Abstract
Endogenous levels of choline and acetylcholine in tetrathyridia of Mesocestoides corti were determined under different environmental conditions. The highest values of choline and ACh were obtained when tetrathyridia were washed in media NCTC 135 and Eagle's Basal Medium. The lowest values were obtained when organisms were washed in 0.85% NaCl, or BME without choline chloride. Incubation of organisms in different concentrations of physostigmine showed an increase in the ACh level. The levels of choline and ACh decreased with decreasing drug concentration. DFP also showed a similar effect on choline and ACh. Oxotremorine increased choline and ACh levels but the increase was considerably lower than in physostigmine or DFP-treated organisms. While the histochemical association of acetylcholinesterase (AChE) with the nervous system has been the primary means of claiming the presence of acetylcholine (ACh) in helminthic tissue, a few workers have demonstrated ACh by biochemical isolation. Bueding (1952) showed that extracts of Schistosoma mansoni contain high levels of AChE and choline acetylase activities. Barker et al. (1966) reported the level of ACh in S. mansoni to be 5.3 jug/g wet weight. Chance and Mansour (1953) demonstrated AChE and choline acetylase activities in the liver fluke, Fasciola hepatica. Furthermore, they reported that this parasite contains an ACh-like substance. The presence of ACh was demonstrated in Ascaris by Mellanby (1955) who showed that the head region contains a much higher level than the body. Several biochemical methods are routinely used for determining ACh-like activity in extremely small amounts, but their reliability has been questioned (Hosein and Orzek, 1966; Stockley, 1969) in view of the fact that their response is not specific to one compound alone. Definitive identification of ACh and choline Received for publication 2 November 1973. * This investigation was aided in part by research grant USPHS AI-11107-01 from the NIH, Bethesda, Maryland. t Recipient of USPHS-NIAID Training Grant No. TOI-A1-00249. + Grand Island Biological Company, 519 Aldo Avenue, Santa Clara, California 95050. was made in tetrathyridia of Mesocestoides corti by means of a combined gas chromatograph-mass spectrometry (GC-MS) system. The functional aspects of ACh in this parasite will be reported elsewhere. MATERIALS AND METHODS Tetrathyridia of Mesocestoides, originally isolated from lizards (Specht and Voge, 1965), were maintained in Swiss albino mice. The animals were killed by cervical dislocation and tetrathyridia were transferred from the body cavity into petri dishes containing GIBCOt NCTC 135, "Eagle's Basal Medium" (BME) with or without choline chloride, or physiological saline solution (0.85% NaCl). Worms were preincubated in BME without choline chloride for a maximum of 1/2 hr before exposure to drug. The number of worms never exceeded 20 per ml of medium. The maximum period of incubation was 1 hr. For each drug concentration, the motility of worms was observed under the dissecting microscope at 15, 30, 45, and 60 min. The effect on choline and ACh levels by 3 different concentrations of physostigmine salicylate and 2 concentrations of oxotremorine and diisopropylfluorophosphate (DFP) was tested. The effect of each concentration was tested 3 times and organisms for each concentration were obtained from a different mouse. After incubation at 37 C, parasites were washed and dried by use of aspiration of fluids or by means of a Millipore filter. Organisms were then removed with a frozen spatula and immediately immersed in liquid nitrogen. The entire process of washing and drying lasted 6 to 8 min. Organisms were pulverized by the method of Campbell et al. (1970). The finely powdered tissue, 0.066 to 1.11 g, was transferred to a tared, chilled glass homogenizer to prevent thawing, and weighed. With few exceptions, the
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