The uptake and metabolism of 5-hydroxytryptamine by tissue slices of the cestode Hymenolepis diminuta

The in vitro uptake of [3H]5HT was investigated in tissue slices of the cestode Hymenolepis diminuta. A concentrative, sodium sensitive, high affinity uptake mechanism (Km 1.43 X 10(-6) M; Vmax 222 fmoles/mg wet wt/min), together with a sodium insensitive component (linear up to 5 X 10(-6) M) were present. In the presence of 2-nitroimipramine the sodium sensitive component was significantly suppressed (Vmax 33 fmoles/mg/wet wt/min) although the Km (1.37 X 10(-6) M) was not affected. Nitroimipramine showed an IC50 of approximately 2 X 10(-6) M. The sodium insensitive component was not affected by nitroimipramine. Biogenic amines and related indoleamines were weak inhibitors of the sodium sensitive and sodium insensitive components of 5HT uptake. The tricyclic antidepressants and fluoxetine were effective inhibitors of the sodium sensitive component of 5-HT uptake; receptor ligands were weak inhibitors or without effect. The metabolism of [3H]5HT in tissue slices of H. diminuta was examined by HPLC. The role of the sodium sensitive uptake and metabolism of 5HT in terms of inactivation and recycling of neurally released 5HT and the possible importance of exogenous recruitment of 5HT are discussed.     

The occurrence and distribution of 5-hydroxytryptamine in Hymenolepis diminuta and H. nana

The presence of 5-HT in Hymenolepis diminuta and Hymenolepis nana was detected by 2 biochemical methods and as yellow fluorescence in a histochemical method. In H. diminuta, 5-HT was found in a concentration of about 1.2 micron/g; this amount did not vary significantly in worms aged 6 to 18 days or more or in various regions of the worm. In H. nana, 5-HT was found in a concentration of about 1.8 micron/g. It was histochemically localized in H. diminuta and H. nana in a pattern similar to that of acetylcholinesterase previously described in these 2 cestodes, and it may be the opposing neuro-transmitter to acetylcholine. The lack of 5-HT in the vestigial rostellum of H. diminuta may be correlated with loss of function of this organ.     

The uptake and metabolism of L-glutamate by tissue slices of the cestode Hymenolepis diminuta

The in vitro uptake of L-[3H]glutamate by tissue slices of the cestode Hymenolepis diminuta, denuded of tegument, was investigated. Two sodium concentration-dependent mechanisms, one of high affinity (Kt 1.8 X 10(-5) M; Vmax 4.76 pMoles/min/mg wet weight) and another of low affinity (Kt 2.2 X 10(-4) M; Vmax 50.7 pMoles/min/mg wet weight), were identified, in addition to a sodium insensitive component. Exchange of preloaded [3H]glutamate did not occur in tissue slices incubated in dilute unlabelled glutamate. Acidic amino acids, imipramine and fluoxetine were effective inhibitors of high and low affinity uptake, while glutamate receptor ligands, neurotransmitters and some antihelminthics generally were not. The concentrations present in, and the metabolism of glutamate by, tissue slices was examined by HPLC. The significance of the three modes of glutamate uptake and their possible role in the physiology of H. diminuta are discussed.     

Demonstration of specific high-affinity binding sites for [3H]5-hydroxytryptamine in the cestode Hymenolepis diminuta

[3H]5-HT (0.16-8.32 nM) exhibited saturable and specific binding in membrane preparations of Hymenolepis diminuta. The saturation data produced a non-linear Scatchard plot which could be resolved into sites having apparent dissociation constants (Kd) of 0.17 and 8.30 nM for the high-affinity and low-affinity components, respectively. Drug displacement studies, using radioligand concentrations of 0.6 and 6 nM, revealed that the two [3H]5-HT binding components are pharmacologically distinct and do not conform to any known class of 5-HT recognition site. The physiological significance of these putative 5-HT receptors and their potential usefulness for the selection of new antiparasitic agents are discussed.     

Hymenolepis diminuta: behavioral effects of 5-hydroxytryptamine, acetylcholine, histamine and somatostatin

Consistent in vitro behavioral patterns were found in the scolex and strobila of adult Hymenolepis diminuta. These patterns were measured with a force transducer and the behavior analyzed with a slow motion closed circuit T.V. Varying concentrations of serotonin (5-HT), acetylcholine (Ach), histamine and somatostatin, in the range of 10(-3) to 10(-9) M, were tested for their influence on the rhythmic patterns of behavior. High concentrations of 5-HT and of Ach decreased scolex motility. While 5-HT significantly increased motility in the anterior-, mid- and posterior regions of the strobila at 10(-3) M, Ach inhibited motility in all 3 regions of the strobila at the same concentrations. At high concentrations, somatostatin had a smaller stimulatory effect on worm motility in the anterior and mid-regions; histamine only significantly affected worm motility in the posterior region of the strobila. Depending on concentration, the action of 5-HT, Ach and histamine can be reversed, particularly in the anterior and posterior regions of the strobila. The in vivo assay for worm migrational responses suggests that the action of the neuromuscular stimulators and inhibitors on worm migration is indirect.     

The embryonic development of the bodywall and nervous system of the cestode flatworm Hymenolepis diminuta

Cestodes (tapeworms) are a derived, parasitic clade of the phylum Platyhelminthes (flatworms). The cestode body wall represents an adaptation to its endoparasitic lifestyle. The epidermis forms a non-ciliated syncytium, and both muscular and nervous system are reduced. Morphological differences between cestodes and free-living flatworms become apparent already during early embryogenesis. Cestodes have a complex life cycle that begins with an infectious larva, called the oncosphere. In regard to cell number, cestode oncospheres are among the simplest multicellular organisms, containing in the order of 50-100 cells. As part of our continuing effort to analyze embryonic development in flatworms, we describe here the staining pattern obtained with acTub in embryos and larvae of the cestode Hymenolepis diminuta and, briefly, the monogenean Neoheterocotyle rhinobatidis. In addition, we labeled the embryonic musculature of Hymenolepis with phalloidin. In Hymenolepis embryos, two different cell types that we interpret as neurons and epidermal gland cells express acTub. There exist only two neurons that develop close to the midline at the anterior pole of the embryo. The axons of these two neurons project posteriorly into the center of the oncosphere, where they innervate the complex of muscles that is attached to the hooklets. In addition to neurons, acTub labels a small and invariant set of epidermal gland cells that develop at superficial positions, anteriorly adjacent to the neurons, in the dorsal midline, and around the posteriorly located hooklets. During late stages of embryogenesis they spread and form a complete covering of the embryo. We discuss these data in the broader context of platyhelminth embryology.     

Amino acid metabolism in the rat tapeworm, Hymenolepis diminuta

1. The amino acid metabolism of the rat tapeworm, Hymenolepis diminuta was investigated. 2. In addition to the characteristic end products of helminth metabolism, H. diminuta also forms substantial amounts of 14C-alanine during incubations in 14C-glucose. 3. Of 10 amino acids tested, only 14C-labelled asparate and, to a lesser extent alanine, generated substantial amounts of 14CO2 when incubated with H. diminuta. 4. 14C-aspartate was incorporated into both succinate and acetate, major products of the worms mitochondrial metabolism, but the rates were low when compared to the metabolism of exogenous glycogen. 5. These results suggest that amino acid metabolism in H. diminuta is very limited.     

The effect of L-glutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta.

The effect of the putative amino acid transmitter, L-glutamate, on adenylate cyclase in crude membrane preparations of the rat tapeworm Hymenolepis diminuta was investigated to determine if glutamate effects the generation of the second messenger cAMP. Addition of glutamate at 10(-3) and 5.5 x 10(-9) M resulted in significant elevations in basal activity of adenylate cyclase, while concentrations in the 10(-5)-10(-7) M range caused significant depressions below basal activity. Assays with glutamate agonists and other acidic compounds showed glutamate to be the only amino acid, dicarboxylic acid, or acidic compound capable of this pattern of stimulation and inhibition. While the response of adenylate cyclase to glutamate agonists suggested that an N-methyl-D-aspartic acid (NMDA) type receptor may be present, glutamate agents acting as NMDA antagonists in vertebrate systems were agonists. Metabolic end products of glycolysis stimulated adenylate cyclase, suggesting that these, along with metabolic glutamate may regulate glycolytic enzymes. Only 10(-3) M L-glutamate significantly stimulated adenylate cyclase activity in tissue slices, and this response was restricted to those slices rich in nervous tissues. L-Glutamate eliminated the 5-hydroxytryptamine (5-HT) stimulated adenylate cyclase response suggesting that glutamate can modulate the 5-HT stimulated elevations in adenylate cyclase activity. The data support the hypothesis that L-glutamate is a neurotransmitter-modulator in the cestode.     

Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.     

Enzymatic antioxidant system in the cestode Hymenolepis diminuta after chronic infection of the rat

Background

The aim of the present paper was to describe the enzymatic antioxidant system in Hymenolepis diminuta collected from rats exposed to chronic cestode invasion.

Methodology

We dissected different tissues of H. diminuta (immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus) and studied activity of: superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (non-Se-dependent GSHPx and Se-dependent GSHPx), glutathione-S-transferase (GST) and glutathione reductase (GSHR), and oxidative stress markers ?? reduced glutathione (GSH), and the lipid peroxidation level (TBARS).

Results

We demonstrated changes in antioxidant enzyme activities and levels of oxidative stress markers in different tissues of the parasite. The levels of TBARS and GSH indicate that oxidative stress occurred in tissues located proximal to the intestine wall. Activity of SOD1 was high in all parts of H. diminuta, but the GST activity was the highest of all studied antioxidant enzymes. SOD2 activity differed significantly in various parts of H. diminuta. Significant differences were observed for nonSeGSHPx and activity of other GSH-dependent enzymes was generally similar in all the tissues.

Conclusions

Our results show that the enzymatic antioxidant system of H. diminuta, allows the parasite to adapt and live under conditions of chronic oxidative stress. It suggests an oxidative-antioxidative balance during interactions between parasite and host.