INTRODUCTION

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THERE IS EVIDENCE SUGGESTING that a wide range of classical and peptidergic neurotransmitters are present in parasitic flatworms. Classical transmitters that have been implicated include acetylcholine, serotonin (5-HT), catecholamines, histamine, and glutamate, and the unstable messenger molecule nitric oxide may also play a regulatory role (5, 8, 15). Structural studies show that these transmitters are localized in the nervous system and can have stimulatory (5-HT, dopamine, glutamate) or inhibitory (acetylcholine, norepinephrine) effects on neuromuscular activity (8, 32). Relatively little is known, however, about the intracellular signaling mechanisms underlying their actions. A number of ion channels have been identified in neuronal and muscle cell membranes in platyhelminths, but none appears to be directly gated through ionotropic receptors (3, 19, 32). This contrasts with the situation in nematodes, in which both chloride channels gated by glutamate and -aminobutyric acid and acetylcholine-gated cation channels have been identified (11, 39). There is evidence, however, that classical transmitters may control flatworm function via metabotropic receptors that activate second messenger pathways through G proteins. For example, glutamate acts via an inositol-1,4,5-trisphosphate (Ins-1,4,5-P₃)-dependent pathway in the tapeworm Hymenolepis diminuta (35, 40), and cAMP has been implicated in the changes in motility (9, 31, 34) and carbohydrate metabolism (17, 22, 36, 37) produced by 5-HT in F. hepatica, Schistosoma mansoni, and H. diminuta.

Although much remains to be learned about the signal transduction mechanisms activated by classical transmitters in flatworms, even less information is available in relation to cellular mechanisms of peptidergic control. These mechanisms merit serious investigation because peptidergic nerves not only constitute a major component of the flatworm nervous system but probably also fulfill what is effectively a hormonal role in these primitive metazoans, which lack either a circulatory system or classical endocrine glands. Immunoreactivities to a large number of vertebrate and invertebrate peptides have been demonstrated in both the central and peripheral nervous systems of flatworms (10), although only a small number of endogenous neuropeptides have been isolated to date (7, 20, 21, 28-30). These peptides are functionally active and have been shown to affect both protein synthesis (10) and motility in the liver fluke F. hepatica (13). In contractility studies, liver fluke muscle strips were most sensitive to stimulation by GYIRFamide, a turbellarian peptide of the FMRFamide-related peptide (FaRP) family, which increased both the frequency and amplitude of contractions at concentrations as low as 50 nM (13). Nothing is known, however, about the transduction pathways involved in FaRP signaling within flatworms. Therefore, the present investigation was designed to investigate some candidate mechanisms using the motility response to GYIRFamide, by testing whether this response was altered in the presence of agents known to activate or inhibit important second messenger pathways. Our results suggest that GYIRFamide acts via a G protein to stimulate phospholipase C (PLC) and thus promotes the actions of protein kinase C (PKC), presumably as a consequence of diacylglycerol (DAG) production.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tissue preparation. Spontaneously active isolated muscle strips were obtained from adult liver flukes (F. hepatica) recovered from the bile ducts of experimentally infected laboratory rats. Flukes were cut horizontally below the ventral sucker, trimmed along the sides and bottom, and sliced into two pieces longitudinally. The resulting strips were suspended vertically in organ baths maintained at 37°C, and isometric tension was recorded as described previously (13, 14). The tissues were continuously superfused with Hédon-Fleig saline buffered using 10 mM HEPES and adjusted to pH 7.4 using 2 M NaOH. All drugs were added in the superfusate, and stable activity was observed under control conditions for at least 20 min before drug application.

Solutions and drugs. Hédon-Fleig saline contained the following components (in mM): 120.7 NaCl, 4 KCl, 1.9 MgSO₄ · 7H₂O, 0.9 CaCl₂ · 2H₂O, 18.5 NaHCO₃, 10 HEPES, and 15 D-glucose, pH 7.4.

Data analysis and presentation. Each experiment was repeated at least five times with strips of tissue from separate flukes. Contraction amplitude and frequency were analyzed separately and only contractions of an amplitude 0.5 mN were counted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Resting activity and controls. Muscle strips from F. hepatica demonstrated spontaneous, phasic contractility under control conditions, with a mean frequency of 7.70 ± 0.57 contractions/3-min time period (n = 6). This activity was unaffected by recirculation of physiological saline solution for periods of up to 1 h, indicating that the recirculation procedure used during prolonged drug applications was not inherently detrimental to the tissue (data not shown). GYIRFamide increased both the rate and amplitude of contraction (Fig. 1), and this effect was repeatable when applications were separated by a 20-min washout period (data not shown), demonstrating that time-dependent decay in the responsiveness to the peptide was not significant for most of the protocols used. In five tissue strips the contraction frequency recovered to 111 ± 7% of the initial control frequency after washout (not significant).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Guanosine 5'-O-(2-thiodiphosphate) (GDPS) inhibited the mechanical responses to GYIRFamide. A: isometric tension recording showing the effect of 0.5 µM GYIRFamide on contractility before (1) and after (2) exposure to 100 µM GDPS. B: graphs displaying the mean GYIRFamide-induced increases in amplitude and frequency, before (solid bars) and after (cross hatched bars) exposure to GDPS (n = 5). Statistically significant reductions in these responses (*P < 0.05, **P < 0.01).

GDPS. The G protein inhibitor GDPS (100 µM) had no effect on spontaneous activity when applied to muscle strips but reduced the subsequent response to 0.5 µM GYIRFamide (n = 5; Fig. 1). The mean GYIRFamide-induced increase in frequency was 10.2 ± 1.16 contractions/3 min during the initial, control application and 1.8 ± 0.66 contractions/3 min after exposure to GDPS (P < 0.01, ANOVA). The corresponding values for contraction amplitude were 0.74 ± 0.16 mN and 0.03 ± 0.17 mN (P < 0.05, ANOVA).

MDL-12330A and H89. The adenylate cyclase inhibitor MDL-12330A (25 µM) appeared to inhibit the excitatory response to 0.5 µM GYIRFamide (Fig. 2). In a series of six muscle strips the effect of GYIRFamide on contraction amplitude was reduced from a mean increase of 0.58 ± 0.26 mN before to an increase of 0.20 ± 0.13 mN after treatment with MDL-12330A (P < 0.05, ANOVA). GYIRFamide produced an increase in frequency of 6.33 ± 2.25 contractions/3 min under control conditions, but after exposure to MDL-12330A, GYIRFamide actually appeared to reduce contraction frequency by 3.33 ± 1.31/3 min (P < 0.01, ANOVA). This was partly due to the stimulation of many small contractions that did not reach the 0.5 mN threshold for analysis (Fig. 2, A2). Interpretation of these results is further complicated by the fact that MDL-12330A itself stimulated mechanical activity (Fig. 2C). MDL-12330A raised the mean frequency from 2.33 ± 0.71 to 8.50 ± 0.96/3 min (P < 0.01, ANOVA), a value very similar to the average frequency during GYIRFamide stimulation under control conditions (8.67 ± 2.72 contractions/3 min). Use of MDL-12330A at a concentration that did not affect spontaneous activity (1 µM) had no effect on the GYIRFamide responses but this concentration also failed to block the excitatory response to forskolin (50 µM), suggesting that adenylate cyclase was not inhibited (data not shown). The feasibility of testing for involvement of cAMP by using a PKA inhibitor (H89, 2 µM) was also explored but this drug failed to block the excitatory effects of cell permeant 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 1 mM) in three muscle strips (data not shown), making it an unsuitable pharmacological tool for this tissue.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of MDL-12330A on spontaneous activity and mechanical responses to GYIRFamide. A: tension recording showing the effect of 0.5 µM GYIRFamide before (1) and after (2) exposure to 25 µM MDL. B: bar charts summarizing the mean increases in frequency and amplitude of contraction due to GYIRFamide before (solid bars) and during (cross-hatched bars) exposure to MDL (n = 5). Statistically significant changes in response following MDL exposure (* P < 0.05, **P < 0.01). C: record showing the excitatory effect of MDL-12330A on spontaneous activity.

Neomycin sulfate. Neomycin sulfate is a PLC inhibitor, and its effect on the contractile response to 0.1 µM GYIRFamide was tested in a total of nine muscle strips (Fig. 3). At 1 mM it had no effect on the spontaneous contractions of the muscle strips but it reduced the frequency and amplitude of the excitatory response to the peptide. GYIRFamide produced a mean increase in frequency of 7.33 ± 1.47 contractions/3 min initially and this was reduced to 2.44 ± 0.92 contractions/3 min after exposure to neomycin (P < 0.01, ANOVA). Corresponding values for contraction amplitude were 1.03 ± 0.19 mN and 0.05 ± 0.33 mN (P < 0.05, ANOVA).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Neomycin inhibited GYIRFamide responses. A: tension recording showing the effect of 0.1 µM GYIRFamide before (1) and after (2) exposure to 1 mM neomycin sulfate. B: graphs displaying the mean GYIRFamide-induced increases in amplitude and frequency, before (solid bars) and after (crosshatched bars) exposure to neomycin sulfate (n = 9). Statistically significant reductions in these responses (*P < 0.05, **P < 0.01).

Chelerythrine chloride. The PKC inhibitor chelerythrine chloride (10 µM) had no effect on spontaneous contractions but inhibited the response to 0.5 µM GYIRFamide (Fig. 4). GYIRFamide produced a mean increase in contraction frequency of 8.40 ± 1.86 contractions/3 min under control conditions but this was reduced to 1.00 ± 0.71 contractions/3 min after exposure to chelerythrine chloride (P < 0.01, ANOVA, n = 5). The corresponding values for contraction amplitude were 1.00 ± 0.16 mN and 0.11 ± 0.12 mN (P < 0.01, ANOVA).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Motor effects of GYIRFamide were blocked by chelerythrine. A: tension recording showing the effect of 0.5 µM GYIRFamide before (1) and after (2) exposure to 10 µM chelerythrine chloride. B: graphs displaying the mean GYIRFamide-induced increases in amplitude and frequency before (solid bars) and after (crosshatched bars) exposure to chelerythrine chloride (n = 5). Statistically significant reductions in these responses (**P < 0.01).

PMA. The effect of the phorbol ester PMA (a PKC activator) on the spontaneous motility of muscle strips was tested using a concentration of 1 µM (Fig. 5, n = 6). Contraction frequency was increased from 2.83 ± 1.33 to 13.17 ± 2.32 contractions/3 min (P < 0.01, Student's paired t-test). In the example shown PMA also increased the amplitude of the phasic activity, but this was not observed in the majority of experiments, and the slight increase in the mean contraction amplitude from 0.68 ± 0.20 to 0.92 ± 0.07 mN was not statistically significant.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   The phorbol ester phorbol 12-myristate 13-acetate (PMA) increased spontaneous activity. A: isometric tension for a single muscle strip exposed to 1 µM PMA (note the break in the record between 10 and 20 min after the start of drug application). B: summarized data covering amplitude (open symbols) and frequency (solid symbols) of contraction for consecutive 3-min time periods covering a 15-min control period and the first 45 min of drug exposure. Contraction frequency was increased after exposure to PMA (P < 0.01, n = 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study provides physiological evidence suggesting that the excitatory response evoked by the endogenous flatworm peptide GYIRFamide is mediated through a G protein in the liver fluke F. hepatica. The data support a model in which activation of PLC stimulates production of DAG, which in turn promotes PKC activity.

The involvement of a G protein in the response to GYIRFamide is suggested by the significant reduction of the response brought about by the G protein inhibitor GDPS (100 µM). In mammals, GDPS binds strongly to the GDP binding site of the G protein, maintaining it in the inactive form and thus inhibiting G protein-dependent processes (12, 33). G protein-linked pathways have been shown to mediate some responses to classical transmitters in flatworm parasites. For example, the response of F. hepatica to 5-HT requires GTP and is mimicked by nonhydrolysable GTP analogs, presumably due to persistent G protein activation (23, 27). This functional evidence is further supported by studies in which proteins analogous to the mammalian G_s and G_i subunits of mammalian G proteins have been identified in both F. hepatica and the nematode Caenorhabditis elegans (1, 27). C. elegans also contains a protein that labels with antibody raised against the subunit complex of mammalian G proteins (1). Thus the involvement of a G protein in the excitatory response to GYIRFamide in F. hepatica is consistent with the presence and physiological activity of G proteins in this and other helminth parasites. Such proteins show pharmacological similarities to each other and also to the G protein signal transduction mechanisms present in their mammalian hosts.

After G protein activation the response to GYIRFamide appears to involve activation of the PLC, DAG, PKC pathway. There are three lines of evidence supporting this suggestion. First, the GYIRFamide response was significantly reduced by neomycin sulfate, a drug that inhibits both PLC and PLD activity, enzymes that normally catalyze the production of DAG from membrane phospholipids. DAG commonly acts to stimulate the activity of PKC, and the feasibility of a PKC-based excitatory pathway was established by using the phorbol ester PMA. Phorbol esters are membrane-permeable and directly activate PKC in vertebrates and invertebrates (2, 25). PMA significantly increased the frequency of contractions in muscle strips from F. hepatica, an observation consistent with studies showing that a variety of phorbol esters stimulate mechanical activity in S. mansoni (4). Those experiments did not investigate which first messengers might modulate schistosomal contractility through PKC, however, so it is of considerable interest that chelerythrine chloride, a potent and selective inhibitor of mammalian PKC (16), inhibited the GYIRFamide response in F. hepatica. As the response to GYIRFamide was almost completely blocked it seems likely that the excitatory action of this neuropeptide is highly dependent on DAG. Involvement of Ins-1,4,5-P₃, the other product of PLC activity, was not directly tested for, however, and cannot be ruled out, especially since Ins-1,4,5-P₃-induced Ca²⁺ release may promote the activity of certain PKC isoforms.

Separate studies have shown that cAMP may well be capable of regulating motility in F. hepatica because an activator of adenylate cyclase (forskolin), a cAMP analog (8-BrcAMP), and phosphodiesterase inhibitors (caffeine and IBMX) all increased spontaneous contractions in fluke muscle strips (14). The present investigation attempted to investigate the possible involvement of a cAMP-dependent pathway in the response to GYIRFamide but few definite conclusions can be drawn from the relevant experiments. MDL-12330A, a cell-permeable, irreversible inhibitor of mammalian adenylate cyclase (26), did appear to block the GYIRFamide-induced excitatory response, but this drug also increased spontaneous activity when used at a concentration adequate to inhibit the response to forskolin. This suggests that MDL-12330A has actions other than simple inhibition of adenylate cyclase in F. hepatica. Similar pharmacological problems frustrated attempts to block PKA, the kinase commonly responsible for cAMP-dependent events, because H89, a selective PKA inhibitor (6), failed to block the response to exogenous, cell permeant 8-BrcAMP. We can, therefore, neither confirm nor exclude a role for cAMP in the GYIRFamide response from the present data, although the efficacy of chelerythrine in inhibiting the GYIRFamide response suggests that any contribution is likely to be upstream of a PKC-dependent step. Observations on the nematodes Ascaris suum and Ascaridia galli indicate that the excitatory effect of the endogenous FaRP, AF-3 (AVPGVLRFamide) is mediated (at least in part) by cAMP, but through inhibition rather than promotion of cAMP production (38). FaRPs are known to operate via a variety of mechanisms in other groups, e.g., in the mollusc, Aplysia, FMRFamide activates both a neuronal K⁺ channel via an arachidonic acid pathway (24) and a neuronal Na⁺ channel via a cAMP-dependent mechanism (18). Diversity of FaRP-activated signaling pathways may well be a feature in platyhelminths as well.