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Electrogenic cation transport across leech caecal epithelium.

Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (RT) and potential difference (VT) were 61.0 +/- 3.5 omega.cm2 and -1.1 +/- 0.2 mV (n = 149), respectively, indicating that leech caecal epithelium is a "leaky" epithelium. Under control conditions short circuit current (ISC) and transepithelial Na+ transport rate (INa) averaged at 22.1 +/- 1.5 microA.cm-2 and 49.7 +/- 2.6 microA.cm-2, respectively. Mucosal application of amiloride (100 mumol.l-1) or benzamil (50 mumol.l-1) influenced neither ISC nor INa. The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na+ concentration. Removal of mucosal Ca2+ increased ISC by about 50% due to an increase of transepithelial Na+ transport. Trivalent cations (La3+ and Tb3+, 1 mmol.l-1 both) added to the mucosal Ringer solution reduced INa by more than 40%. Serosal ouabain (1 mmol.l-1) almost halved ISC and INa while 0.1% (= 5.4 mmol.l-1) DNP decreased INa to 11.8 +/- 5.1% of initial values. Serosal addition of cAMP increased both ISC and INa whereas the neurotransmitters. FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced INa markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca2+ but insensitive to amiloride. Basolaterally Na+ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na+ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown.

Animals↗

Study of the effect of leeching on plasma endothelin and soluble interleukin-2 receptor in patients with systemic lupus erythematosus.

OBJECTIVE: to explore the mechanism of leeching in treating systemic lupus erythematosus (SLE). METHODS: Forty-four patients with SLE were randomly divided into conventional corticosteroid treated group (control group, n = 20) and conventional treatment group with leeching intervention added (leeching group, n = 24). Before and after treatment the concentration of plasma endothelin (ET) and soluble interleukin-2 receptor (sIL-2R) were determined. RESULTS: Before treatment the level of plasma ET and sIL-2R in the SLE patients were all higher than those in the normal healthy group, (P < 0.01). But after treatment the level of these in both groups were significantly improved than those of before treatment (P < 0.05), and comparison between these two treated groups showed that the difference between them was significant (P < 0.05). CONCLUSION: Leeching added to conventional treatment of SLE could be more effective in improving the level of plasma ET and sIL-2R, and ameliorating the impairment of renal tissues.

Adult↗

Entrainment of leech swimming activity by the ventral stretch receptor.

Rhythmic animal movements originate in CNS oscillator circuits; however, sensory inputs play an important role in shaping motor output. Our recent studies demonstrated that leeches with severed nerve cords swim with excellent coordination between the two ends, indicating that sensory inputs are sufficient for maintaining intersegmental coordination. In this study, we examined the neuronal substrates that underlie intersegmental coordination via sensory mechanisms. Among the identified sensory neurons in the leech, we found the ventral stretch receptor (VSR) to be the best candidate for our study because of its sensitivity to tension in longitudinal muscle. Our experiments demonstrate that (1) the membrane potential of the VSR is depolarized during swimming and oscillates with an amplitude of 1.5-5.0 mV, (2) rhythmic currents injected into the VSR can entrain ongoing swimming over a large frequency range (0.9-1.8 Hz), and (3) large current pulses injected into the VSR shift the phase of the swimming rhythm. These results suggest that VSRs play an important role in generating and modulating the swim rhythm. We propose that coordinated swimming in leech preparations with severed nerve cords results from mutual entrainment between the two ends of the leech mediated by stretch receptors.

Action Potentials↗

Molecular characterization and embryonic expression of innexins in the leech Hirudo medicinalis.

Gap junctions are direct intercellular channels that permit the passage of ions and small signaling molecules. The temporal and spatial regulation of gap junctional communication is, thus, one mechanism by which cell interactions, and hence cell properties and cell fate, may be regulated during development. The nervous system of the leech, Hirudo medicinalis, is a particularly advantageous system in which to study developmental mechanisms involving gap junctions because interactions between identified cells may be studied in vivo in both the embryo and the adult. As in most invertebrates, gap junctions in the leech are composed of innexin proteins, which are distantly related to the vertebrate pannexins and are encoded by a multi-gene family. We have cloned ten novel leech innexins and describe the expression of these, plus two other previously reported members of this gene family, in the leech embryo between embryonic days 6 and 12, a period during which the main features of the central nervous system are established. Four innexins are expressed in neurons and two in glia, while several innexins are expressed in the excretory, circulatory, and reproductive organs. Of particular interest is Hm-inx6, whose expression appears to be restricted to the characterized S cell and two other neurons putatively identified as presynaptic to this cell. Two other innexins also show highly restricted expressions in neurons and may be developmentally regulated.

Animals↗

Differential expression of the EF-hand calcium-binding protein calsensin in the central nervous system of hirudinid leeches.

By immunocytochemistry the distribution and developmental expression of the small EF-hand calcium-binding protein calsensin in the peripheral (PNS) and central nervous system (CNS) of the three hirudinid leech species Haemopis, Hirudo, and Macrobdella was compared. Labeling with calsensin-specific antibodies demonstrated that there was a pronounced difference in the distribution of calsensin immunoreactivity in the CNS of these leeches. In Haemopis more than 70 neurons were labeled, whereas the number in Hirudo was 51 and in Macrobdella only 8. Furthermore, the expression of calsensin in identified cells common to all three leech species also differed. Immunoblot analysis indicated that this variability was not likely to be due to multiple proteins or isoforms being recognized by the calsensin antibody. Labeling of embryos in various stages of development shows that the ontogeny of calsensin expression in the CNS is a gradual process with some neurons expressing calsensin immediately after completion of neurogenesis, about one-third of the way through embryogenesis, and others expressing calsensin only postembryonically. In contrast to the variability in the pattern and temporal expression by CNS neurons, the early embryonic calsensin expression in a small subgroup of sensillar PNS neurons was a shared feature by all three leech species. These findings suggest that calsensin may have different functional properties in CNS and PNS neurons.

Animals↗

Antibody staining reveals novel aspects of segmentation within the leech central nervous system.

The leech is a segmented annelid with a well characterized central nervous system. In this report, we use antibodies to map the distribution of neurons confined to selected segmental ganglia in the mud leech Haemopis marmorata. The distribution of these neurons suggest 3 novel aspects of segmentation in the leech nervous system: (1) neurons are assigned to even-numbered ganglia through a mechanism which effectively counts through the leech segmental body plan by units of 2, (2) neurons are assigned to ganglia 7 and 14 through a mechanism which effectively counts in units of 7 and (3) neurons are assigned to the 2nd and 4 fused head ganglia and to the 2nd of 21 unfused midbody ganglia through a mechanism which effectively counts units from the origin of these 2 ganglionic series. These 3 hypothetical counting mechanisms divide the central nervous system (CNS) into supersegmental units. Neurons used to define these supersegmental units have been injected with tracer and identified as interganglionic interneurons. Competitive interactions among embryonic precursors of these neurons may directly eliminate their homologs from intervening ganglia, and thus sculpture supersegmental patterns into the mature nervous system.

Animals↗

Oxytocin-like peptide: a novel epitope colocalized with the FMRFamide-like peptide in the supernumerary neurons of the sex segmental ganglia of leeches--morphological and biochemical characterization; putative anti-diuretic function.

A large number of oxytocin (OT)-like neurons were detected in the sex segmental ganglia (SG5, SG6) of three species of leeches belonging to different orders: Theromyzon tessulatum, Hirudo medicinalis and Erpobdella octoculata. In this latter species, an epitope close to the vertebrate OT by its C-terminal part (MSH release inhibiting factor: MIF), localized in granules of a size diameter of ca 120 nm and colocalized with FMRFamide(FMRFa)-like material was demonstrated. With reverse phase-high performance liquid chromatography, evidence was given that the two epitopes (OT and FMRFa) colocalized in the same neurons were biochemically different. A titration of OT per SG indicated that the OT-like amount was considerably higher in sex SG than in non-sex SG (ca. 5 pmol vs. ca. 0.5 pmol). Moreover, at the level of sex SG, this amount was ca. 3-fold higher in immature leeches than in mature specimens. Injections of extracts of SG of E. octoculata and of fragments of OT (Tocinoic acid or MIF) to T. tessulatum, indicated that MIF (the epitope found in the sex SG) and sex SG have the same anti-diuretic effect on the leeches injected. These results pointed to an anti-diuretic role of the leech OT-like substance.

Animals↗

Inhibition of plasma kallikrein. Kininase and kinin-like activities of preparations from the medicinal leeches.

The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of blood coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as well as kinin-like effects of extracts heated at 100 degrees C have been detected. The last one is correlated with the hyperalgetic property of the heated extract. An analgetic effect was observed with the unheated extract but not with leech saliva after intranasal administration to rats.

Amino Acid Sequence↗

Metabolism of enkephalins in head membranes of the leech Theromyzon tessulatum by peptidases: isolation of an enkephalin-degrading aminopeptidase.

Metabolism of leucine and methionine enkephalins by enzyme preparations from head parts of the leech Theromyzon tessulatum was investigated. Leech homogenate degraded enkephalins by cleavage of the Tyr1-Gly2 and Gly3-Phe4 bonds. The Tyr1-Gly2-Gly3 was detected as a major metabolite when amastatin (aminopeptidase inhibitor) was present to prevent Tyr1-Gly2 breakdown. Around 50% of enkephalin-degrading activity was isolated in a 20000 x g membrane fraction and was shown to be almost entirely due to an aminopeptidase activity. This enzyme, a homodimer of approx. 70 kDa, has been purified to homogeneity by a combined approach including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC. This enkephalin-degrading aminopeptidase is a typical integral membrane 'zincin' metalloprotein with an apparent k(m) of 30 microM, a specific activity of 12 nmol GGFM min-1 mg protein-1 and a catalytic efficiency (kcat/k(m)) of 46 x 10(6) mol-1 min-1. This enzyme is specifically inhibited by amastatin (IC50 = 0.5 microM), but not by bestatin and actinonin. In leech membranes, the other degrading activities performed at the same time were due to a neuropeptide-endopeptidase (NEP)-like enzyme attack, inhibited by phosphoramidon (IC50 = 0.1 microM) and in the case of the Met-enkephalin by a combined action of an angiotensin-converting-like enzyme, inhibited by captopril (IC50 = 0.2 microM) and the NEP-like enzyme. These two enzymes were previously isolated from head membranes of T. tessulatum and possess towards Met-enkephalin a catalytic efficiency (kcat/k(m)) of, respectively, 12 x 10(6) mol-1 min-1 and 78 x 10(6) mol-1 min-1. These findings constitute the first report in leeches on the nature and the sites of attack of the membrane peptidases involved in the metabolism of enkephalins and also the first biochemical evidence for a novel member of the aminopeptidase family.

Aminopeptidases↗

Stretch receptors and body wall muscle in leeches.

1. A new kind of muscle receptor has been described in the medicinal leech, the first stretch receptor in a soft-bodied invertebrate to be identified and characterized. 2. The sensory responses to stretch in these receptors are different from those of other more well known muscle receptors such as vertebrate spindles or crustacean stretch receptors. 3. The leech receptors provide the sensory innervation for the tubular muscle layers of the body wall, and they respond to stretch of the muscle with hyperpolarizing potentials that are conducted passively to the CNS. 4. This review summarizes their unusual morphology, membrane properties and sensory responses to stretch; their association with particular muscle fibre types in leech body wall; their synaptic connections; and their role in a well characterized rhythmic behaviour in the leech-swimming.

Animals↗

Proton magnetic resonance spectroscopy of leech muscle and nervous system.

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.

3-Hydroxybutyric Acid↗

The effects of procaine, strychnine and penicillin on nociceptive neurons in leech segmental ganglia.

Procaine, strychnine and penicillin selectively depolarized the membrane potential and prolonged the action potential recorded in the lateral but not the medial nociceptive (N) cell in the hirudinid leech Macrobdella decora. In contrast, procaine did not differentiate between medial and lateral N cells in two other hirudinid leeches Hirudo medicinalis and Haemopis marmorata. In these species, the drug equally decreased the amplitude of action potentials in both types of N cells without effecting their resting membrane properties. In the nociceptive neurons of the glossiphoniid leech Haementeria ghilianii which possesses only one type of N cell, procaine produced a depolarization and prolonged the action potential. This finding indicates that the single pair of N cells in Haementeria is of the lateral type. The results suggest that the lateral type N cell in Macrobdella and Haementeria share a unique Na+-dependent conductance which is selectively opened by the local anesthetics procaine and strychnine as well as by penicillin. This conductance is either not present or insensitive to the drugs in the homologous N cells in the two other leech species examined.

Animals↗

A new lineage of trypanosomes from Australian vertebrates and terrestrial bloodsucking leeches (Haemadipsidae).

Little is known about the trypanosomes of indigenous Australian vertebrates and their vectors. We surveyed a range of vertebrates and blood-feeding invertebrates for trypanosomes by parasitological and PCR-based methods using primers specific to the small subunit ribosomal RNA (SSU rRNA) gene of genus Trypanosoma. Trypanosome isolates were obtained in culture from two common wombats, one swamp wallaby and an Australian bird (Strepera sp.). By PCR, blood samples from three wombats, one brush-tailed wallaby, three platypuses and a frog were positive for trypanosome DNA. All the blood-sucking invertebrates screened were negative for trypanosomes both by microscopy and PCR, except for specimens of terrestrial leeches (Haemadipsidae). Of the latter, two Micobdella sp. specimens from Victoria and 18 Philaemon sp. specimens from Queensland were positive by PCR. Four Haemadipsa zeylanica specimens from Sri Lanka and three Leiobdella jawarerensis specimens from Papua New Guinea were also PCR positive for trypanosome DNA. We sequenced the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes in order to determine the phylogenetic positions of the new vertebrate and terrestrial leech trypanosomes. In trees based on these genes, Australian vertebrate trypanosomes fell in several distinct clades, for the most part being more closely related to trypanosomes outside Australia than to each other. Two previously undescribed wallaby trypanosomes fell in a clade with Trypanosoma theileri, the cosmopolitan bovid trypanosome, and Trypanosoma cyclops from a Malaysian primate. The terrestrial leech trypanosomes were closely related to the wallaby trypanosomes, T. cyclops and a trypanosome from an Australian frog. We suggest that haemadipsid leeches may be significant and widespread vectors of trypanosomes in Australia and Asia.

Amphibians↗

Neuronal control of leech behavior.

The medicinal leech has served as an important experimental preparation for neuroscience research since the late 19th century. Initial anatomical and developmental studies dating back more than 100 years ago were followed by behavioral and electrophysiological investigations in the first half of the 20th century. More recently, intense studies of the neuronal mechanisms underlying leech movements have resulted in detailed descriptions of six behaviors described in this review; namely, heartbeat, local bending, shortening, swimming, crawling, and feeding. Neuroethological studies in leeches are particularly tractable because the CNS is distributed and metameric, with only 400 identifiable, mostly paired neurons in segmental ganglia. An interesting, yet limited, set of discrete movements allows students of leech behavior not only to describe the underlying neuronal circuits, but also interactions among circuits and behaviors. This review provides descriptions of six behaviors including their origins within neuronal circuits, their modification by feedback loops and neuromodulators, and interactions between circuits underlying with these behaviors.

Animals↗

Molecular phylogeny of selected predaceous leeches with reference to the evolution of body size and terrestrialism.

The phylogenetic relationships of erpobdellid leeches collected throughout Europe were investigated using newly obtained mitochondrial cytochrome c oxidase subunit I (CO-I) gene sequence data from 10 taxa. Monophyly of the five European Erpobdella species (sub-family Erpobdellinae) was supported, but a newly discovered leech, E. wuttkei Kutschera, 2004 (the smallest member of its genus, discovered in an aquarium) was only distantly related to this clade. Three members of the semiaquatic Trochetinae were included in this study. The largest European leech species discovered so far, Trocheta haskonis Grosser, 2000, was found to be a terrestrial predator that feeds on earthworms. The rare species T. haskonis is the sister taxon of T. bykowskii Gedroyc, 1913, a well-known amphibious leech. Based on a comparison of body sizes and a phylogenetic tree the evolution of terrestrialism in the family Erpobdellidae is discussed.

Aging↗

Invertebrate proenkephalin: delta opioid binding sites in leech ganglia and immunocytes.

The leech Theromyzon tessulatum and the marine mussel Mytilus edulis immunocytes contain a mammalian-like proenkephalin molecule. The opioid precursor was purified by gel permeation chromatography, anti-Met- and Leu-enkephalin-affinity column separation and then by reversed-phase HPLC. The amino acid sequence analysis, determined by Edman degradation, enzymatic treatments and matrix assisted laser desorption time of flight. The structure of the leech proenkephalin material demonstrates considerable amino acid sequence similarity with amphibian proenkephalin (e.g. 25.4% with Xenopus laevis) but it is smaller, 15 kDa vs. 30 kDa. In contrast, Mytilus proenkephalin is not only larger (26 kDa) but it exhibits a higher sequence identity with guinea pig proenkephalin (50%). Both of the invertebrate materials possess Met-enkephalin and Leu-enkephalin in a ratio of 3:1 for Mytilus and 1:2 in the leech. They also contain Met-enkephalin-Arg-Gly-Leu and Met-enkephalin-Arg-Phe sequences that are flanked by dibasic amino acid residues, demonstrating cleavage sites. Furthermore, using sequence comparison with bovine proenkephalin A (209-237), enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM), an antibacterial peptide is found in the proenkephalin of both animals and it exhibits a 98% sequence identity with mammalian material. Finally, opioid binding experiments demonstrate the presence in leech ganglia and immunocytes of delta1 and delta2 opioid receptor subtypes as also found human and Mytilus immune cells. This report constitutes the first complete biochemical characterization of mammalian proenkephalin in invertebrates, demonstrating its origin in simpler animals.

Amino Acid Sequence↗

Behavioral hierarchy in the medicinal leech, Hirudo medicinalis: feeding as a dominant behavior.

The effect of feeding behavior on other behaviors (swimming, crawling and shortening) was investigated in the leech, Hirudo medicinalis. The stimulus locations and intensities required to produce mechanically elicited behaviors were first determined in the non-feeding leech. Stimuli were delivered while the leech was in various body positions to determine whether stimulus location affected behavioral response. Response thresholds were determined for the mechanically elicited behaviors. The same stimuli were then applied to feeding leeches to determine if response thresholds had changed. A solution with NaCl and arginine was used to elicit feeding. The same sets of stimuli were applied at intervals for an hour after feeding, to determine the duration of feeding-induced changes in behavior. Depending on the body position and stimulus location, stimuli produced different combinations of behaviors that included shortening, swimming and crawling. Anterior stimuli generally elicited shortening, whereas posterior stimuli generally elicited crawling and swimming, with swimming more likely to ventral stimulation than to dorsal stimulation. Having the front sucker attached changed these behavioral patterns. During feeding, the response thresholds changed dramatically, from 3-5 V to greater than 9 V. This increase in threshold began with the start of feeding, even before ingestion commenced. Suppression of the behaviors lasted up to 1 h after the end of feeding, with the effect on swimming being the most pronounced and longest lasting.

Animals↗

Leech egg-laying-like hormone: structure, neuronal distribution and phylogeny.

Cells immunoreactive to antisera specifically directed against Lymnaea stagnalis caudo dorsal cells egg-laying hormone (CDCH) or against alpha- and beta-peptides (CDCP), encoded on the egg-laying hormone precursor, were detected in central nervous system (CNS) of the rhynchobdellid leech Theromyzon tessulatum. A co-localization of the CDC-like hormone and CDC-like peptides was found in T. tessulatum as in L. stagnalis CNS. approximately 45 immunoreactive cells to the anti-CDCH were detected in leech brain but this number varies according to the stage of the animal life cycle, i.e. it reaches a maximum just before egg-laying while after it decreases to 2-3 cells. CDCH and alpha-CDCP epitopes recognized by anti-CDCH and anti-alpha-CDCP were contained in neurosecretory granules. Following an extensive purification, including HPGPC and reverse-phase HPLC, the CDC-like hormone contained in the T. tessulatum CNA was isolated. The sequence (GSGVSNGGTEMIQLSHIRERQRYWAQDNLRRRFLEK-amide) was established by a combination of automated Edman degradation, arginyl-endopeptidase digestion, electrospray mass spectrometry measurement and carboxypeptidase A treatment. The results demonstrate that the peptide recognized by the anti-CDCH in the leech CNS possesses 27.8, 37.2 and 47.2% sequence identity with Aplysia parvula, Lymnaea stagnalis and Aplysia californica ELH, respectively. This molecule was named the leech egg-laying-like hormone (L-ELH). The secondary structure prediction of the L-ELH and all mollusks ELH, revealed the existence of a conserved segment (segment 29-34) in a strong helicoidal bend that might be important for receptor recognition and/or activation. This finding constitutes the first biochemical characterization of an egg-laying hormone in other invertebrates than mollusks.

Amino Acid Sequence↗