Drug effects on the 5-HT response of Schistosoma mansoni

Comp. Biochem. Physiol. Vol. 77C, No. 1, pp. 199-203, 1984 Printed in Great Britain

0306-4492/84 $3.00 + 0.00 © 1984 Pergamon Press Ltd

D R U G EFFECTS ON THE 5-HT RESPONSE OF S C H I S T O S O M A M A N S O N I WM. S. WILLCOCKSON and GILBERT R. HILLMAN Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX 77550, USA (Received 24 May 1983)

Several vertebrate 5-HT antagonists at concentrations around 0.1 mM reduced 5-HT-induced increases in the motor activity of the parasitic blood fluke Sehistosoma rnansoni. The order of potency for 5-HT response antagonism was haloperidol > cyproheptadine > mianserin > trazodone > spiperone > methysergide. 2. Nisoxetine, a 5-HT uptake inhibitor in vertebrate preparations, was also a potent antagonist of the 5-HT response in schistosomes. 3. The potent antischistosomal praziquantel reduced the 5-HT response similarly to the other antiserotonergic drugs, but at much lower concentrations, beginning around 0.1 pM. 4. The 5-HT agonist quipazine stimulated worm activity at 1~.1 mM when applied alone, but reduced the 1 mM 5-HT response when quipazine and 5-HT were administered concurrently. 5. Dopamine (DA) alone had no effect on the overall activity of S. mansoni. 6. Although no drug was found to have absolute species specificity, quantitative differences were observed between the relative activity of drugs in schistosomes and vertebrates. Abstract--l.

INTRODUCTION Serotonin (5-HT) produces a marked increase in the motor activity of the blood fluke S c h i s t o s o m a m a n soni (Barker et al., 1966). 5-HT has been located histochemically throughout the schistosome (Bennett et al., 1969; Bennett and Bueding, 1971; Machado et al., 1972). Tryptamine has been found equipotent to 5-HT in stimulating motor activity (Nimmo-Smith and Raison, 1968; Tomosky et al., 1974). The 5-HT response can be partially antagonized by methysergide and dihydroergotamine (Hillman et al., 1974) and more completely by bromolysergic acid diethylamide and metergoline, but not by LSD (Tomosky et al., 1974). There have been several recent attempts to characterize 5-HT receptors, primarily in the central nervous system of vertebrates (Aghajanian and Wang, 1978; Peroutka and Snyder, 1979). Although, in general, drugs acting on 5-HT receptors in the periphery also act at 5-HT receptors in the brain, there are suggestions that 5-HT receptors of different conformation and vulnerability may exist. Some of the compounds used in this study which may aid in characterizing 5-HT receptors in schistosomes include: quipazine, a 5-HT agonist (Hong et al., 1969; Rodriguez et al., 1973; Green et al., 1976); several 5-HT antagonists including cyproheptadine (Stone et al., 1961), mianserin (Vargraftig et al., 1971; Maj et al., 1978), methysergide (Gyermak, 1961; K a u a et al., 1961), trazodone (Maj et al., 1979), and haloperidol and spiroperidol (Maj et al., 1978; Peroutka and Snyder, 1979); nisoxetine, a 5-HT uptake inhibitor (Maggi et al., 1980); and 5,6-dihydroxytryptamine, an agent toxic to 5-HT neurons (Baumgarten et al., 1971, 1972) and which can also block 5-HT receptors (Carruba, 1974; Peroutka and Snyder, 1979). M a n y of these drugs are known to affect other receptor 199

systems as well. Antidopaminergic agents sulpiride and metoclopramide were also tested for comparison. Dopamine (DA) was also tested on the 5-HT response to determine possible interrelationships of D A and 5-HT action. These compounds were tested on the molar activity responses of S. m a n s o n i in order to further characterize similarities and differences in 5-HT receptors of schistosomes and vertebrates.

METHODS

Preparation of schistosomes Female CD1 mice infected with Puerto Rican strain Schistosomo mansoni were obtained from a mouse-snail cycle maintained by Drs S. K. File and J. H. Smith of the University of Texas Medical Branch Pathology Department, Galveston, Texas. The adult paired schistosomes were removed 45-60 days post inoculation. The mice were injected with heparin and anaesthetized with ether before being sacrificed by cervical dislocation. Using gentle hook dissection, the adult paired schistosomes were removed from the portal and mesenteric veins. The worms were maintained at 3T'C and pH 7.45 in Fischer's Cell Culture Medium (FM) (Grand Island Biological Co.), with 15 pg of streptomycin and 15 units of penicillin per ml added to retard bacterial growth, and 1 mg NaHCO and 18mg tricine per ml for buffering (Hillman and Senti, 1973). Motor activity studies Motor responses of paired S. mansoni to the compounds were measured in a specially constructed motor activity monitor (Hillman and Senft, 1973; Hillman, 1979). The device contained four flow chambers mounted in a temperature regulated block. Two worm pairs were placed in each glass-bottomed cell located above an array of fiberoptics connected to photocells. Light interruptions caused by the schistosomes were translated electronically into "counts" which were proportional to the total amount

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WM. S. WILLCOCKSON and GILBERT R. HILLMAN

of movement. The counts for each chamber were accumulated for 2-min intervals and the data were automatically transferred to a computer for plotting and statistical reduction. A typical experiment involved the use of two cells as control chambers and two cells as test chambers. The control chambers were perfused with FM with no drug (for basal activity), followed by FM containing 5-HT (for stimulation), and then FM containing carbachol (CCh) (for paralysis) in the absence of the test compound. The test chambers were perfused with the same drugs after exposure to the test compound. The effects of various concentrations and lengths of exposure to the c o m p o u n d s were recorded. The results represented data obtained from at least four experiments on different worm pairs tested on at least two different occasions.

concentration was tested in triplicate in at least two separate experiments.

Compounds Drugs used in the study came from the following sources: cyproheptadine HCI--Merck, Sharp and Dohme: 5,6-dihydroxytryptamine Regis; dopamine HCI and serotonin creatinine sulfate--Sigma; haloperidol McNeil; methysergide m a l e a t ~ S a n d o z ; metoclopramide HCI-Robins; sulpiride--gift of Dr O. Steinsland; mianserin HCI Organon; nisoxetine HCI--LilIy; trazodone HCI Meade-Johnson; praziquantel--Bayer; and spiroperidol-Janssen. All compounds, except spiroperidol and praziquantel, were made up at or near the desired concentration in FM just prior to use. Spiroperidol was converted to an acetate salt at 0.I M before making serial dilutions in FM. Praziquantel was dissolved in dimethyl sulfoxide at 1 m M before serial dilution in FM. The pH of all drug solutions applied to schistosomes was adjusted to 7.4.

5-HT Uptake studies ]4C-Serotonin binoxalate (New England Nuclear) was diluted with unlabelled 5-HT to achieve final concentrations of 10 or 100/xM. Live schistosomes were incubated for 5 min at 3 7 C in FM containing ]4C-5HT (10-100 /~M) and DA (10/~M, 100/tM and 1 mM). The schistosomes were then recovered, washed briefly (5 sec) in non-radioactive FM at 2 5 C , and then placed in 100/~1 Protosol (New England Nuclear). After complete solubilization, 3 ml of scintillation fluid was added (Aquasol, New England Nuclear). Following dark adaptation, samples were counted in a liquid scintillation counter (Senft et al., 1976). Each

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Log molar [SHT] F'ig. 1. Effects of various drugs on the 5-HT d o s e - m o t o r activity response curve of S. mansoni. Ordinate shows the number of movements counted per 2-rain interval expressed as a percentage of the maximal response taken as the control response to 1 m M 5-HT. The concentration of each drug plotted was 0.1 mM, except for praziquantel which is represented here at a concentration of 3 x 10 7M. Values represent the mean of greater than four experiments on different worm pairs on at least two different occasions _+ standard deviations. *Indicates a significant difference from control by at least P < 0.05 as measured by Student's t-test.

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R e s p o n s e o f Schistosoma to 5 - H T

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Table 1. Compound Cypropheptadine Mianserin Trazodone Nisoxetine Methysergide Haloperidol Spiroperidol Praziquantel Quipuziue Dopamine

Conc (M) 10 4 10 4 10 4 10 4 10 4 10 4 10 4 3 x 10 7 10 4 10 4

Response alone* (P§)

On 1 mM 5HT responset (P)

NS NS NS NS +310% (<0.05) NS +700% (<0.01) NS +513% (<0.005) NS

~ 8 5 % (<0.001) - 4 1 % (<0.05) - 3 2 % (<0.05) - 4 2 % (<0.025) - 2 0 % (<0.05) - 9 2 % (<0.001) 33~ (<0.05) - 6 1 ~ (<0.001) - 4 5 ~ o (<0.001) +120% 11 (<0.025)

On 0.3 mM CCh response~ (P) NS NS NS NS NS - 16'~o (<0.05) -557~ (<0.01) NS NS NS

*Response [(motility after d r u g - m o t i l i t y in FM)/motility in FM] × 100. +Effect - [(response after drug with 1 mM 5 - H T - response after 1 mM 5-HT alone)/response after 1 mM 5-HT alone] x 100. ++Effect [(response to drug with 0.3 mM C C h - response after CCh alone)/response after CCh alone] × 100. §Probability that drug had no effect on response; from nonpaired student's t-test. ]IDA greatly enhanced the 5-HT response to 10 5 10 4M 5-HT, but could not increase activity further at 10 3 M 5-HT. The 12(P~, increase reflects the DA effect at a 5-HT concentration of 10-~M instead of 10-3M (see Fig. 1). NS - No significant difference between test and control.

The antischistosomal drug praziquantel reduced the 5-HT response beginning at 0.1/~M. Sulpiride, 5,6-dihydroxytryptamine and metoclopramide had no effect on the 5-HT response of schistosomes at a concentration of 0.1 mM. The 5-HT agonist quipazine (0.01 1 mM) stimulated the worms when applied alone, but was also effective in reducing the 1 mM 5-I-1T response when quipazine and 5-HT were applied together. Of the compounds tested only haloperidol, spiroperidol and quipazine caused significant stimulation of worm motor activity when administered alone. The paralytic response to carbachol was also tested following 5-HT stimulation during exposure to each of the compounds. Compounds that reduced CCh-induced paralysis included haloperidol and spiroperidol. DA, when applied with 5-HT, caused a large increase in activity over 5-HT given alone (Fig. 1). This effect was seen at DA concentrations of 0.01-1 mM and 5-HT concentrations 0.01 0.1 mM, but not 1 raM. DA antagonists having no effect on the 5-HT response of schistosomes did not reduce the DA-induced enhancement of the 5-HT response up to concentrations of 0.1 raM, with the exception of sulpiride which caused a 56% reduction (P < 0.05) of the DA effect. DA had no apparent effect on overall worm motility when administered in the absence of 5-HT. DA was also tested for possible effects on 14C-5-HT uptake. At concentrations of DA and 5-HT which caused the motor activity effects (0.01, 0.1 mM DA; 0.01, 0.1 mM 5-HT), there was no significant increase in 5-HT uptake over control induced by DA in any experimental trial. Nisoxetine at 0.1 mM was also tested in 14C-5-HT uptake studies and was found to have no significant effect on 5-HT (0.01 raM) uptake.

DISCUSSION

It is apparent from these studies that many compounds that have been shown in vertebrate studies to act at serotonergic sites have effects on the 5-HT response of schistosomes. Vertebrate 5-HT antagonists cyproheptadine, mianserin and methysergide all

antagonized the 5-HT response of schistosomes. Haloperidol, spiroperidol and trazodone, which have effects in vertebrates on both DA and 5-HT receptors, also antagonized 5-HT responses. Other antidopaminergic drugs, such as sulpiride and metoclopramide, had little effect on the 5-HT response suggesting some structural discrimination. The vertebrate 5-HT agonist quipazine mimicked the 5-HT effect in schistosomes, causing a doserelated stimulation of motor activity. However, quipazine appeared to be a partial agonist in schistosomes due to its ability to reduce the l mM response to 5-HT when applied simultaneously with 5-HT at concentrations of 0.1-1 mM. It is uncertain whether quipazine is a partial agonist at postsynaptic sites in vertebrates; however, quipazine appears to act presynaptically on serotonin neurons in vertebrates (Fuller et al., 1976; Hamon et al., 1976). Presynaptic inhibition might also be occurring in schistosomes. Methysergide and spiroperidol also caused a transient stimulation of motor activity in schistosomes when applied alone. The mechanism of this effect is unknown at this time. Cholinergic antagonists often cause a transitory stimulation of schistosomes in addition to reducing carbachol-induced paralysis (Barker et al., 1966). Spiroperidol was potent in reducing the carbachol response and, therefore, its stimulatory effects could be explained by an anticholinergic action. Butyrophenones are known to act at cholinergic receptors in vertebrates (Snyder and Yamamura, 1977; Snyder et al,, 1978). Haloperidol also reduced the carbachol response in schistosomes, although it was less potent than spiroperidol. This difference in potency may explain why no stimulatory effects were seen after application of ha[operidol alone. The stimulatory effects of methysergide do not appear to be due to an anticholinergic effect, since it had no effect on the carbachol response. The stimulatory effects of methysergide are not as easily explained. Methysergide may have some agonist activity at 5-HT sites in schistosomes as well as its more predominant antagonist effect. Nisoxetine, a 5-HT uptake inhibitor in vertebrates, was a potent 5-HT antagonist in schistosomes. It had no significant effect on 5-HT uptake in schistosomes, and therefore must act by a different mechanism in

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this parasite. Fluoxetine, a compound related to nisoxetine and a 5-HT uptake inhibitor in vertebrates, has also been found to antagonize the stimulatory effects of 5-HT on the musculature of S. mansoni (Pax et al., 1979). Fluoxetine does inhibit 5-HT uptake in S. mansoni schistosomules (Catto and Ottesen, 1979) and adult schistosomes (Hillman, unpublished results). The results of these studies also suggest some differences between 5-HT receptors of schistosomes and vertebrates. While it is difficult to classify several of the compounds tested because of their effects on multiple receptor systems in vertebrates, it appears that the order of potency for 5-HT antagonism in schistosomes is somewhat different than 5-HT antagonism in vertebrates. The study by Peroutka and Snyder (1979), for example, on brain 5-HT receptor binding showed the order of antagonism to be methysergide > spiroperidol > manserin > cyproheptadine > haloperidol. This order of potency for 5-HT antagonism in brain binding is by no means exemplary of 5-HT antagonism in all vertebrate 5-HT receptor systems. There still remains no universally accepted criteria of 5-HT receptor antagonism even within the brain (Fuller, 1980). Peroutka and Snyder also found, for example, that at 5-HT receptors, i.e. those that more readily bind spiroperidol than 5-HT, the order of spiroperidol binding antagonism in rat brain is spiroperidol > cyproheptadine > methysergide > mianserin > haloperidol. Differences in 5-HT antagonism within the brain have also been detected in electrophysiological work with 5-HT antagonists (Haigler and Aghajanian, 1977). The order of 5-HT response antagonism in schistosomes does not coincide exactly with any of the vertebrate 5-HT receptor systems described in the literature. The high potency of haloperidol and nisoxetine (a 5-HT uptake inhibitor in vertebrates) and the low potency of spiroperidol are the most notable exceptions to the results of vertebrate 5-HT antagonist studies. While these findings cannot suggest a structural conformation of the schistosome 5-HT receptor, they do reflect different selectivities of drug action. Tomosky et al. (1974) have postulated that the schistosome 5-HT receptor more resembles a tryptaminergic receptor, since tryptamine is equipotent to 5-HT in stimulating worm activity. Differences in 5-HT receptor conformation of schistosomes and their hosts would provide a promising point of attack for therapeutic antischistosomal drugs. While its site of therapeutic action cannot be demonstrated by these experiments, it may be relevant that praziquantel was found to be the most potent of all compounds tested in reducing the 5-HT response. It is known, however, that praziquantel has other actions in the schistosome; for example, it may alter ion permeability in the tegument of the worm (Pax et al., 1978; Nechay et al., 1980). At present we cannot offer a mechanism to explain the fact that D A increased the response to 5-HT. The uptake of HC-5-HT was not significantly influenced by DA, and DA antagonists, with the exception of sulpiride, had no effect on the DA-induced increase of 5-HT stimulation. Further studies may determine whether this effect of D A is due to actions at DA, 5-HT, or some other receptor.

Acknowledgements--The authors thank Drs S. J. Enna and

O. S. Steinsland for suggestions and advice. This research was supported by USPHS grant No. AI 15536. REFERENCES

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