Manus J. Donahue

Serotonin (5-hydroxytryptamine): A possible regulator of glycogenolysis in perfused muscle segments of Ascarissuum

Summary When isolated muscle segments from the parasite Ascaris suum were perfused with varying concentrations of serotonin (5-hydroxytryptamine), the level of cyclic AMP increased three-fold. Maximal levels of cyclic AMP were produced by the muscle at serotonin concentrations of 50 μM. Perfusion of saturating levels of serotonin through isolated muscle segments for various time periods resulted in an inactivation of glycogen synthase and an activation of phosphorylase with a concomitant rise in cyclic AMP values. Collectively, the results support the concept that serotonin may be functioning as a regulatory hormone of carbohydrate metabolism in this worm.

Serotonin receptors in the tissues of adult Ascaris suum

Serotonin (5-hydroxytryptamine, 5-HT) receptors in the muscle and intestinal tissues of adult Ascaris suum have been investigated. [3H] lysergic acid diethylamide (LSD) exhibited specific and saturable binding to membranes prepared from both intestine and muscle. The intestinal tissue membranes had an equilibrium dissociation constant (Kd) of 2.70 nM for LSD and a Kd of 2.50 microM for 5-HT. As compared to the intestine, the muscle membranes had comparatively higher affinity for both LSD (Kd = 1.80 nM) and 5-HT (Kd = 0.68 microM). The muscle membranes also had a high binding affinity for ketanserin, a 5-HT2 antagonist, (Kd = 16.7 nM) whereas intestinal membranes exhibited no specific binding of ketanserin. Serotonin significantly inhibited the binding of LSD to the intestinal and muscle tissue membranes while adrenergic and cholinergic drugs and histamine did not. This suggested that the binding of LSD, 5-HT and ketanserin to the parasite membranes was specific. Collectively, the data demonstrated the presence of a serotonin receptor in the muscle and intestinal tissues of the adult A. suum. The receptor in the muscle was pharmacologically similar to the mammalian serotonin type 2 receptor.

GLYCOGEN METABOLIZING ENZYMES DURING STARVATION AND FEEDING OF ASCARIS SUUM MAINTAINED IN A PERFUSION CHAMBER

The glycogen content of muscle was correlated with the activity of glycogen synthase and glycogen phosphorylase from the parasitic roundworm Ascaris suum maintained in vitro. Adult female worms were maintained in the laboratory in a perfusion system during periods of starvation and feeding. During starvation, the levels of glucogen decreased at a rate of 0.1 to 0.2 mumoles/min/g wet weight of muscle-cuticle. During this time, 95% of the glycogen synthase (E.C. 2.4.1.11) was in the active D-form, and 48% of the phosphorylase (E.C. 2.4.1.1) was in the active a-form. Upon feeding, the rate of incorporation of glycosyl residues into glycogen proceeded at a rate of 0.75 to 1.0 mumoles/min/g muscle-cuticle. Glycogen synthase was 22% in the active I-form and phosphorylase a-levels remained virtually unchanged at 41% as compared with the starved worm. Total levels of both enzymes remained constant over the starvation-feeding period with 3.9 units/g phosphorylase and 0.4 units/g glycogen synthase. The apparent Km value for the substrate UDPG for glycogen synthase was 0.22 +/- 0.02 mM. For glycogen phosphorylase the Km value for G-1-P was 1.76 +/- 0.38 mM.

Evidence for the absorption and synthesis of 5-hydroxytryptamine in perfused muscle and intestinal tissue and whole worms of adult Ascaris suum.

The metabolites of 5-hydroxytryptamine (5-HT, serotonin) namely, L-tryptophan, 5-hydroxytryptophan, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were measured in perfused tissue and whole worms from adult female Ascaris suum using reversed-phase liquid chromatography with electrochemical detection. The intracellular levels of each metabolite were quantitated in response to several physiological effectors but only L-tryptophan (TRP) caused dose-dependent changes in these metabolites. Serotonin itself could also be absorbed by perfused A. suum muscle and intestinal tissue. When live A. suum were tied at the anterior and posterior regions to restrict TRP absorption by the intestine, TRP was absorbed through the cuticle and converted into 5-HT by the muscle tissue. In united live parasites TRP absorption was observed in both muscle and intestinal tissue. Collectively, the data indicated that 5-HT may be either absorbed directly or synthesized de novo from absorbed TRP in the isolated tissue of A. suum. The 5-HT, in the adult female A. suum, can be synthesized de novo from TRP, or 5-HT can be absorbed from the environment both through the cuticle and by the intestine of living parasites. Data also indicated that there was preferential sequestering of 5-HT and the metabolites of 5-HT in the anterior tissues of the worms.

THE BACTERIAL FLORA OF THE INTESTINE OF ASCARIS SUUM AND 5-HYDROXYTRYPTAMINE PRODUCTION

Representative facultative anaerobes of the bacterial flora from the intestine of female Ascaris suum were isolated and identified. The number of bacteria in the intestine was approximately 4 X 10(9) per g wet weight of intestine. Seventeen of 19 of the isolated colonies were found to secrete 5-hydroxytryptamine in culture. Holding A. suum in an antibiotic-containing medium did not affect the levels of 5-hydroxytryptamine in the worm, which were 231 +/- 14 ng/g in antibiotic-media as compared to 250 +/- 16 ng/g in control media. This implied that the bacteria may not be contributing to the level of 5-hydroxytryptamine in the tissues of A. suum.

Regulation of glycogenolysis by adenosine 3′,5′-monophosphate in Ascaris suum muscle

Abstract 1. 1. Glycogen metabolism in Ascaris suum muscle was correlated with increase cyclic AMP accumulation, and glycogen phosphorylase activity, and decreased glycogen synthase activity. 2. 2. A cyclic AMP-dependent protein kinase was partially purified from Ascaris muscle. 3. 3. The activation by cyclic AMP, chromatographic properties, inhibition by the heat-stable inhibitor protein, and substrate specificity of the enzyme indicates that the principal cyclic AMP-dependent protein kinase in Ascaris muscle is Isozyme I. 4. 4. Glycogen metabolism in Ascaris suum muscle appears to be regulated by a cascade mechanism initiated by cyclic AMP.

ACTIVITY OF ENZYMES REGULATING GLYCOGEN METABOLISM IN PERFUSED MUSCLE-CUTICLE SECTIONS OF ASCARIS SUUM (NEMATODA)

A new perfusion system has been developed in which muscle-cuticle sections of Ascaris suum were perfused, enabling study of enzymes in vitro. Using this technique the activity of the regulatory enzymes glycogen synthase and glycogen phosphorylase was determined, and the level of glycogen in the muscle was assessed. During starvation, 98% of glycogen synthase was in the inactive D-form, and 80% of the glycogen phosphorylase activity was in the active a-form. When the ascarid muscle section was perfused with 27 mM glucose, 13.1% of the glycogen synthase was in the active I-form, whereas phosphorylase a-levels dropped to 46% and glycogen was synthesized at a linear rate of 12 mg/g/hr or 1.23 mumoles/min/g muscle-cuticle. ATP levels (3.71 +/- 0.32 mM) remained unchanged over a 4-hr perfusion period with an adenylate energy charge of 0.82. Fructose supported glycogen synthesis, though not as well as glucose. Galactose, mannose, and trehalose did not support glycogen synthesis. The new perfusion system should be useful in future, similar studies on Ascaris.

Graphical modeling and computer animation of tensile deformation in polymer liquid crystals (PLCs)

Abstract We create polymer liquid crystals (PLCs) on a computer and subject them to constant-force tensile deformations. Molecular dynamics simulation procedure is used and graphical models and animations of crack formation and propagation as a function of time are generated. Special attention is given to realistic rigid LC island geometry and the island spatial distribution in the material. Internal fracture processes can now be easily studied. As a result of the numerous animations recorded, clear patterns in PLC crack initiation and propagation emerge. With the help of the animations, the structure of PLCs may be understood without having to resort to costly laboratory experimentation.

Identification of a novel 5-HTN (nematoda) receptor from Ascaris suum muscle

1. The abilities of various serotonergic drugs to bind with the 5-HT receptor of Ascaris suum muscle and to affect cyclic AMP levels in muscle tissue were examined. 2. Ligands which selectively interact with either the 5-HT1 or the 5-HT2 receptor in mammalian systems interact with the 5-HT receptor from A. suum muscle and increase cyclic AMP levels. 3. No binding of 5-HT3 ligands to 5-HT receptors from A. suum muscle was observed. 4. The 5-HT receptor of A. suum muscle should be called the 5-HTN (for Nematoda) receptor because its pharmacological and biochemical behaviors were different from those of mammalian 5-HT receptors.

Bacterial flora, a possible source of serotonin in the intestine of adult female Ascaris suum.

The effect of intestinal bacteria on 5-hydroxytryptamine (5-HT, serotonin) level and 5-HT turnover rates in Ascaris suum intestine are presented. Ascaris suum were incubated in media containing antibiotics for 24 hr, and the bacterial flora in the anterior regions of the intestine of A. suum was eliminated. The bacteria were significantly reduced (> 99%) but not eliminated in the middle and posterior segments of the worm. The 5-HT level decreased in the intestine after 24 hr incubation in antibiotics, whereas the 5-HT turnover rate increased (131 ng/mg protein/hr). Two possible sources of 5-HT from the intestine were examined: the intestinal tissue itself and the microflora inhabiting the intestine. The 5-HT level in the microflora was 30% higher (72.6 ng/g) than the intestinal tissue (43.3 ng/g) in control samples (0 hr, no antibiotics). These values decreased significantly after 24 hr incubation in A. suum saline. The 5-HT values decreased to 18.6 ng/g in the presence and 28.6 ng/g in the absence of antibiotics. The 5-HT turnover rate during this time period indicated that as the number of bacteria declined, the 5-HT turnover rate also declined in the microflora, but the 5-HT turnover rate in the intestinal tissues increased. Results from these studies suggest that bacterial 5-HT may be contributing to the 5-HT level in A. suum intestinal tissue.