Naunyn-Schmiedeberg's
Archivesof Pharmacology
Naanyn-Schmiedeberg's Arch. Pharmacol. 304, 309-315 (1978)
9 by Springer-Verlag 1978
A Benzodiazepine Derivative and Praziquantel: Effects on Musculature of Schistosoma mansoni and Schistosoma japonicum R. Pax, J. L. Bennett, a n d R. F e t t e r e r Departments of Pharmacology and Zoology, Michigan State University, East Lansing, Michigan 48824, USA
Summary. T h e b e n z o d i a z e p i n e derivative ( R o 11-3128) which has central n e r v o u s effects similar to o t h er benzodiazepines, a n d p r a z i q u a n t e l (PZ), are t wo new a n t i s c h i s t o s o m a l drugs. A t lo w c o n c e n t r a t i o n s these drugs will p r o d u c e a m a r k e d spastic paralysis o f male S c h i s t o s o m a m a n s o n i musculature. A n analysis o f the action o f these drugs o n the parasite's m u s c u l a t u r e shows t h a t R o 11-3128 a n d P Z p r o d u c e d a rapid rise in the tension o f the m u s c u l a t u r e o f male schistosomes. V a r i o u s c o m p o u n d s k n o w n to interact with the s c h i s t o s o m e ' s n e u r o r e c e p t i v e sites did n o t b lo ck or p o t e n t i a t e the action o f these drugs. R e m o v a l o f Ca 2 + or a d d i t i o n o f M g 2+ to the i n c u b a t i o n m e d i u m b l o c k e d the action o f these drugs on the s c h i s to s o m e ' s m u s c u l a ture. U p t a k e studies o f i n o r g a n i c cations by m a l e sc h i s t o s o m e' s indicate that R o 11-3128 a n d P Z decrease the influx o f K + but stimulate the influx o f C a 2 + a n d N a + into the male schistosome. It is suggested t h a t this interference with i n o r g a n i c i o n t r a n s p o r t m e c h a nisms causes the c o n t r a c t i o n o f the schistosome musculature.
Key words: B e n z o d i a z e p i n e - P r a z i q u a n t e l - Schistosomes - C a t i o n s p o r t o f cations.
M u s c u l a t u r e - M e m b r a n e trans-
Introduction Th e b e n z o d i a z e p i n e derivative, R o 11-3128, a n d praziq u a n t e l (PZ), are p o t e n t a n t i s c h i s t o s o m a l c o m p o u n d s ; a single oral dose given to infected mice is lethal to ad u l t a n d d e v e l o p i n g stages o f the parasite (Stohler, 1978; G 6 n n e r t an d A n d r e w s , 1977). These a u t h o r s n o t e d t h a t these two c o m p o u n d s p r o d u c e d a spastic paralysis o f the parasite's musculature. This o b s e r v a t i o n p r o m p t e d
Send offprint requests to J. L. Bennett at the above address
US to analyze the action o f these drugs on the s c h i s t o s o m e ' s m u s c u l a t u r e using a t e c h n i q u e cap abl e o f directly m e a s u r i n g the m e c h a n i c a l properties o f those muscles which are responsible for the parasite's m o t o r activity.
Materials and Methods Isolation of Parasites. Male Schistosomes (S. mansoni, St. Lucian strain and S. japonicum, Chinese strain) were isolated from infected mice as previously described (Bennett and Seed, i977) and then incubated at 37~C in Earles balanced salts (EBS) solution (Gibco, Long Island, New York) containing 10 % heat-inactivated human serum (Gibco), buffered at pH 7.4 with 0.02M Hepes (Sigma Chemical Co., St. Louis, Mo.). Description of Instrument Used to Analyze Effects of Drugs. The effects of the benzodiazepines and PZ on the musculature of single male schistosomes was determined by connecting the parasites to an apparatus (Fig. 1) capable of measuring the mechanical activity of its musculature. In brief, the parasite was suspended in the incubation medium by two suction pipettes. One pipette was very inflexible and attached to the tail while the other was very flexible and attached 1.0-2.0 mm from the inflexible pipette. Shortening or lengthening movements by the parasite caused movement of the flexible pipette which was converted to movement of a balanced wire system connected to a black acetate strip which acted as a shutter for a phototube in a myograph transducer. Since the amount of tension developed depends on the length of worm over which measurements are made, all tension measurements are given in terms of mg tension/ram of worm. With this system we can reliably measure shortening or lengthening movements as small as 30 ~tm and tension changes as small as 0.10 mg. The capabilities of this instrument are described in detail by Fetterer et al. (1977). Most experiments, using this apparatus, were performed at 37~ in a polyethylene dish containing 2.5 ml of Earles balanced salt (EBS) solution buffered at pH 7.4 with 0.02 M Hepes. Drugs. Praziquantel (MW = 312) and Ro 11-3128 [(+)-5-(o-chlorophenyI)-l,3-dihydro-3-methyI-7-nitro-2H-1,4-benzodiazepine-2one; MW = 322)] (Fig. 2) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 2 M and diluted to the desired concentration with distilled water. 2.5 gl of the drug was then added to the recording chamber fluid (2.5 mI) to give the final concentration. These drugs, when given orally to infected mice, were suspended in Emulphor EL-620 (Gaf Corp.).
0028-1298/78/0304/0309/$01.40
310
Naunyn-Schmiedeberg's Arch. Pharmacol. 304 (1978)
Pipette Shutter
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Ro 11-3128 Fig. 1. Schematic diagram of apparatus used to analyze the mechanical activity of male S. mansoni
," C--O
PRAZIQUANTE L
1 /'-c"3
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N
RoU- 3128
Fig.2. Structure of praziquantel and Ro 11-3128
Other drugs used were: dopamine (3-hydroxytyramine HC1), 5HT (5-hydroxytryptamine creatinine sulfate), carbachol (carbamylcholine chloride), atropine, pentobarbital (all Sigma Chemical Co., St. Louis, Mo.), spiroperidol (Janssen Pharmaceuticals), BOL (bromlysergic acid diethylamide; Sandoz, Corp.), X-537A (Hoffmann-La Roche) and A-23187 (Eli Lilly Co.). Praziquantel was supplied by Dr. P. Andrews of the Bayer Company, while Ro 11-3128 was supplied by Dr. W. H. Scott and Dr. H. Stohler of Hoffmann-La Roche.
4SCa2+ Uptake Studies. To study the effects of Ro 11-3128 or PZ on uptake of 45Ca2 + (New England Nuclear, 30 Ci/g calcium) by male schistosomes, we preincubated 15 parasites in two rtCi o f 4 5 C a 2 + and EBS at 37~ C for 30 rain. (After 30 min the tissue to medium ratio of 45Ca 2 + remained constant.) Following this incubation, Ro 11-3128 or PZ was added and incubated for varying periods. After incubation with a drug the parasites were separated from the medium by filtration over a 25121153_glass fiber filter (Wanman GF/B) placed under vacuum on a millipore filtration apparatus (Millipore Company, Model XX10-024-00). The trapped parasites were then washed three times in 5ml of EBS containing 6raM CaCI 2. Three washes were found to be adequate since additional washes did not lower the amount of radioactivity bound to the parasite. The parasites were then weighed, placed in a vial containing 0.5 ml of tissue solubilizer (NCS, Amersham Corp.) and then incubated at 50~ for I h. The solubilized parasites were then placed in a scintillation vial containing 10ml of scintillation fluid consisting of one liter 95 ~ ethanol, 21 toluene, 0.3 g of POPOP, and 12 g of POP. The vials were then placed in a scintillation spectrometer, (Beckman Model LS-t00) where the counting efficiency was approximately 32 ~ for 45Ca2 +. *2K+ or 22Na+ Uptake Studies. Uptake of radioactive 42K+ (Michigan State University Nuclear Reactor Lab, 10 mCi/g potassium) or Z2Na+ (New England Nuclear, Carrier Free) by male schistosomes was performed as described above for 45Ca z + uptake,
Fig.3, Effects of 10-6M Ro 11-3128 on longitudinal contractile activity and tension of a male schistosome. Horizontal calibration = 20 s; vertical calibration = 0.5 mg of tension
except for the following. Our procedure for determining the effects of PZ or Ro 11-3128 and K + and Na § uptake consisted ofa 1 - 30 min incubation of the parasites in 1 pCi of the radionuclides in the presence or absence of PZ or Ro 11-3128. The filtered parasites incubated in 4ZK + were washed 3 times in EBS containing 10 mM K + while parasites incubated in 22Na+ were washed in EBS. After weighing the parasites we placed them in a small vial which in turn was placed into a gamma counter (Packard Inst., Model 578).
Results F i g u r e 3 d e m o n s t r a t e s t h e effects o f 1 0 - 6 M R o 113128 o n t h e m e c h a n i c a l a c t i v i t y o f a m a l e s c h i s t o s o m e . B e f o r e the d r u g was a d d e d the w o r m was c o n t r a c t i n g w h i l e t e n s i o n r e m a i n e d c o n s t a n t . W i t h i n 10 s after the addition of the drug, spontaneous contractions began to d i s a p p e a r a n d t e n s i o n b e g a n to rise. P Z , at 10 7 M , p r o d u c e d s i m i l a r effects o n the m a l e s c h i s t o s o m e . P Z o r R o l l - 3 1 2 8 - i n d u c e d effects w e r e r e v e r s i b l e since ten s i o n a n d c o n t r a c t i l e r a t e r e t u r n e d to n o r m a l 2 0 m i n a f t e r r e m o v a l o f d r u g f t o m the i n c u b a t i o n m e d i u m . Since the effect o f these d r u g s o n the t e n s i o n o f t h e p a r a s i t e ' s m u s c u l a t u r e c o u l d be easily q u a n t i f i e d , we e x a m i n e d t h e effects o f v a r i o u s c o n c e n t r a t i o n s o f P Z o r R o 11-3128 o n the t e n s i o n o f the m u s c u l a t u r e o f m a l e S. mansoni o r S. japonicum. F i g u r e 4 s h o w s t h a t P Z , at l o w c o n c e n t r a t i o n s , p r o d u c e d a m a r k e d effect o n the tens i o n o f the m u s c u l a t u r e o f the m a l e S. mansoni o r S. japonicum. T h i s was n o t the case w i t h R o 11-3128 (Fig. 4) since this c o m p o u n d was f o u n d to be e f f e c t i v e a g a i n s t m a l e S. mansoni b u t n o t a g a i n s t m a l e S. japonicum. T h i s o b s e r v a t i o n c o r r e l a t e s w i t h a r e p o r t b y S t o h l e r (1978), d e m o n s t r a t i n g t h a t R o 11-3128 c o u l d c u r e m i c e i n f e c t e d w i t h S. mansoni b u t n o t w i t h S.
japonicum. T h e d a t a r e p o r t e d b y G 6 n n e r t a n d A n d r e w s (1977) a n d b y S t o h l e r (1978) s u g g e s t t h a t it t a k e s a b o u t 4 - 5 t i m e s m o r e P Z t h a n R o 11-3128 to c u r e m i c e i n f e c t e d w i t h S. mansoni. In a n a t t e m p t to e x p l a i n w h y P Z was less e f f e c t i v e t h a n R o 11-3128 in c u r i n g i n f e c t e d mice, we p e r f o r m e d t h e f o l l o w i n g e x p e r i m e n t . We a d m i n i s t e r e d 80 m g / k g o f P Z o r R o 11-3128 to i n f e c t e d m i c e a n d
R. Pax et al. : Response of S. mansoni to Praziquantel and a Benzodiazepine Derivative
311
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o.o Fig. 4 Effects of various concentrations of Ro I 1-3128 or PZ on the tension produced by the musculature of S. japonicum (B - I ) or S. mansoni (0 -0)
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o Fig.5A and B Response of musculature of S. mansoni to 0.1 ml serum (A) or 25 gl or urine (B) removed from infected mice at various intervals after the administration of 80mg/kg Ro 11-3128 ( ) or PZ
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then at various intervals, after drug administration, we collected urine and isolated plasma from these mice. Samples of urine or plasma from mice treated with PZ, Ro 11-3128, or vehicle were placed in the presence of male schistosomes connected to our apparatus. These schistosomes were isolated from mice not treated with these drugs. Results (Fig.5) indicate that PZ or PZrelated material is rapidly removed from the blood by the kidney, but not Ro 11-3128 or Ro 11-3128 related material. In an attempt to determine the mode of the antischistosomal action of PZ and Ro tl-3128, we examined the interaction between these drugs and putative neurotransmitters of this parasite. For example, Tomosky et al. (1974) demonstrated that dopamine is one of these putative neurotransmitters which, when incubated in the presence of male schistosomes, will lengthen the parasite, i.e., a response opposite to that induced by PZ or Ro 11-3128. Thus, to determine whether dopamine would block PZ or Ro 11-3128induced rise in the tension of male S. mansoni we preincubated the worm in dopamine for 20 rain and then added PZ or Ro 11-3128. From Fig. 6 it can be seen that dopamine did not block the PZ-induced rise in
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8 Time (h)
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Fig.6. Effect of dopamine (DA) on the PZ-induced rise in the tension of the musculature of male S. mansoni. Horizontal calibration = 1 rain; vertical calibration = 0.5 mg of tension
tension, nor did it block (not shown) the effect o f R o 113128. In addition to dopamine we attempted to block the effect of PZ or Ro 11-3128 with other compounds that are known to interact with other neuroreceptive sites in S. mansoni, i.e., 5-hydroxytryptamine, carbachol, spiroperidol, brom-lysergic acid diethylamide, and atropine (Barker et al., 1966; Tomosky et al., 1974). None of these compounds (at 10 -4 M) blocked the PZ or Ro ll-3128-induced increase in tension in
Naunyn-Schmiedeberg'sArch. Pharmacol. 304 (1978)
312 male S. mansoni. We also attempted to block, without success, the PZ-induced rise with 1 0 - 3 M pentobarbital, a compound that lengthens and paralyzes the schistosome. During the above studies we observed great variability in the time required to detect a particular drug's ability to induce changes in the schistosome's mechanical activity. To determine if there was a difference in the time required for a drug to induce a response in male schistosomes we determined the time required to see the half maximal effect (i. e., increased contraction rate) of 1 0 - 5 M 5-hydroxytryptamine and compared it to the time required to see the half maximal effect (i.e., increase in tension) of 1 0 - 6 M PZ or 1 0 - 5 M Ro 113128. The time required for half maximal effect of 5hydroxytryptamine was 37 _+ 5 s while for PZ or Ro 113128 it was 11.2 _+ 1.4 and 15.6 _+ 2.3s, respectively. These results combined with the inability of neurotransmitter agonist or antagonist t0 block the action of PZ or Ro 11-3128 on the musculataure of male S. mansoni suggested to us that these drugs may be acting directly on mechanisms (e.g., transport of inorganic ions) that regulate muscle contractility. To determine if inorganic ions play a vital role in mediating the action of PZ or Ro 11-3128 we performed some ion-substitution experiments. Mechanical activity of male S. rnansoni was measured as described in methods except that Hanks balanced salts solution (HBS), buffered at pH 7.4 with 0.01 M Tris, was used rather than EBS. The response of male schistosomes to PZ or Ro 11-3128 in HBS was qualitatively and quantitatively identical to that observed when the parasites were incubated in EBS. When schistosomes were preincubated for 15 rain in Ca 2 + free HBS we did not observe the PZ or Ro ll-3128-induced rise in the tension on the musculature of male schistosomes. When Ca 2+ was added back to the Ca 2 + free HBS, containing PZ, the schistosomes would contract. Incubation of the parasites in Ca 2 + free HBS produced a slight rise in tension of the musculature of the parasite and a reduction in the number of spontaneous contractions. Since high concentrations of Mg a+ have been shown to attenuate biological mechanisms which depend upon Ca 2§ (del Castillo and Katz, 1954), we studied the effect of various concentrations of Ca 2 § and Mg 2 + on PZ or Ro 11-3128 induced-contraction of the male schistosome musculature. When male schistosomes were preincubated in HBS containing 0.4 m M Ca 2 + and 30 m M Mg 2 +, we noted no change in tension and a slight decrease in spontaneous contractile activity; when PZ (Fig.7) or Ro 11-3128 (not shown) was added we observed a transient rise in tension. This same experiment was performed in 1.4 mM Ca 2 § and I m M Mg 2§ or 1.4raM Ca 2+ and 3 0 m M Mg 2§ In 1.4mM
"•1.6 O '_~ .8
.5
1.0
2,0
2,5
Time {minl
Fig.7. Effectof various concentrations o f C a 2 + and Mg2+ on the PZinduced rise in tension of male S. mansoni. The arrow points to the time at which 10-6M PZ was added to parasites preincubated in normal HBS (e e), in HBS containing 1.4mM Ca2+ and 30raM Mg2+ (D [21), or in HBS containing 0.4mM Ca2+ and 30 mM Mg2+ (9
Table 1. Percent increase in tension of the musculature of male S. mansoni when exposed to the ionophores X537A or A23187 Ionophore
Concentrationsof ionophore 10 4
X537A
252 _+25 ~
A23187
232+ 19~o
10-5 0.0 ~ 193_+17~
10-6 0.0 132_+14~
Ca 2+ and l m M Mg z+ PZ produced a sustained increase in the tension on the male schistosome while the PZ-induced increase in tension was transient when the worm was incubated in 1.4 mM Ca z + and 30 m M MgZ + To determine if the PZ-induced transient rise in tension could be performed twice on the same male schistosome we performed the following experiment. We induced this transient rise in tension with PZ on male schistosomes incubated in HBS containing 0 . 4 m M Ca 2 + and 30raM Mg 2 + and then incubated these parasites for 30 rain in the modified HBS containing no PZ; this was enough time to reverse the effects of PZ on the parasite's musculature. We then added PZ back to the incubation medium. Under these conditions, PZ did not induce a transient rise in tension when reapplied to the incubation medium. This same experiment was performed in 1.4 mM Ca 2 + and 1 mM Mg 2 + ; under these conditions PZ, upon reapplication, induced a marked increase in tension. Since extracellular Ca z + seemed to be involved in the action of PZ or Ro 11-3128 on S. mansoni we examined the response of male worms to ionophores, i.e., compounds that can transport cations, C a 2 + in particular, across membranes and increase cytoplasmic Ca z+ concentration (Reed and Lardy, 1972). When
R, Pax et al. : Response of S. mansoni to Praziquantei and a Benzodiazepine Derivative
3001
Table2. Translocation of Ca 2+ or Na + into bulk organic phase
Compound 500/aM
CaCI2
313
NaCI I
laM cation in organic phase 200-
Control Praziquantel X537A
4.0 + 0.3 6.4 + 0.6 92.0+3.0
3.2 _+ 0,1 3.0 + 0,1 198 _+12
Each compound was dissolved in 30 ~ Butanol-70 ~ toluene and 1 ml was added to test tubes containing 1/2ml of 40 mM Tris pH 7.5 and eitb,er 20mM ~5CaC12 (1 Ci/rM) or 20 mM ZZNaCI (:I Ci/mI). The tubes were shaken for 5 min, centrifuged at 2,500 rpm for 5 rain and then 0.5 m] of the organic phase was analyzed for radioactivity. Data represent mean _+ S.E.M. of 4 determinations
+; D~ 100
lb Incubation Time (min)
Fig.9. Uptake of 2ZNa+ by male schistosomes in the absence (O O) and presence of 10-6M PZ ( A - - Z x ) or Ro 11-3128 (11 --). Drugs and radionuclide were added to the incubation medium at the same time
170
3000
~
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~
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30
-
2000 130
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~ 1000
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10
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Incubation Time (rain)
to-
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Incubation Time [min }
Fig.8, Effect of 10-~M PZ (9 or ]0 SM Ro 1~-3128 (O O) on the uptake of 4SCaz+ by male schistosomes. Parasites were preincubated in ~SCaZ+ for 30 min then the drugs were added (see arrow on figure)
(e @) and presence of l0 6M PZ ( Z x - - • or Ro 11-3128 (l~), Drugs and radionuclide were added to the incubation medium at the same time
male schistosomes were incubated in the presence of various concentrations ofX537 or A23187 we observed an increase in the tension of the parasite's musculature (Table 1). At low concentrations, A23187 induced a greater response than X537A. These results prompted us to determine if PZ and Ro 11-3128 could, Iike ionophores, transfer cations from an aqueous medium to an organic phase. Compared to X537A, Ro 11-3128 and PZ did not transfer N a + or Ca 2 + from an aqueous medium into an organic phase (Table 2). Since Ca z + appears to play an important role in mediating the effects of PZ or Ro 11-3128 we attempted to determine if alterations in the concentration of other inorganic ions would affect the action of these drugs on the musculature of S. mansoni. We observed that decreasing the concentration of K +, N a + or C I - in
HBS had little if any effect upon the Ro 11-3128 or PZinduced rise in tension of the schistosome musculature. To more accurately assess the role that inorganic ions may play in the action of PZ or Ro 11-3128 on the schistosome's musculature we studied the effects of these drugs on the influx of 4~Ca2 +, 22Na +, and r + into the parasite. To study the effects of PZ or Ro 113128 on the transport of Ca z + by male schistosomes, we preincubated the parasites in r + for 30 min and then added PZ or Ro 11-3128 to the incubation medium. The addition of these drugs resulted in a rapid accumulation of 45Ca2+ by the male schistosome (Fig. 8). In addition to Ca 2 +, we analyzed the effects of PZ or Ro 11-3128 on the uptake of K + and Na + by male schistosome. F r o m Fig.9 and 10 it can be observed that PZ or Ro 11-3128 stimulated the uptake
Fig.tO. Uptake of 4ZK+ by male schistosomes m the absence
314 o f 2 2 N a + but inhibited the uptake of 42K+ by the
parasite. Discussion
Our results confirm previous observations (Stohler, 1978; G6nnert and Andrews, 1977) which demonstrated that incubation of S. mansoni with PZ or Ro 113128 produces a spastic paralysis of the parasite's musculature. This drug-induced spastic paralysis appeared to be independent of any action the drug may have on the parasites' neurotransmitter receptors, since we observed that the PZ or Ro 11-3128-induced response could not be blocked by compounds known to interact with these receptors. Since the drug did not appear to interact with schistosome neuroreceptive sites, we determined if the action of these drugs was dependent upon the presence of inorganic ions. Since pre-incubation of the worm in Ca 2 + free HBS blocked the action of these drugs on the schistosome, it would appear that PZ and Ro 11-3128 were directly or indirectly activating a Ca 2 § dependent contraction of the parasite's musculature. A similar conclusion with respect to PZ was made by investigators at Imperial Chemical Industries (G. C. Coles, unpublished observations). This conclusion was supported by the rapid increase in the influx of45Ca2 § into the worm after exposure to PZ or Ro 11-3128 and the response of the parasites to the Ca 2 § inophore A23187. From these results it would appear that extracellular Ca 2§ may play an essential role in the contractile activity of the schistosome musculature. Thus, like vertebrate smooth muscle, but unlike vertebrate skeletal muscle (Carafoli et al., 1975), the schistosome muscle may not be able to store small amounts of Ca 2 +. For example, when we pre-incubated schistosomes in a medium containing a low concentration of Ca 2 § and a high concentration of Mg 2+, we observed only a transient rise in the tension of the parasite's musculature when PZ was added to the medium. When PZ was removed from the medium and then reapplied 30 min later (the time required for the tension of the parasite's musculature to return to normal) the schistosome did not respond. When this experiment was repeated in a medium containing physiological levels of Ca 2 + and Mg 2§ we observed that reapplication of PZ would induce a rise in the tension of the musculature of the schistosome. F r o m these experiments we conclude that the transient PZ-induced rise in the tension of the parasite's musculature, incubated in low Ca 2 + and high Mg 2§ is due to the release of a small amount of sequestered Ca 2§ that is not displaced by the high Mg 2§ Once this stored Ca 2 § is released, due to an activation of the parasite's contractile elements by PZ, it is not replaced because Mg 2§ blocks or competes
Naunyn-Schmiedeberg's Arch. Pharmacol. 304 (1978)
with Ca 2 + for this Ca 2 + storage site. Further studies using 45Ca2 § are presently being carried out to verify the presence of this Ca 2§ storage site within male S. mansoni.
As with most contractile processes in animals it is not suprising to observe that the action of PZ and Ro 11-3128 on the musculature of S. mansoni can be blocked or attenuated by altering the concentration of inorganic ions within the medium, especially Ca 2§ since it is intimately associated with the contraction of muscle in various animals (Prosser, 1973). Thus, although Ca 2 § is necessary for the action of these drugs the important question, as to how PZ and Ro 11-3128 bring about this rapid rise in the tension of the musculature of S. mansoni, still needs to be answered. A possible mechanism that could explain the action of PZ or Ro 11-3128 on the musculature of S. mansoni could be due to a disruption of the resting membrane potential of the parasite's muscle cells. For example, these drugs could perturb the membrane of these cells and disrupt mechanisms which regulate ion fluxes, e. g., inhibit enzymes involved in maintaining inorganic ion gradients and/or fluidize the membrane such that it becomes more permeable to inorganic ions. If these drugs were in fact depolarizing the schistosome's muscle cells then one would predict that K + ions should flow out of these cells and Na + ions should flow in; this assumes that the resting membrane potential of the schistosome's muscle cells is maintained in a manner similar to other animals. Our results would suggest that this may be the mechanism since PZ and Ro 11-3128 stimulated the uptake of 22Na+ but inhibited the uptake of 4 2 K + . Recent electrophysiological studies performed in our lab (Fetterer et al., 1978) strongly suggest that these drugs are depolarizing the cells of this parasite. With respect to the possibility that these drugs are inhibiting enzymes that regulate parasite ion fluxes, it is known that ATPases are located in the invaginations along the epidermis of male S. mansoni (Ernst, 1977). Unfortunately, very little is known about the physiological function of these enzymes although in other animals they are often associated with the translocation of ions across membranes (Skou, 1965). Since more needs to be known about these enzymes it would be interesting to determine if these drugs can inhibit the parasite's membrane bound Na +, K +ATPase. Another possibility is that PZ and Ro 11-3128 may be chelating C a 2 + or Na + ions and then transporting these ions into the cells of S. mansoni. Since we observed that these drugs could not combine with these ions and carry them into a non-polar organic phase (a property common to ionophores) we dismissed the idea that they were acting like ionophores. Acknowledgements. This research has been supported by the Edna McConnell Clark Foundation, World Health Organization, and
R. Pax et al. : Response of S. mansoni to Praziquantel and a Benzodiazepine Derivative Hoffmann-La Roche Company. Materials used in this study were provided by the U.S.-Japan Cooperative Medical Science ProgramNIAID.
References Barker, L. R., Bueding, E. B., Timms, A. R.: The possible role of acetylcholine in Schistosoma mansoni. Br. J. Pharmacol. Chemother. 26, 656-665 (1966) Bennett, J. L., Seed, J. L.: Characterization and isolation of concanavalin. A binding sites from the epidermis of S. mansoni. J. Parasitol. 63, 250-258 (1977) Carafoli, E., Clementi, F., Drabikowski, W., Margreth, A. : Calcium transport in contraction and secretion. Amsterdam: NorthHolland Publ. Co. 1975 del Castillo, J., Katz, B.: Quantal components of the end plate potential. J. Physiol. (Lond.) 124, 560-573 (1954) Ernst, S. C.: Biochemical and cytochemical studies of alkaline phosphatase activity in Schistosoma mansoni. Rice Univ. Studies 62, 8 1 - 9 5 (J977)
315
Fetterer, R. H., Pax, R. A., Bennett, J. L.: Schistosoma ma~lsoni. Direct method for simultaneous recording of electrical and motor activity. Exp. Parasitol. 43, 286-294 (1977) Fetterer, R. H., Pax, R. A., Bennett, J. L.: Clonazepam and praziquantel: Mode of antischistosomal action. Fed. Proc. 37, 604 (1978) G6nnert, R., Andrews, P.: Praziquantel, a new broad-spectrum antischistosomal agent. Z. Parasitenk. 52, 129-150 (1977) Prosser, C. L. : Comparative animal physiology. 3rd ed. Philadelphia: W. B. Saunders 1973 Reed, P. W., Lardy, H. A. : A23187: A divalent cation ionophore. J. Biol. Chem. 247, 6970-6977 (I972) Skou, J. C. : Active transport by cell membranes. Physiol. Rev. 45, 596-617 (1965) Stohler, H. R. : Ro 11-3128 - A novel schistosomacidal compound. Congress of Chemotherapy, September I 8 - 2 3 , 1977. Zurich, Switzerland, in press, (1978) Tomosky, T., Bennett, J. L., Bueding, E. B. : Characterization of the tryptaminergic amd dopaminergic receptors in S. mansoni. J. Pharmacol. Exp. Ther. 190, 260-271 (1974) Received April 3~Accepted July 5, 1978
Archivesof Pharmacology
Naanyn-Schmiedeberg's Arch. Pharmacol. 304, 309-315 (1978)
9 by Springer-Verlag 1978
A Benzodiazepine Derivative and Praziquantel: Effects on Musculature of Schistosoma mansoni and Schistosoma japonicum R. Pax, J. L. Bennett, a n d R. F e t t e r e r Departments of Pharmacology and Zoology, Michigan State University, East Lansing, Michigan 48824, USA
Summary. T h e b e n z o d i a z e p i n e derivative ( R o 11-3128) which has central n e r v o u s effects similar to o t h er benzodiazepines, a n d p r a z i q u a n t e l (PZ), are t wo new a n t i s c h i s t o s o m a l drugs. A t lo w c o n c e n t r a t i o n s these drugs will p r o d u c e a m a r k e d spastic paralysis o f male S c h i s t o s o m a m a n s o n i musculature. A n analysis o f the action o f these drugs o n the parasite's m u s c u l a t u r e shows t h a t R o 11-3128 a n d P Z p r o d u c e d a rapid rise in the tension o f the m u s c u l a t u r e o f male schistosomes. V a r i o u s c o m p o u n d s k n o w n to interact with the s c h i s t o s o m e ' s n e u r o r e c e p t i v e sites did n o t b lo ck or p o t e n t i a t e the action o f these drugs. R e m o v a l o f Ca 2 + or a d d i t i o n o f M g 2+ to the i n c u b a t i o n m e d i u m b l o c k e d the action o f these drugs on the s c h i s to s o m e ' s m u s c u l a ture. U p t a k e studies o f i n o r g a n i c cations by m a l e sc h i s t o s o m e' s indicate that R o 11-3128 a n d P Z decrease the influx o f K + but stimulate the influx o f C a 2 + a n d N a + into the male schistosome. It is suggested t h a t this interference with i n o r g a n i c i o n t r a n s p o r t m e c h a nisms causes the c o n t r a c t i o n o f the schistosome musculature.
Key words: B e n z o d i a z e p i n e - P r a z i q u a n t e l - Schistosomes - C a t i o n s p o r t o f cations.
M u s c u l a t u r e - M e m b r a n e trans-
Introduction Th e b e n z o d i a z e p i n e derivative, R o 11-3128, a n d praziq u a n t e l (PZ), are p o t e n t a n t i s c h i s t o s o m a l c o m p o u n d s ; a single oral dose given to infected mice is lethal to ad u l t a n d d e v e l o p i n g stages o f the parasite (Stohler, 1978; G 6 n n e r t an d A n d r e w s , 1977). These a u t h o r s n o t e d t h a t these two c o m p o u n d s p r o d u c e d a spastic paralysis o f the parasite's musculature. This o b s e r v a t i o n p r o m p t e d
Send offprint requests to J. L. Bennett at the above address
US to analyze the action o f these drugs on the s c h i s t o s o m e ' s m u s c u l a t u r e using a t e c h n i q u e cap abl e o f directly m e a s u r i n g the m e c h a n i c a l properties o f those muscles which are responsible for the parasite's m o t o r activity.
Materials and Methods Isolation of Parasites. Male Schistosomes (S. mansoni, St. Lucian strain and S. japonicum, Chinese strain) were isolated from infected mice as previously described (Bennett and Seed, i977) and then incubated at 37~C in Earles balanced salts (EBS) solution (Gibco, Long Island, New York) containing 10 % heat-inactivated human serum (Gibco), buffered at pH 7.4 with 0.02M Hepes (Sigma Chemical Co., St. Louis, Mo.). Description of Instrument Used to Analyze Effects of Drugs. The effects of the benzodiazepines and PZ on the musculature of single male schistosomes was determined by connecting the parasites to an apparatus (Fig. 1) capable of measuring the mechanical activity of its musculature. In brief, the parasite was suspended in the incubation medium by two suction pipettes. One pipette was very inflexible and attached to the tail while the other was very flexible and attached 1.0-2.0 mm from the inflexible pipette. Shortening or lengthening movements by the parasite caused movement of the flexible pipette which was converted to movement of a balanced wire system connected to a black acetate strip which acted as a shutter for a phototube in a myograph transducer. Since the amount of tension developed depends on the length of worm over which measurements are made, all tension measurements are given in terms of mg tension/ram of worm. With this system we can reliably measure shortening or lengthening movements as small as 30 ~tm and tension changes as small as 0.10 mg. The capabilities of this instrument are described in detail by Fetterer et al. (1977). Most experiments, using this apparatus, were performed at 37~ in a polyethylene dish containing 2.5 ml of Earles balanced salt (EBS) solution buffered at pH 7.4 with 0.02 M Hepes. Drugs. Praziquantel (MW = 312) and Ro 11-3128 [(+)-5-(o-chlorophenyI)-l,3-dihydro-3-methyI-7-nitro-2H-1,4-benzodiazepine-2one; MW = 322)] (Fig. 2) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 2 M and diluted to the desired concentration with distilled water. 2.5 gl of the drug was then added to the recording chamber fluid (2.5 mI) to give the final concentration. These drugs, when given orally to infected mice, were suspended in Emulphor EL-620 (Gaf Corp.).
0028-1298/78/0304/0309/$01.40
310
Naunyn-Schmiedeberg's Arch. Pharmacol. 304 (1978)
Pipette Shutter
\ I
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Ro 11-3128 Fig. 1. Schematic diagram of apparatus used to analyze the mechanical activity of male S. mansoni
," C--O
PRAZIQUANTE L
1 /'-c"3
^..l 2u N ~ :
N
RoU- 3128
Fig.2. Structure of praziquantel and Ro 11-3128
Other drugs used were: dopamine (3-hydroxytyramine HC1), 5HT (5-hydroxytryptamine creatinine sulfate), carbachol (carbamylcholine chloride), atropine, pentobarbital (all Sigma Chemical Co., St. Louis, Mo.), spiroperidol (Janssen Pharmaceuticals), BOL (bromlysergic acid diethylamide; Sandoz, Corp.), X-537A (Hoffmann-La Roche) and A-23187 (Eli Lilly Co.). Praziquantel was supplied by Dr. P. Andrews of the Bayer Company, while Ro 11-3128 was supplied by Dr. W. H. Scott and Dr. H. Stohler of Hoffmann-La Roche.
4SCa2+ Uptake Studies. To study the effects of Ro 11-3128 or PZ on uptake of 45Ca2 + (New England Nuclear, 30 Ci/g calcium) by male schistosomes, we preincubated 15 parasites in two rtCi o f 4 5 C a 2 + and EBS at 37~ C for 30 rain. (After 30 min the tissue to medium ratio of 45Ca 2 + remained constant.) Following this incubation, Ro 11-3128 or PZ was added and incubated for varying periods. After incubation with a drug the parasites were separated from the medium by filtration over a 25121153_glass fiber filter (Wanman GF/B) placed under vacuum on a millipore filtration apparatus (Millipore Company, Model XX10-024-00). The trapped parasites were then washed three times in 5ml of EBS containing 6raM CaCI 2. Three washes were found to be adequate since additional washes did not lower the amount of radioactivity bound to the parasite. The parasites were then weighed, placed in a vial containing 0.5 ml of tissue solubilizer (NCS, Amersham Corp.) and then incubated at 50~ for I h. The solubilized parasites were then placed in a scintillation vial containing 10ml of scintillation fluid consisting of one liter 95 ~ ethanol, 21 toluene, 0.3 g of POPOP, and 12 g of POP. The vials were then placed in a scintillation spectrometer, (Beckman Model LS-t00) where the counting efficiency was approximately 32 ~ for 45Ca2 +. *2K+ or 22Na+ Uptake Studies. Uptake of radioactive 42K+ (Michigan State University Nuclear Reactor Lab, 10 mCi/g potassium) or Z2Na+ (New England Nuclear, Carrier Free) by male schistosomes was performed as described above for 45Ca z + uptake,
Fig.3, Effects of 10-6M Ro 11-3128 on longitudinal contractile activity and tension of a male schistosome. Horizontal calibration = 20 s; vertical calibration = 0.5 mg of tension
except for the following. Our procedure for determining the effects of PZ or Ro 11-3128 and K + and Na § uptake consisted ofa 1 - 30 min incubation of the parasites in 1 pCi of the radionuclides in the presence or absence of PZ or Ro 11-3128. The filtered parasites incubated in 4ZK + were washed 3 times in EBS containing 10 mM K + while parasites incubated in 22Na+ were washed in EBS. After weighing the parasites we placed them in a small vial which in turn was placed into a gamma counter (Packard Inst., Model 578).
Results F i g u r e 3 d e m o n s t r a t e s t h e effects o f 1 0 - 6 M R o 113128 o n t h e m e c h a n i c a l a c t i v i t y o f a m a l e s c h i s t o s o m e . B e f o r e the d r u g was a d d e d the w o r m was c o n t r a c t i n g w h i l e t e n s i o n r e m a i n e d c o n s t a n t . W i t h i n 10 s after the addition of the drug, spontaneous contractions began to d i s a p p e a r a n d t e n s i o n b e g a n to rise. P Z , at 10 7 M , p r o d u c e d s i m i l a r effects o n the m a l e s c h i s t o s o m e . P Z o r R o l l - 3 1 2 8 - i n d u c e d effects w e r e r e v e r s i b l e since ten s i o n a n d c o n t r a c t i l e r a t e r e t u r n e d to n o r m a l 2 0 m i n a f t e r r e m o v a l o f d r u g f t o m the i n c u b a t i o n m e d i u m . Since the effect o f these d r u g s o n the t e n s i o n o f t h e p a r a s i t e ' s m u s c u l a t u r e c o u l d be easily q u a n t i f i e d , we e x a m i n e d t h e effects o f v a r i o u s c o n c e n t r a t i o n s o f P Z o r R o 11-3128 o n the t e n s i o n o f the m u s c u l a t u r e o f m a l e S. mansoni o r S. japonicum. F i g u r e 4 s h o w s t h a t P Z , at l o w c o n c e n t r a t i o n s , p r o d u c e d a m a r k e d effect o n the tens i o n o f the m u s c u l a t u r e o f the m a l e S. mansoni o r S. japonicum. T h i s was n o t the case w i t h R o 11-3128 (Fig. 4) since this c o m p o u n d was f o u n d to be e f f e c t i v e a g a i n s t m a l e S. mansoni b u t n o t a g a i n s t m a l e S. japonicum. T h i s o b s e r v a t i o n c o r r e l a t e s w i t h a r e p o r t b y S t o h l e r (1978), d e m o n s t r a t i n g t h a t R o 11-3128 c o u l d c u r e m i c e i n f e c t e d w i t h S. mansoni b u t n o t w i t h S.
japonicum. T h e d a t a r e p o r t e d b y G 6 n n e r t a n d A n d r e w s (1977) a n d b y S t o h l e r (1978) s u g g e s t t h a t it t a k e s a b o u t 4 - 5 t i m e s m o r e P Z t h a n R o 11-3128 to c u r e m i c e i n f e c t e d w i t h S. mansoni. In a n a t t e m p t to e x p l a i n w h y P Z was less e f f e c t i v e t h a n R o 11-3128 in c u r i n g i n f e c t e d mice, we p e r f o r m e d t h e f o l l o w i n g e x p e r i m e n t . We a d m i n i s t e r e d 80 m g / k g o f P Z o r R o 11-3128 to i n f e c t e d m i c e a n d
R. Pax et al. : Response of S. mansoni to Praziquantel and a Benzodiazepine Derivative
311
4.0
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o.o Fig. 4 Effects of various concentrations of Ro I 1-3128 or PZ on the tension produced by the musculature of S. japonicum (B - I ) or S. mansoni (0 -0)
{
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o Fig.5A and B Response of musculature of S. mansoni to 0.1 ml serum (A) or 25 gl or urine (B) removed from infected mice at various intervals after the administration of 80mg/kg Ro 11-3128 ( ) or PZ
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then at various intervals, after drug administration, we collected urine and isolated plasma from these mice. Samples of urine or plasma from mice treated with PZ, Ro 11-3128, or vehicle were placed in the presence of male schistosomes connected to our apparatus. These schistosomes were isolated from mice not treated with these drugs. Results (Fig.5) indicate that PZ or PZrelated material is rapidly removed from the blood by the kidney, but not Ro 11-3128 or Ro 11-3128 related material. In an attempt to determine the mode of the antischistosomal action of PZ and Ro tl-3128, we examined the interaction between these drugs and putative neurotransmitters of this parasite. For example, Tomosky et al. (1974) demonstrated that dopamine is one of these putative neurotransmitters which, when incubated in the presence of male schistosomes, will lengthen the parasite, i.e., a response opposite to that induced by PZ or Ro 11-3128. Thus, to determine whether dopamine would block PZ or Ro 11-3128induced rise in the tension of male S. mansoni we preincubated the worm in dopamine for 20 rain and then added PZ or Ro 11-3128. From Fig. 6 it can be seen that dopamine did not block the PZ-induced rise in
\',1i- -
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8 Time (h)
DA 10"4M
I
2Z-,
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i ......
1
4
8
2/-,
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Fig.6. Effect of dopamine (DA) on the PZ-induced rise in the tension of the musculature of male S. mansoni. Horizontal calibration = 1 rain; vertical calibration = 0.5 mg of tension
tension, nor did it block (not shown) the effect o f R o 113128. In addition to dopamine we attempted to block the effect of PZ or Ro 11-3128 with other compounds that are known to interact with other neuroreceptive sites in S. mansoni, i.e., 5-hydroxytryptamine, carbachol, spiroperidol, brom-lysergic acid diethylamide, and atropine (Barker et al., 1966; Tomosky et al., 1974). None of these compounds (at 10 -4 M) blocked the PZ or Ro ll-3128-induced increase in tension in
Naunyn-Schmiedeberg'sArch. Pharmacol. 304 (1978)
312 male S. mansoni. We also attempted to block, without success, the PZ-induced rise with 1 0 - 3 M pentobarbital, a compound that lengthens and paralyzes the schistosome. During the above studies we observed great variability in the time required to detect a particular drug's ability to induce changes in the schistosome's mechanical activity. To determine if there was a difference in the time required for a drug to induce a response in male schistosomes we determined the time required to see the half maximal effect (i. e., increased contraction rate) of 1 0 - 5 M 5-hydroxytryptamine and compared it to the time required to see the half maximal effect (i.e., increase in tension) of 1 0 - 6 M PZ or 1 0 - 5 M Ro 113128. The time required for half maximal effect of 5hydroxytryptamine was 37 _+ 5 s while for PZ or Ro 113128 it was 11.2 _+ 1.4 and 15.6 _+ 2.3s, respectively. These results combined with the inability of neurotransmitter agonist or antagonist t0 block the action of PZ or Ro 11-3128 on the musculataure of male S. mansoni suggested to us that these drugs may be acting directly on mechanisms (e.g., transport of inorganic ions) that regulate muscle contractility. To determine if inorganic ions play a vital role in mediating the action of PZ or Ro 11-3128 we performed some ion-substitution experiments. Mechanical activity of male S. rnansoni was measured as described in methods except that Hanks balanced salts solution (HBS), buffered at pH 7.4 with 0.01 M Tris, was used rather than EBS. The response of male schistosomes to PZ or Ro 11-3128 in HBS was qualitatively and quantitatively identical to that observed when the parasites were incubated in EBS. When schistosomes were preincubated for 15 rain in Ca 2 + free HBS we did not observe the PZ or Ro ll-3128-induced rise in the tension on the musculature of male schistosomes. When Ca 2+ was added back to the Ca 2 + free HBS, containing PZ, the schistosomes would contract. Incubation of the parasites in Ca 2 + free HBS produced a slight rise in tension of the musculature of the parasite and a reduction in the number of spontaneous contractions. Since high concentrations of Mg a+ have been shown to attenuate biological mechanisms which depend upon Ca 2§ (del Castillo and Katz, 1954), we studied the effect of various concentrations of Ca 2 § and Mg 2 + on PZ or Ro 11-3128 induced-contraction of the male schistosome musculature. When male schistosomes were preincubated in HBS containing 0.4 m M Ca 2 + and 30 m M Mg 2 +, we noted no change in tension and a slight decrease in spontaneous contractile activity; when PZ (Fig.7) or Ro 11-3128 (not shown) was added we observed a transient rise in tension. This same experiment was performed in 1.4 mM Ca 2 § and I m M Mg 2§ or 1.4raM Ca 2+ and 3 0 m M Mg 2§ In 1.4mM
"•1.6 O '_~ .8
.5
1.0
2,0
2,5
Time {minl
Fig.7. Effectof various concentrations o f C a 2 + and Mg2+ on the PZinduced rise in tension of male S. mansoni. The arrow points to the time at which 10-6M PZ was added to parasites preincubated in normal HBS (e e), in HBS containing 1.4mM Ca2+ and 30raM Mg2+ (D [21), or in HBS containing 0.4mM Ca2+ and 30 mM Mg2+ (9
Table 1. Percent increase in tension of the musculature of male S. mansoni when exposed to the ionophores X537A or A23187 Ionophore
Concentrationsof ionophore 10 4
X537A
252 _+25 ~
A23187
232+ 19~o
10-5 0.0 ~ 193_+17~
10-6 0.0 132_+14~
Ca 2+ and l m M Mg z+ PZ produced a sustained increase in the tension on the male schistosome while the PZ-induced increase in tension was transient when the worm was incubated in 1.4 mM Ca z + and 30 m M MgZ + To determine if the PZ-induced transient rise in tension could be performed twice on the same male schistosome we performed the following experiment. We induced this transient rise in tension with PZ on male schistosomes incubated in HBS containing 0 . 4 m M Ca 2 + and 30raM Mg 2 + and then incubated these parasites for 30 rain in the modified HBS containing no PZ; this was enough time to reverse the effects of PZ on the parasite's musculature. We then added PZ back to the incubation medium. Under these conditions, PZ did not induce a transient rise in tension when reapplied to the incubation medium. This same experiment was performed in 1.4 mM Ca 2 + and 1 mM Mg 2 + ; under these conditions PZ, upon reapplication, induced a marked increase in tension. Since extracellular Ca z + seemed to be involved in the action of PZ or Ro 11-3128 on S. mansoni we examined the response of male worms to ionophores, i.e., compounds that can transport cations, C a 2 + in particular, across membranes and increase cytoplasmic Ca z+ concentration (Reed and Lardy, 1972). When
R, Pax et al. : Response of S. mansoni to Praziquantei and a Benzodiazepine Derivative
3001
Table2. Translocation of Ca 2+ or Na + into bulk organic phase
Compound 500/aM
CaCI2
313
NaCI I
laM cation in organic phase 200-
Control Praziquantel X537A
4.0 + 0.3 6.4 + 0.6 92.0+3.0
3.2 _+ 0,1 3.0 + 0,1 198 _+12
Each compound was dissolved in 30 ~ Butanol-70 ~ toluene and 1 ml was added to test tubes containing 1/2ml of 40 mM Tris pH 7.5 and eitb,er 20mM ~5CaC12 (1 Ci/rM) or 20 mM ZZNaCI (:I Ci/mI). The tubes were shaken for 5 min, centrifuged at 2,500 rpm for 5 rain and then 0.5 m] of the organic phase was analyzed for radioactivity. Data represent mean _+ S.E.M. of 4 determinations
+; D~ 100
lb Incubation Time (min)
Fig.9. Uptake of 2ZNa+ by male schistosomes in the absence (O O) and presence of 10-6M PZ ( A - - Z x ) or Ro 11-3128 (11 --). Drugs and radionuclide were added to the incubation medium at the same time
170
3000
~
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~
140-
30
-
2000 130
~ '~
120"
~ 1000
110"
10
2'0
30
Incubation Time (rain)
to-
3o
Incubation Time [min }
Fig.8, Effect of 10-~M PZ (9 or ]0 SM Ro 1~-3128 (O O) on the uptake of 4SCaz+ by male schistosomes. Parasites were preincubated in ~SCaZ+ for 30 min then the drugs were added (see arrow on figure)
(e @) and presence of l0 6M PZ ( Z x - - • or Ro 11-3128 (l~), Drugs and radionuclide were added to the incubation medium at the same time
male schistosomes were incubated in the presence of various concentrations ofX537 or A23187 we observed an increase in the tension of the parasite's musculature (Table 1). At low concentrations, A23187 induced a greater response than X537A. These results prompted us to determine if PZ and Ro 11-3128 could, Iike ionophores, transfer cations from an aqueous medium to an organic phase. Compared to X537A, Ro 11-3128 and PZ did not transfer N a + or Ca 2 + from an aqueous medium into an organic phase (Table 2). Since Ca z + appears to play an important role in mediating the effects of PZ or Ro 11-3128 we attempted to determine if alterations in the concentration of other inorganic ions would affect the action of these drugs on the musculature of S. mansoni. We observed that decreasing the concentration of K +, N a + or C I - in
HBS had little if any effect upon the Ro 11-3128 or PZinduced rise in tension of the schistosome musculature. To more accurately assess the role that inorganic ions may play in the action of PZ or Ro 11-3128 on the schistosome's musculature we studied the effects of these drugs on the influx of 4~Ca2 +, 22Na +, and r + into the parasite. To study the effects of PZ or Ro 113128 on the transport of Ca z + by male schistosomes, we preincubated the parasites in r + for 30 min and then added PZ or Ro 11-3128 to the incubation medium. The addition of these drugs resulted in a rapid accumulation of 45Ca2+ by the male schistosome (Fig. 8). In addition to Ca 2 +, we analyzed the effects of PZ or Ro 11-3128 on the uptake of K + and Na + by male schistosome. F r o m Fig.9 and 10 it can be observed that PZ or Ro 11-3128 stimulated the uptake
Fig.tO. Uptake of 4ZK+ by male schistosomes m the absence
314 o f 2 2 N a + but inhibited the uptake of 42K+ by the
parasite. Discussion
Our results confirm previous observations (Stohler, 1978; G6nnert and Andrews, 1977) which demonstrated that incubation of S. mansoni with PZ or Ro 113128 produces a spastic paralysis of the parasite's musculature. This drug-induced spastic paralysis appeared to be independent of any action the drug may have on the parasites' neurotransmitter receptors, since we observed that the PZ or Ro 11-3128-induced response could not be blocked by compounds known to interact with these receptors. Since the drug did not appear to interact with schistosome neuroreceptive sites, we determined if the action of these drugs was dependent upon the presence of inorganic ions. Since pre-incubation of the worm in Ca 2 + free HBS blocked the action of these drugs on the schistosome, it would appear that PZ and Ro 11-3128 were directly or indirectly activating a Ca 2 § dependent contraction of the parasite's musculature. A similar conclusion with respect to PZ was made by investigators at Imperial Chemical Industries (G. C. Coles, unpublished observations). This conclusion was supported by the rapid increase in the influx of45Ca2 § into the worm after exposure to PZ or Ro 11-3128 and the response of the parasites to the Ca 2 § inophore A23187. From these results it would appear that extracellular Ca 2§ may play an essential role in the contractile activity of the schistosome musculature. Thus, like vertebrate smooth muscle, but unlike vertebrate skeletal muscle (Carafoli et al., 1975), the schistosome muscle may not be able to store small amounts of Ca 2 +. For example, when we pre-incubated schistosomes in a medium containing a low concentration of Ca 2 § and a high concentration of Mg 2+, we observed only a transient rise in the tension of the parasite's musculature when PZ was added to the medium. When PZ was removed from the medium and then reapplied 30 min later (the time required for the tension of the parasite's musculature to return to normal) the schistosome did not respond. When this experiment was repeated in a medium containing physiological levels of Ca 2 + and Mg 2§ we observed that reapplication of PZ would induce a rise in the tension of the musculature of the schistosome. F r o m these experiments we conclude that the transient PZ-induced rise in the tension of the parasite's musculature, incubated in low Ca 2 + and high Mg 2§ is due to the release of a small amount of sequestered Ca 2§ that is not displaced by the high Mg 2§ Once this stored Ca 2 § is released, due to an activation of the parasite's contractile elements by PZ, it is not replaced because Mg 2§ blocks or competes
Naunyn-Schmiedeberg's Arch. Pharmacol. 304 (1978)
with Ca 2 + for this Ca 2 + storage site. Further studies using 45Ca2 § are presently being carried out to verify the presence of this Ca 2§ storage site within male S. mansoni.
As with most contractile processes in animals it is not suprising to observe that the action of PZ and Ro 11-3128 on the musculature of S. mansoni can be blocked or attenuated by altering the concentration of inorganic ions within the medium, especially Ca 2§ since it is intimately associated with the contraction of muscle in various animals (Prosser, 1973). Thus, although Ca 2 § is necessary for the action of these drugs the important question, as to how PZ and Ro 11-3128 bring about this rapid rise in the tension of the musculature of S. mansoni, still needs to be answered. A possible mechanism that could explain the action of PZ or Ro 11-3128 on the musculature of S. mansoni could be due to a disruption of the resting membrane potential of the parasite's muscle cells. For example, these drugs could perturb the membrane of these cells and disrupt mechanisms which regulate ion fluxes, e. g., inhibit enzymes involved in maintaining inorganic ion gradients and/or fluidize the membrane such that it becomes more permeable to inorganic ions. If these drugs were in fact depolarizing the schistosome's muscle cells then one would predict that K + ions should flow out of these cells and Na + ions should flow in; this assumes that the resting membrane potential of the schistosome's muscle cells is maintained in a manner similar to other animals. Our results would suggest that this may be the mechanism since PZ and Ro 11-3128 stimulated the uptake of 22Na+ but inhibited the uptake of 4 2 K + . Recent electrophysiological studies performed in our lab (Fetterer et al., 1978) strongly suggest that these drugs are depolarizing the cells of this parasite. With respect to the possibility that these drugs are inhibiting enzymes that regulate parasite ion fluxes, it is known that ATPases are located in the invaginations along the epidermis of male S. mansoni (Ernst, 1977). Unfortunately, very little is known about the physiological function of these enzymes although in other animals they are often associated with the translocation of ions across membranes (Skou, 1965). Since more needs to be known about these enzymes it would be interesting to determine if these drugs can inhibit the parasite's membrane bound Na +, K +ATPase. Another possibility is that PZ and Ro 11-3128 may be chelating C a 2 + or Na + ions and then transporting these ions into the cells of S. mansoni. Since we observed that these drugs could not combine with these ions and carry them into a non-polar organic phase (a property common to ionophores) we dismissed the idea that they were acting like ionophores. Acknowledgements. This research has been supported by the Edna McConnell Clark Foundation, World Health Organization, and
R. Pax et al. : Response of S. mansoni to Praziquantel and a Benzodiazepine Derivative Hoffmann-La Roche Company. Materials used in this study were provided by the U.S.-Japan Cooperative Medical Science ProgramNIAID.
References Barker, L. R., Bueding, E. B., Timms, A. R.: The possible role of acetylcholine in Schistosoma mansoni. Br. J. Pharmacol. Chemother. 26, 656-665 (1966) Bennett, J. L., Seed, J. L.: Characterization and isolation of concanavalin. A binding sites from the epidermis of S. mansoni. J. Parasitol. 63, 250-258 (1977) Carafoli, E., Clementi, F., Drabikowski, W., Margreth, A. : Calcium transport in contraction and secretion. Amsterdam: NorthHolland Publ. Co. 1975 del Castillo, J., Katz, B.: Quantal components of the end plate potential. J. Physiol. (Lond.) 124, 560-573 (1954) Ernst, S. C.: Biochemical and cytochemical studies of alkaline phosphatase activity in Schistosoma mansoni. Rice Univ. Studies 62, 8 1 - 9 5 (J977)
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Fetterer, R. H., Pax, R. A., Bennett, J. L.: Schistosoma ma~lsoni. Direct method for simultaneous recording of electrical and motor activity. Exp. Parasitol. 43, 286-294 (1977) Fetterer, R. H., Pax, R. A., Bennett, J. L.: Clonazepam and praziquantel: Mode of antischistosomal action. Fed. Proc. 37, 604 (1978) G6nnert, R., Andrews, P.: Praziquantel, a new broad-spectrum antischistosomal agent. Z. Parasitenk. 52, 129-150 (1977) Prosser, C. L. : Comparative animal physiology. 3rd ed. Philadelphia: W. B. Saunders 1973 Reed, P. W., Lardy, H. A. : A23187: A divalent cation ionophore. J. Biol. Chem. 247, 6970-6977 (I972) Skou, J. C. : Active transport by cell membranes. Physiol. Rev. 45, 596-617 (1965) Stohler, H. R. : Ro 11-3128 - A novel schistosomacidal compound. Congress of Chemotherapy, September I 8 - 2 3 , 1977. Zurich, Switzerland, in press, (1978) Tomosky, T., Bennett, J. L., Bueding, E. B. : Characterization of the tryptaminergic amd dopaminergic receptors in S. mansoni. J. Pharmacol. Exp. Ther. 190, 260-271 (1974) Received April 3~Accepted July 5, 1978
