Drugs. Caffeine, 1,7-dimethylxanthine (DMX), theophyl- line, theobromine, ryanodine, ruthenium red and neomycin were all obtained from Sigma.
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Functional ryanodine receptor channels in flatworm muscle fibres T. A. D A Y "*, J. H A I T H C O C K ", M. K I M B E R # and A. G. M A U L E # " Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA # Parasitology Research Group, The Queen’s University of Belfast, Belfast, Northern Ireland, UK (Received 4 August 1999 ; revised 14 October 1999 ; accepted 14 October 1999)
Caffeine, which stimulates intracellular Ca#+ release channels known as ryanodine receptor (RyR) channels, induces contraction of individual muscle fibres dissociated from the trematode Schistosoma mansoni, and the turbellarians Dugesia tigrina and Procerodes littoralis. Caffeine is much more potent on S. mansoni fibres (EC 0n7 m) than those from D. &! tigrina or P. littoralis (3n2 m and 4n6 m, respectively). These caffeine-induced contractions are blocked by ryanodine, confirming the presence of functional RyR channels in these flatworm muscles. However, the contractions are not blocked by typical RyR channel blockers ruthenium red or neomycin, indicating that there may be important pharmacological differences between the RyR channels in this early-diverging phylum and those of later animals. These studies demonstrate that RyR channels are present in the muscle of these flatworms, and that the sarcoplasmic reticulum stores sufficient Ca#+ to support contraction. Key words : Platyhelminthes, Trematoda, Turbellaria, schistosome, planaria, muscle, ryanodine, ryanodine receptor, caffeine.
Early electron microscopy revealed the presence of sarcoplasmic reticulum (SR) in the muscle of flatworms, including turbellarians (MacRae, 1963, 1965), trematodes (Silk & Spence, 1969) and cestodes (Lumsden & Byram, 1967). However, the SR was described as ‘ poorly defined ’ (Silk & Spence, 1969) in schistosomes and, in general, the role of the SR in Ca#+ storage and release in the muscle of platyhelminths has not been clear. Recently, a number of the components vital for Ca#+ storage and release from the SR have been demonstrated in schistosomes. Sarco(endo)plasmic reticulum Ca#+-ATPases (SERCAs) are the intracellular pumps which sequester Ca#+ into the SR and, based on physiological evidence (Cunha, Reis & Noe$ l, 1996) and molecular cloning (de Mendonca et al. 1995 ; Talla et al. 1998), at least 2 SERCAs are present in schistosomes. These SERCAs are distinct structurally and pharmacologically from the Ca#+ATPases that extrude Ca#+ across the sarcolemma. Ryanodine receptors (RyR) are channels present on the SR which mediate the release of stored Ca#+, in the case of smooth muscle the RyR channels are triggered by Ca#+, mediating a Ca#+-induced Ca#+ release. The RyR channels are distinct from the inositol triphosphate (IP ) receptor channels which $ * Corresponding author : Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA. Tel : j517 432 4075. Fax : j517 353 8915. E-mail : dayt!msu.edu Parasitology (2000), 120, 417–422.
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also mediate the release of intracellularly stored Ca#+. Based on the binding of [$H]ryanodine, RyR channels are present in schistosomes (Silva et al. 1998). Further, the SERCA pumps and the RyR channels are similarly distributed in crude subcellular fractions, suggesting that they may be expressed in the same subcellular location in vivo (Silva et al. 1998). These data lead to the hypothesis that the SR plays a role in sequestering (via SERCAs) and releasing (via RyR channels) Ca#+ in schistosome muscle and, therefore, also in the muscle of other platyhelminths. If the SR of flatworm muscle stores Ca#+ sufficient to evoke contraction, and if RyR channels are present in the SR membrane, then RyR channel agonists should be able to elicit contraction of muscle fibres. Here, we report the physiological effects of various RyR channel pharmacological modulators on muscle fibres from 3 flatworms : the trematode parasite of humans Schistosoma mansoni, the freshwater planarian Dugesia tigrina and the brackish water triclad turbellarian Procerodes littoralis. The objective was to test if Ca#+ released by the opening of RyR channels is sufficient to induce contraction, and to examine the pharmacology of those contractions.
Worms S. mansoni used in these experiments were Puerto Rican strain maintained in the laboratory. Adult " 2000 Cambridge University Press
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Table 1. Specifics of dispersal conditions Schistosoma mansoni
Dugesia tigrina
Procerodes littoralis
Number of worms Dissociation medium Enzyme
25–40 Dissociation DMEM 1 mg\ml papain*
5–6 Dissociation DMEM 1 mg\ml papain*
Temp. during dissociation (mC) Duration in enzyme (h) Incubation medium
35 0n75 Modified DMEM
RT 0n75 Modified DMEM
25–30 Dissociation worm saline 0n15 mg\ml collagenase† and 0n15 mg\ml protease‡ 4 12 Worm saline
* BoehringerkMannheim. † Sigma type 1A Clostridiopeptidase, from C. hystoliticum. ‡ Sigma type XIV, from Streptomyces griseus.
parasites were recovered from the hepatic and mesenteric vasculature of female Swiss Webster mice 45–60 days post-infection. D. tigrina were obtained from Ward’s Biology and were maintained in a continuously filtered, aerated aquarium at 18–22 mC in artificial spring water (µ) : 351 KH PO , 14(NH ) SO , 102 MgSO , 248 CaCl , 0n5 # % %# % % # FeCl , pH to 7n2 with NaOH). Live specimens of P. $ littoralis were collected at low tide from Portavogie harbour, Northern Ireland. Worms were transferred to the laboratory in brackish water and maintained in a 50 % solution of artificial sea water ((m) : 231 NaCl, 1n2 MgSO , 6n1 KCl, 1n2 NaHCO , 4n9 MgCl , % $ # 5n7 CaCl ) which was changed every 2–3 days. # Dissociated muscle preparations The muscle fibres were dissociated from all 3 species of worms using the same basic protocols, based on those that have been described for S. mansoni (Day, Bennett & Pax, 1994 ; Day et al. 1994). In each case, a collection of worms were first placed on a glass slide and chopped approximately 100 times with a sterile razor, resulting in worm pieces. After incubation in an enzyme-containing dissociation medium (see Table 1), the worm pieces were rinsed 3 times with enzyme-free dissociation medium and then, to facilitate dissociation, triturated through a fire-polished Pasteur pipette until a turbid solution was produced. This solution, which invariably contained many dissociated muscle fibres, was then plated onto plastic Petri dishes and allowed 1 h for settling and adherence. After 1 h, the dissociation medium was exchanged for incubation medium in which the contraction assays were performed. The details of this procedure were different for all 3 species, and the specifics are delineated in Table 1.
Media Media for the work with the S. mansoni and the D. tigrina muscle fibres was based on Dulbecco’s
Modified Eagle’s Medium (DMEM w\o Na HPO # % or NaHCO , Sigma). The incubation medium was a $ modified DMEM, which was DMEM reduced to 67 % of normal concentration to which was added (m) 2n2 CaCl , 2n7 MgSO , 0n04 Na HPO , 61n1 # % # % glucose, 1n0 dithiothreitol (DTT), 0n01 serotonin, 15 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (Hepes) (pH 7n4), and 1 % (v\v) antibiotic\ antimycotic solution (Gibco). The dissociation DMEM was this incubation medium to which had been added 1 m ethyleneglycol-bis-(β-aminoethyl ether) tetraacetic acid (EGTA), 1 m ethylenediamine tetraacetic acid (EDTA), and 0n1 % bovine serum albumin. The P. littoralis muscle fibres were dissociated and incubated in a worm saline which consisted of (m) : 13n6 CaCl , 13n4 KCl, 458 NaCl, 9n8 MgCl , 13n6 # # Na SO , 10 Hepes, 10 glucose, and 1 % (v\v) # % antibiotic\antimycotic solution (Gibco).
Contraction assay The muscle contraction data are based on visual observation of contractions of individual muscle fibres in response to microperfusion with test medium via a micropipette. Each fibre selected for testing was identified as quiescent and fit the general morphological characteristics of being either a ‘ frayed ’ or a ‘ spindle-shaped ’ fibre (see Day et al. 1992). After selection of a fibre, the micropipette was brought to within 50 µm of the cell and test medium was pressure ejected directly onto the fibre. All of the observations were recorded to videotape for later analysis. In each 35 mm Petri dish, 15–20 of the muscle fibres were exposed to the agent and during analysis, each was scored as either a contraction or a noncontraction. Formally, a muscle fibre was deemed to have contracted when it shortened to less than 50 % of its original length, but this criterion was rarely necessary. Since the fibres have no imposed load, the contractions were all or none.
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Fig. 1. Concentration–response relationships for caffeine contraction of flatworm muscle. The curves displayed were generated by fitting the raw data with the Boltzmann equation, which in each case yielded χ# 0n3, and EC of 0n7 n for Schistosoma mansoni &! ( ), 3n2 m for Dugesia tigrina (>) and 4n6 m for Procerodes littoralis ($).
Data are presented as percentage of fibres which contract in response to the test medium. Each datum presented is the meanp... for 6 dishes (15–20 fibres tested in each dish) prepared on at least 3 different days. The concentration–response data were fit using the Boltzmann equation in the form, %Contraction l
(maxVmin) 1jexp((xVEC )\k) &!
using Origin 5.0 (Microcal Software Inc., Northampton MA), where max and min represent the maximal and minimal percentage of contractions, x is the concentration on the x-axis and EC is the &! concentration at which the curve would predict a half-maximal response, and k is a factor describing the slope of the curve. Comparisons of EC (p..) &! were made with the Student’s t-test. Drugs Caffeine, 1,7-dimethylxanthine (DMX), theophylline, theobromine, ryanodine, ruthenium red and neomycin were all obtained from Sigma. Caffeine and caffeine analogues The RyR channel agonist caffeine (1,3,7-trimethylxanthine) elicited immediate contractions of individual muscle fibres derived from all 3 of the flatworms used in this study (Fig. 1). Depolarization with elevated extracellular K+ also elicits contractions of the individual muscle fibres from all 3 species, and the caffeine-induced contractions were
Fig. 2. Contractile responses to RyR channel agonists and caffeine tested at 1 and 10 m.
qualitatively similar in all cases. Specifically, the contractions were definitive and complete, almost never partial or graded. Different from the high K+induced contractions, the caffeine-induced contractions frequently included some slight twitching preceding the complete contraction. The concentration–response relationships were similar for all 3 of the muscle types. In all 3 cases the maximum percentage of fibres responding was very similar, ranging from 86 to 88 %. The concentration–response relationships were also all similarly quite steep ; in each case, the response went from minimal to maximal over a single order of magnitude of caffeine concentrations. When the data are described as a Boltzmann distribution, the k values, which reflect the steepness of the curve,
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420 A
Fig. 3. Ryanodine block of contractions induced by 10 m caffeine in (>) Dugesia tigrina and ($) Procerodes littoralis. The y-axis is standardized as a percentage of the response obtained with 10 m caffeine and no ryanodine. The curves displayed are generated from raw data using the Boltzmann equation, which in both cases yielded χ# 0n3, and IC of 72 n for D. &! tigrina and 530 n for P. littoralis.
range from 2n0 to 4n5. The most significant difference in the caffeine responses of the 3 species was that the S. mansoni muscle fibres were significantly more sensitive (P 0n001). The EC was 0n7 m for the &! S. mansoni fibres, as compared to 3n2 and 4n6 m for D. tigrina and P. littoralis, respectively. A number of caffeine analogues also elicited concentration-dependent contractions of muscle from all 3 flatworms. The contractions elicited by these analogues appeared as those elicited by caffeine – definitive and complete. The analogue 1,7dimethylxanthine (DMX) was equally or more effective than caffeine on muscle from all 3 worms (Fig. 2). With the S. mansoni muscle, for example, 1 m DMX produced a 67p5 % response, compared to 61p5 % for caffeine. And, while 1 m caffeine contracted only 8p1 % of the Dugesia muscles, the same concentration of DMX elicited a 44p4 % response. Theophylline (1,3-dimethylxanthine) was also effective on all 3 muscle types and, in each case, was essentially as effective as caffeine in almost every case, with the only exception being at 1 m on S. mansoni muscle where it was significantly less effective. Theobromine (3,7dimethylxanthine) also induced contractions but was less effective than caffeine on S. mansoni and D. tigrina at the concentrations tested here. Ryanodine and other blockers In many cases, ryanodine is known to act as an agonist at RyR at low concentrations, stimulating Ca#+ release from the SR, and as a blocker at higher concentrations. But, ryanodine alone did not have any detectable effects on these platyhelminth muscle
B
Fig. 4. (A) Ryanodine block of caffeine-induced contractions in Schistosoma mansoni. Contractions were elicited by ($) 1 m, (>) 3 m and ( ) 10 m caffeine, and the results on the y-axis are standardized as the percentage contraction elicited by that concentration of caffeine with no ryanodine present. The curves displayed are generated from raw data using the Boltzmann equation, which in each case yielded χ# 0n7. (B) The relationship between the concentration of caffeine used to stimulate the fibres and the amount of ryanodine needed to block the contractions. The IC &! were derived from the Boltzmann description of the data, with limits set at 0 and 100 %. The slope of the line comparing the ∆ log (ryanodine IC )\∆ log &! ([caffeine]) is 2n2p0n1, signifying that increasing concentrations of stimulatory caffeine demanded even greater increases in the ryanodine to achieve equivalent blockade. Specifically, over this range, the increase in ryanodine required was 2-fold greater than the increase in caffeine.
fibres. It did not elicit contractions of the individual muscle fibres at concentrations ranging from 1 n to 100 µ. Also, there was no noticeable change in the muscle fibres when these concentrations of ryanodine were included in the incubation medium for as long as 15 min. Ryanodine potently inhibited the caffeine-induced contractions in muscles derived from all 3 worms (Figs 3 and 4). Specifically, when the muscle fibres
Ryanodine receptors in flatworms
were pre-incubated in various concentrations of ryanodine for 5 min, there was a concentrationdependent inhibition of the contractions elicited by caffeine. In D. tigrina and P. littoralis, where caffeine was essentially equipotent, ryanodine was tested against 10 m caffeine (Fig. 3). The concentration– inhibition relationships for ryanodine were similar in both cases, with IC of 72 n for D. tigrina and &! 530 n for P. littoralis. For S. mansoni, where caffeine was more potent, more ryanodine was required to block contractions elicited by 10 m caffeine so that the IC was significantly higher &! (22n5 µ). Otherwise, the inhibition curve for S. mansoni was similar to those obtained with the other worms. In order to examine further the relationship between caffeine and ryanodine, ryanodine was also tested on S. mansoni muscle against other concentrations of caffeine. Over the range examined, there was a direct relationship between the amount of caffeine used to stimulate the fibres and the amount of ryanodine needed to block those contractions (Fig. 4). Increases in the caffeine required a greater increase in the amount of ryanodine to achieve blockade. Ryanodine was also effective at blocking the contractions elicited by 1,7-DMX and theophylline. Some compounds which are effective blockers of other ryanodine receptors did not block the caffeine response in these platyhelminth muscle fibres. At concentrations as high as 10 µ, neither ruthenium red nor neomycin had any inhibitory effect on contractions of D. tigrina or S. mansoni muscles elicited by 10 m or 1 m caffeine. Both of these compounds were also ineffective against contractions elicited by 1,7-DMX, theophylline and theobromine. These results demonstrate that flatworm muscle fibres contain functional RyR channels that control intracellular Ca#+ stores sufficient to support contraction. The presence of functional RyR channels in schistosomes was previously demonstrated by [$H]ryanodine binding in homogenates of worms and ryanodine-stimulated release of %&Ca#+ from microsomes (Silva et al. 1998). These studies show that, in schistosomes and other flatworms, one location of these RyR channels is the muscle, and demonstrate the physiological function of these receptors in this early-diverging phylum. For the most part, this study reveals a general uniformity in pharmacological properties of the RyR channels in these 3 different flatworms. Specifically, they are all stimulated by caffeine, DMX, theophylline and theobromine, while they are all blocked by ryanodine and not ruthenium red or neomycin. The most notable difference amongst them is that
421
the schistosome muscle fibres are more sensitive to caffeine than are those from the other flatworms, which may be attributable to the dramatically smaller size of the individual fibres rather than to any pharmacological differences in the RyR channels between the species. The potency of caffeine on these flatworm muscles, EC ranging from 1–5 m, is within the range &! reported for RyR channels in preparations derived from other animals (Zucchi & Ronca-Testoni, 1997). Also, the steep concentration–response relationship for these caffeine-induced contractions is expected based on the Ca#+-dependence of Ca#+ release from the SR. The release of some Ca#+ induces a positive feedback mechanism leading to the further release of Ca#+. There are pharmacological differences between the flatworm RyR channels described here and those of other animals. For example, here the methylxanthines theophylline and theobromine were equally or less potent than caffeine, whereas they are more potent in rabbit skeletal muscle (Rousseau et al. 1988). The most striking difference, however, is the apparent insensitivity of the flatworm RyR channels to some blockers. Notably, the inorganic polyamine ruthenium red had no detectable effect in this assay. Aminoglycosides are also widely reported to inhibit RyR channels, but the most potent of these, neomycin (Zimanyi & Pessah, 1991), had no effect on the flatworm muscle. These apparent pharmacological differences could signal important structural differences in the RyR channels of flatworms from those of later animals. In other preparations, the effects of ryanodine on RyR channels is complex. Low concentrations stimulate Ca#+ release, while high concentrations block release (Fairhurst, 1974 ; Sutko et al. 1979). Likewise, RyR channel binding of ryanodine is complex, with both high- and low-affinity binding observed for many channels (Lai et al. 1989 ; Wang, Needleman & Hamilton, 1993). The stimulation of Ca#+ release has been associated with a high-affinity binding site, and the blockade has been attributed to a low-affinity binding site (Pessah & Zimanyi, 1991 ; Sutko et al. 1997). In these flatworm muscles, this assay reveals no evidence of a high-affinity stimulation of the flatworm muscle RyR channels by ryanodine – that is, the dispersed muscles did not contract to ryanodine alone at any concentration. The only observed effect of ryanodine is a blockade of contraction, typically attributed to a low-affinity binding site. However, studies of ryanodine binding in schistosome homogenates reveal only a single binding site and, with a dissociation constant of 7 n, it would be classified as a high-affinity site (Silva et al. 1998). It is possible that the single inhibitory effect observed with ryanodine is being mediated by the high-affinity binding that has been observed, but it is also possible that the binding
T. A. Day and others
studies missed the low-affinity sites (Silva et al. 1998) and that the contraction assays used in the present studies are not sensitive enough to measure stimulation of Ca#+ release mediated by high-affinity sites. Based on a number of structural and physiological observations, the somatic muscle of platyhelminths is considered most comparable to smooth muscle of later animals. In these other types of smooth muscle, the physiological stimulus for RyR channel Ca#+ release is elevated intracellular Ca#+, and the RyR channels are often found located on cisternae of the SR in close apposition to the sarcolemma. This spatial arrangement is hypothesized to increase the efficiency of stimulation of the RyR channels by the Ca#+ influxes across the sarcolemma. Cisternae of the SR have likewise been found to be arranged close to the sarcolemma in many platyhelminth muscles, including the planarians D. tigrina (MacRae, 1963) and Notoplana (MacRae, 1965), the trematode S. mansoni, and many cestodes (Lumsden & Byram, 1967). With the establishment of functional RyR channels in the muscle of platyhelminths, it is reasonable to hypothesize that these RyR channels reside in these SR cisternae close to the sarcolemma. The work at MSU was supported in part by NIH grant AI30465. The work at QUB was supported in part by a GM Williams award to M K. We would like to thank Natasha Kreshchenko of QUB for assistance, and Dr François Noe$ l of Universidade Federal do Rio de Janeiro for many helpful suggestions.
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