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Cu. ‘: ‘é 7“: r-.-- ‘ 4‘.\_.':-'~:. .. 1x 015;.‘7'3x‘fi‘? :‘izzct-' :‘:\t -‘- . fig; .1, 1'32.qu I ' . _‘ ‘n CUTOU‘ 1: ‘9”3," ..:‘~ LIBRARY Michigan State University This is to certify that the dissertation entitled PHYSIOLOGICAL RESPONSES TO CHOLINERGIC COMPOUNDS IN ADULT MALE SCHISTOSOMA MANSONI presented by David Robert Semeyn has been accepted towards fulfillment of the requirements for doctoral degreein Neurosciences and Zoology IZJZ (2 Z; Major professor Date (36 Et/L‘ I? K? Ilflli-n-AkM—n‘ ' V" 1"" - g - . 0.12771 IVAESI_J BEIURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from 4—3—- your record. FINES will be charged if book is returned after the date stamped below. PHYSIOLOGICAL RESPONSES TO CHOLINERGIC COMPOUNDS IN ADULT MALE SCHISTOSOMA MANSONI BY David Robert Semeyn A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Neuroscience Program and Department of Zoology 1987 ABSTRACT Physiological Responses to Cholinergic Compounds in Adult Male Schistosoma mansoni by David Robert Semeyn Cholinomimetic agents exert predominantly inhibitory actions on the body musculature of adult male Schistosoma mansoni. The actions of cholinergic compounds were evaluated in this parasitic trematode using physiological and biochemical techniques to gain a better understanding of the role played by the cholinergic component of the animal's nervous system. The agonists arecoline, carbachol and nicotine, and the cholinesterase inhibitor eserine produced signifi- cant inhibitory effects on mechanical activity. A slight hyperpolarization of the parasite's tegumental and muscle membranes was produced by these same compounds. Lobeline produced an increase in mechanical activity and depolariza- tion of the parasite's membranes. The antagonists benzoquinonium, atropine, benactyzine and propantheline were effective to varying degrees in blocking the actions of inhibitory compounds. Mechanical and membrane responses to inhibi- tory agonists were not altered in the presence of altered external ion concentra- tions. Antimony potassium tartrate pretreatment or exposure to low temperature blocked or attenuated mechanical responses to inhibitory cholinergic agents, indicating the possible involvement of energy-dependent processes in mediating their actions. David Robert Semeyn Inhibitors of CaZ+-ATPase and Na+/K+-ATPase produced an acute contrac- ture of the parasite's longitudinal musculature. Schistosomes exposed to these treatments demonstrated marked decreases in responsiveness to inhibitory choli- nergic agents. 5 Z Efflux of 4 Ca + from schistosomes appears to be enhanced in the presence of inhibitory cholinergic agents. These compounds also decrease steady-state 45 levels of Ca2+ in the animal. This action can be blocked by cholinergic antagonists, ouabain or trifluoperazine. A Ca2+lMgz+-dependent ATPase can be identified in whole worm homogen- 2 ates which has optimal activity at pH 8.0 in the presence of 40 11M Ca +, 5 mM Mgz+ and 5 mM ATP. Exogenous calmodulin and cholinergic agents producing inhibition of mechanical activity cause concentration-dependent activation of this enzyme. Inhibitory cholinergic agents do not appear to activate schistosomal Na+/K+-ATPase. These results suggest that one manner by which cholinergic receptor activation may induce inhibition of the parasite's mechanical activity is by enhancing the removal of calcium from the animal through the activation of: (1) an active calcium pump, and/or (2) a sodium/calcium exchange mechanism. to Katherine O. 11. ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. Ralph A. Fax (Dept. of Zoology/Neuroscience Program), my major professor, for his guidance and con- structive criticism throughout this investigation. Special thanks to Dr. James L. Bennett (Dept. Pharmacology/Toxicology) and Dr. Philip Gerhardt (College of Osteopathic Medicine, Director, Medical Scientist Training Program) for assist- ance in obtaining funding, and for providing creative insights during these investigations. Thanks also to Dr. Neil Band (Dept. of Zoology), Dr. Gerard Gebber (Dept. of Pharmacology/Toxicology), Dr. Richard Rech (Dept. of Pharmacology/Toxico- logy, and Dr. John Thornburg (College of Osteopathic Medicine and Dept. of Pharmacology/Toxicology) for helpfully serving as committee members and con- sultants. I would also like to thank Dr. Glenn Hatton (Dept. of Psychology, Director, Neuroscience Program) for enthusiastically loaning equipment as well as providing creative insights on numerous occasions. Thanks and best wishes to my colleagues in parasite research, Connie Bricker, Helen Cirrito, George Cheng, Joe Depenbusch, Tim Martin, Dan Morri- son, Carla Siefker, John and Beth VandeWaa, Kevin Blair and Carolyn Lane. Finally, very special thanks to Russell D. Peterson, D.O., for helping me to keep things in perspective and providing much needed breaks from the world of research. iii TABLE OF CONTENTS Page LIST OF TABLES -------------------------------------------------- vii LIST OF FIGURES ------------------------------------------------- ix GENERAL INTRODUCTION ----------------------------------------- 1 A. Schistosoma mansoni: Parasite, life-cycle and disease ------------- 2 B. Anatomical and physiological characteristics of _S_. mansoni --------- 6 1. Generaly anatomy ---------------------------------------- 6 2. The tegument ------------------------------------------- 8 3. Physiology of the tegument -------------------------------- 12 4. The musculature ----------------------------------------- 15 5. Physiology of the musculature ----------------------------- 16 6. The nervous system --------------------------------------- 17 7. Schistosome neurobiology ---------------------------------- 18 Acetylcholine -------------------------------------- l9 Catecholamines ------------------------------------- 20 5-Hydroxytryptamine -------------------------------- 21 8. Energy metabolism --------------------------------------- Z3 OBJECTIVES ------------------------------------------------------ 25 SECTION I ------------------------------------------------------- 2? Physiological responses to cholinergic agonists and antagonists in adult male Schistosoma mansoni ------------------------------------------ 27 1. Summary ---------------------------------------------------- 27 II. Introduction -------------------------------------------------- 28 III. Materials and Methods ----------------------------------------- 30 A. Experimental animals and media --------------------------- 30 1. Source and maintenance of animals ------------------- 30 2. Recording media ----------------------------------- 30 Potassium ------------------------------------ 30 Chloride ------------------------------------- 31 Magnesium ----------------------------------- 31 Sodium --------------------------------------- 31 iv TABLE OF CONTENTS (continued) Page Calcium ------------------------------------- 3f Low Sodium/0 Calcium -------------------------- 31 B. Mechanical activity recordings ----------------------------- 31 C. Microelectrode recordings -------------------------------- 35 D. Pharmacological agents ----------------------------------- 36 Cholinergic agents ---------------------------------- 36 Ion channel blockers and activators -------------------- 36 E. Statistical procedures ------------------------------------- 36 IV. Results ...................................................... 37 A. Mechanical activity ..................................... 37 1. Normal contractile activity .......................... 37 2. Response to “nicotinic” compounds .................... 37 Agonists ------------------------------------- 37 Antagonists ----------------------------------- 42 3. Response to "muscarinic" compounds .................. 42 Agonists ------------------------------------- 42 Antagonists ----------------------------------- 42 4. Response to "mixed" compounds ---------------------- 42 Agonists ..................................... 42 5. Antagonism of inhibitory effects --------------------- 45 6. Effect of low temperature --------------------------- 47 7. Effect of antimony --------------------------------- 48 8. Effect of altered ion concentrations ------------------- 48 9. Response to ion channel blockers/activators ------------ 52 K++channels ----------------------------------- 52 Na2+channels ---------------------------------- 52 Ca channels --------------------------------- 53 Inorganic agents ------------------------------ 55 Organic agents -------------------------------- 55 10. Effect f ionophore A-23187 ------------------------- 6O 11. High K -induced contractures ------------------------ 64 B. Membrane potentials ------------------------------------- 66 V. Discussion --------------------------------------------------- 76 h TABLE OF CONTENTS (continued) Page SECTION II ------------------------------------------------------ 85 Ionic mechanisms involved in mediation of cholinergically-induced inhibition of mechanical activity in adult male Schistosoma mansoni ------ 85 L Summary ---------------------------------------------------- 85 II. Introduction -------------------------------------------------- 87 III. Materials and Methods ----------------------------------------- 88 A. Experimental animals and media --------------------------- 88 B. Mechanical activity recordings --------------------------- 89 C. Measurement of ion fluxes -------------------------------- 90 D. Measurement of ATPase activity --------------------------- 92 1. Cai+lMgz+-ATPase -------------------------------- 92 2. Na [K -ATPase ------------------------------------- 93 E. Pharmacological agents ----------------------------------- 93 F. Statistical procedures ------------------------------------- 94 IV. Results ---------------------------------------------------- 94 A. Mechanical activity -------------------------------------- 94 1. Gait TPase inhibitors ------------------------------ 94 2. Na [K -ATPase inhibitor --------------------------- 97 B. 450.1.2+ fluxes ------------------------------------------ 101 C. ATPase measurements ------------------------------------ 110 1. Caz+/Mg2+-A'I'Pase --------------------------------- 110 Effect of Ca2+ and Mg2+ concentration ----------- 110 Effect of ATP, pH and temperature -------------- 113 Effect of A'I'Pase inhibitors --------------------- 113 Effect of exogenous calmodulin ------------------ 118 Effect of cholinergic agents ------------------ -- 118 + Effect of other agents -------------------------- 122 2. Na [K -ATPase ------------------------------------- 122 V. Discussion --------------------------------------------------- 122 GENERAL DISCUSSION -------------------------------------------- 129 SUMMARY -------------------------------------------------------- 132 APPENDIX ------------------------------------------------------- 135 Composition of RPMI-164O medium ----------------------------- 135 BIBLIOGRAPHY -------------------------------------------------- 137 vi Table 10 11 12 LIST OF TABLES Effect of nicotinic compounds on resting tension in adult male _S_. mansoni ........................................ Effect of muscarinic compounds on resting tension in adult male _S_. mansoni ---------------------------------------- Effect of mixed compounds on resting tension in adult male S. mansoni --------------------------------------------- Effect of cholinergic antagonists on resting tension re- sponses to cholinergic agonists in adult male _S_. mansoni ------ Effect of antimony potassium tartrate pretreatment on resting responses to cholinergic agonists in adult male _S_. mansoni ---------------------------------------------- Resting tension responses to inhibitory cholinergic agonists in altered external ion concentration ---------------------- Effect of compounds acting on Ca2+ channels on longitudi- nal muscle tension in adult male S. mansoni ................ Effect of pretreatment with inhibitory choliner ic agents on longitudinal muscle tension responses to high K medium ---- Response to tension decreasing agents in the presence or absence of high K medium .............................. Effect of cholinergic compounds on tegumental and muscle membrane potentials of adult male §_. mansoni .............. Effect of altered ion concentration on membrane potentials and membrane responses to tension decreasing agents ------- Change in longitudinal muscle tension responsiveness to ch + er 'c agonists following pretreatment with Ca /Mg +-ATPase inhibitors ---------------------------- vii Page 41 43 46 50 51 65 67 69 71 98 5-. LIST OF TABLES (Continued) Table 13 14 15 16 17 18 19 Change in longitudinal muscle tension responsiveness to cholinergic age ts +and D-600 following pretreatment with inhibitors of Na /K -A'I'Pase ............................. Effect of tension decreasing agents on rate of 4SCaz+ accumulation and efflux in adult male _S_. mansoni ------------ Alteration of steady-state 4SCaZ+ levels by various pharma- cological agents affecting schistosomal mechanical activity -- Alteration of steady-state 45Caz+ levels by cholinergic agents and trifluoperazine in adult male _S. PM ......... Caz+lMgz+-A'I'Pase activity: Effect of calmodulin antago- nists -------------------------------------------------- 2+ 2+-A'I'Pase activity: Effect of exogenous calmodu- +-A'I'Pase activity: Effect of cholinergic agents --- viii Page 103 106 107 111 119 120 121 10 ll 12 LIST OF FIGURES Life cycle of S. mansoni ................................. Gross morphology of paired S. mansoni ..................... - Diagram of tegumental structure of S. mansoni ------------- Electrical potentials in adult male S. mansoni ............... Schematic of apparatus used to record mechanical activity in adult male S. mansoni ................................. Chart recordings flowing effects of distilled water, 10-6M arecoline and 10 M nicotine on mechanical activity in adult male S. mansoni ................................... Chart recordings sharing effects of 10-4M lobeline, 10-5M benactyzine and 10 M benactyzine on mechanical activity in adult male S. mansoni ................................. Chart recordings_§howing effects of distilled water, lO-4M carbachol and 10 M eserine on mechanical activity in adult male §_- mansoni ........................................ Effects of preincubation in 10-4M antimony potassium tar- trate on longitudinal muscle tensjgn responses to 10 M arecoline, 10 M carbachol and 10 M eserine in adult male .8: mansoni ............................................. Chart recordings showing effects of Ca2+ channel blockers on mechanical activity in adult male S. mansoni ............ Chart recordings showing effects of ionophore A-23187 on mechanical activity in adult male S. mansoni ............... Chart recordings showing effects of ionophore A-23187 on mechanical activity responses to tension decreasing agents in adult male S. mansoni ................................. ix Page 4 7 9 14 33 38 39 40 49 56 61 62 LIST OF FIGURES (Continued) Figge 13 14 15 16 17 18 19 20 21 22 23 24 Chart recording showing effect of ionophore A-23187 0 response to 10 M carbachol in the presence of high K medium ------------------------------------------------ Effect of high K+ medium and carbachol on membrane potentials and longitudinal muscle tension in adult male S. mansoni ----------------------------------------------- Effect of high K+ medium and D-6OO on membrane poten- tials and longitudinal muscle tension in adult male S. man- soni ------------------------------------------------- Acute responsze+ of ail+ult male S. mansoni longitudinal muscle tension to Ca /Mg -ATPase inhibitors ------------------- Acute responae of adult male S. mansoni longitudinal muscle tension to Na [K -A'I‘Pase inhibitors ...................... Accumulation of 45Ca2+ in adult male S. mansoni ----------- Efflux Of 45Ca2+ from adult male S. mansoni --------------- Effect of divalent cations on schistosomal ATPase activity: Enzyme activity as a function of free Mg concentration ---- Effect of divalent cations on schistosomal Cay/Mg“- A ase activity: Enzyme activity as a function of free Ca concentration ------------------------------------- Effect of ATP concentration on schistosomal Caz+/Mgz+- ATPase activity ........................................ Effect of H+ concentration on schistosomal Ca2+/Mgz+- ATPase activity ---------------------------------------- Effect of temperature on schistosomal Ca2+/Mg2+-A'I'Pase activity ------------------------------------------------ 68 72 74 95 100 103 104 112 114 115 116 117 GENERAL INTRODUCTION Schistosomiasis is a waterborne parasitic disease which continues to be a major medical problem in tropical and subtropical regions of the world. The world Health Organization (WHO) ranks this infectious disease second only to malaria in terms of socioeconomic and public health importance. Schisosomiasis is endemic in 74 countries, many of them Third World nations, with as many as 600 million people currently exposed to infection (Iarotski and Davis, 1981). Numerous biological and ecological methods have been employed in attempts to control the disease, but such methods have suffered from many limitations, and have, therefore, been only marginally successful (Jordan at 9.1., 1978; Polderman, 1984). the ultimate means of control for this disease will presumably be development of a vaccine designed to prevent its transmission. Despite recent progress in this area using monoclonal antibody techniques (Harn, Mitsuyama and David, 1984), the observation by Taylor (1980), that development of a practical human vaccine is probably far in the future, still holds true today. Accordingly, it seems that chemotherapy will play an ever increasing and more crucial role in controlling schistosomiasis. Furthermore, it appears that chemotherapy will remain the mainstay of treating individual patients already suffering from the disease (Archer, 1985). One of the major targets of present and future chemotherapeutic agents used to treat this disease may be the parasite's nervous system. It appears that several agents employed in recent years may be acting by interfering with the animal's normal neurochemical processes. For example, dichlorvos, metrifonate, and oxamniquine are thought to affect the motor activity of S. mansoni by effects on the cholinergic component of the animal's nervous system (Reiner at a_l_., 1980; Kaye, 1984). The precise mode of action of most antischistosomals currently in wide use, however, is either unknown or not clearly defined. Many possibilities exist for the development of safe and efficacious anti- schistosomals based on the ability of a drug to either block or mimic the actions of the parasite's neurotransmitters. Therefore, it becomes extremely important to first characterize and define the actions of the neurotransmitters utilized within the animal's nervous system. Numerous investigators have succeeded in demonstrating that certain putative neurotransmitter substances and their analo- gues affect schistosome motility, and that these substances can be identified within the animal's neuronal elements. An extremely large gap (currently exists, however, in our understanding of the precise role played by the various compo- nents of the parasite's nervous system, and in how the putative neurotransmitters exert their modulatory actions on mechanical activity. In light of this, I have taken the first steps towards characterizing the inhibitory role played by the cholinergic component of the schistosome's nervous system and determining how acetylcholine exerts its actions on this parasite's motility. A. Schistosoma mansoni: Parasite, Life-Cycle and Disease The blood fluke, Schistosoma mansoni, is a digenetic trematode which infects humans during one stage of its polymorphic life-cycle. Schistosomiasis has a long history, with the first description of the disease being found in the Kahun Papyrus, written about 1900 B.C. (Jordan and Webbe, 1969). However, it was not until relatively recently, in 1851, that the German pathologist, Theodor Bilharz, actually identified the schistosome as the causative agent of the disease. Four major species of schistosomes have now been identified as the causative agents in human schistosomiasis. There are numerous differences among these four species, including geographic distribution, gross morphology, intermediate hosts, disease patterns and responses to antischistosomal agents. S. mansoni, which causes intestinal schistosomiasis, is distributed throughout Africa, the eastern Mediterranean, the Caribbean and South America. S. haematobium, which causes urinary schistosomiasis, is found in Africa and Cyprus, and is also common in various parts of the Middle East. Two other species, S. japonicum, which was first identified by Katsurda in 1904, and S. mekoagi, first described by 'Voge, Bruckner and Bruce (1978), are reSponsible for the intestinal form of the disease found in Southeast Asia and the Western Pacific. The life-cycle of schistosomes is complex (Figure 1), and involves an aquatic snail intermediate host, the human definitive host, and the mutual presence of both snail and man in the environment in which transmission of the disease occurs. Paired adult flukes live in the blood vessels supplying the intestines, liver or bladder. While adult parasites can produce large nubmers of eggs (between 300 and 3,000 per day), they do not multiply within the human host (Warren, 1980, 1984). Ova pass out of the body via feces or urine, and, provided they reach fresh water, hatch into a free-swimming larval stage, the miracidium. Miracidia then have only several hours in which to find and penetrate the appropriate freshwater snail, which serves as the intermediate host. After 25 to 40 days within the snail, during which the miracidia develop into primary sporocysts, and then secondary sporocysts, under the proper conditions large numbers of infective, free-swimming cercariae emerge. These cercariae, which remain infective for up to 24 hours after being shed from the snail, burrow through the skin of humans they encounter in the water. Agitation of infested water during such activities as bathing, MAMMAI- FRESH WATER @999 (in i9c9s) // . . 6‘ 9 \®113:33:9, 9 paired adults ( m9s9n09ri9s ) @spo sporocysts T ’T‘( (in snails) ! ( lung stage I schistosomulum \ cercaria @‘m skin.“ (free-living) skin stage schistosomulum Figure 1. Life-cycle of S. mansoni. fishing, planting or recreation stimulates cercarial motility. Recent evidence indicates that fatty acids in human skin, particularly linoleic and linolenic acid, serve to attract cercarie and promote penetration (Haas and Schmidt, 1982). Following penetration of the skin, the cercariae begin the schistosomulum stage of development. After migrating along the lymph ducts into the venous system and lungs, they eventually reach the liver, where they mature into adult schistosomes within 5 to 6 weeks. The female parasite normally lies enclosed within the ventrally located gynec0phoric canal of the male, leaving only for brief periods to enter the narrowest capillaries of the intestine and rectum during oviposition (Belding, 1965). The symptoms of human schistosomiasis proceed in three stages, and the severity of the disease is related to the intensity of the parasite burden. Soon after the cercariae penetrate the skin the first stage occurs, producing a rash known as swimmer's itch. Three to nine weeks following infection, the second or acute stage of the disease occurs, causing complications which include diarrhea, hematochezia and hepatosplenomegaly (Jordan and Webbe, 1969). A chronic stage of infection develops in patients which have large parasite burdens of long duration. Most of the pathology associated with this stage of the disease is due to the backflow of deposited ova into the liver and spleen. Eggs trapped in these tissues release enzymes and other antigenic substaces that stimulate an immime response by activating host lymphocytes and other immune system cells. Ulti- mately, extensive granulomatous pseudotubercles surround each ova, which in turn cause various physical complications (Laughlin, 1984). As mentioned previously, the number of persons suffering from schistoso- miasis world-wide is estimated at 250 to 600 million, with approximately 1.5 billion believed to be at risk. The disease has been steadily increasing during recent decades, particularly in regions where major efforts have been made to improve irrigation of tillable land, e.g., along the Nile River. As a result, areas in which schistosomiasis had previously been endemic are now becoming hyperende- mic zones. Unfortunately, the current drug of choice for treating human schistosomiasis, praziquantel, is too expensive to be made available to Third World inhabitants on a population-wide basis, especially since the chance of re- infection is very high (Kolata, 1985). B. Anatomical and Physiological Characteristics of Schistosoma mansoni 1. General anatch Schistosoma mansoni is a sexually dimorphic plathyhelminth. The flat body of the larger male is curved to form a ventral gynecophoral canal running the length of its body, within which the more slender female normally lies during capulation (Figure 2). Adult male S. mansoni are approximately 1 cm long, 0.10 cm wide and have a wet weight of 0.8 mg. Adult female S. mansoni are slightly longer (1.5 cm) and much thinner (0.2 mm) than the adult male. Rostrally located ventral and anterior (oral) suckers are present in both sexes. The body of adult schistosomes possesses a superficial cortex covering the body musculature, the parenchyma, and the major nerve cards. This cortex consists of a tegumental epithelium, with an outer anuclear layer connected to a deeper layer of cell bodies. The tegumental surface of the male is rougher than that of the female. Beneath the tegument lies layers of circular and longitudinal muscles (Smith, Reynolds and von Lichtenberg, 1969; Senft, Philpott and Pelofsky, 1961). Subjacent to the muscle layers lie the digestive, reproductive and excretory systems. Both male and female digestive systems Open at the oral sucker. The mouth, pharynx and esophagus lead into an intestine which bifurcates in the region of the ventral sucker. The cecum reunites and ends blindly at the posterior Figure 2. Drawing illustrating the external characterstics of male and female S. mansoni. OS, oral sucker; VS, ventrla sucker; GC, gynecophoral canal. portion of the parasite. The cecum and intestine are often brown in appearance due to ingested hemoglobin (Erasmus, 1972). The male reproductive organs consist of 4-8 testes situated dorsal to the ventral sucker. These lead via the efferent duct, vas deferens and seminal vesicle, to the genital pore just posterior to the ventral sucker. The female reproductive system includes an elongate ovary situated in the anterior half of its body. The oviduct runs from the ovary and joins the vitelline duct at the ootype, where fertilization occurs. The uterus runs anteriorly from the ootype, and opens to the genital pore posterior to the ventral sucker (Noble and Noble, 1976). The excretory system of the schistosome is protonephridial in nature. Diffusely distributed flame cells empty via two longitudinal collecting tubules into a posteriorly located bladder and excretory pore (Meglitsch, 1972). 2. The tagument The outer covering of Schistosoma mansoni is a rough surfaced structure which appears to be an anatomic syncytium of anuclear material (Silk, Spence and Gear, 1969) (Figure 3). The outer surface of the tegument is highly invaginated, forming a series of pits or channels which project up to 0.5 um into the cytoplasm of the tegumental epithelium. The thickness of the tegumental epithelium ranges from 1 to 5 pm, varying with the contractile state of the animal and the region in which it is measured, in general being somewhat thicker on the dorsal surface. In both juvenile and mature parasites, the tegument contains several distinct structures: (1) crystalline spines which extend from the surface mem- brane of the tegument to the basal lamina; (2) poorly developed mitochondria; and (3) various membrane-bound inchisions known as multilaminate vesicles, spherical bOdies and elongate bodies (Morris and Threadgold, 1968; Smith 9; 9a., 1969; Silk a g” 1969; Wilson and Barnes, 1974a). There is also anatomical evidence .5308 "333833 .nS—H radon 6089830 .mm $033.89 858.5808 52 $39: .4 “3038 839:0 .80 “3038 "08393383 .24 «08% .m 3883 Manna. .15 «05.5808 8088 :3 ”08.5808 80:5 .20 «$3 .m “£08.30 £83335 .00 “page no 8.03:: .2 “sauna ~0808=m0u .OH. “833580 unua085m0u .9 .a .w mo 08505.5» gnun08nw0u no 8num0mQ .m 0.3th 10 III") {M alluv. HMS: s \ u\~\\ )Vq s—h. [Ii \in.\l.-§.:§? C . 11 indicating that innervated sensory structures are located in the tegument (Morris and Threadgold, 1967, 1968). The function of these sensory structures may be to detect the direction of flow of the surrounding medium. Though not shown in Figure 3, the inner plasma membrane is thrown into numerous narrow invagina- tions which project upward into the cytoplasm of the tegumental epithelium. The surface channels and basal invaginations may be separated from each other by as little as 0.25 pm (Wilson and Barnes, 1974b). The outer anuclear epithelium is in cytoplasmic continuity via inter- nuncial processes with cell bodies, called cytons, which are situated beneath the Imderlying musculature (Silk and Spence, 1969a; Fetterer _e_t_ al_., 1978). In contrast to the anuclear epithelium, the tegumental cytons contain an abundance of cellular organelles which are usually associated with secretory processes (e.g., endoplasmic reticulum, golgi apparatus, and polysomes) (Wilson and Barnes, 1974a; Silk at; g” 1969). These nucleus-containing cells manufacture and secrete the matrix of the tegument, and the internuncial processes originating from them pass through several layers, including: (1) the basal lamina, (2) the interstitial layer, and (3) the inner longitudinal and outer circularly-arranged muscle layers (Silk 91 3.1., 1969; Smith §_t_ al_., 1969; Smith and Van Lichtenberg, 1974). The membranes bounding the tegument are complex. The distal border of the outer tegument is a heptalaminate membrane which freeze-fracture studies have revealed consists of two adjacent lipid bilayers, and has a thickness of approximately 11 nm (Hockley 93 al_., 1975; Torpier, Capron and Capron, 1977; McClaren 91 a}, 1978). On the basal surface there is a classical trilaminate membrane 8-10 nm thick which is continuous with the trilaminate membrane of the internuncial processes and cytons (Hockley and McClaren, 1973; Wilson and Barnes, 1974a). 12 3. Physiolgy of the tegument The tegument of the schistosome has been implicated in a number of physiologically important processes. In addition to forming a host-parasite interface that is highly resistant to host degradative enzymes, carrier systems for the transport of various nutrients have also been demonstrated. Absorption of various hexoses (Fripp, 1967b; Isseroff, Ertel and Levy, 1976; Rogers and Bueding, 1975; Uglem and Read, 1975; Cornford and Oldendorf, 1979), amino acids (Senft, 1968; Chappell, 1974; Asch and Read, 1975a,b; Isseroff _e_t g, 1976) and purine and pyrimidine compounds (Leavy and Read, 197 5a,b) have been described. The outer tegumental membrane of S. mansoni as well as many other trematodes is invested with a superficially positioned glycocalyx, which varies considerably in morphology and chemistry between different developmental stages (Lumsden, 1975). This surface coat is typically inconspicuous, usually present as only a thin line (Bogitsh and Krupa, 1971). Histo- and cytochemical methods indicate the presence of acid mucopolysaccharides in the glycocalyx (Stein and Lumsden, 1973). Although the physiological role of this glycocalyx is at present imclear, a number of possible functions have been preposed. It has been suggested that the fixed negative charges associated with the glycocalyx of the schisto- some's tegument may play a role in the adsorption of cellular elements from the host, and thereby serve to disguise the worm from immunological recognition (Clegg, 1972; Smithers and Terry, 1969). The surface of the outer tegumental membrane has also been shown to contain various enzymes which may aid in absorption and digestion of nutrients from the host's plasma (Pappas and Read, 1975; Lumsden, 1975; Ernst, 1977). Recent work conducted by Fetterer, Pax and Bennett (1980a) has demonstrated that a well defined tegumental potential (Eteg) of about -60 mV exists that can be altered by changing physical and/or chemical qualities of the 13 parasite's environment (Figure 4). Elevated concentrations of external K+, reduced Ca2+, cardiac glycosides, and reduced temperature all induce depolariza- tion of the tegument, indicating that some form of active Na+-K+ transport may be involved in maintaining this potential (Fetterer at a_l., 1980a). Fetterer g al. (1980a) employed iontophoretic injection of horseradish peroxidase (HRP) as a marker for determining the anatomical location of the recording electrode while gathering these data. During all penetrations in which potentials in the range of -60 mV were recorded, HRP was subsequently localized in only those compart- ments directly associated with the tegument (e.g., the outer tegument itself, cytoplasmic channels or tegumental cytons). Recordings made in the tegument of Imanesthetized parasites revealed spontaneous depolarizations that ranged from 3 to 15 mV in amplitude with 20 to 100 msec durations. Most of this activity was abolished by anesthesia with sodium pentobarbital or carbamylcholine (Fetterer g a_l., 1980a). Other evidence suggesting that the tegument may actively propagate electrical activity has been reported in microelectrode (Thompson, Pax and Bennett, 1982a) and surface electrical (Fetterer, Pax and Bennett, 1977; Semeyn, Pax and Bennett, 1982) recordings. Alternatively, the electrical transients recorded in these studies could be volume conducted depolarizations of the imderlying muscle elements which, based on microelectrophysiological experiments, are electrically coupled to the tegumental syncytium, with a coupling ratio of approximately 0.85 (Thompson e_t a_l., 1982). A possible morphological substrate for electrical coupling between these tissues has been observed in electron micrographs. Plasma membranes of integumentary cells appear to form junctional complexes with neighboring muscle cells that closely resemble those fomd connecting smooth muscle cells (Silk g a}, 1969). 14 1 M Figure 4. Potential profile obtained as a microelectrode is advanced into an adult male S. mansoni. The initial vertical drop represents penetration of the tegument. The first upward deflection represents Em , and the second upward deflection E3. ' Calibration: vertical bar equals 10 a??? horizontal bar equals 2 sec. 15 4. The musculature In general, the musculature of the schitsosome resembles that of most invertebrate muscle, appearing to be of the smooth variety (Lowy and Hansen, 1962). It is located immediately beneath the basement membrane of the tegument and consists primarily of an outer circular, and inner longitudinal muscle layer. Radially oriented muscle fibers are also present, but are well developed only in the proximal and distal areas of the acetabulum (Silk and Spence, 1969a). The female musculature is not as well developed as that of the male (Smith at a_l., 1969; Silk and Spence, 1969a). In the adult parasites, the longitudinal muscle layer is most prominent, especially on the dorsal surface (Smith 215110 1969). Ultrastructural studies show the myofibrils to consist of arrays of thick (18—40 nm diameter) myofilaments surrounded by a relatively large number (8-14) of thin (5 nm diameter) filaments (Silk and Spence, 1969a). This ratio of thin to thick filaments resembles that normally observed in vertebrate smooth muscle (Perry and Grand, 1979). The thin filaments show considerable branching and cross-linking between thick and thin filaments, while the thick filaments are arranged primarily in parallel arrays (Silk and Spence, 1969a). Ovoid nuclei are normally separate from and located deeper than the muscle fiber bundles, and are connected to them via cytoplasmic processes. - The sarcoplasmic reticulum is poorly developed or absent, but rough elements can be found at scattered intervals. There is also a lack of transverse tubules and microtubules, which is characteristic of other smooth muscles. Mitochondria appear in sac-like distentions of the sarcoplasm along myofibril bimdles. Lipid globules as well as alpha- and beta-glycogen particles are distributed throughout the muscle cells (Silk and Spence, 1969a). 16 Jimctional compelxes are observed between muscle cells, with the outer layers of adjoining sarcolemmas being closely apposed at distances of 7 to 9 nm. These junctions are of variable length, but seldom cover the total length of apposing sarcolemmas. Similar junctional complexes exist between muscle cells and the tegumental cytons (Figure 3) (Silk and Spence, 1969a). 5. Physiolog of the musculature Fetterer a; a1. (1977) have developed a technique for monitoring schistosome muscle activity directly, employing suction pipettes in circuit with a force transducer. This method is, in some instances, as much as 100X more sensitive than those previously described for measuring mechanical responses of the parasite. In addition to serving as a sensitive monitor for the effects of various pharmacological agents, this method has demonstrated that the contrac- tile properties of the schistosome musculature are much like those reported for other invertebrate as well as vertebrate muscle preparations. For example, elevated concentrations of K+ induce a pronounced, well maintained contracture (Fetterer e_t a_l., 1977) and hyperosmotic sucrose solutions induce paralysis (Pax at a_l., 1981). Bricker, Pax and Bennett (1982), using iontophoretically injected HRP as a marker, have recently identified a second compartment of electrical potential in the schistosome which is endemic to muscle tissue. Resting membrane potentials recorded intramuscularly are generally about -30 mV, or about one-half the magnitude of the potential recorded in the overlying tegumen- tal regions (Figure 4) (Bricker e_t Q” 1982; Thompson SE a_l., 1982). Electrophysio- logical recordings using multiple electrodes have revealed that the muscle compartment in schistosomes, like the tegument, is an electrical syncytium, and that low resistance pathways connect these two tissue compartments in a nonrectifying manner (Thompson 3 a_l., 1982). 17 6. The nervous ystem The nervous system of Schistosoma mansoni follows the same basic pattern of other trematodes as has been described by Bullock and Horridge (1965). Cholinesterase staining reveals the nervous system to consist of two pairs of anteriorly located central ganglia lying on either side of the esophagus which are joined by circumesophageal commissures. Two major pairs of nerve trunks, dorsal and ventral, extend longitudinally from the ganglia to both the anterior and posterior portions of the parasite (Fripp, 1967a). The nerve cords follow the lateral contours of the animal and are located within the parenchyma. The cords converge at the posterior end of the animal, and are connected at intervals throughout their length by numerous dorsal and ventral transverse commissures. Small transverse branches of the cords project peripherally and terminate near the subtegumentally located muscle layers. Although no direct innervation of the tegument or the region immediately below the tegument has been reported, presumed sensory structures located within the tegument have been described (Morris and Threadgold, 196?; Silk and Spence, 1969b). Anteriorly, a small nerve cord projects from the central ganglion to innervate the oral sucker, and two branches of the ventral nerve cords innervate the ventral sucker (Fripp, 1967a). The primary study describing the ultrastructure of the nervous system of the schistosome was conducted by Silk and Spence (1969b). The central ganglia contain cells with large nuclei and prominent nucleoli. These cells give rise to groups of closely packed non-myelinated axons of variable shape, size and content. Adjacent axolemmas are separated from each other, the surrounding musculature, and other parenchymal cells by a 11-12 nm uniform layer of weakly osmOphilic cement substance. A variety of organelles are present within the predominantly electron-lucent ax0plasm. Large, dense granules of 100-160 nm enclosed by a single membrane which may be neurosecretory in nature are often 18 seen. These granules are often present in single rows or small aggregations within nerve processes investing muscle or other cells. Large, ovoid clear axoplasmic vesicles 50-150 nm in length often accompany these large granules. Other dense osmophilic granules 30-90 nm in diameter found within the axoplasm appear to be similar to those believed to contain catecholamines in other neural tissues. This type of granule is often found in association with collections of clear synaptic vesicles 20-50 nm in diameter. Other osmophilic granules found within the axoplasm include stellate clusters resembling alpha-glycogen and individual 20 nm granules with the apeparance of beta-glycogen. Synapses between axons are observed within the central ganglia and in other regions of the nervous system. These synapses are characterized by accumulations of the clear, 20-50 nm diameter type vesicles attached to the presynaptic membrane, which is separated from the postsynaptic membrane by a 10-20 nm synaptic cleft. Postsynaptic membranes are relatively devoid of cytoplasmic inclusions and appear more thickened and osmophilic than the presynaptic membranes. Neuromuscular jtmctions are essentially similar to the axo-axonal synapses just described. The junctional cleft is approximately 10 nm wide and is symmetrically thickened. Postsynaptic elements consist of broadened, osmophilic sarcolemmas of the adjoining muscle cells. hi addition to the clear synaptic vesicles associated with the smaller dense granules, the large osmophilic neuro- secretory granules and their associated clear axOplasmic vesicles are also observed within the sarcoplasm of the muscle cells. 7. Schistosome neurobiology A number of compmmds known to fimction as neurotransmitters in other nervous system have been identified via biochemical and histochemical techniques in the schistosome. In particular, acetylcholine, serotonin (5-hydroxy 19 tryptamine, S-HT), dopamine and norepinephrine have been identified and impli- cated to be serving as neurotransmtiter substances in S. mansoni. Acetylcholine. The identification of acetylcholinesterase in 1952 by Bueding, and later its histochemical localization within the nervous system of adult schistosomes (Frip, 1967b), led early on to the suggestion that acetylcholine may play an important physiological role in this parasite. Acetylcholinesterase staining techniques have also been utilized to study the nervous system of immature stages of S. mansoni (Bruckner and Voge, 1974). The characteristics of acetylcholinesterase present in four species of schistosomes have been compared by Gear and Fripp (1974). A personal communication appearing in Bueding's 1952 article reported the presence in S. mansoni of choline acetyltransferase, the enzyme which catalyzes the synthesis of acetylcholine from the precursors acetyl-CoA and choline. Numerous studies have presented evidence indicating the presence of cholinergic receptors in schistosomes. However, it has not been possible to characterize these receptors pharmacologically as being either nicotinic or muscarinic based on experiments observing parasite motility (Barker 3 a1., 1966). Cholinomimetic agents such as the non-hydrolyzable agonist carbamylcholine (carbachol) have been reported to inhibit spontaneous contractions, decrease circular and longitudinal muscle tone, and decrease the level of spontaneous electrical activity recorded from the surface of the parasite (Barker, Bueding and Timms, 1966; Hillman and Senft, 1973; Fetterer e_t_ g” 1977; Semeyn g g” 1982; Siefker, Pax and Bennett, 1983; Pax a; a_l., 1984). Carbachol and the cholinester- ase inhibitors eserine (physostigmine) and metrifonate, block electrically-induced contractions of longitudinal musculature (Pax 91 a_l., 1981). Significant inhibition of cholinesterases present in extracts of adult male and female S. mansoni has been demonstrated by eserine, metrifonate and dichlorvos (Bueding, Liu and 20 Rogers, 1972; Gear, 1976). The antischistosomals hycanthone and praziquantel have also been found to be effective inhibitors of parasite cholinesterases (Kim 32 a}, 1981). Exposure of S. mansoni to the muscarinic receptor antagonist atropine induces a marked increase in contractile activity, suggesting the existence of an endogenous excitatory neurotransmitter whose actions are modulated by acetyl- choline (Barker g a_l., 1966). These effects of cholinergic compounds have led to the hypothesis that acetylcholine may be functioning as an inhibitory transmitter in this parasite. The presence of specific cholinergic receptors in S. mansoni has been suggested based on binding experiments employing dansylated choline derivatives as fluorescent markers (Hillman and Gibler, 1975; Hillman, Gibler and Anderson, 1978; Willcockson, Ahmed and Hillman, 1983). However, dansyl choline has been reported by Tomosky-Sykes and Bueding (1977) to lack specificity for cholinergic receptors, based on their findings demonstrating the inability of any cholinergic compOImd to effectively antagonize its binding to tissues, and because identical fluorescence staining patterns were observed with the non-cholinergic compmmds dansic acid and dansyl chloride. Characterization and localization of specific cholinergic receptor sites within the parasite employing more conventional receptor-binding techniques have yet to be reported in the literature. Catecholamines. The presence of the catecholamines dopamine and norepinephrine within S. mansoni have been demonstrated by fluorescence histo- chemistry and radioenzymatic techniques (Bennett and Bueding, 1971; Chou, Bennett and Bueding, 1972; Machado, Machado and Pellegrino, 1972; Gianutsos and Bennett, 1977). Bennett and Bueding have presented evidence that schisto- somes are able to synthesize dopamine from the precursor L-DOPA, indicating the parasite possesses a L-aromatic acid decarboxylase similar to that found in vertebrate species. The existence of such an enzyme in homogenates of adult and 21 larVal schistosomes has been confirmed by Catto (1981). Other steps of the catecholamine synthetic pathway have not been examined in this parasite. Schistosomes appear to obtain at least a portion of their norepinephrine from the host's plasma via an uptake mechanism specific for this catecholamine (Bennett and Bueding, 1973). Monoamine oxidase (MAO), one of the enzymes involved with degradation of biogenic amines, has been identified in schistosome homogenates (Nimmo-Smith and Raison, 1968). Exposure of schistosomes to dopamine or epinephrine produces a characteristic lengthening response of the animal's musculature with a concurrent decrease in spontaneous contractile activity, effects believed to be mediated via catecholaminergic receptors located on the parasite's musculature (Tomosky, Bennett and Bueding, 1974). The catecholaminergic compounds epinephrine and apomorphine produce similar responses, while oct0pamine, clonidine and isopro- terenol have no effect on the contractile activity of the animal. Alpha- and beta- adrenergic blocking agents produce, at best, only a partial antagonism of the lengthening responses produced by dopamine, norepinephrine, epinephrine or apomorphine. Spontaneous surface electrical activity is significantly decreased by concentrations of dopamine as low as 1x10"8 M (Semeyn _e_t; a1” 1982), even though no effect on motility is observed at this dose. The existence of catecholaminergic receptors in the parasite is to date based solely on pharmacological studies examining motility responses and surface electrical activity. Studies concerning the characterization and localization of specific catecholamine receptors have not been reported. S-Hydroxytryptamine. The identification of serotonin (5-HT) within schistosomes was first reported by Bennett 3 a1. (1969). The distribution of serotonin within the schistosome has been revealed using fluorescence histo- chemistry (Bennett and Bueding, 1971). The ultrastructural localization of 22 radiolabeled S-HT in adult S. mansoni was reported by Dei-Cas at al_. (1979). Tritiated 5-HT was found primarily in three areas of the parasite: (1) at the level of the central ganglia, (2) directly beneath the longitudinal muscle layer, and (3) within the major nerve trunks. No label was observed within the muscle fibers or the digestive and excretory organs. Synthesis of 5-HT' from tryptophan via the enzyme tryptophan hydroxylase could not be demonstrated, although it appears the schistosome is capable of decarboxylating S-hydroxytryptophan (S-HTP) to form 5-HT (Bennett and Bueding, 1973; Catto, 1981). In addition to S-HT, schistosomes are capable of taking up tryptamine and 5-HTP from the surrounding medium (Bennett and Bueding, 1973; Bueding _e_t g, 1974; Chou e_t_ al., 1973). A similar uptake mechanism apepars to be present in the schistosomulum stage of S. mansoni (Catto and Ottsesen, 1979). Both 5-HT and tryptamine have been shown to be good substrates for monoamine oxidase present in homogenates of adult S. mansoni (Nimmo-Smith and Raison, 1968). Tryptamine was reported to be deaminated at a rate five times greater than S-HT by this degradative enzyme. Deamination of 5-HT by schistosome MAO was more sensitive to inhibition by nialamide and iproniazid than was tryptamine, while the opposite was found with the MAO inhibitor phenelzine. Several observations have led to the suggestion that 5-HT, or a closely related compound, may be acting as an excitatory neurotransmitter in this parasite. A number of studies hve demonstrated the ability of S-H'I'P, 5-HT and tryptamine to increase the level of contractile activity in adult male schistosomes (Barker at a}, 1966; Tomosky e_t_ al_., 1974; Fetterer 93. al_., 1977 ; Willcockson and Hillman, 1984), as well as in schistosomules (Catto and Ottsen, 1979). Fluoxetine and imipramine, two compatmds known to inhibit the 5-HT reuptake mechanism in mammalian central nervous system neurons (Fuller and Wong, 1977), produce decreases in longitudinal muscle tension and spontaneous contractile activity 23 (Pax, Fetterer and Bennett, 1979). 5-l-lT also produces an increase in the level of spontaneous surface electrical activity which corresponds with the increased mechanical activity observed in response to this amine (Semeyn at g, 1982; Thompson at a_l., 1982). It should be noted that although these putative neurotransmitters appear most concentrated in or aromd neural tissues, their distribution within this parasite is not limited to the nervous system. Whether or not these substances act directly on the musculature or on structures which innervate the musculature is difficult to determine with presently available experimental techniques. It is possible that these substances may have fimctions in addition to neurotransmis- sion. For example, the catecholamine dopamine is a good substrate for phenoloxi- dase, and may be involved with regulation of egg production by the female (Seed and Bennett, 1978). 8. En_ergy metabolism The outstanding feature of carbohydrate metabolism in the adult schistosome is the rapid rate of glucose uptake and utilization, and the subsequent high levels of lactic acid produced. The adult may use up to 26% of its dry weight in glucose per hour in lactate fermentation (Bueding, 1950). Most energy needs of the adult schistosome are met anaerobically, so the adult stage does not appear to have a strict requirment for oxygen in energy production. Evidence for the lack of a role for oxygen in the energy metabolism of adult parasites is presented primarily in the work of Bueding _e_t_ al. (1950, 1959, 1969, 1972, 1982) and Schiller Si a_l. (1975). Their studies have shown that the survival of S. mansoni in; Lita-g is not adversely affected by the absence of oxygen (Schiller 3 al_., 1975; Bueding 3 al_., 1972). Glucose utilization and production of lactic acid has been shown to be similar under aerobic and anaerobic conditions, indicating the absence of a Pasteur effect. Furthermore, ATP levels in the parasite are not reduced 24 following incubation under nitrogen, even after periods of increased carbohydrate metabolism induced by exposure to 5-HT (Bueding, 1972). In previously tested vertebrate and invertebrate preparations that show a Pasteur effect, phoshofruc- tokinase (PFK) activity, a rate-limiting factor in glycolysis, is inhibited by citrate, an important intermediate of respiration (Passeneau and Lowry, 1964). It is believed that the mechanism underlying the Pasteur effect is based on the reduction of citrate brought about by anaerobiosis and the sbuseuent disinhibition of PFK. Unless the organism possesses a cyanide-insensitive terminal oxidase, a Pasteur effect is also brought about by inhibition of the terminal oxidase of electron transport by cyanide. Alternate oxidases have not been reported in schistosomes (Coles, 1972b). Production of ATP by glycoslysis is inhibited in schistosomes by the presence of trivalent antimonials (Coles, 1973). Schistosomes exposed :12 33532 or _ia _v_iv_o to trivalent antimonials show elevated levels of glucose-6-phosphate and fructose-6-phosphate, and reduced levels of fructose-1,6-diphosphate. These sugar phosphate levels return to normal when the drug is withdrawn or the worms are transferred to untreated hosts (Bueding and Fisher, 1966). This information has been used to argue that antimony compounds are toxic to schistosomes because of their inhibition of PFK (Bueding and Fisher, 1966; Bueding, 1959). More recent studies using worm extracts have confirmed this view (Coles and Chappell, 1979). Shen _e_t_ a_l_. (1959) have demonstrated that antimonials also inhibit glycolysis in S. japonicum. Their studies, however, indicate that these compounds may also inhibit schistosome glutamic-pyruvic transaminase, suggest- ing another possible mode of action for antimonials. OBJECTTVES The general goal of the present study is to gain a better understanding of the physiology of the schistosome's cholienrgic nervous system. To date, most research concerning the neurobiology of S. mansoni has focused on identification and localization of putative neurotransmitter substances, and the subjective evaluation of their actions on parasite motility. This is especially true with regard to the cholinergic component of the parasite's nervous system. While histochemical studies have identified enzymes associated with acetylcholine metabolism within the animal's neural elements, few investigations have evalu- ated the physiological actions of Cholinomimetic agents with other than subjective methods. One major objective of the present study is, therefore, to systematical- ly evaluate the physiological responses of S. mansoni to cholinergic agonists and antagonists in an objective and quantifiable manner. This will be accomplished by examining the effects of these compounds on contractile activity, longitudinal muscle tension, and membrane potentials. A second major objective of the present study is to examine the ionic basis of cholinergically-induced alterations in parasite motility. Cholinergic compounds are known to influence ion movement across biological membranes in numerous other vertebrate and invertebrate preparations. However, studies addressing cholinergically-induced alterations of ion movement in schistosomes are non- existent. A final major objective of the present study is to gain information concerning the cellular mechanisms by which cholinergic compounds may be 25 26 modulating parasite motility. Many neurotransmitter substances are currently thought to affect ion movement in other tissues by modulation of various ion channels, as well as membrane-bound ion transport mechanisms. Similar actions of cholinergic compounds in S. mansoni have not been investigated. The present study is divided into two sections, each of which addresses one or more of the preceding objectives. Each section contains a Summary, Introduction, Materials and Methods, Results and Discussion. It is hoped that the information obtained through these studies will make possible increasingly ration- al approaches to the development of safe and efficacious antichistosomal drugs, and ultimately the clinical control of the disease schistosomiasis. SECTION I PhysiolOJical Responses to Cholinergic Agonists and Antagonists in Adult Male Schistosoma mansoni I. Summary The physiological effects of various cholinergic compmmds were evaluated in adult male Schistosoma mansoni. Agonists producing significant time- and dose-dependent decreases in muscle tension and contractile activity included arecoline, carbachol, eserine, nicotine, butyrylcholine, propionylcholine and the cholinesterase inhibitor eserine. Unlike the other agonists evaluated, the nicoti- nic compound lobeline produced significant time- and dose-dependent increases in resting tension and contractile activity. Antagonists producing significant in- creases in resting tension, and in some cases contractile activity, included atropine, benactyzine, benzoquinonium, d-tubocurarine, gallamine, propantheline and scopolamine. Several nicotinic and muscarinic antagonists were tested for their ability to block the inhibitory effects of the agonists arecoline, carbachol, eserine and nicotine. Benzoquinonium significantly attenuated the actions of carbachol and eserine. Two muscarinic antagonists were found to be effective. Atropine attenuated the actions of carbachol and eserine, while benactyzine attenuated the actions of all four agonists tested. Propantheline, a mixed- function antagonist, inhibited the actions of all agonists but arecoline. The effects of Cholinomimetic agents on the parasite's tegumental and muscle membrane potentials were also investigated. Arecoline, carbachol, 27 28 eserine and nicotine produced hyperpolarization of these membranes, while benactyzine and benzoquinonium produced depolarization. Mechanical and membrane responses to inhibitory agonists were evaluated in the presence of altered external ion concentrations. Exposure to 0 mM K+, 60 2' 2'4’, 30 mM Na+ or 0 mM Ca2+/30 mM mM K+, 40 mM of, 0 mM Mg *, 0 mM Ca Na+ media for 30 seconds did not significantly alter the time course or magnitude of mechanical responses. Membrane potentials were not significantly altered in response to these agents in the presence of 0 mM K+, 60 mM K+ or 40 mM Cl- media. The energy-dependence of mechanical responses to inhibitory agents was evaluated by pretreatment of parasites with low temperature (5°C) or antimony potassium tartrate. These pretreatments either blocked or significantly attenu- ated responses to these agents. Mechanical responses to various ion channel blockers and activators were also examined. The ability of these compounds to mimic or alter responses to inhibitory cholinomimetics was evaluated. The results of these experiments indicate that inhibitory cholinergic agents appear to exert their effects in this parasite through mechanisms which are largely energey-dependent. While membrane permeability to several iOns may be altered by cholinergic agents, it appears that Ca2+ may be the one of most importance. 11. Introduction The presence of acetylcholinesterase (Bueding, 1952), and later the histo- chemical localization of this enzyme within the nervous system of immature and adult schistosome (Fripp, 1967a) suggests that acetylcholine may be playing an important physiological role in this parasite. Numerous studies have presented 29 evidence supporting the existence of cholinergic receptors within schistosomes, specifically Schistosoma mansoni. Cholinomimetic agents such as carbachol have been reported to inhibit spontaneous contractile activity (Barker at al_., 1966; Hillman and Senft, 1973), decrease longitudinal and circular muscle tension (Fetterer at; g” 1977; Pax g a}, 1981; Depenbusch e_t_ a}, 1983; Pax 2121.» 1984), reduce the level of spontaneous electrical activity recorded from the surface of the parasite (Semeyn fl a_l., 1982; Thompson 3 al_., 1982; Mellin _e_t; al_., 1983) and produce hyperpolarization of the parasite's membranes (Bricker, 1981). Eserine, metrifonate and carbachol block electrically-induced contractions of the longitu- dinal musculature in adult male schistosomes (Pax a: a_l., 1981). In the studies of Barker 3 a_l. (1966), observational methods were used to investigate motility responses of S. mansoni to a variety of “nicotinic” and "muscarinic” cholinomime- tic agents as well as several cholinesterase inhibitors. These experiments demonstrated paralysis of the parasite's musculature by a number of these compounds. Furthermore, several cholinergic antagonists were shown to have the ability to partially reverse the paralysis induced by these agents. These effects indicate that acetylcholine, or some acetylcholine-like compound may be acting as an inhibitory modulator of contractile activity in schistosomes. In this section the results of experiments designed to quantifiably evaluate two important physiological responses of adult male Schistosoma mansoni to various "nicotinic", "muscarinic" and "mixed" cholinergic agonists and antagonists are reported. By quantitatively analyzing the manner in which these agents affect contractile activity, as well as their effects on the parasite's membrane potentials, we have attempted to more clearly understand the function of the putative cholinergic transmitter in this parasite. The results of these studies indicate that one action of inhibitory cholinomimetic agents may be to alter calcium ion availability to contractile elements within the parasite's musculature. 30 111. Materials and Methods A. Experimental Animals and Media 1. Source and Maintenance of Animals Female laboratory mice (Mus musculus) injected intraperitoneal- 1y with 200-300 cercariae of Schistosoma mansoni (St. Lucian strain) were obtained from the laboratory of Dr. J.L. Bennett, Department of Pharmacology and Toxicology, Michigan State University. Mature parasites were dissected from the portal and mesenteric veins of the mice 45-60 days post-infection as previously described (Bennett and Seed, 1977). Paired schistosomes were maintained at 37°C in either Hank's balanced salt solution (HBS; final 2+ 2 concentrations of constituents: Na+ 138 mM, K+ 5.9 mM, Ca 1.4 mM, Mg + 0.5 mM, p0 0.5 mM, 01' 147 mM, so 0.5 mM, glucose 5 mM) or RPMI-164O (Grand 4 4 Island Biological; see addendum for composition). Both media were buffered at pH 7.4 with 20 mM Hepes (N-2-hydroxyethylpiperazine N-Z-ethane sulphonic acid, Sigma Chemical Co.) and contained 100 units/ml penicillin-streptomycin (GIBCO). Parasites were used within 8 hours following removal from mice. 2. Recording Media Either HBS or RPMI-l640 was utilized as the recording medium in all experiments. The effects of altered external ion concentrations on mechanical activity and membrane potentials were determined by measuring responses from parasites bathed in normal HBS, and then exchanging the HBS for a modified saline containing the altered ion concentration. The manner in which the HBS was altered differed for each of the ions studied but in general the methods of Fetterer g g. (1978) were followed. Potassium: Concentrations of 0 and 60 mM were used. The concentration of potassium in the modified HBS was adjusted by adding or 31 deleting KCl. The chloride concentration was held constant by adding or deleting NaCl. Chloride: A chloride concentration of 44 mM was used. The chloride concentration was altered by decreasing NaCl and replacing Na+ with NazSO4. Magaesium: A 0 mM Mg2+ concentration was used. This solution was obtained by deleting MgSO4. _S_oajliga: A sodium concentration of 30 mM was obtained by lowering the NaCl concentration and adding Tris HCl (2-amino-2(hydroxymethyl)- 1,3-pr0pandiol; Sigma Chemical Co.) as a substitute. The concentration of other constituents was the same as normal HBS except that Hepes was omitted and Tris was used as the buffer (pH 7.4). 2 Calcium: A 0 mM Ca + HBS was obtained by deleting CaCl 4 2. This solution also contained 5x10- M EGTA (ethyleneglycol-bis( B-amino-ethyl- ether)-N,N,N',N'-tetraacetic acid; Sigma Chemical Co.). 2 Low Sodiumjo Calcium: A 30 mM Na+IO mM Ca + HBS was obtained by preparing 30 mM Na+ HBS without adding CaCl EGTA (5x10-4M) 2' was also added to this solution. Cobalt and lanthanum ions were introduced in the form of chloride salts (Fischer Scientific). B. Mechanical Activity RecordLan Recording of longitudinal muscle tension and contractile activity was performed as previously described by Fetterer at a_l. (1977, 1978). Briefly, worms were placed in a recording chamber containing 2.5 ml medium maintained at 37°C. Tension was measured by means of suction pipets made of polyethylene tubing (i.d. 0.38 mm; o.d. 1.0 mm) drawn out to give an inside tip diameter of 125 um. A nonflexible pipet was attached at the tail end of the parasite. A second flexible 32 pipet was attached 1.5 to 2.0 mm anterior to the nonflexible pipet. In this way any shortening movements between the pipets caused movement of the flexible pipet relative to the nonflexible one. The movement was monitored by means of a modified Narco Bio-Systems (Houston, TX) "A" myograph in which the steel spring and shutter were removed and replaced by a balance arm system constructed from 0.25 mm steel wire and an acetate shutter. A fine steel wire (0.25 mm) attached near the tip of the flexible pipet transmitted movments to the balance arm system and acetate shutter (Figure 5). The system was calibrated by adding known weights to the balance arm and determining pipet deflection as manifested by pen deflections on the chart recorder. All measurements of contractile activity were made with a 4 mg load on the balance arm. The force exerted by the parasite's musculature depended on the length of warm over which the measurements were taken. Since this length varied from animal to animal, all tension measurements were referenced to the amount of tension developed over a 1 mm length of warm by dividing the measured tension change in each case by the length of worm over which the measurements were taken. After the initial hook-up of parasites to the recording system, a 10-minute equilibration period was allowed before the application of any treatment. The effect of lowered temperature on contractile activity responses was determined in animals bathed in HBS. The temperature of the bathing media was altered by methods previously described (Fetterer 9; al_., 1978). Unless otherwise noted, data concerning contractile activity responses to pharmacological agents, presented in tables and elsewhere in the text, give the change in tension 10 minutes after addition of the agent as compared to the tension just prior to its introduction or, for controls, addition of vehicle alone. In cases where two or more treatments were given sequentially, data presented 33 480808 08003033 308 #390 8 8383 0338 "0885383 080 53300 0:30.588 c.8008 3 000: 03080990 mo 8030800080.“ «0 03080nom .m 08mg 34 m 290$ 09. n6 05(04— I 5.230.3/ 8.833. - _TJ _ \\ p03 0.— 1 Fr, a q TX ” XNIIIL 3:23 <11 4/ 32.8: >235; 35 represent the change in tension after 10 minutes exposure to the second treatment as compared to the tension just prior to introduction of the second treatment. C. Microelectrode Recordiiiga Microelectrodes were made from 1.5 mm capillary tubing (Kwik Fill; WP Instruments, New Haven, CT) with a horizontal electrode puller (Narashige Instruments). Electrodes prepared in this manner were filled with 3 M potassium chloride and had resistances of 15-30 megaohms. A Leitz micromanipulator was used to position the electrodes. The electrodes were connected via a silver wire and lead wire to a preamplifier (M-4A, WP Instruments) and the resulting signal displayed on an oscilloscope (Tektronix 5118) and chart recorder (Gould Model 220). A silver/silver chloride wire or a KCl/agar bridge placed in the recording bath served as ground. The recording chamber consisted of a 10 ml glass petri dish, the bottom of which was lined with Sylgard resin (Dow Corning, Midland, MI). The temperature of solutions placed in the chamber was maintained at 37°C with a thermoelectric heater placed under the recording chamber. Temperature of solutions in the chamber was monitored with a thermistor. Resting tegumental and muscle membrane potentials were measured by penetrating the dorsal surface of parasites immobilized in HBS or RPMI-1640 containing 50 mg% sodium pentobarbital (Sigma Chemical Co.) as previously described by Thompson g__t_ a_l_. (1982). A minimum of 10 penetrations along the length of the dorsal surface of each parasite were made during the control period. The test drug was then added, and after 10 minutes exposure, a minimum of 10 additional penetrations per worm were made. 36 D. Pharmacoflical Agents Cholinergic agents: Drugs used were acetylcholine chloride (ACh), arecoline hydrobromide (Arec), atropine sulfate (Atro), benactyzine (benzilic acid B-diethylaminoethyl ester; Ben), benzoquinonium choline (qu), benzoylcholine chloride (Bcc), butyrylcholine chloride (Btc), carbachol (carbamylcholine chloride; Carb), eserine sulfate (Eser), gallamine triethiodide (Gal), hexamethnonium bro- mide (Hex), lobeline hydrochloride (Lob), muscarine chloride (Musc), methacholine (acetyl-B-methylcholine; Mech), nicotine hydrogen tartrate (Nict), oxotremorine sesquifumarate (Oxo), pilocarpine hydrochloride (Pilo), propantheline bromide (Prop), propionylcholine chloride (PCC), scopolamine chloride (Scop) and succinyl- choline chloride (Scc), all from Sigma Chemical Co. Ion channel blockers and activators: Compounds used were A-23187, 4-aminopyridine, D-600, diltiazem, funarazine, nicardipine, nifedipine, tetraethyl- ammonium chloride (TEA), tetrodotoxin (TTX), verapamil, veratridine (all from Sigma Chemical Co.) and Bay K-8644 (Bayer Pharmaceuticals, Germany). 3"; Fisher Scientific, Fairlawn, NJ) Antimony potassium tartrate (Sb was used as an inhibitor of schistosome glycolysis. All drugs were dissolved immediately prior to use in either double glass distilled water (D.W.), 100% dimethylsulfoxide (DMSO; Sigma Chemical Co.) or a 50/50 v/v mixture of D.W.lDMSO, and added to the recording chamber in 25 111 aliquots to achieve the final concentration desired. Vehicle control trials were performed by adding 25 ul aliquots of the appropriate drug solvent to the recording chamber. E. Statistical Procedures All data are given as the mean i 1 standard error of the mean (SEM). Unless otherwise noted, a minimum of six replications was used to determine each data point. All tests for significance of difference between means were 37 performed using Students' i test (two-tailed), or ANOVA with Dunnett's i-test where appropriate. IV. Results A. Mechanical Activity 1. Normal contractile activity Parasites incubated in either HBS or RPMI-1640 exhibited con- tractions ranging in size from those just detectable (shortenings as little as 0.02 mm) up to levels representing decreases in length of about 20% (shortenings as great as 0.4 mm). Large contractions appeared alone or consisted of a variable number of superimposed smaller contractions. Smaller contractions also often appeared alone. The rate at which these contractions occurred varied over time in any particular animal, with periods of relatively lesser activity interrupted by periods of greater activity. In general, there was little or no shift in tonus over the duration of control recordings (Figures 6, 7, and 8). 2. Response to nicotinic compounds Agonists. Nicotine produced rapid time- and dose-dependent decreases in both muscle tension and contractile activity. Significant effects were seen at concentrations of 1x10-6M or greater (Table 1). Approximately 2 to 4 minutes following exposure to 1x10"4 or lxlo-SM nicotine, isolated rapid contractions developed and occurred with a frequency of about 10 per minute (Figure 6). Contractions of this type were not observed in parasites exposed to 1x10-6M nicotine. In contrast to the tension decreasing effects of nicotine, lobeline increased longitudinal muscle tension and contractile activity (Table 1). 4 At a concentration of 1x10- M, lobeline induced a rapid tonic contraction of the parasite's musculature that was maintained for at least 30 minutes (Figure 7). At 38 D. W. t AREC NICT Figure 6. Sigiart recordings showing the effects of _2‘5 111 of distilled water (upper trace) 1x10 M arecoline (middle trace), and 1x10 M nicotine (lower trace) on muscle tension and contractile activity in adult male S. mansoni incubated in RPMI-1640. 39 , lOB ‘ 2mg I llmin 1* BEN ‘1 sea Figure 7. art recordings showing the effects of 1 10-4M lobeline (upper trace), 1x10- M benactyzine (middle trace) and 1x10- M benactyzine (lower trace) on muscle tension and contractile activity in adult male S. mansoni incubated in RPMI-l640. 40 (1V! CARI ESE! —" fiv Figure 8. Chart recordings showing the effects of 23 ul of distilled water (upper trace), 1x10 M carbachol (middle trace) and 1x10 M eserine (lower trace) on muscle tension and contractile activity in adult male S. mansoni incubated in RPMI'1640. 41 Table 1 Effect of Nicotinic Compounds on Resting Tension in Adult Male Schistosoma mansoni“ Treatment Tmax (mg) t1 ,2 m ax (secs) CONTROL (D.W.) -0.60:O.31 284:42 AGONISTS Nicotine 10 _:M -5.4z:0.16: 16: 1 10 6_M -3.43I0.zz: 33: 1 10 _M7 -1.47+0.32 d 230124 10 M -0.S4E0.28 278136 Lobeline 10 :‘gM 6. 6310.23: 14: 1 10 _6M 4.35%.“: 34:19 10 _7M 1.70+0.75c 228123 10 M 0.4 4.4I0.z6c 259130 ANTAGONISTS Benzoquinonium 103M 1. 9810.22: 128:22 10 M 0. 54I0.35c 213:33 d-Tubocurarine 10'4M 0.99:0.19a 150113 Gallamine 10'4M 0.63:0.16b 152121 Hexamethonium 10'4M -0.023~_0.23‘l 296:39 a b c d .001 _6). 45 produced by arecoline or nicotine, but was well maintained for periods of at least 60 minutes (Figure 8). At concentrations greater than 1x10-8M, the cholinesterase inhibitor eserine produced significant decreases in tension and abolished contractions (Table 3). Eserine had a much slower onset of action when compared to the other inhibitory agents at equipotent concentrations (Figure 8). 4M the choline derivatives acetylcholine, benzoylcholine and succinyl- At 1x10' choline had minimal effects on resting tension and contractile activity, while propionylcholine and butyrylcholine caused significant decreases in mechanical activity (Tale 3). The effect of propionylcholine was well maintained, while that of butyrylcholine was transient, reaching its maximum at approximately 5 minutes following application and then returning to control levels within 10 minutes. 6 Following pretreatment with lxlO- M eserine (2 minutes exposure) the effects of acetylcholine, butyrylcholine and benzoylcholine became much more pronounced (Table 3). A lO-minute exposure to these agents in the presence of eserine produced decreases in mechanical activity substantially greater than by eserine alone. Eserine pretreatment did not alter the response to succinylcholine. Propantheline, a mixed function antagonist, produced a signifi- cant rise in resting tension at 1x10-4M. This occurred without discernable effects on contractile activity. 5. Ant_agonism of inhibitory effects The various antagonists just described were tested for their ability to alter the mechanical activity responses to arecoline (1x10-6M), carba- chol (1x10-4M), eserine (1x10-5M) and nicotine (1x10-4M). These concentrations of agonists were used because of their equipotent effects on muscle tension and contractile activity. Parasites were pretreated for 10 minutes with the antago- nist prior to addition of the agonist. Longitudinal muscle tension was monitored for 10 minutes following application of the agonist, and the maximum effect in Effect of Cholinergic Antagonists on Resting Tension Responses to Cholinergic Agonists in Adult Male Schistosoma mansoni" 46 Table 4 Treatment Tmax (mg) t1,z max (sec) Control (D.W.) + Arecoline 10:4M -6.10I0.1z 12I 1 + Carbachol 10_5M -5.7410.29 351 7 + Eserine 10_4M -5.4810.29 123114 + Nicotine 10 M -5.4210.l6 161 l Benzoquinonium 10-4141 + Arecoline -5.6510.20b 111 1 + Carbachol -3.2510.55 215138 + Eserine -1.77I0.512 237126 + Nicotine -5.4910.21 161 2 Atrcmme 10’4M + Arecoline -5.8410.73 121 l + Carbachol -z.13I0.412 137I39 + Eserine -0.9410.54 234116 + Nicotine -5.99I0.35 15I z Benamzine 10'4M + Arecoline 45410.44: 266123 + Carbachol -3.41I0.31 224I32 + Eserine -1.43I0.45a 326I40 + Nicotine -1.99I0.53“‘ 14I 1 Prgpantheline 10-41% + Arecoline -5.7810.12b 121 1 + Carbachol -3.6410.15 180142 + Eserine -4.29I0.34c 135I21 + Nicotine -z.05I0.sza 13I 2 3.001 __6). 55 Inorganic agents. A number of inorganic cations can interact with calcium channels in a direct and reversible manner. Cobalt ion (002+) and lanthanum ion (La3+) behave as potent competitive inhibitors of ion permeation through calcium channels, and are known to inhibit ionic currents conducted by these channels (Imai and Takeda, 1967; Godfraind, 1976; Marier, Putney and Van den Walle, 1978). In experiments testing the actions of these ions in schistosomes, control recordings were made from animals in normal HBS. The desired cation was then added to teh recording bath. Cobalt ion (10 mM) produced a gradual increase in muscle tension (1-2 mg) and contractile activity during the first two minutes following application, and then a gradual decrease so that by 10 minutes tension was approximately 1 mg below the original baseline level. A similar pattern of responsiveness was seen with 1 mM Coz+, except that tension and contractile activity remained elevated over the 10-minute test period. Lanthanum ion (10 mM) 'mitially caused a transient increase in muscle tension and contractility following its application, but, in contrast to 2+ Co , then produced a more rapid and significant decrease in muscle tension. Following exposure to 1 mM La3+, no alteration in tension was seen, but contraction amplitudes were somewhat reduced. 2+ channel blockers are considered 2+ _O_rganic agents. Organic Ca to be highly selective compounds for inhibiting Ca influx in numerous cardiac and smooth muscle preparations (Kroeger e_t_ Q" 1975; Cauvin gt 93., 1983; Snyder 2'*'-channel blockers had and Reynolds, 1985; Triggle, 1982). Several of these Ca notable effects on parasite mechanical activity (Figure 10, Table 7). Following addition of 1x10-4M verapamil or its derivative D-600 tension showed a slight initial elevation, but then declined rapidly. Verapamil produced a greater tension decrease than did D-600. Application of these two 56 05 3 @033 0.83 0395.83: 2 no": no 0:398:53 2 :35 5:89.32, 2 .395 65 3. 33.3.3 3 8283 3858 .w sums 33E 3 rumba—om w 3038 amazon—flamed no 29.003 #09530 83013 «o $00.20 mfiBOAm mmszououou «3A0 don—Bonn mfivuouou 2.: 585 a -2: mucouunoo 93 03:3 .3 users 57 wEu S 0.3m; «(U-2 58 drugs also caused contractions, which during the control period were relatively irregular in rate and amplitude, to become very regular and spike-like with greatly increased amplitudes (Figure 10). At 13:10-5 M, both compounds still initially caused tension to increase slightly, but the subsequent decline in tone was of a smaller magnitude and had a more prolonged time-course. The rhythmic, spike-like contractions were not observed following exposure to the lower concentration of either D-600 or verapamil. 4M or 1x10-5M, often Flunarazine, at concentrations of 1x10- produced a triphasic effect on muscle tension. Immediately following its application, a brief (IO-20 second), distinct decrease in tension was often seen. This was followed by a more prolonged slight elevation of tone, which then decayed to levels significantly below baseline. Spike-like contractures similar to those produced by D-600 or verapamil were observed in only one animal (out of 12) treated with fltmarazine. Both concentrations of this drug consistently caused contraction amplitudes to decrease, especially after 5 minutes of exposure. Addition of 1x10-5M nicardipine induced a biphasic effect on muscle tension similar to that of D-600 and verapamil, i.e., in the first two minutes following its application tension was consistently elevated, but would then decline fairly rapidly. Early in the phase of declining muscle tension, spike- like contractions were often seen, but were not as rhythmic or of as great an amplitude as those induced by verapamil or D-600. Contractions seen in the presence of nicardipine often consisted of multiple smaller spikes superimposed upon a more prolonged tension increase, in contrast to the single, well isolated spikes seen with verapamil or D-600. Like flunarazine, nicardipine consistently decreased contraction amplitudes after 5 minutes exposure, and after 10 minutes exposure virtually all active contractions were abolished. 59 Nifedipine (1x10-5M) produced no distinct changes in contractile activity other than a gradual decline in muscle tension. The character of active 2 contractions was unaffected by this Ca + channel blocker. In contrast to the effect of other blocking agents tested, 4M longitudinal muscle tension gradually increased following addition of 1x10- diltiazem and remained significantly elevated during the lO-minute test period. At a concentration of 1x10-5M, diltiazem produced a gradual decline in tension of marginal significance. As with nifedipine, both doses of diltiazem had no effect on the character of active contractions. Compound Bay K-8644, a dihydropyridine-derivative which has been shown to enhance rather than block current flow through calcium channels (Schramm g al_., 1983; Garcia, Sala and Reig, 1984; Kokob1m and Reuter, 1984), also had distinct effects on schistosome mechanical activity. Application of 1x10-'4 5 M Bay K-8644 produced a response identical in charcter to that of 1x10- M nicardipine, i.e., initially tension was elevated followed by a rapid decline with occasional spike-like contractions of progressively smaller amplitude. All active 4M contractions were abolished within 10 minutes following exposure to a 1x10- concentration of this compound. At 1x10-5M, Bay K-8644 did not cause an initial elevation in tension, but did induce a rapid decline in longitudinal muscle tone. Contractions also tended to become more regular and of greater amplitude, but this effect was distinctly different from that seen with D-600 or verapamil. In several animals, 1x10-4M carbachol or 1x10-5M eserine was applied following a lS—minute exposure to 1x10-5 M Bay K-8644. While these cholinergic agents did produce small further decreases in muscle tension, they failed to abolish the active contractions seen in the presence of the Bay compound. 60 10. Effect of A-23187 The free intracellular calcium ion concentration is responsible for determining the contractile state of all muscles. Since modulation of free intracellular Ca2+ concentration is probably the means by which cholinergic agents ultimately influence motility in §. mansoni, I examined responses of adult male worms to cholinergic drugs in the presence of the ionophore A-23187, a compound that can transport cations, Ca2+ in particular, across membranes and increase cytpolasmic Ca2+ concentration (Pressman and Fahim, 1982; Bowman, Rodger and Shahid, 1985). IonOphore A-23187 alone produced dose-dependent increases in tension of the parasite's longitudinal musculature (Figure 11). A concentration of 1x10-5M induced a rapid substantial contracture ”max = 5.80:0.29 mg, p < .001; t1/2max = 57:11 sec) that was well maintained for at least 30 minutes. A 1x10-6M concentration also produced a significant increase in tension, although of a smaller magnitude and slower time-course ”max = 2.82:0.30 mg, .001

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TENSION (mg/mm) Em(mV) 73 50 44 34 24 ‘1 04 04 mi 5 3: 3+ 60 K“ 3+CARs fl j j 5 IO 15 mas (Mm) Figure 14 01 74 Figure 15. Effect of high K+ and D-600 on membrane potentials and longitudinal muscle tension in adult male _S_. mansoni. Parasites were first incq‘bated in normal HBS. At tiquero, the normal HBS was exchanged for 60 mM K HBS. After 10 minutes, 1x10 M D-600 was added to the recording chamber. In the upper panel, closed circles represent tegumental membrane potential and open circles repre- sent muscle membrane potential. Data points in the lower panel represent change in longitudinal muscle tension. Vertical lines represent 3 1 SEM (n36). TENSION (mg/mm) 1b 1'5 2'0 0‘ U! t+ 60 K+ 3+D-600 lb TIME (MIN) Figure 15 76 V. Discussion The presence in schistosomes of a specific acetylcholinesterase (Bueding, 1952), the demonstration of choline acetyltransferase, the enzyme which cata- lyzes the synthesis of acetylcholine from choline and acetate in the presence of ATP and Coenzyme A (Nachmansohn g a_l., 1949), and the apparent presence of acetylcholine in a neutralized perchloric acid extract of _S_. mansoni (Barker e_t_ g, 1966), suggests that acetylcholine may play a functional role in this parasite. Evidence presented in the current study further supports the existence of cholino- ceptive receptors in _S_. mansoni. However, these receptors mediate physiological actions which differ substantially from those mediated by cholinoceptive recep- tors existing in their mammalian host. Furthermore, the receptors present in the schistosome differ in their sensitivity to various cholinergic agonists and antago- nists. Autonomic cholinergically innervated effector organs of vertebrates, i.e., those with the "muscarinic" type of receptor, are stimulated by arecoline, muscarine, methacholine, oxotremorine and pilocarpine, while the parasite's longitudinal musculature is flaccidly paralyzed in response to arecoline, but not by the four other agents. The muscarinic antagonsits atropine and benactyzine produce stimulation of parasite contractile activity and increased longitudinal muscle tone, while scopolamine has marginally similar effects. The autonomic ganglia agonists nicotine and lobeline produce opposite effects on the mechanical activity of §. mansoni. At concentrations as low as 1x10-7M, nicotine produces a substantial decrease in frequency of active contractions and longitudinal muscle tone, whereas lobeline increases contractile activity and muscle tone. These actions of lobeline and nicotine on schistosome motility have not been reported previously in the literature. Of the nicotinic antagonists examined, benzoquino- nium, d-tubocurarine and gallamine produce varying degrees of stimulation and increased longitudinal muscle tension, while hexamethonium is without effect. 77 The non-hydrolyzable cholinergic agonist carbachol, and the cholinesterase inhibi- tor eserine, induce a flaccid paralysis of the parasite's musculature similar to that observed with arecoline. The hydrolyzable choline esters acetylcholine, butyrylcholine, benzoylcho- line and succinylcholine, when administered alone, produce marginal effects on the parasite's mechanical activity (Table 3). However, in the presence of the cholinesterase inhibitor eserine the actions of these compounds become quite pronounced, inducing a flaccid paralysis similar to that produced by arecoline, carbachol and eserine alone. These results support the existence of an active degradative enzyme for choline esters within the animal. Furthermore, the fact that eserine alone induces a significant flaccid paralysis of the longitudinal musculature supports the presence of an endogenous inhibitory cholinergic com- pound within the parasite. Of the various cholinergic antagonists examined only four were found to be effective blockers of the inhibitory responses produced by arecoline, carbachol, eserine and nicotine on mechanical activity (Table 4). The "nicotinic" agent benzoquinonium and the "muscarinic" agent atr0pine significantly attenuated only the responses to carbachol and eserine. Propantheline, a ”mixed“ function cholinergic antagonist, was effective against all agonists tested except arecoline. The "muscarinic" blocker benactyzine was the most effective antagonist tested, being able to significantly attenuate mechanical responses to all four inhibitory agonists examined. The fact that agonists of the same type produce opposite effects on the schistosome's mechanical activity, e.g., nicotine and lobeline, and that the parasite exhibits significant responses to cholinergic agents of the ”muscarinic", "nicotinic" and "mixed" type, suggests that possible presence within the animal of more than one class of cholinergic receptor. These widely differing responses to 78 cholinergic agents of different classes may be a function of the anatomical location of the cholinergic receptors, i.e., peripheral versus central, and/or the ability of the different agents to penetrate the various membranes of the parasite to reach these receptors. Furthermore, these receptors seem to differ substan- tially from those found in mammalian systems, indicating the possibility, at least theoretically, of designing cholinomimetic compounds as therapeutic agents which are highly selective for this parasite. It should also be noted that the cholinergic agonists and antagonists exmained also have a number of non-cholinergic actions in other tissue preparations, e.g., stimulation or inhibition of catecholamine release and local anesthetic properties. Inhibitory cholinergic agents appear to exert their effects through mecha- nisms which are largely energy-dependent. Exposure of parasites to low tempera- ture or preincubation in trivalent antimonials either abolishes or greatly attenu- ates the tension decreasing responses normally observed with arecoline, carba- chol, eserine or eserine plus Ach. Furthermore, the attenuation of the relaxing action of cholinergic agents becomes more pronounced the longer the exposure to antimony (Figure 9, Table 5). Most of the energy needs of the adult schistosome, i.e., production of ATP, are met anaerobically by metabolism of glucose to lactate via the glycolytic pathway. ATP production by glycolysis is blocked in schistosomes in the presence of trivalent antimonials (Coles, 1978) due to inhibition of the enzyme PFK by these compounds (Bueding, 1959; Bueding and Fisher, 1966). Therefore, exposure of parasites to antimonials could be expected to inhibit or slow ATP-dependent processes within the animal, such as active membrane transport mechanisms. Exposure to low temperature is also known to interfere with membrane transport mechanisms (Lehninger, 1975). Evidence that an active transport process, specifically one involved with Ca2+ movement, is important in mediating relaxation of the schistosome's longitudinal musculature 79 has been presented by WoldeMussie g g. (1982). Results presented in the current study indicate that a similar mechanism may also be involved in mediating the inhibitory actions of cholinergic compmmds on the parasite's musculature. An alteration in membrane fluidity, which is a function of lipid composition, may also explain the lack of effect of cholinergic agents in parasites exposed to low temperature media. Responses mediated by muscarinic receptors in "neurone-like" murine neuroblastoma cells (specifically clone NlE-lIS) do not occur below temperatures of 21°C, which is in the vicinity of the transition temperature for lipid bilayers (El-Fakahany and Richelson, 1980). It is interesting to note that schistosomes exposed to 4°C and cholinergic agonists show a marked relaxation of maintained tension when, upon rewarming, the recording bath temperature reaches 20° to 22°C. In examining how cholinergic agents exert their inhibitory actions on schitsosomal longitudinal muscle, many possibilities exist. It is now well established that the free ionized calcium concentration within the sarcoplasm plays a central role in modulating contractile activity in all types of invertebrate and vertebrate muscle. Therefore, in considering the mechanisms by which cholinergic compounds mediate relaxation of the musculature in schistosomes, processes which ultimately bring about a reduction in calcium availability to the contractile elements must be emphasized. The primary mechanisms by which cholinergic receptor stimulation may decrease sarcoplasmic Ca2+ concentration, and thus mediate inhibition of con- tractile activity and reduction of tone, mclude: (a) increased Ca2+ sequestration into intracellular storages sites (e.g., sarcoplasmic reticula and mitochondria); (b) 2+ 2+ decreased Ca influx into schistosome muscle cells; and (c) increased Ca removal from schistosome muscle cells. 80 Ultrastructural studies reveal that the sarc0plasmic reticulum of schistoso- mal muscle is either poorly developed or absent. This would tend to rule out possibility (a) above as being a primary site of cholinergically mediated effects. However, mitochondria do appear to be present in sac-like distentions of the sarcolemma in schistosome muscle cells, and may therefore play a role in cholinergic responses. Decreased Ca2+ influx 'mto schistosome muscle cells could be mediated by cholinergic effects on ion channels located within the parasite's membranes. Cholinergically-induced increases in permeability to K+ and/or Cl- would be expected to cause hyperpolarization of membranes with a subsequent inactivation of voltage-dependent Ca2+ channels, thereby decreasing Ca2+ entrance. Acetyl- choline is known to produce membrane hyperpolarization by altering permeability to K+ and CI. in several other tissues. Iontophoretically applied ACh induces hyperpolarization of neurons in the parietal and visceral ganglia of the snails Helix aspersa or Cryptophallus agiersa by increasing membrane conductance to c1“ (Kerkut _e_t al_., 1975; Chiarandini and Gerschenfeld, 1967). Kehoe (1967, 1972a) demonstrated that ACh produces a dual component inhibitory post-synaptic potential (IPSP) in medial cells of the Aplysia pleural ganglion. The initial (phasic) component was found to be chloride-dependent, while the second, more slowly developing component was identified to be potassium-dependent. In vertebrate cardiac muscle, ACh-induced hyperpolarization results from an in- creased potassium conductance (Krnjevic, 1974). Inhibitory cholinergic agonists could also act by directly decreasing mem- brane permeability to other cations, i.e. Ca2+ and/or Na+. As noted previously, contractile activity in numerous invertebrate and vertebrate muscles is modulated by sarcolemmal action potentials, which are dependent to varying degrees upon sodium and calcium ions (Hagiwara, 1981; Hagiwara and Byerly, l98la,b; Spitzer, 81 1979). Mobilization of intracellular Ca2+ for contractile activity via Nat and/or Cay-induced release mechanisms (so-called ”trigger" Na+ or Ca2+) is also thought to be important in regulation of contractile activity in vertebrate and invertebrate muscle systems (Fabiato, 1981; Miyamoto and Racker, 1980). In several vertebrate cardiac muscle preparations, acetylcholine has been demon- strated to markedly reduce inward currents carried by Cami/Na+ (Giles and Noble, 1976; Giles and Tsien, 1975; Ikemoto and Goto, 1975; Ten Eik gt 9., 1976). Increased competition for Ca2+ binding sites on myofilaments secondary to an 2+ increased concentration of Mg within the sarcoplasm could also be brought about by cholinergic compounds acting to increase sarcolemmal permeability to Mgz+. The third primary way in which cholinergic agents may bring about a decrease in sarcoplasmic Ca2+ concentration in the schistosome is by activating Ca2+ removal mechanisms. Biological membranes appear to have developed two primary means by which to transport calcium ion against its electrochemical gradient: ATP-dependent Ca2+ pumps (Sulakhe and St. Louis, 1980; Schuurmanis- Stekhoven and Bonting, 1981), and Na+/Ca2+ exchange systems (Sulakhe and St. Louis, 1980; Reuter, 1982; van Breemen at al_., 1979; Requena and Mullins, 1979), which utilize the sodium ion electrochemical gradient existing across the mem- brane to transport calcium in the opposite direction. Results concerning these two transport mechanisms as they relate to the actions of cholinergic agents in the schistosome are not directly addressed in this section, but are dealt with in detail in Section II. The results of many experiments presented in this section point towards Caz + as being the primary ion whose movement is being modulated by cholinergic receptor activation in the schistosome. The data presented in Table 6 demon- strate that the flaccid paralysis induced by arecoline, carbachol, eserine or 82 eserine plus ACh is not significantly altered by the external ionic environment of the parasite. Membrane potentials recorded in various altered ion concentrations are also not significantly affected by addition of inhibitory cholinergic agents (Table 11). These results seem to indicate that cholinergic receptor activation in this parasite is not selectively altering membrane permeability to 11+, C1- or M82+ . The slight hyperpolarization of parasite membranes observed in response to inhibitory cholinergic agents imder control conditions may be the result of activation of some electrogenic transport process rather than an alteration in conductance to K+ and/or 01-. Inhibitory cholinergic agents may be acting to directly decrease Ca2+ permeability in the schistosome by interacting with Ca2+ channels. Several of the 2+—channel blocking agents examined produce reductions in longitudinal muscle Ca tension that are similar to those observed with cholinergic compounds (Table 7; Figure 10). However, important qualitative and quantitative differences exist between the mechanical responses induced by these two types of agents. In general, cholinergic compounds induce a rapid, flaccid paralysis of the parasite's longitudinal musculature, whereas the CaZ+-charmel blockers produce a more gradual reduction in muscle tone of a much lesser magnitude, and, with the exception of nicardipine, active contractions usually persist. The active contrac- tions observed following addition of Caz+-channel blockers are either abolished or have their frequency of occurrence sharply decreased upon exposure to inhibitory cholinergic compounds. Therefore, while an action similar to that exerted by 2 Ca +-channel blockers cannot be ruled out based on these results, it appears that cholinergic receptor stimulation produces additional effects on schistosomal muscle. If cholinergic receptor activation is in fact utlimately bringing about a 2+ reduction in the free intracellular Ca concentration to induce muscular 83 relaxation as hypothesized, it would seem logical that a compound which causes intracellular Ca2 + to rise would reverse the actions of inhibitory cholinergic agents. Experiments performed in this section proved this to be true. Compound A-23187, a cation ionophore highly selective for calcium ion (Reed and Lardy, 1972) was able to reverse the tension decreasing effects of arecoline, carbachol and eserine, as well as D-600 (Figure 12). Altough not shown in Figure 12, active contractile activity also returned in most animals within 10 minutes following addition of the ionophore. Exposure of schistosomes to A-23187 alone produced dose-dependent increases in both contractile activity and longitudinal muscle tension (Figure 11). Although not shown in Figure 11, addition of inhibitory cholinergic agonists readily reversed the actions of A-23187. Further evidence supporting the idea that cholinergic compomds may be blocking Ca2+ entrance and/or enhancing Ca2+ efflux from the parasite comes from examining the ability of these agents to block and reverse the contracture of the longitudinal musculature induced by exposure to high K+. Contractures induced by high K+ in the schistosome presumably are due to depolarization of the sarcolemmal membrane and consequent release of intramuscular Ca2+ stores and/or entrance of externally derived Ca2+ into the sarcoplasm. Arecoline, carbachol and eserine appear to have the ability to prevent and/or reverse these events associated with exposure of the parasite to high K... §. mansoni pretreated with these cholinergic agonists exhibit only minimal increases in longitudinal muscle tension upon exposure to 60 mM K+ medium (Table 8). Similar responses 2 have been observed in animals pretreated with the Ca + channel blockers D-600, 2+ or La3+ (Fetterer e_t a_l., 1980; Depenbusch e_t Q” 1983). Co Arecoline, carbachol, eserine and D-600 are also able to reverse the tonic contracture induced by exposure to high K+ medium (Table 9, Figure 13). It is interesting to note that, while addition of carbachol or D-600 to parasites 84 contracted by 60 mM K+ results in a rapid decline in longitudinal muscle tension, no corresponding repolarization of either the tegumental or muscle membrane is observed (Figures 14 and 15). A similar dissociation between membrane potential and muscle tension has been observed in vertebrate smooth muscle (Bell, 1969; Diamond and Marshall, 1969; Marshall and Kroeger, 1973; Ito gt al_., 1979). Such responses could be attributed to these compounds decreasing Ca2+ availability to contractile elements by mechanisms which would not necessarily alter membrane 2+ 2+ polarization, i.e., prevention of Ca entrance and/or enhanced Ca sequestra- tion or removal. SECTION II Ionic Mechanisms Involved in Mediation of Cholinergigally- Induced Inhibition of Mechanical Activity in Adult Male Schistosoma mansoni 1. Summary The effects of various inhibitors of ion transport mechanisms on mechanical activity in adult male Schistosoma mansoni were evaluated. The Caz+-ATPase inhibitors NAP-taurine, calmidazolium, compound 48/80 and trifluoperazine pro- duced significant time- and dose-dependent increases in longitudinal muscle ten- sion, while 1 mM La3+ and trifluoperazine sulfoxide, an inactive derivative of trifluoperazine, were without effect. The tension decreasing effects of arecoline, carbachol and eserine were significantly attenuated in parasites preincubated in 3* for 20 to 60 calmidazolium, compound 48/80, trifluoperazine or 1 mM La minutes. Responses to inhibitory cholinergic compounds were not altered by preincubation of parasites in trifluoperazine sulfoxide. The Na+lK+-A'I'Pase inhibitors ouabain, 138 mM Li+ and 0 mM K" produced an acute, biphasic contracture of schistosome longitudinal muscle. As with inhibitors of Ca2+—ATPase, preincubation of parasites in these Na+/K+-A'I'Pase inhibitors caused a marked decrease in responsiveness to inhibitory cholinergic 2+-channel blocking agent D-600 produced a rapid agents. In contrast, the Ca reversal of the increased longitudinal muscle tension observed in response to ouabain, Li+ or o K+. 85 86 45Ca2+ were evaluated under control conditions and in Uptake and efflux of the presence of inhibitory cholinergic agonists or dopamine. Under both control and drug-treatment conditions, biphasic rates of uptake and efflux were observed, with a rapid phase occurring during the first two minutes, followed by a slower phase during the next 30-40 minutes. Arecoline, carbachol, eserine and dopamine 2+ movement, with the produced time- and dose-dependent alterations in 45Ca most pronounced effects seen on the initial rates of uptake and efflux. Arecoline, carbachol and eserine also produced dose-dependent decreases in steady-state 45 Ca2+ levels within the parasite which could be partially blocked by the cholinergic antagonists atrOpine and benactyzine. Exposure of animals to ouabain or trifluoperazine also antagonized the ability of cholinergic agonists to decrease steady-state 45Caz+ levels. A Ca2+lMg2+-A‘I'Pase was identified in whole worm homogenates obtained from adult male parasites. Optimal activity of this enzyme was observed in 50 Z 2* and 5 mM ATP at 37°C and mM TRIS buffer containing 5 mM Mg +, 40 11M Ca pH 8.0. Ouabain had no effect on this schistosomal CaZ+IMg2+-ATPase activity, while calmidazolium, triflu0perazine, chlorpromazine, NAP-taurine and compound W-7 produced dose-dependent inhibition. Addition of exogenous calmodulin induced a concentration-dependent activation of this enzyme. Cholinergic agents causing inhibition of schistosomal mechanical activity produced dose-dependent activation of this Ca2+/Mg2+-ATPase which could be antagonized by atropine, calmidazolium or trifluoperazine. These same inhibitory cholinergic agents had no effect on ouabain-sensitive Na+lK+-ATPase activity measured in whole worm homogenates. These results suggest that one manner by which cholinergic agents cause inhibition of schistosome mechanical activity may be via activation of a C32+IMgz+-dependent ATPase, which results in a net efflux of Ca2+ from the 87 parasite and subsequent muscular relaxation. Preliminary results also suggest the 2 possible participation of a Na+/Ca +-exchange mechanism in the actions of inhibitory cholinergic agonists on schistosomal longitudinal muscle. II. htroduction Acetylcholine is considered to be an inhibitory modulator of contractile activity in adult schistosomes. Cholinomimetic agents, such as carbacol and eserine, have a number of well documented actions on parasite mechanical activity including inhibition of spontaneous contractile activity (Barker e_t_ a_l., 1966; Hilman and Senft, 1973), tension decreasing effects on longitudinal and circular muscle (Fetterer £11., 1977;'Pax e_t_ a_l., 1981, 1984), and antagonism of electricallydinduced contractions (Pax g g” 1981). However, the ionic mecha- nisms mediating these effects of cholinomimetic agents in this parasite have received little attention. Recent experiments performed in our laboratory indicate inhibitory cholinergic compounds may be exerting their actions by ultimately altering calcium availability at the level of the animal's musculature. Previous studies have shown that tje normal, spontaneous contractile activity of the schistosome is dependent upon extracellular Ca2+. Exposure of parasites to Caztfree incubation medium reduces contraction amplitudes by 76% within 15 minutes (Pax g g” 1979). The response of schistosome musculature to various tension-inducing treatments also appears to depend on external Ca2+ availability. The tension increasing actions of 60 mM K+, 2,4-dinitrophenol (DNP), the antischistosomal praziquantel (P2), cardiac glycosides, and low temperature (4°C) can be blocked by preincubation of the parasite in Caz+-free 2+-channel blocking agents D-600 medium, as well as by pretreating with the Ca and lanthanum ion (La3+) (Fetterer gt a_l., 1980). Maintenance of contractures induced by various treatments also appears to depend on the presence of Ca2+ in 88 2 the animal's external environment. Removal of Ca + from the bathing medium of parasites contracted with 60 mM K+, DNP or PZ results in relaxation of the 2+ from the medium of longitudinal musculature. However, removal of Ca parasites previously contracted with ouabain or low temperature has virtually no effect on maintained tension levels (Wolde Mussie gt 9, 1982). These findings indicate the presence of active Ca2+ transport processes within the schistosome. The existence of a Ca2+- and Mgztdependent A'I'Pase in whole worm homogenates and tegumental fractions of _S_. mansoni has been reported by several investigators (Rogers, 1976; Nechay e_t a_l., 1980; Cesari g g, 1981; Podesta and McDiarmid, 1982; Huang g _al., 1984). However, a detailed characterization of this enzyme was not performed by these investigators. In this section experiments designed to investigate the possibility that cholinergic compounds may be exerting their inhibitory actions on parasite motility by altering Ca2+ availability at the level of the animal's contractile elements are described. Results indicate that one mechanism by which this may z"h-dependent A'I'Pase present within the 2+_ and M824; occur is by activation of a Ca2+- and Mg parasite's membranes. A detailed characterization of a Ca activated A'I'Pase present in whole worm homogenates of adult male S. mansoni is presented. The possible involvement of a Na+/Caz+-exchange mechanism in mediating the actions of inhibitory cholinergic compounds is also discussed. III. Materials and Methods A. Experimental Animals and Media Adult Schistosoma mansoni (St. Lucian strain) were isolated by gentle dissection from the portal and mesenteric veins of mice 45-60 days post-infection as previously described (Bennett and Seed, 1977). Paired animals were maintained at 37°C in either Hank's balanced salt solution (HBS; final concentrations of 89 2 constituents: Na+ 138 mM, K+ 5.9 mM, cs2+ 1.4 mM, Mg + 0.5 mM, P0 0.5 mM, 4 Cl" 147 mM, so 0.5 mM, glucose 5 mM) or RPMI-1640 (Grand Island Biological; 4 see addendum for composition). Both media were buffered at pH 7.4 with 20 mM Hepes (N-Z-hydroxyethylpiperazine N-2-ethane sulphonic acid, Sigma Chemical Co., St. Louis, MO) and contained 100 units/ml penicillin-streptomycin (GIBCO). Parasites were used within 8 hours following removal from mice. All experiments were performed at 37°C in either HBS or RPMI—1640 as indicated, unless otherwise stated. High K+ HBS consisted of HBS to which 60 mM KCl and 85 mM NaCl were added instead of the usual concentrations of these two salts. A 0 mM K+ HBS was obtained by deleting KCl. The chloride concentration of this solution was held constant by adding NaCl. HBS from which CaCl was omitted and to which was added 5x10-4M EGTA (Ethyleneglycol-bis( B- 2 amino-ethyl ether)-N,N,N',N'-tetraacetic acid; Sigma Chemical Co.) was used to make up the zero CaZ+/EGTA HBS. 138 mM Li+ HBS was made by completely substituting LiCl for NaCl. B. Mechanical Activity Recordirgg Recording of longitudinal muscle tension and contractile activity was performed as previously described by Fetterer _e_t_ a_l. (1977, 1978). After the initial attachment of parasites to the recording apparatus, a 10-minute equilibra- tion period was allowed before the application of any experimental treatment. Unless otherwise noted, data concerning contractile activity responses to various treatments, presented in tables and elsewhere in the text, give the change in tension 10 minutes after treatment application relative to the average level of tension recorded for 4 minutes before its application. In cases where two or more treatments were given sequentially, data presented represent the change in tension after 10 minutes exposure to the second treatment as compared to the tension just prior to introduction of the second treatment. Since tension change is 90 relative to the length of parasite over which the change occurs, tension data are referenced to the amount of tension developed over a 1 mm length of worm. C. Measurement of Ion Fluxes Following removal from mice, isolated parasites were placed in RPMI- 1640 containing 50 mg% sodium pentobarbital (PB; Sigma Chemical Co.) to facilitate separation of male and female worms. The female worms were then discarded. Male parasites were rinsed three times with 10 ml HBS (pH 7.4; 37°C) to allow for recovery from the anesthetic effects of the PB. Worms were then inspected under a dissecting microscope (0.9X magnification), and all visibly damaged animals were discarded. Groups of 10-15 parasites were then placed in small (7 ml) glass scintillation vials containing 2.5 m1 of fresh HBS. The vials were capped and incubated at 37°C for 30 minutes prior to the start of the experiment. 45 2+ For Ca uptake experiments, the 2.5 ml of HBS was decanted and replaced with 1.8 ml of fresh label-free HBS (37°C). Uptake was initiated by 2+ adding 0.2 ml of HBS containing 4 uCi of 450a (as calcium chloride, radionuclide and radiochemical purity 99%; specific activity 24.61 mCi/mg; New England Nuclear, Boston, MA) along with the desired drug or vehicle control, and allowing the incubation to proceed for varying times (0.5-55 minutes) at 37°C. Parasites were prepared for studies of 45Ca2+ efflux by removing the label-free HBS and preincubating them in 2 ml of HBS containing 4 uCi 45Ca2+ for 35 minutes at 37°C. After preincubation, efflux was initiated by removing the HBS containing 45Ca2+, washing the parasites two times with 5 ml of ice-cold label-free, drug-free HBS, and returning them to 2 ml of warmed (37°C) HBS containing the desired drug or appropriate vehicle. The parasites were then incubated for various periods (0.5-30 minutes). 91 In some experiments, the ability of certain treatments to alter steady- state 45Ca2+ content of parasites was evaluated. Worms were prepared for these 45 2" for 35 or 60 studies by preincubation in 2 ml HBS containing 4 uCi Ca minutes. Pharmacological agents or appropriate vehicles were then added to the vials, and the incubations allowed to continue for additional 10 minute periods. In all experiments the final 45Ca2+ concentration in each vial was approximately 3 11M. At the end of desired incubation periods, vial contents were poured over Whatman GF—B glass fiber filters placed on a Millipore filter apparatus (Millipore Inc., Model XX1-024-00). Trapped parasites were immediately washed 2 three times with 4.0 ml aliquots of ice-cold, zero-Ca + HBS containing 0.5 mM 4SCa2+ EGTA to remove externally bound . After filtration worms were weighed, removed from the filters with forceps, placed in large (20 ml) borosilicate (low K+) glass scintillation vials (Rochester Scientific Co., Inc., Rochester, NY) containing 1.0 ml of 0.6 N tissue solubilizer (NCS, Amersham Corp., Arlington Heights, IL), and incubated for 1 hour at 50°C in a water bath. Following removal from the water bath and cooling to room temperature, 0.5 m1 of 100% glacial acetic acid (Sigma Chemical Co.) was added to neutralize the solubilizing fluid. A 10 ml volume of aqueous counting scintillant (ACS, Amersham) was added to each 45Ca2+ activity determined using a liquid scintillation spectrometer vial, and (Beckman LS 7000, Beckman Instruments, Inc., Fullerton, CA) with a calculated counting efficiency of 90 to 95%. Analysis of 45Ca2+ accumulation and efflux data was performed using a Tektronix 4052 computer plotting system (courtesy of the Michigan State University Carcinogenesis Laboratory, Department of Bio- chemistry). 92 D. Measurement of A'I'Pase Activity 1. Caz+/Mgz+-A'I'Pase Ca2+- and Mg2+-dependent A‘I'Pase activities were determined using the procedure described by Sulakhe g 51. (1973), and Martin (1982). Briefly, whole worm homogenates (from adult male parasites) containing 200-400 ug membrane protein were prepared in 50 mM Tris (2-amino-2(hydroxymethyl)-l,3- propandiol; Sigma Chemical Co.) medium containing various concentrations of CaCl and MgClz in a final volume of 2.5 m1. In some experiments, a calcium- 2 EDTA (0.5 mM EDTA; ethylenediamine tetraacetic acid, free acid; Sigma Chemical Co.) or calcium-EGTA (0.5 mM EGTA; free acid) equilibrium system was employed (Portzehl g g” 1964). Calcium and magnesium concentrations are stated as free ion present in the incubation solution. Calculated concentrations of free divalent cations were confirmed using flame spectrophotometry. Pharmaco- logical agents were added 10 minutes prior to reaction initiation unless otherwise noted. To evaluate the effects of exogenous calmodulin (purified from extracts of bovine brain; Boehringer Mannheim, Indianapolis, IN) on CaZ+IMg2+- A'I'Pase activity, homogenates were prepared in 50 mM Tris buffer containing 5 mM MgCl and 0.5 mM EGTA at 4°C. The suspension was centrifuged and the 2 pellet resuspended in the Tris/EGTA buffer two times. After the final centrifuga- tion, the pellet was resuspended in 50 mM Tris buffer containing 5 mM MgC12 and 40 11M CaClz (pH 7.4). Exogenous calmodulin (1-50 ug/ml final concentration) was added, and the suspensions were allowed to incubate at 37°C for 15 minutes prior to reaction initiation. To avoid addition of unwanted cations, the reaction was intitiated by addition of Tris-ATP (adenosine 5'-triphosphate, Tris(hydroxymethyl)-aminometh- ane salt, ”vanadium-free"; Sigma Chemical Co.) and allowed to proceed for 93 exactly 10 minutes. The reaction was terminated by addition of 1 ml of 12% trichloroacetic acid (TCA; Sigma Chemical Co.). Following low speed (10,000 rpm) centrifugation for 15 minutes (at 4°C) to remove denatured protein, two 1 ml aliquots of supernatant were assayed for inorganic phosphate by the method of Fiske and Subbarow (1929) using a spectrophotometer (Spectronic 20, Bausch and Lomb). Protein in homogenates was quantified by the method of Lowry 3 9.1. (1951), using crystalline bovine serum albumin (Sigma Chemical Co.) as the standard. 2. Na+/K+-ATPase Na+/K+-ATPase activity was determined by the methods of Fetterer e_t_ al. (1981). Homogenates were prepared and suspended in 0.25 M sucrose containing 5 mM histidine and 1 mM EGTA. Samples containing 200- 400 11g protein were preincubated at 37°C in medium containing 50 mM Tris (pH 7.8), 100 mM NaCl, 10 mM KCl and 5 mM MgC12 with or without 1.0 mM ouabain (Sigma Chemical Co.) for 20 minutes. ATP (final concentration, 5 mM) was added, and the incubation allowed to continue for exactly 10 minutes. The reaction was terminated by addition of ice-cold 0.8 N perchloric acid (Pierce Chemical Co.). The precipitate was removed by centrifugation at 4°C, and aliquots of the supernatant assayed for inorganic phosphate as stated previously. Ouabain-sensitive A'I'Pase activity was determined by subtracting the A'I'Pase activity in the presence of ouabain from that in the absence of ouabain. All A'I'Pase activities are expressed as 11M P04 released per hour per milligram of protein. All determinations were performed a minimum of 6 times. E. Pharmacggical Agents Drugs used were d-amphetamine sulfate, arecoline, carbachol (carba- mylcholine chloride), chlorpromazine, compound 48/80, compound W-7 (N-(6- aminohexyl)-5-chloro-1dnaphthalenesulfonamide), dOpamine (3-hydroxytyramine 94 HCl), eserine (physostigmine), ouabain, procaine HCl, quercetin, serotonin (5- hydroxytryptamine; S-HT), sodium pentobarbital (PB) and trifluoperazine (TFP), all from Sigma Chemical Co.; calmidazolium (Janssen Pharmaceutical, Beerse, Belgium), D-600 (Knoll A.G., Ludwigshafen am Rhein, Germany), imipramine (CIBA-Geigy Pharmaceuticals), NAP-Taurine (N-(4-azido-Z-nitrophenyl)-2-amino ethylsulfonate; Pierce Chemical Co., Rockford, IL) and praziquantel (PZ), kindly supplied by Drs. P. Andrews and H. Thomas of the Bayer Co. All drugs, dissolved first in double distilled water or dimethylsulfoxide (DMSO, Mallinkrodt Chemical Co.) at a concentration of 10 mM, were added at concentrations and time points specified. The final concentration of DMSO present in the experimental medium did not exceed 0.05% in any assay. F. Statistical Procedures All data are given as the mean i 1 standard error of the mean (SEM). Unless otherwise noted, a minimum of six replications was used to determine each data point for muscle tension measurements, and a minimum of five replications to determine each 45Caz+ data point. All tests for significance of difference between means were performed using Students' t—test (two-tailed), or ANOVA with Dunnet's t—test where appropriate. IV. Results A. Mechanical Activity 2 1. Ca +-ATPase inhibitors 2 The acute effects of Ca +-ATPase inhibitors on longitudinal muscle tension in schistosomes are presented in Figure 16. Following application of lxlo‘4 M NAP-taurine, a gradually developing, well maintained contracture was recorded. 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