The presence of a proteolytic enzyme has been demonstrated in ground-up preparations of Schistosoma mansoni. A twenty-fold purification has been achieved by ultracentrifugation at pH 3.0; the enzyme has an optimum pH of 3.9 and a marked substrate specificity for haemoglobin. No significant proteolysis was observed either with whole serum at pH values of 3.9, 6.0 or 8.0, or with isolated serum proteins at pH ranges between 2.5 and 7.7. The evidence is discussed that this enzyme may be located in the intestine of the schistosomes and that it is, at least in part, responsible for the supply of amino acids to the organisms.Little is known about the protein metabolism of Schistosoma mansoni.Rogers (1940) reported that the black pigment present in the intestine of S. mansoni is probably haematin formed by the digestion of haemoglobin from the host. The parasite must obtain nutrient materials, including amino acids, from the blood of the host, and it is conceivable that there is a mechanism for the degradation of one or more blood proteins, following ingestion into the intestine of the parasite. Halawani, Hafez, Newsome, and Cooper (1949) observed that haemoglobin disappeared from the medium in which S. mansoni survived in vitro.If it were known precisely what amino acids are required by S. mansoni and by what mechanism the parasite obtains them, an opportunity might become available for the development of schistosomicidal drugs. An initial approach to this problem is reported in this paper, in which the demonstration, partial purification and properties of a proteolytic enzyme from S. mansoni are described. The enzyme shows a marked specificity for haemoglobin (Timms and Bueding, 1958).METHODS AND MATERIALS Survival Studies.-Survival of schistosomes in a synthetic medium was studied by the use of procedures reported previously (Ross and Bueding, 1950). The synthetic medium used in the present investigation contained the following constituents in
It has been demonstrated that extracts of Schistosoma mansoni contain high acetylcholinesterase and choline acetylase activities (Bueding, 1952). Subsequently, acetylcholine esterase and choline acetylase activities were found by Chance & Mansour (1953) in another trematode, the liver fluke Fasciola hepatica. Furthermore, it has been reported that Fasciola hepatica contains an acetylcholine-like substance and that a variety of cholinomimetic agents exert an inhibitory effect on the muscular activity of this parasite (Chance & Mansour, 1953). Similarly, inhibitory effects of some cholinomimetic agents on the motor activity of Schistosoma mansoni have been observed (Bueding, 1962). This report deals with motor responses of Schistosoma mansoni to cholinomimetic and other pharmacological agents and with experiments designed to determine the presence of acetylcholine in this parasite. Observations are reported indicating that cholinomimetic agents and cholinesterase inhibitors produce a paralysis of the worms, which is reversed by certain antagonists of acetylcholine-like drugs. Furthermore, S. mansoni contains a material the properties of which are similar to those of acetylcholine. METHODSIndividual adult worm pairs of Schistosoma mansoni (Puerto Rican strain) were incubated in 75%,' horse serum in distilled water at 37' C in cylindrical glass vessels (17 mm inside diameter, 12 mm high). Motility of the worms was observed under a dissecting microscope at predetermined time intervals (5 min, 15 min, thereafter every 15 min, for 1 hr, and thereafter every hour for two to four hours). At least four worm pairs (i.e., four males and four females) were used at any given concentration of a tested compound. Normal motor activity consisted of waves of contraction along the entire body, with occasional whipping motion and of frequent contractions of the oral sucker and acetabulum. Activity of the acetabulum may also result in adhesion to the bottom of the vessel with occasional moving along the glass. Paralysis and stimulation are readily observable deviations from this motor behaviour. The activity of control worms in 75% horse serum did not change significantly during 5 hr, the maximum period of observation.With two exceptions (see below) all compounds were added in an aqueous solution to 0.75 ml. of horse serum and enough distilled water was added to bring the total volume to 1.0 ml. The two water-insoluble compounds, 0,0-dimethyl-0-(2,4,5-trichlorophenyl) phosphorothioate (Ronnel) and its oxygen analogue (McCollister et al., 1959) were dissolved in 95% ethanol and 0.02 ml. of this solution were added to a mixture of 0.75 ml. of horse serum and 0.23 ml. of water. The motor activity of schistosomes was not affected in 2% ethanol.
The pharmacological effects of a series of fatty acid-choline esters have been studied on the isolated rabbit heart, the isolated guinea-pig ileum and the rat stomach. The effect changed with increasing chain length, and three different types of response were observed. The shortchain compounds produced depression of the isolated rabbit heart and spasm of the isolated guinea-pig ileum. Only one, butyrylcholine, had an erratic stimulating effect on hydrochloric acid secretion by the partially vagotomized rat stomach. Medium-chain compounds had a stimulating effect on the isolated rabbit heart, a mixed spasmogenic and relaxant effect on the isolated guinea-pig ileum, and no effect on the rat stomach. Long-chain compounds blocked the effect of acetylcholine on the isolated rabbit heart and the isolated guinea-pig ileum; they also depressed spontaneous hydrochloric acid secretion by the rat stomach. The nature of these three types of response is discussed.The present investigation is concerned with the pharmacology of the choline esters of an homologous series of saturated straight-chain monobasic fatty acids.Previous work was mainly concerned with the effect of these compounds on blood pressure. Hunt and Taveau (1911), in their extensive investigation of the pharmacology of a large number of choline derivatives, included the esters from acetylcholine (ACh) to pentanoylcholine and in addition palmitoylcholine, all of which occur in the series used in this work. They found that with increasing chain length the depressor effect declined, and a pressor effect was observed which in some cases became manifest after atropine but in others without atropine. These findings were confirmed and elaborated by le Heux (1921), Simonart (1932), Chang and Gaddum (1933, and Bovet and BovetNitti (1948). Fourneau and Page (1914) synthesized a series of these esters with even numbers of carbon atoms in the chain. Haemolytic action was most pronounced with the palmitic and stearic acid esters; it diminished with decreasing chain length, and was no longer found with members below dodecanoylcholine. In the present investigation an almost complete series of these esters was available. The effect of the chain length upon the pharmacological action was examined on the isolated mammalian heart, the motility of the small intestine, and the secretory response of the stomach. METHODSIsolated Rabbit Heart.-The usual Langendorff type of preparation with retrograde aortic perfusion was used. The composition of the perfusion solution used was: NaCl 9 g., KC1 0.42 g., CaCl2 0.24 g., NaHCO3 0.5 g., dextrose 1.0 g., distilled water 1 litre.The compounds tested were injected through the rubber-capped side-arm of the perfusion apparatus.Isolated Guinea-pig Ileum.-The ability of some of the compounds to induce spasm was expressed in terms of the concentration which caused an effect equal to that produced by 10'.5 M-ACh. This was determined upon the isolated guinea-pig ileum suspended in the 2.0 ml. bath of an automatic assay apparatus, similar to that desc...
Axelsson, J., S. G . R. Hogberg and A. R. Timms. The effect of removing and readmitting glucose on the electrical and mechanical activity and glucose and glycogen content of intestinal smooth muscle from the taenia coli o f the guinea-pig. Acta physiol. scand. 1965. 64. 28-42. The effects of changing the
Antihistamines such as 2(N-benzyl-N-phenylaminomethyl)-iminazoline hydrochloride (antazoline hydrochloride) and N-dimethyl-aminoethyl benzhydryl ether hydrochloride (diphenhydramine hydrochloride), have been shown to possess many sideeffects (Dutta, 1949), including local anaesthetic effects, antagonism of the depressant action of acetylcholine on the heart, depression of the maximal rate at which the isolated rabbit auricles will respond to electrical stimulation, antagonism of acetylcholine on isolated frog rectus abdominis muscle, and depression of the contraction of the nictitating membrane in response to preganglionic stimulation. Often, the concentrations of antihistamines required to produce such effects considerably exceed those necessary to produce the more specific effects against histamine, so that their modes of action are obscure. Marshall (1955a) claimed to have shown that the antagonism between many antihistamines and histamine on the guineapig ileum is competitive, although the criteria upon which competition was judged are not in entire agreement with those adopted in this paper.A report is presented upon a preliminary investigation into the mode of action of two series of antihistamines as antagonists of acetylcholine (ACh) on the isolated guinea-pig ileum. METHODSThe structure of the two series of compounds used in this work appear in Tables I and II. The lever used throughout this work was constructed according to the dimensions suggested by Schild (1947), and was not allowed to move more than 30°from the horizontal. These precautions ensure that all measurements of the heights of excursions from the kymograph paper are linearly related to the actual shortening of the muszle.Measurement of Anti-Acetylcholine Activity.-In order to determine the anti-ACh activity of these compounds, pA2 andpA10 values (Schild, 1947) were measured against ACh on the isolated guinea-pig ileum. The 'tissue was suspended in an oxygenated Tyrode solution which was contained in the 2.0 ml. bath of an automatic assay apparatus of the type described by Boura, Mongar, and Schild (1954). Doses of ACh bromide (usually 0.0125 pg. base/ml. as basic dose) were added every 2 min. and antagonists were allowed to remain in contact for 10 min., represented by 5 complete cycles of the apparatus. pA2 and pALO values for each compound were not always determined on the same sample of ileum. Samples of ileum which did not recover fully from the effects of the antagonists were discarded, and fresh samples were used for the next test (Schild, 1947). Four determinations of pA2 and four of pA10 were carried out for each substance using samples of intestine from different guinea-pigs.Concentration-action Curves.-In order to determine the validity of the pA method as applied to the activedrug/antagonist systems studied, concentration-action curves were plotted for the active substance, ACh or histamine, first alone and then after equilibration to a fixed concentration of antagonist (Schild, 1949). The importance of such curves is discusse...
S a n d o z P h a r m a c e u t i c a l s , R e s e a r c h Department, Pharmacology S e c t i o n , Hanover, N. J .T h e t a e n i a coli is a narrow band of smooth muscle running longitudinally on t h e ventral and dorsal s u r f a c e s of the caecum in certain s p e c i e s . T h e purpose of t h i s presentation is t o review t h e e x i s t i n g information on t h e physiology and biochemistry of t h e taenia coli of t h e guinea pig. T h e information available r e s u l t s largely from t h e work of E. Biilbring in collaboration with E. Bueding, J.Axelsson and P. J. Goodford. U n l e s s otherwise stipulated, t h e material in t h i s paper refers exclusively to the t a e n i a c o l i of the guinea Pig.In 1954, Bulbring reported on a s e r i e s of experiments in which simultaneous measurements of membrane potential, action potent i a l s and isometric t e n s i o n of taenia coli were made. It w a s found that the membrane potential averaged about 60 mV when the muscle w a s a t i t s i n situ length and fell t o 43 m V when the muscle w a s stretched. T h e s e v a l u e s were found t o b e the mean v a l u e s of t h e membrane potential which w a s fluctuating in a manner related to the spontaneous rhythm. P e r i o d s of higher tension w e r e found to b e a s s o c i a t e d with lower membrane potentials and increased r a t e s of s p i k e discharge, while periods of lower tension were a s s o c i a t e d with higher membrane potentials and reduced s p i k e frequency. T h e s e measurements were made using intracellular recording techniques.In 1958, Burnstock and Straub4 described an adaption of t h e s u c r o s e gap method of StPmpfil,' using external electrodes, t o t h e study of membrane and action potentials of smooth muscle s t r i p s from t h e taenia coli. V a l u e s for the resting membrane potential obtained by t h i s method were comparable t o t h o s e previously obtained with microelectrodes, and the method offered the advantage that quick changes of t h e bathing solution were p o s s i b l e so that continuous recordings could b e made of the e n t i r e s e q u e n c e of c h a n g e s c a u s e d by t e s t solutions. T h e chamber containing the muscle w a s further modified by Bulbring, Burnstock and Holman6 so t h a t t h e muscle could b e stimulated electrically and nieasurements of isometric tension could b e made simultaneously.' *Thin paper, illustrated with slide8. wan Presented at a meeting of t h e Seetion on May 11, 1964. 902
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