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Histopathological changes after induced injury in leeches.

Cells involved in leech inflammatory responses have been characterized by morphological, histochemical, and immunohistochemical methods. Macrophage-like cells, NK-like cells, and granulocytes migrated shortly after injury by pricking with bacterial lipopolysaccharide. Inflammatory responses increased progressively and provoked cell migration to the body wall and then to wound surfaces. Macrophages, NK cells, and granulocytes display similar features and behavior traits in invertebrates and vertebrates.

Animals↗

Crystallization and preliminary crystallographic analysis of antistasin, a leech-derived inhibitor of blood coagulation factor Xa.

The salivary gland of the Mexican leech Haementeria officinalis contains a 15 kDa protein which is a potent and selective inhibitor of factor Xa. It inhibits not only blood coagulation, but also metastasis. A gene, coding for a sequence similar to published antistasin sequences, has been synthesized and expressed in Chinese hamster ovary (CHO) cells. The recombinant protein was purified and crystallized at pH 6.0, using 31% ammonium sulfate as a precipitant. The crystals diffract at least to 2.8 A. The spacegroup is I422 with a = b = 77.7 A and c = 88.4 A. The crystals contain 42% solvent and one protein molecule in the asymmetric unit. A search for heavy atom derivatives is in progress.

Animals↗

The 1.8 A structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism.

Intramolecular trans-sialidase from leech (Macrobdella decora) is the first member of the sialidase superfamily found to exhibit strict specificity towards the cleavage of terminal Neu5Acalpha2-->3Gal linkage in sialoglycoconjugates. Its release of 2,7-anhydro-Neu5Ac instead of Neu5Ac indicates that it catalyzes an intramolecular trans-sialosyl reaction. Crystal structures of its complexes with an inactive substrate analogue 2-propenyl-Neu5Ac, and with the product 2,7-anhydro-Neu5Ac, have been determined to 1.8 A resolution. The boat conformation of the pyranose observed in the complexes supports the proposed enzymatic mechanism that O7 of an axial 6-glycerol group attacks the positively charged C2 of the intermediate. A generalized mechanism is proposed for the sialidase superfamily.

Amino Acid Sequence↗

Promotion of lung tumor colonization in mice by the synthetic thrombin inhibitor (no. 805) and its reversal by leech salivary gland extracts.

The role of anticoagulation per se in the reduction of experimental or spontaneous metastasis still remains to be determined, as shown by the conflicting results reported by the literature using different conventional anticoagulants. A new compound has been synthesized (compound no. 805) which prolongs or suppresses coagulation via specific inhibition of thrombin and its possible use in a model of experimental metastasis to clarify the role of anticoagulants in tumor spread was investigated. Contrary to our expectations, this compound increased rather than decreased the number of lung colonies induced by intravenous injections of a variety of murine neoplasias. Studies of the mechanism of this effect indicated that the compound increases retention of tumor cells by the lung without apparent impairment of the natural cell immune system, suggesting that the synthetic thrombin inhibitor may enhance vascular attachment of tumor cells. The promoting effect of compound no. 805 on metastasis was totally reversed by the administration of leech salivary gland extracts, which appear to protect capillaries from damage produced by cyclophosphamide, as revealed by other studies.

Animals↗

Modulation of swimming behavior in the medicinal leech. II. Ionic conductances underlying serotonergic modulation of swim-gating cell 204.

In the previous paper we showed that serotonin had several effects on the electrical properties of swim-gating neurons (cells 204) of the leech. These included membrane potential depolarization, induction of a sag voltage response, and enhancement of rebound responses. Here we investigate the ionic basis of these changes by comparing responses of cell 204 to injected current pulses in experimental salines containing modified concentrations of Na+, K+, Ca2+, or Cl-. Our data indicate that serotonin modulates multiple conductances in cell 204. However, most effects of serotonin can be explained by enhancement of two Na(+)-dependent conductances, a Cs(+)-sensitive cation conductance (gh) and a persistent Na+ conductance (gNaS). Both conductances contribute to the resting potential depolarization and increased amplitude of postinhibitory rebound responses induced by serotonin. In addition, enhanced gh underlies a sag potential elicited by hyperpolarizing current pulses in serotonin-treated cells. Hyperpolarizing rebound responses following depolarizing current pulses are composed of Na(+)-dependent and Na(+)-independent components, both of which are enhanced by serotonin. Activation of an electrogenic Na+/K+ pump may underlie the prolonged Na(+)-dependent component. The Na(+)-independent component decays within 1 s and may be produced by a voltage- or Ca(2+)-activated K+ conductance.

Animals↗

A persistent sodium current contributes to oscillatory activity in heart interneurons of the medicinal leech.

1. Normal activity in bilateral pairs of heart interneurons, from ganglia 3 or 4, in the medicinal leech (Hirudo medicinalis) is antiphasic due to their reciprocally inhibitory connections. However, Ca(++)-free Co(++)-containing salines lead to synchronous oscillations in these neurons. 2. Internal TEA+ allows expression of full plateaus during Co++ induced oscillations in heart interneurons; these plateaus are not blocked by Cs+. Similar plateaus are also observed with internal TEA+ alone, but under these conditions activity in heart interneurons from ganglia 3 or 4 is antiphasic. 3. Plateaus in heart interneurons induced by Co++ and internal TEA+ involve a conductance increase. 4. A voltage-dependent inward current, IP, showing little inactivation, was isolated using single-electrode voltage-clamp in heart interneurons. This current is carried at least in part by Na+; the current is reduced when external Na+ is reduced and is carried by Li++ when substituted for Na+. 5. Calcium channel blockers such as La3+ and Co++ block neither the TEA+ induced plateaus nor IP, suggesting that Na+ is not using Ca++ channels. Moreover, IP is enhanced by Ca(++)-free CO(++)-containing salines. Thus, IP is correlated with the TEA(+)- and Co(++)-induced plateau behavior.

Animals↗

Modulation of K(+)-channels in p-neurones of the leech CNS by phosphorylation.

Two types of potassium channels of identified (p-) neurones of the leech (Hirudo medicinalis) were investigated by using the patch-clamp technique. The open-state probability of these channels in cell-attached patches can be reduced by addition of 5-hydroxytryptamine to the bath solution. After excising the patches the application of alkaline phosphatase to the cytosolic face of the patch increases the open probability. The 5-HT1A-receptor agonist buspirone mimics the effect of 5-HT. Our experiments show that the effect of 5-HT might be due to a channel phosphorylation via a 5-HT1A-receptor subtype.

Alkaline Phosphatase↗

Termination of leech swimming activity by a previously identified swim trigger neuron.

Cell Tr2 is a neuron in the subesophageal ganglion of the leech that can trigger swim episodes. In this report, we describe the ability of Tr2 to terminate ongoing swim episodes as well as to trigger swimming. Stimulation of Tr2 terminated ongoing swim episodes in nearly every preparation tested, while Tr2 stimulation triggered swim episodes in only a minority of the preparations. We suggest that the primary role of Tr2 is in the termination rather than the initiation of swimming activity. The swim trigger neuron Tr3 and a swim-gating neuron, cell 21, hyperpolarized during Tr2-induced swim termination. Another swim-gating neuron, cell 204 was sometimes slightly excited, but more often, hyperpolarized during Tr2-induced swim termination. In contrast to these cells, Tr2 stimulation excited another swim-gating neuron, cell 61. The responses of the swim-gating cells were variable in amplitude and sometimes not evident during Tr2-induced swim termination. Hence, the effects of Tr2 stimulation on swim-gating neurons seem unlikely to be the direct cause of swim termination. Oscillator cells examined during Tr2-induced swim termination include: 27, 28, 33, 60, 115, and 208. The largest effect seen in an oscillator neuron was in cell 208, which was repolarized by up to 10 mV during Tr2 stimulation. Tr2 stimulation did not produce any obvious synaptic effects in motor neurons DI-1, VI-1, and DE-3. Our findings indicate that other, yet undiscovered, connections are likely to be important in Tr2-induced swim termination. Therefore, we propose that cell Tr2 is probably a member of a distributed neural network involved in swim termination.

Animals↗

Mechanism of Cl- sensitivity in internal ion receptors of the leech: an inward current gated off by Cl- in the nephridial nerve cells.

The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl- can be replaced by Br-, but not by organic mono- or divalent anions or inorganic divalent anions. At physiological Cl- concentrations (36 mM extracellular Cl-), the nephridial nerve cell has a high resting conductance for Cl- and the membrane potential is governed by Cl-. In high extracellular Cl- concentrations (110-130 mM), membrane conductance is low, most likely due to the gating off of Cl- channels. Under these conditions, membrane potential is dominated by the K+ distribution and the nephridial nerve cell hyperpolarizes towards EK.

Animals↗

Two forms of sensitization of the local bending reflex of the medicinal leech.

Sensitization of the local bending reflex of the medicinal leech Hirudo medicinalis was studied in a semi-intact preparation in which behavioral and electrophysiological recordings were made simultaneously. 1. Sensitization of local bending could be produced in two ways: by repeated stimulation of the mechanoreceptor sensitive to pressure (the P cell), and by stimulation of the mechanoreceptor sensitive to noxious stimuli (the N cell). 2. Both forms of sensitization produced a central neuronal change, measured as an increase in the number of stimulus-evoked action potentials in cell 3 (an excitor of dorsal longitudinal muscles). 3. Intracellular stimulation of serotonin-containing neurons 21 and 61 mimicked the sensitizing stimuli, but stimulation of the Retzius cell, which also contains serotonin, did not. 4. Stimulation of the Leydig cell, which releases octopamine, decreased the strength of local bending.

Action Potentials↗

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis.

The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCPB. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCPA and SCPB.

Animals↗

Modulation of inwardly rectifying Na(+)-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech, Haementeria.

The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about -70 mV were associated with the activation of a slow inward current (Ih) which showed no inactivation with time. The time course of activation of Ih was described by a single-exponential function and was strongly voltage dependent. The activation curve of Ih ranged from -72 to -118 mV, with half-activation occurring at -100 mV. Ion-substitution experiments indicated that Ih is carried by both Na+ and K+ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of Ih and its rate of activation. It also produced a positive shift of the activation curve of the conductance underlying Ih (Gh) without altering the slope factor, thus indicating that the voltage dependence of Ih was modulated by 5-HT. Cs+ blocked both Ih and the 5-HT-potentiated current in a voltage-independent manner, whereas Ba2+ had little effect. It is concluded that 5-HT increases Ih by modulating the inwardly rectifying Na(+)-K+ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since Ih was increased by 8-bromo-cyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. In contrast, Ih was reduced by 8-bromo-cyclic GMP and by zaprinast (an inhibitor of cyclic GMP-sensitive phosphodiesterase). Cyclic GMP itself also reduced Ih, and the effect was specific to the 3',5' form; 2',3'-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMP.

Animals↗

Similarities and differences in the structure of segmentally homologous neurons that control the hearts of the leech, Hirudo medicinalis.

The neural circuit that controls the hearts in the leech comprises an ensemble of synaptically interconnected cardiac motor neurons (HE cells) and cardiac interneurons (HN cells). Both the HE cells and the HN cells constitute segmentally homologous sets. We have investigated the structure of these neurons by iontophoretic injection of Lucifer Yellow dye. Bilateral pairs of HE cells have been identified in segmental ganglia 3-19 of the nerve cord. Their structure was found to be nearly identical from ganglion to ganglion and from animal to animal. Bilateral pairs of HN cells have been identified in segmental ganglia 1-7 of the nerve cord. Their dendritic structure was found to vary from ganglion to ganglion. These segmental differences among HN cells were observed consistently from animal to animal. Some of the segmental differences in HN cell structure correlate with previously described physiological differences.

Animals↗

Protease inhibitors in the alimentary tract of the medicinal leech Hirudo medicinalis: in vivo and in vitro studies.

In individual leeches the flux of labeled serum through the digestive tract was monitored to measure the rate of digestion. A mean value of 10 mg of the original serum (or 2-3 mg of the contents of the foregut) per individual per day was found, which was constant during 10 weeks. On average the serum remained in the intestinum for 20 days. Occurrence and concentrations of eglin and bdellin, specific proteinase inhibitors of Hirudo, were analyzed after various periods following the ingestion of a meal. In the foregut they were present immediately after feeding. Their quantities increased several-fold within a few weeks. In the intestinum the tests for these inhibitors were always negative. The inhibition of the proteolytic activity of intestinum preparations by eglin, bdellin, and foregut extract was tested in vitro. Using azo-albumin as (an unspecific) substrate, inhibition by eglin was maximally 25% and by bdellin 60%. When the quantitative relations presumably representing in vivo conditions were applied, only a slight inhibition of proteolysis occurred. A hypothetical role of the inhibitors in the preservation of the blood stored in the foregut is discussed.

Animals↗

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia.

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N2 bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0-8 ,h) and long-term severe hypoxia (8-72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.

Aerobiosis↗

Recovery after anaerobic metabolism in the leech (Hirudo medicinalis L.).

Medicinal leeches (Hirudo medicinalis L.) responded to self-induced hypoxia (72 h) with typical anaerobic metabolism characterized by a decrease in adenylate energy charge, utilization of the substrates glycogen and malate, and accumulation of the main anaerobic end-products succinate and propionate. Propionate was also excreted into the medium. Ammonia excretion was suppressed. Aerobic recovery resulted in a profound O2 debt. Resynthesis of ATP was completed within 30 min. Disposal of succinate and restoring of malate required 2-3 h, and clearance of propionate and recharging of glycogen 6-12 h. Ammonia excretion did not exceed normoxic rates and excretion of propionate during recovery accounted for only 10% of total propionate accumulated during hypoxia. It is postulated that the clearance of succinate and propionate involves oxidation but also resynthesis of malate and glycogen. During hypoxia and recovery blood osmolality remained constant. The Na+ and Cl- ion concentrations in blood, the decrease of which was nearly equimolar during hypoxia, were re-established following different time-courses. Na+ concentration returned to normoxic levels after 2-3 h. The delayed increase in Cl- concentration, however, correlating with 6-12 h necessary to clear blood propionate, is interpreted as an anion regulating effect.

Acetates↗

Ultrastructure of an identified array of growth cones and possible substrates for guidance in the embryonic medicinal leech, Hirudo medicinalis.

The oblique muscle organizer (Comb- or C-cell) in the embryonic medicinal leech, Hirudo medicinalis, provides an amenable situation to examine growth cone navigation in vivo. Each of the segmentally iterated C-cells extends an array of growth cones through the body wall along oblique trajectories. C-cell growth cones undergo an early, relatively slow period of extension followed by later, protracted and rapid directed outgrowth. During such transitions in extension, guidance might be mediated by a number of factors, including intrinsic constraints on polarity, spatially and temporally regulated cell and matrix interactions, physical constraints imposed by the environment, or guidance along particular cells in advance of the growth cones. Growth cones and their environment were examined by transmission electron microscopy to define those factors that might play a significant role in migration and guidance in this system. The ultrastructural examination has made the possibility very unlikely that simple, physical constraints play a prominent role in guiding C-cell growth cones. No anatomically defined paths or obliquely aligned channels were found in advance of these growth cones, and there were no identifiable physical boundaries, which might constrain young growth cones to a particular location in the body wall before rapid extension. There were diverse associations with many matrices and basement membranes located above, below, and within the layer in which growth cones appear to extend at the light level. Additionally, a preliminary examination of myocyte assembly upon processes proximal to the growth cones further implicates a role for matrix-associated interactions in muscle histogenesis as well as process outgrowth during embryonic development.

Animals↗

Cell- and tissue-specific expression of putative protein kinase mRNAs in the embryonic leech, Hirudo medicinalis.

Protein kinases play important roles in various cellular interactions underlying metazoan development. To complement existing analyses of protein kinase function in the development of members of the three phyla, Chordata, Arthropoda, and Nematoda, we have begun to examine the cell- and tissue-specific localization of protein kinases in another metazoan phylum, the Annelida. For this purpose, we used the polymerase chain reaction to amplify putative protein kinase catalytic domain cDNAs from the medicinal leech, Hirudo medicinalis. This strategy allowed us to identify 11 cytoplasmic and receptor tyrosine kinase catalytic domains, and 2 cytoplasmic serine/threonine kinase catalytic domains. Using these cDNAs as probes for nonradioactive whole-mount in situ hybridization, we examined the embryonic expression pattern of each of the corresponding putative kinase mRNAs. As has been found in other species, most of the Hirudo protein kinase mRNAs were expressed in a highly specific manner in certain embryonic cells and tissues. We found both neuron- and glia-specific kinases within the nervous system, as well as kinases expressed in non-nervous tissues, such as the haemocoelomic, muscular, and excretory systems. These kinase cDNAs encode proteins likely to be critical for proper development, and can be used as cell- and tissue-specific histological probes for the analysis of Hirudo embryogenesis.

Amino Acid Sequence↗