stagnalis haemocytes (Walker and Plows, 2003; Plows et al.,2004).

Raw data were collected by performing a series of independentassays. Data describing the effects of treatment on NO produc-tion in haemocytes were then analysed by ANOVA and post-hocmultiple-comparison tests (Tukey) using the statistical softwarepackage SPSS. In experiments using NOS or pathway inhibi-tors, mean fluorescence levels in stimulated cells in the presenceof the inhibitor were compared with those obtained without,and the latter were assigned a value of 100%.

Determination of PKC and ERK activationHaemocyte monolayers were prepared as described above, ex-cept that 24-well tissue culture plates were used (500 µl ofdiluted haemolymph per well and 500 µl of SSS used forwashes). Haemocytes were then challenged with laminarin(10 mg/ml), PMA (1 µM) or vehicle, and at various time points(0–60 min) were lysed with boiling SDS/PAGE sample buffer,

and the lysates were processed immediately for SDS/PAGE andWestern blotting or stored at −20◦C. Where appropriate, PKCor ERK inhibitors were preincubated with haemocytes for30 min prior to challenge. Samples containing equal amounts ofprotein were loaded on to discontinuous SDS/PAGE gels, whichcontained 10% acrylamide in the resolving gel. After electro-phoresis, proteins were electroblotted on to Hybond nitrocellu-lose membrane (0.45 µm) using a semi-dry transfer unit, andhomogeneous transfer was confirmed by staining with PonceauS. Membranes were then blocked for 1 h at room temperaturewith 5% (w/v) non-fat dried milk in TBS (Tris-buffered sa-line) containing 0.1% (v/v) Tween 20. Membranes were incu-bated overnight at 4◦C with agitation with anti-(phospho-p44/42 MAPK) (ERK1/2) (1:1000), anti-phospho-PKC (pan)(1:1000) or anti-actin (1:5000) primary antibodies. The pro-teins were revealed, after exposure to horseradish-peroxidase-conjugated secondary antibody for 1 h at 25◦C, with ECL re-agent. Membranes were then stripped using a commercialreagent and were re-probed with anti-actin antibody (1:5000) toconfirm equal loading of protein.

Visualizing NO within haemocytesFollowing haemolymph extraction and dilution in SSS, haemo-cytes were allowed to bind to individual coverslips (200 µl ofdiluted haemolymph per coverslip) for 30 min. The coverslipswere then washed three times with 1 ml of SSS to remove haemo-lymph and dead/non-adherent cells before being inverted on toParafilm with DAF-FM diacetate (5 µM) and were incubated inthe dark for 1 h. Following two washes in 1 ml of SSS, coverslipswere placed in 1 ml of SSS for 30 min to de-esterify intracellularDAF-FM diacetate. Cells were then challenged by incubatingcoverslips in optimal concentrations of PMA (10 µM), lamin-arin (10 mg/ml) or vehicle for 60 min. To visualize the effectsof signalling pathway inhibitors on NO production in haemo-cytes, 200 µl of calphostin C (10 µM), GF109203X (10 µM),PD98059 (100 µM) or U0126 (10 µM) were incubated withhaemocytes at the start of the de-esterification stage (i.e. 30 minprior to challenge). All incubations were done at 22◦C in hu-midified chambers. Following challenge, haemocytes were fixedwith 3.7% formaldehyde for 10 min, briefly washed in SSS andmounted on to slides in Vectashield to preserve the fluorescencesignal; coverslips were then sealed with clear nail varnish. Intra-cellular fluorescence was visualized using a Zeiss Axiophot20 photomicroscope equipped with a triple filter; excitationwavelengths were 410, 505 and 585 nm (beamsplitters: 395,485, 560 nm; barriers: 460, 530 and 610 nm respectively). Im-ages of the haemocytes were then captured using a Nikon DN100camera linked to a Nikon Eclipse Net image analysis softwarepackage.

AcknowledgmentWe are grateful to Dr Richard Cook for help with thestatistical analyses.

ReferencesAdema, C.M., van Deutekom-Mulder, E.C., van der Knapp, W.P.W.

and Sminia, T. (1994) Schistosomicidal activities of Lymnaeastagnalis haemocytes: the role of oxygen radicals. Parasitology109, 479–485



Arumugam, M., Romestand, B., Torreilles, J. and Roch, P. (2000)In vitro production of superoxide and nitric oxide (as nitrite andnitrate) by Mytilus galloprovincialis haemocytes upon incubationwith PMA or laminarin or during yeast phagocytosis. Eur. J.Cell Biol. 79, 513–519

Arundine, M., Sanelli, T., He, B.P. and Strong, M.J. (2003) NMDAinduces NOS 1 translocation to the cell membrane inNGF-differentiated PC12 cells. Brain Res. 976, 149–158

Blos, M., Schleicher, U., Rocha, F.J.S., Meissner, U., Rollinghoff, M.and Bogdan, C. (2003) Organ-specific and stage-dependentcontrol of Leishmania major infection by inducible nitric oxidesynthase and phagocyte NADPH oxidase. Eur. J. Immunol. 33,1224–1234

Bodnarova, M., Martasek, P. and Moroz, L.L. (2005)Calcium/calmodulin-dependent nitric oxide synthase activity in theCNS of Aplysia californica: biochemical characterization and link tocGMP pathways. J. Inorg. Biochem. 99, 922–928

Canesi, L., Betti, M., Ciacci, C., Scarpato, A., Citterio, B., Pruzzo, C.and Gallo, G. (2002) Signaling pathways involved in thephysiological response of mussel hemocytes to bacterialchallenge: the role of stress-activated p38 MAP kinases.Dev. Comp. Immunol. 26, 325–334

Chan, E.D. and Riches, D.W.H. (2001) IFN-γ + LPS induction of iNOSis modulated by ERK, JNK/SAPK, and p38mapk in a mousemacrophage cell line. Am. J. Physiol. Cell Physiol. 280,C441–C450

Chen, C.-C. and Wang, J.-K. (1999) p38 but not p44/42mitogen-activated protein kinase is required for nitric oxidesynthase induction mediated by lipopolysaccharide in RAW 264.7macrophages. Mol. Pharmacol. 55, 481–488

Chio, C.-C., Chang, Y.-H., Hsu, Y.-W., Chi, K.-H. and Lin, W.W. (2004)PKA-dependent activation of PKC, p38 MAPK and IKK inmacrophage: implication in the induction of inducible nitric oxidesynthase and interleukin-6 by dibutyryl cAMP. Cell. Signal. 16,565–575

Conte, A. and Ottaviani, E. (1995) Nitric oxide synthase activity inmolluscan hemocytes. FEBS Lett. 365, 120–124

Dikkeboom, R., Bayne, C.J., van der Knapp, W.P.W. and Tijnagel,J.M.G.H. (1988) Possible role of reactive forms of oxygen in in vitrokilling of Schistosoma mansoni sporocysts by haemocytes ofLymnaea stagnalis. Parasitol. Res. 75, 148–154

Franchini, A., Conte, A. and Ottaviani, E. (1995) Nitric oxide: anancestral immunocyte effector molecule. Adv. Neuroimmunol. 5,463–478

Garvey, P.E., Oplinger, A.J., Furfine, S.E., Kiff, J.R., Laszlo, F., Whittle,J.R.B. and Knowles, G.R. (1997) 1400W is a slow, tight-binding,and highly selective inhibitor of inducible nitric-oxide synthasein vitro and in vivo. J. Biol. Chem. 272, 4956–4963

Gourdon, I., Guerin, M.-C., Torreilles, J. and Roch, P. (2001) Nitricoxide generation by haemocytes of the mussel Mytilusgalloprovincialis. Nitric Oxide 5, 1–6

Griffith, W.O. and Stuehr, J.D. (1995) Nitric oxide synthases:properties and catalytic mechanism. Annu. Rev. Physiol. 57,707–736

Hahn, U.K., Bender, R.C. and Bayne, C.J. (2000) Production ofreactive oxygen species by hemocytes of Biomphalaria glabrata:carbohydrate-specific stimulation. Dev. Comp. Immunol. 24,531–541

Hahn, U.K., Bender, R.C. and Bayne, C.J. (2001) Involvement of nitricoxide in killing of Schistosoma mansoni sporocysts by haemocytesfrom resistant Biomphalaria glabrata. J. Parasitol. 87, 778–785

He, H. and Kogut, M.H. (2003) CpG-ODN-induced nitric oxideproduction is mediated through clathrin-dependent endocytosis,endosomal maturation, and activation of PKC, MEK1/2 and p38MAPK, and NF-κB pathways in avian macrophage cells (HD11).Cell. Signal. 15, 911–917

Hoffmann, J.A., Kafatos, F.C., Janeway, C.A. and Ezekowitz, R.A.(1999) Phylogenetic perspectives in innate immunity.Science (Washington, D.C.) 284, 1313–1318

James, S.L., Cheever, A.W., Caspar, P. and Wynn, T.A. (1998)Inducible nitric oxide synthase-deficient mice develop enhancedtype 1 cytokine associated cellular and humoral immune responseafter vaccination with attenuated Schistosoma mansoni cercariaebut display partially reduced resistance. Infect. Immun. 66,3510–3518

Kleinert, H., Pautz, A., Linker, K. and Schwarz, P.M. (2004)Regulation of the expression of inducible nitric oxide synthase.Eur. J. Pharmacol. 500, 255–266

Korneev, S.A., Piper, M.R., Picot, J., Phillips, R., Korneeva, E.I. andO’Shea, M. (1998) Molecular characterization of NOS in a mollusc:expression in a giant modulatory neuron. J. Neurobiol. 35,65–76

Korneev, S.A., Kemenes, I., Straub, V., Staras, K., Korneeva, E.I.,Kemenes, G., Benjamin, P.R. and O’Shea, M. (2002) Supression ofnitric oxide (NO)-dependent behaviour by double-strandedRNA-mediated silencing of a neuronal NO synthase gene.J. Neurosci. 22, RC227, 1–5

Lopez-Figueroa, M.O., Caamano, C., Marin, R., Guerra, B.,Alonso, R., Morano, M.I., Akil, H. and Watson, S.J. (2001)Characterization of basal nitric oxide production in living cells.Biochim. Biophys. Acta 1540, 253–264

Matsubara, M., Hayashi, N., Jing, T. and Titani, K. (2003) Regulationof endothelial nitric oxide synthase by protein kinase C.J. Biochem. (Tokyo) 133, 773–781

Nakane, M., Pollock, S.J., Klinghofer, V., Basha, F., Marsden, P.A.,Hokari, A., Ogura, T., Esumi, H. and Carter, W.G. (1995) Functionalexpression of three isoforms of human nitric oxide synthase inbaculovirus-infected insect cells. Biochem. Biophys.Res. Commun. 206, 511–517

Nakatsubo, N., Kojima, H., Kikuchi, K., Nagoshi, H., Hirata, Y.,Maeda, D., Imai, Y., Irimura, T. and Nagano, T. (1998) Directevidence of nitric oxide production from bovine aortic endothelialcells using new fluorescence indicators: diaminofluoresceins.FEBS Lett. 427, 263–266

Nakayama, K. and Maruyama, T. (1998) Differential production ofactive oxygen species in photo-symbiotic and non-symbioticbivalves. Dev. Comp. Immunol. 22, 151–159

Nathan, C. and Xie, Q.-W. (1994) Nitric oxide synthases: roles, tolls,and controls. Cell (Cambridge, Mass.) 78, 915–918

Novas, A., Cao, A., Barcia, R. and Ramos-Martinez, J.I. (2004) Nitricoxide release by hemocytes of the mussel Mytilus galloprovincialisLmk was provoked by interleukin-2 but not by lipopolysaccharide.Int. J. Biochem. Cell Biol. 36, 390–394

Nunez, P.E., Adema, C.M. and de Jong-Brink, M. (1994) Modulationof the bacterial clearance activity of haemocytes from thefreshwater mollusc, Lymnaea stagnalis, by the avian schistosome,Trichobilharzia ocellata. Parasitology 109, 299–310

Ottaviani, E., Barbieri, D., Malagoli, D. and Franchini, A. (2001) Nitricoxide induces apoptosis in the fat body cell line IPLB-LdFB fromthe insect Lymantria dispar. Comp. Biochem. Physiol., Part B:Biochem. Mol. Biol. 128, 247–254

Pinge-Filho, P., Peron, J.P.S., de Moura, T.R., Menolli, R.A., Graca, V.,Estevao, D., Tadokoro, C.E., Jankevicius, J. and Rizzo, L.V. (2005)Protective immunity against Trypanosoma cruzi provided by oralimmunization with Phytomonas serpens: role for nitric oxide.Immunol. Lett. 96, 283–290

Plows, L.D., Cook, R.T., Davies, A.J. and Walker, A.J. (2004)Activation of extracellular-signal regulated kinase is requiredfor phagocytosis by Lymnaea stagnalis haemocytes.Biochim. Biophys. Acta 1692, 25–33

Plows, L.D., Cook, R.T., Davies, A.J. and Walker, A.J. (2005)Carbohydrates that mimic schistosome surface coat componentsaffect ERK and PKC signalling in Lymnaea stagnalis haemocytes.Int. J. Parasitol. 35, 293–302



Ribiere, C., Jaubert, A.-M., Sabourault, D., Lacasa, D. andGiudicelli, Y. (2002) Insulin stimulates nitric oxide production in ratadipocytes. Biochem. Biophys. Res. Commun. 291, 394–399

Schonhoff, C.M., Bulseco, D.A., Branncho, D.M., Parada, L.F. andRoss, A.H. (2001) The Ras–ERK pathway is required for theinduction of neuronal nitric oxide synthase in differentiating PC12cells. J. Neurochem. 78, 631–639

Sminia, T. (1972) Structure and function of blood and connectivetissue cells of the freshwater pulmonate Lymnaea stagnalis studiesby electron microscopy and enzyme histochemistry. Z ZellforschMikrosk. Anat. 130, 497–526

Stefano, G.B. and Ottaviani, E. (2002) The biochemical substrate ofnitric oxide signalling is present in primitive non-cognitiveorganisms. Brain Res. 924, 82–89

Tafalla, C., Novoa, B. and Figueras, A. (2002) Production of nitricoxide by mussel (Mytilus galloprovincialis) hemocytes and effect ofexogenous nitric oxide on phagocytic functions. Comp. Biochem.Physiol., Part B: Biochem. Mol. Biol. 132, 423–431

Tafalla, C., Gomez-Leon, J., Novoa, B. and Figueras, A. (2003) Nitricoxide production by carpet shell clam (Ruditapes decussatus)hemocytes. Dev. Comp. Immunol. 27, 197–205

van der Knaap, W.P.W., Adema, C.M. and Sminia, T. (1993)Invertebrate blood cells: morphological and functional aspectsof the haemocytes in the pond snail Lymnaea stagnalis.Comp. Haematol. Int. 3, 20–26

Walker, A.J. and Plows, L.D. (2003) Bacterial lipopolysaccharidemodulates protein kinase C signalling in Lymnaea stagnalishaemocytes. Biol. Cell 95, 527–533

Yoshino, T.P. and Vasta, G.R. (1996) Parasite – invertebrate hostimmune interactions. Adv. Comp. Environ. Physiol. 24,125–167

Zelck, U.E., Janje, B. and Schneider, O. (2005) Superoxide dismutaseexpression and H2O2 production by haemocytes of the trematodeintermediate host Lymnaea stagnalis. Dev. Comp. Immunol. 29,305–314

Received 28 July 2005/28 October 2005; accepted 17 November 2005

Published as Immediate Publication 17 November 2005, doi:10.1042/BC20050066


regulation of nitric oxide production in snail (lymnaea stagnalis) defence cells: a role for pkc and...

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