59
Praziquantel: physiological evidence for its site(s) of action in magnesium-paralysed Schistosoma mansoni K. L. BLAIR1, J. L. BENNETT 2 and R. A. PAX1* 1
Department of Zoology and Neuroscience Program, 2 Department of Pharmacology and Toxicology, Michigan University, East Lansing, MI 48824-1115, USA
State
(Received 10 May 1991; revised 19 July 1991; accepted 20 July 1991) SUMMARY
The mechanism whereby praziquantel produces a contraction and subsequent flaccid paralysis (a loss of sensitivity to subsequent stimuli) of Schistosoma mansoni in a medium containing an elevated Mg2+:Ca2+ ratio was investigated. In RPMI, praziquantel produced a concentration-dependent tonic contraction of the parasite with an EC50 of 200 nM. Magnesium inhibited the contraction in such a manner as to convert the tonic contraction to a phasic one without altering the peak force generated. The Mg2+-dependent block was non-competitive with praziquantel but was competitive with extracellular Ca2+, ratios of 7-5:1; Mg2+: Ca2+ being needed to inhibit the tonic contraction and to induce flaccid paralysis. Flaccid paralysis was associated with a reduced ability of the parasite to take up 45Ca2+ from the bath compared to parasites that had not entered into flaccid paralysis and flaccid paralysis was reversible. Recovery from flaccid paralysis was accelerated by treatments that are expected to increase Ca2+ uptake by the parasite. At a concentration of 500 nM, praziquantel produced 2 distinct phasic contractions in intact parasites incubated in an elevated [Mg2+] medium but only 1 phasic contraction in parasites lacking their surface tegumental membranes. In zero Ca2+ I-RPMI, 10 /im praziquantel produced a phasic contraction of intact parasites but did not stimulate contraction of detegumented parasites until Ca2+ was reintroduced into the bath. These results indicate that praziquantel interacts with specific Ca2+-permeable sites in the tegumental and sarcoplasmic membranes of the parasite and that under these conditions of elevated Mg2+:Ca2+ ratios, these sites become blocked by Mg2+, leading to flaccid paralysis of the parasite. Key words: Schistosoma mansoni, magnesium, praziquantel, muscle, calcium channel, flatworm.
INTRODUCTION
Praziquantel is a selective anthelmintic with trematocidal and cestocidal activity but no apparent activity against higher organisms such as nematodes (Thomas & Andrews, 1977; Thomas & Gonnert, 1977; Campbell, 1986). Currently, praziquantel is the drug of choice for treating schistosomiasis in humans (Campbell, 1986). Schistosome physiology is affected in many ways by praziquantel (Andrews, 1985; Harnett, 1988). Major among the in vitro effects is contraction of the musculature (Pax, Bennett & Fetterer, 1978) and there is also blebbing and disruption of the parasite's tegument (Bricker et al. 1983 ; Xiao et al. 1984). Both effects occur at concentrations of praziquantel reached during therapeutic treatment (Andrews, 1985). The in vivo and in vitro effects of praziquantel show the same stereo-specificity (Andrews, 1985; Xiao & Catto, 1989), thus, each of these effects may be initiated by praziquantel binding to a specific receptor site within the parasite (Laduron, 1988). Further evidence that the in vitro responses (muscle contraction and tegumental blebbing) rely on com* Reprint requests to: Dr Ralph A. Pax, Department of Zoology, Michigan State University, East Lansing, MI 48824-1115, USA. Parasitology (1992), 104, 59-66 Printed in Great Britain
mon or similar mechanisms of action are (1) each is dependent on the presence of Ca2+ in the bath (Wolde Mussie et al. 1982; Xiao et al. 1984) and (2) each is prevented when the parasites are incubated in a medium containing 30 mM Mg2+ and 0-4 HIM Ca2+ (Bricker et al. 1983; Pax et al. 1978). In this 30 mM Mg2+ medium, the parasites do contract in response to praziquantel but the contraction is not tonic, rather it is phasic and once the parasites relax they enter a state of flaccid paralysis in which they are not responsive to other stimuli that are expected to cause contraction, such as depolarization with 60 mM K+ (Fetterer et al. 1980) or exposure to 10 fiM 3-methyl clonazepam (Pax et al. 1978, 1983). Schistosomes bathed in the 30 mM Mg2+ medium and induced to contract with 60 mM K+ (Fetterer et al. 1980) or 10 fiM 3-methyl clonazepam (Pax et al. 1978, 1983) do not enter the state of flaccid paralysis. These results demonstrate that in a medium with a higher Mg2+: Ca2+ ratio, praziquantel alters the Ca2+ flux mechanisms that are involved in muscle contraction and tegumental blebbing of the schistosome in some unique manner, a manner that might give insight into the site and mechanism of action of praziquantel. In the following muscle physiology studies, we have characterized the effects of altered Mg 2+ : Ca2+
K. L. Blair, J. L. Bennett and R. A. Pax ratios of incubation media on praziquantelstimulated muscle contraction. We provide evidence indicating that praziquantel is acting at the level of the tegument and at the saroplasmic membrane. The nature of these interactions suggests that praziquantel, in these altered media, causes a reduction in the Ca2+ permeability of the parasite which leads to the state of flaccid paralysis.
60
flexible pipette couples movements of the portion of the worm between the pipette to a modified E & M type A myograph transducer. The tethered worms were allowed to acclimate to the system for 10—15 min before the initiation of experiments. For the 45Ca2+ uptake studies, 5 parasites each were incubated in 12x75 mm glass culture tubes containing 1 ml of medium at 37 °C. The parasites were maintained in RPMI until the beginning of the experiment. First, the RPMI was replaced with 1 ml of I-RPMI in which the concentration of Mg2+ was MATERIALS AND METHODS increased to 30 mM (30 mM Mg2+ I-RPMI). After 15 min in the 30 mM Mg2+ I-RPMI, praziquantel Adult male S. mansoni (St Lucian strain), 40 days or (10IIM) or dimethylsulphoxide (Me2SO) (001%) older were isolated from mice and used throughout. was added to the culture tube and the parasites were After removal from the host, the parasites were incubated for another 5 min. After this 5 min maintained at 37 °C in RPMI-1640 (Gibco, Grand incubation, the medium was replaced with 1-0 ml of Island, NY) plus 20 mM 4-iV(2-hydroxyethyl)-lI-RPMI to which was added 2/tCi/ml 45Ca2+ piperazine-ethane sulphonic acid (HEPES) buffer (pH 7-4) and 100 units/ml each of penicillin and (36-7 Ci/g, New England Nuclear). The parasites were incubated in the labelled medium for 15 sstreptomycin. This medium will be referred to 10 min and then the uptake was stopped by the hereafter as RPMI. addition of 3 ml of I-RPMI containing 10 mM Cd2+ Detegumented parasites were prepared according and then the parasites were trapped on a nylon to the method of Oaks et al. (1981) with minor screen (60 jjxn mesh) under vacuum. The parasites modifications as follows. Parasites (10-20) were were washed twice more with 3 ml of the 10 mM Cd2+ incubated in 3 ml of ice-cold 02 % Triton X-100 in I-RPMI and weighed. The dpm/mg parasite wet RPMI for 5 min and then vortexed at low speed weight was determined by standard liquid scintil(Vortex Genie) for 30 s. The detergent solution was lation spectroscopy. removed and the parasites washed 8-10 times with All data were analysed by one-way analysis of 3—4 ml of RPMI plus 0-1 % bovine serum albumin variance. Comparisons for significance against a (BSA) at 37 °C. The parasites were allowed to control were made using Dunnett's procedure. recover for at least 1 h in the 01 % BSA-RPMI Multiple comparisons were made with the Student before testing. Detegumentation was confirmed by Newman Keul procedure. Kinetic determinations the inability of the treated parasites to exclude 0-1 % were made using the computer program Dose Effect trypan blue and their lack of a tegumental membrane Analysis with Micro-Computers (Elsevier Publishpotential and an intact sarcolemmal potential of — 25 ing). Numerical data in the text are presented as to - 3 0 mV (Depenbusch et al. 1983). means+ S.D., n > 6 while graphical data are When the ionic composition of the medium was means+ S.E. mean, n > 6. Presented physiograph altered, the inorganic, ionic equivalent of RPMI chart recordings are representative of the responses (mM): NaCl, 102; KC1, 5-6; Ca(NO3)2, 0 4 ; MgSO4> observed. Each time-trial in the 45Ca2+ uptake studies 0-4; Na 2 HPO 4 , 5-4; HEPES (pH 74), 20) plus was performed in triplicate and replicated at least glucose (11 1 mM) and glutamine (5 mM) was used 3 times. and is referred to as I-RPMI (Lane, Pax & Bennett, Praziquantel, ionophore A-23187 (free acid), and 1987). For the studies involving increased Mg2+, 3-methyl clonazepam were prepared as 10~2 M stock Ca2+ or K+, Na+ was reduced isotonically. For the solutions in Me2SO and diluted in Me2SO or distilled altered Ca2+ studies and the Cd2+ I-RPMI wash, the water to the appropriate concentration for testing. polyvalent anions were replaced with Cl~ to prevent The final concentration of Me2SO never exceeded precipitation. The zero Ca2+ I-RPMI was prepared 0-1 %. Praziquantel was a gift from Drs P. Andrews by omitting Ca(NO 3 ) 2 and adding 5 mM ethyleneand H. Thomas of Bayer Co. The 3-methyl clonaglycol-6w-(/?-amino-ethyl ether) iV,iV-tetraacetic acid (EGTA). Preliminary observations and pre- zepam was donated by Dr W. H. Scott and Dr H. Stohler of Hoffman-La Roche. All other drugs were vious studies (Fetterer et al. 1978) demonstrate the purchased from Sigma Chemical Co., St Louis, MO, schistosome's muscle activity and membrane poUSA. tential to be insensitive to these changes in Na+ and Cl~ concentration. To measure muscle activity, parasites were attached to a transducer system as described by Fetterer, Pax & Bennett (1977). Briefly, the posterior 1*5—2#5 mm of the parasite was attached between two suction pipettes; one inflexible and oneflexible.The
Action of praziquantel on Schistosoma mansoni A
B 30 mM Mg 2 + I-RPMI
RPMI
10/iM
Fig. 1. Chart recordings showing concentration dependency of praziquantel-induced (|) contractions of Schistosoma mansoni in RPMI (A) and how the contractions are modified when the parasites are exposed to 30 mM Mg2+ I-RPMI (B) for 15 min prior to the addition of praziquantel. Calibration: vertical, 2 mg; horizontal, 1 min.
RESULTS
Responses of intact parasites to praziquantel in RPMI or in altered magnesium media When attached to the transducer system, parasites bathed in RPMI exhibited rhythmic contractile activity superimposed upon some level of muscle tone. Praziquantel caused a concentration-dependent increase in the muscle tone with an EC 50 of 200 nM (Fig. 1 A). This effect was observed within 15—20 sec of adding praziquantel to the bath. The spontaneous contractile activity of the muscle was not affected at concentrations of praziquantel from 30 to 180 nM but at concentrations of 300 nM or greater, spontaneous muscle activity decreased as muscle tone increased. Maximal tone (7-1 + 1-6 mg) was obtained with 1 fiM praziquantel and all spontaneous contractions ceased. Once a maximal tonic contraction was obtained, it was maintained for at least 15 min, making the peak and tonic contractions equal in magnitude. When parasites were exposed to 30 mM Mg 2+ IRPMI, i.e. a medium in which the [Mg 2+ ] was elevated from 0 4 to 30 mM, underlying muscle tone decreased by 4-2 + 0-7 mg within 5-10 min. If praziquantel was then added to the bathing medium, the peak change in tone in response to praziquantel was the same as in animals in RPMI (Fig. IB) but the responses were now phasic. This was the case at all praziquantel concentrations tested. At concentrations of praziquantel from 180 nM to 1 fiM, 2 distinct phasic contractions were elicited (Fig. 1B, only 300 nM and 1 fiM shown). The first contraction
61
occurred 15-20 sec after the addition of praziquantel, just as was observed for parasites incubated in RPMI. Before the first contraction relaxed fully, the second contraction began. At 1 fiM praziquantel, the initiation of this second contraction was sufficiently rapid to be almost indistinguishable from the first. At 10 fiM, the two contractions could no longer be distinguished from each other. The time to | relaxation (£i) of this phasic contraction was also dependent on the concentration of praziquantel. That is, the parasites relaxed more quickly as the concentration of praziquantel was increased, dropping from greater than 2 min with 300 nM praziquantel to about 40 sec with 10 /IM praziquantel. As described in the Introduction section, once parasites are treated with praziquantel in the presence of high magnesium, they enter a state of flaccid paralysis in which they are not responsive to agents normally expected to cause contractions of the musculature. When the parasites were incubated in 30 mM M g " I-RPMI for 15 min and then contracted in response to the addition of praziquantel to the bath, the time to the onset of this flaccid paralysis relative to the initial exposure to praziquantel was also dependent on the concentration of praziquantel used. Parasites treated with less than 180 nM praziquantel did not enter this state within 15-30 min of observation. Parasites treated with 300 nM praziquantel entered flaccid paralysis in roughly 15 min, while parasites exposed to 10 fiM praziquantel became unresponsive within 5 min. Once in the flaccid paralysis state, parasites were refractory to: praziquantel (10/
Praziquantel: physiological evidence for its site(s) of action in magnesium-paralysed Schistosoma mansoni K. L. BLAIR1, J. L. BENNETT 2 and R. A. PAX1* 1
Department of Zoology and Neuroscience Program, 2 Department of Pharmacology and Toxicology, Michigan University, East Lansing, MI 48824-1115, USA
State
(Received 10 May 1991; revised 19 July 1991; accepted 20 July 1991) SUMMARY
The mechanism whereby praziquantel produces a contraction and subsequent flaccid paralysis (a loss of sensitivity to subsequent stimuli) of Schistosoma mansoni in a medium containing an elevated Mg2+:Ca2+ ratio was investigated. In RPMI, praziquantel produced a concentration-dependent tonic contraction of the parasite with an EC50 of 200 nM. Magnesium inhibited the contraction in such a manner as to convert the tonic contraction to a phasic one without altering the peak force generated. The Mg2+-dependent block was non-competitive with praziquantel but was competitive with extracellular Ca2+, ratios of 7-5:1; Mg2+: Ca2+ being needed to inhibit the tonic contraction and to induce flaccid paralysis. Flaccid paralysis was associated with a reduced ability of the parasite to take up 45Ca2+ from the bath compared to parasites that had not entered into flaccid paralysis and flaccid paralysis was reversible. Recovery from flaccid paralysis was accelerated by treatments that are expected to increase Ca2+ uptake by the parasite. At a concentration of 500 nM, praziquantel produced 2 distinct phasic contractions in intact parasites incubated in an elevated [Mg2+] medium but only 1 phasic contraction in parasites lacking their surface tegumental membranes. In zero Ca2+ I-RPMI, 10 /im praziquantel produced a phasic contraction of intact parasites but did not stimulate contraction of detegumented parasites until Ca2+ was reintroduced into the bath. These results indicate that praziquantel interacts with specific Ca2+-permeable sites in the tegumental and sarcoplasmic membranes of the parasite and that under these conditions of elevated Mg2+:Ca2+ ratios, these sites become blocked by Mg2+, leading to flaccid paralysis of the parasite. Key words: Schistosoma mansoni, magnesium, praziquantel, muscle, calcium channel, flatworm.
INTRODUCTION
Praziquantel is a selective anthelmintic with trematocidal and cestocidal activity but no apparent activity against higher organisms such as nematodes (Thomas & Andrews, 1977; Thomas & Gonnert, 1977; Campbell, 1986). Currently, praziquantel is the drug of choice for treating schistosomiasis in humans (Campbell, 1986). Schistosome physiology is affected in many ways by praziquantel (Andrews, 1985; Harnett, 1988). Major among the in vitro effects is contraction of the musculature (Pax, Bennett & Fetterer, 1978) and there is also blebbing and disruption of the parasite's tegument (Bricker et al. 1983 ; Xiao et al. 1984). Both effects occur at concentrations of praziquantel reached during therapeutic treatment (Andrews, 1985). The in vivo and in vitro effects of praziquantel show the same stereo-specificity (Andrews, 1985; Xiao & Catto, 1989), thus, each of these effects may be initiated by praziquantel binding to a specific receptor site within the parasite (Laduron, 1988). Further evidence that the in vitro responses (muscle contraction and tegumental blebbing) rely on com* Reprint requests to: Dr Ralph A. Pax, Department of Zoology, Michigan State University, East Lansing, MI 48824-1115, USA. Parasitology (1992), 104, 59-66 Printed in Great Britain
mon or similar mechanisms of action are (1) each is dependent on the presence of Ca2+ in the bath (Wolde Mussie et al. 1982; Xiao et al. 1984) and (2) each is prevented when the parasites are incubated in a medium containing 30 mM Mg2+ and 0-4 HIM Ca2+ (Bricker et al. 1983; Pax et al. 1978). In this 30 mM Mg2+ medium, the parasites do contract in response to praziquantel but the contraction is not tonic, rather it is phasic and once the parasites relax they enter a state of flaccid paralysis in which they are not responsive to other stimuli that are expected to cause contraction, such as depolarization with 60 mM K+ (Fetterer et al. 1980) or exposure to 10 fiM 3-methyl clonazepam (Pax et al. 1978, 1983). Schistosomes bathed in the 30 mM Mg2+ medium and induced to contract with 60 mM K+ (Fetterer et al. 1980) or 10 fiM 3-methyl clonazepam (Pax et al. 1978, 1983) do not enter the state of flaccid paralysis. These results demonstrate that in a medium with a higher Mg2+: Ca2+ ratio, praziquantel alters the Ca2+ flux mechanisms that are involved in muscle contraction and tegumental blebbing of the schistosome in some unique manner, a manner that might give insight into the site and mechanism of action of praziquantel. In the following muscle physiology studies, we have characterized the effects of altered Mg 2+ : Ca2+
K. L. Blair, J. L. Bennett and R. A. Pax ratios of incubation media on praziquantelstimulated muscle contraction. We provide evidence indicating that praziquantel is acting at the level of the tegument and at the saroplasmic membrane. The nature of these interactions suggests that praziquantel, in these altered media, causes a reduction in the Ca2+ permeability of the parasite which leads to the state of flaccid paralysis.
60
flexible pipette couples movements of the portion of the worm between the pipette to a modified E & M type A myograph transducer. The tethered worms were allowed to acclimate to the system for 10—15 min before the initiation of experiments. For the 45Ca2+ uptake studies, 5 parasites each were incubated in 12x75 mm glass culture tubes containing 1 ml of medium at 37 °C. The parasites were maintained in RPMI until the beginning of the experiment. First, the RPMI was replaced with 1 ml of I-RPMI in which the concentration of Mg2+ was MATERIALS AND METHODS increased to 30 mM (30 mM Mg2+ I-RPMI). After 15 min in the 30 mM Mg2+ I-RPMI, praziquantel Adult male S. mansoni (St Lucian strain), 40 days or (10IIM) or dimethylsulphoxide (Me2SO) (001%) older were isolated from mice and used throughout. was added to the culture tube and the parasites were After removal from the host, the parasites were incubated for another 5 min. After this 5 min maintained at 37 °C in RPMI-1640 (Gibco, Grand incubation, the medium was replaced with 1-0 ml of Island, NY) plus 20 mM 4-iV(2-hydroxyethyl)-lI-RPMI to which was added 2/tCi/ml 45Ca2+ piperazine-ethane sulphonic acid (HEPES) buffer (pH 7-4) and 100 units/ml each of penicillin and (36-7 Ci/g, New England Nuclear). The parasites were incubated in the labelled medium for 15 sstreptomycin. This medium will be referred to 10 min and then the uptake was stopped by the hereafter as RPMI. addition of 3 ml of I-RPMI containing 10 mM Cd2+ Detegumented parasites were prepared according and then the parasites were trapped on a nylon to the method of Oaks et al. (1981) with minor screen (60 jjxn mesh) under vacuum. The parasites modifications as follows. Parasites (10-20) were were washed twice more with 3 ml of the 10 mM Cd2+ incubated in 3 ml of ice-cold 02 % Triton X-100 in I-RPMI and weighed. The dpm/mg parasite wet RPMI for 5 min and then vortexed at low speed weight was determined by standard liquid scintil(Vortex Genie) for 30 s. The detergent solution was lation spectroscopy. removed and the parasites washed 8-10 times with All data were analysed by one-way analysis of 3—4 ml of RPMI plus 0-1 % bovine serum albumin variance. Comparisons for significance against a (BSA) at 37 °C. The parasites were allowed to control were made using Dunnett's procedure. recover for at least 1 h in the 01 % BSA-RPMI Multiple comparisons were made with the Student before testing. Detegumentation was confirmed by Newman Keul procedure. Kinetic determinations the inability of the treated parasites to exclude 0-1 % were made using the computer program Dose Effect trypan blue and their lack of a tegumental membrane Analysis with Micro-Computers (Elsevier Publishpotential and an intact sarcolemmal potential of — 25 ing). Numerical data in the text are presented as to - 3 0 mV (Depenbusch et al. 1983). means+ S.D., n > 6 while graphical data are When the ionic composition of the medium was means+ S.E. mean, n > 6. Presented physiograph altered, the inorganic, ionic equivalent of RPMI chart recordings are representative of the responses (mM): NaCl, 102; KC1, 5-6; Ca(NO3)2, 0 4 ; MgSO4> observed. Each time-trial in the 45Ca2+ uptake studies 0-4; Na 2 HPO 4 , 5-4; HEPES (pH 74), 20) plus was performed in triplicate and replicated at least glucose (11 1 mM) and glutamine (5 mM) was used 3 times. and is referred to as I-RPMI (Lane, Pax & Bennett, Praziquantel, ionophore A-23187 (free acid), and 1987). For the studies involving increased Mg2+, 3-methyl clonazepam were prepared as 10~2 M stock Ca2+ or K+, Na+ was reduced isotonically. For the solutions in Me2SO and diluted in Me2SO or distilled altered Ca2+ studies and the Cd2+ I-RPMI wash, the water to the appropriate concentration for testing. polyvalent anions were replaced with Cl~ to prevent The final concentration of Me2SO never exceeded precipitation. The zero Ca2+ I-RPMI was prepared 0-1 %. Praziquantel was a gift from Drs P. Andrews by omitting Ca(NO 3 ) 2 and adding 5 mM ethyleneand H. Thomas of Bayer Co. The 3-methyl clonaglycol-6w-(/?-amino-ethyl ether) iV,iV-tetraacetic acid (EGTA). Preliminary observations and pre- zepam was donated by Dr W. H. Scott and Dr H. Stohler of Hoffman-La Roche. All other drugs were vious studies (Fetterer et al. 1978) demonstrate the purchased from Sigma Chemical Co., St Louis, MO, schistosome's muscle activity and membrane poUSA. tential to be insensitive to these changes in Na+ and Cl~ concentration. To measure muscle activity, parasites were attached to a transducer system as described by Fetterer, Pax & Bennett (1977). Briefly, the posterior 1*5—2#5 mm of the parasite was attached between two suction pipettes; one inflexible and oneflexible.The
Action of praziquantel on Schistosoma mansoni A
B 30 mM Mg 2 + I-RPMI
RPMI
10/iM
Fig. 1. Chart recordings showing concentration dependency of praziquantel-induced (|) contractions of Schistosoma mansoni in RPMI (A) and how the contractions are modified when the parasites are exposed to 30 mM Mg2+ I-RPMI (B) for 15 min prior to the addition of praziquantel. Calibration: vertical, 2 mg; horizontal, 1 min.
RESULTS
Responses of intact parasites to praziquantel in RPMI or in altered magnesium media When attached to the transducer system, parasites bathed in RPMI exhibited rhythmic contractile activity superimposed upon some level of muscle tone. Praziquantel caused a concentration-dependent increase in the muscle tone with an EC 50 of 200 nM (Fig. 1 A). This effect was observed within 15—20 sec of adding praziquantel to the bath. The spontaneous contractile activity of the muscle was not affected at concentrations of praziquantel from 30 to 180 nM but at concentrations of 300 nM or greater, spontaneous muscle activity decreased as muscle tone increased. Maximal tone (7-1 + 1-6 mg) was obtained with 1 fiM praziquantel and all spontaneous contractions ceased. Once a maximal tonic contraction was obtained, it was maintained for at least 15 min, making the peak and tonic contractions equal in magnitude. When parasites were exposed to 30 mM Mg 2+ IRPMI, i.e. a medium in which the [Mg 2+ ] was elevated from 0 4 to 30 mM, underlying muscle tone decreased by 4-2 + 0-7 mg within 5-10 min. If praziquantel was then added to the bathing medium, the peak change in tone in response to praziquantel was the same as in animals in RPMI (Fig. IB) but the responses were now phasic. This was the case at all praziquantel concentrations tested. At concentrations of praziquantel from 180 nM to 1 fiM, 2 distinct phasic contractions were elicited (Fig. 1B, only 300 nM and 1 fiM shown). The first contraction
61
occurred 15-20 sec after the addition of praziquantel, just as was observed for parasites incubated in RPMI. Before the first contraction relaxed fully, the second contraction began. At 1 fiM praziquantel, the initiation of this second contraction was sufficiently rapid to be almost indistinguishable from the first. At 10 fiM, the two contractions could no longer be distinguished from each other. The time to | relaxation (£i) of this phasic contraction was also dependent on the concentration of praziquantel. That is, the parasites relaxed more quickly as the concentration of praziquantel was increased, dropping from greater than 2 min with 300 nM praziquantel to about 40 sec with 10 /IM praziquantel. As described in the Introduction section, once parasites are treated with praziquantel in the presence of high magnesium, they enter a state of flaccid paralysis in which they are not responsive to agents normally expected to cause contractions of the musculature. When the parasites were incubated in 30 mM M g " I-RPMI for 15 min and then contracted in response to the addition of praziquantel to the bath, the time to the onset of this flaccid paralysis relative to the initial exposure to praziquantel was also dependent on the concentration of praziquantel used. Parasites treated with less than 180 nM praziquantel did not enter this state within 15-30 min of observation. Parasites treated with 300 nM praziquantel entered flaccid paralysis in roughly 15 min, while parasites exposed to 10 fiM praziquantel became unresponsive within 5 min. Once in the flaccid paralysis state, parasites were refractory to: praziquantel (10/
