Martine Verger-Bocquet

Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.

Proenkephalin A-derived peptides in invertebrate innate immune processes

Lipopolysaccharides (LPS) injection into the coelomic fluid of the leech Theromyzon tessulatum stimulates release of proenkephalin A (PEA)-derived peptides as determined by immunoprecipitation and Western blot analyses. This release occurs in the first 15 min after LPS exposure and yields a 5.3-kDa peptide fragment corresponding to the C-terminal part of the precursor. This fragment is then cleaved to free an antibacterial peptide related to mammals arginine phenylalanine extended enkelytin: the peptide B. These PEA processing peptides were characterized using a combination of techniques including reversed-phase HPLC, microsequencing and mass spectrometry. The isolated invertebrate peptide B presents a high sequence homology with the bovines and the same activity against Gram+bacteria. Titrations revealed the simultaneous appearance of Methionine-enkephalin (ME) and peptide B in invertebrates after stimulation by LPS (in a dose-dependent manner), surgical trauma or electrical stimulations to neural tissues of the mussel. Furthermore, peptide B processing in vitro yields Methionine-enkephalin arginine phenylalanine (MERF), which exhibits via the delta receptors, immunocyte excitatory properties, i.e., movement and conformational changes, but no antibacterial activity. We surmise that this unified response to the various stimuli is a survival strategy for organism by providing immediate antibacterial activity and immunocyte stimulation, thereby reducing any immune latency period needed for an adequate immune response.

Isolation and structural characterization of enkephalins in the brain of the Rhynchobdellid leech Theromyzon tessulatum

This paper reports the purification of four peptides related to enkephalins from the brain of the leech Theromyzon tessulatum. After reverse‐phase HPLC purification, the sequence of the enkephalins (YGGFM, YGGFL, FM, FL) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and co‐elution experiments in reverse‐phase HPLC with synthetic peptides. ELISA titrations performed on each purified peptide indicated that the major amount was borne by the leucine‐enkephalin. The ratio of leucine‐enkephalin and methionine‐enkephalin of 2:1 is in line with previous immunocytochemical data obtained on T. tessulatum brains. The presence of enkephalins in T. tessulatum, an animal belonging to the oldest group of coelomate metazoans (the Annelida) establishes the very ancient phylogenetic origin of opioids and their conservation in the course of evolution.

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.

Elements of angiotensin system are involved in leeches and mollusks immune response modulation

We present immunocytochemical, biochemical and cellular evidences for the presence of a renin-angiotensin system (RAS) in coelomocytes of invertebrates (leech, Theromyzon tessulatum and mollusk Mytilus edulis). Leech coelomocytes are immunoreactive to polyclonal antisera raised against the T. tessulatum angiotensin-converting enzyme (ACE) and leech brain angiotensin II (AII) and a commercial anti-AT1 receptor. Biochemically, renin, ACE- and AT1-like receptor were identified in the leech immune cells. We further demonstrate that leech AII (10(-6) M) alone does not initiate nitric oxide (NO) release in invertebrate immunocytes but does only after pre-exposing the cells to IL-1 (15.9+/-2.6 nM; P<0.005 vs. 1.1 nM when AII is added alone). Similar results were obtained with human leukocytes (14.5+/-2.7 nM; P<0.005 IL-1+AII vs. 0.9 nM when AII is added alone). Then, an immunocytochemical study performed at the structural and ultrastructural levels confirmed the presence in same immune cells all the molecules of the renin-angiotensin system (RAS) in leeches as epitopes to IL-1-like protein and IL-1-like receptor. This is the first report in invertebrates and of a co-action between cytokines like substances and neuropeptides in an immune process and the involvement of the RAS in modulation of the immune response.

Biochemical identification and ganglionic localization of leech angiotensin-converting enzymes

We demonstrate the presence of a membrane and soluble form of leech Theromyzon tessulatum angiotensin-converting enzyme (ACE). Four steps in the purification of this enzyme include gel-permeation, captopril-sepharose affinity and anion-exchange chromatography followed by a reverse-phase HPLC. The peptidyl dipeptidases (of approximately 120 and approximately 100 kDa) are glycosylated enzymes hydrolysing the Phe8-His9 bond of angiotensin I, exhibiting the same specific activity and Km whereas the soluble ACE exhibits a higher catalytic efficiency. This hydrolysis is inhibited by the ACE-specific antagonist captopril. Western blot analysis of a polyclonal antiserum raised against the first 11 amino-acid residues of the membrane ACE and the N-terminal sequence of the soluble molecule also demonstrates the presence of two ACE enzymes. Anti-ACE immunocytochemistry also supports the presence of two forms of ACE. This material is found in neurons and glia. We demonstrate for the first time the cellular localization and biochemical characterization of ACEs in the central nervous system of an invertebrate. Thus, the leech brain may represent a simple model for the study of these enzymes.

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.

Structural characterization of osmoregulator peptides from the brain of the leech Theromyzon tessulatum : IPEPYVWD and IPEPYVWD-amide

Neurons immunoreactive to an antiserum (a-OT) directed specifically against the C-terminal part (prolyl-leucyl-glycinamide) of vertebrate oxytocin (OT) were detected in the brain of the leech Theromyzon tessulatum. With high pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts, evidence was given of the presence of three peptides (P1, P2, P3) immunoreactive to a-OT. Results of injection experiments in T. tessulatum and of titrations of each peptide at the different physiological stages of the animals which showed a peak in peptide P1 amount at stage 3B, indicated that P1 is the active OT-like peptide. Using three steps of reversed-phase HPLC, Edman degradation and electrospray mass spectrometry, two sequences for P1 (IPEPYVWD and IPEPYVWD-amide) were found. These peptides differ from peptides to the oxytocin/vasopressin family and are unique in the animal kingdom. Confirmation of their action on the hydric balance and their distribution in the CNS were presented.

Putative leech dopamine1-like receptor molecular characterization: sequence homologies between dopamine and serotonin leech CNS receptors explain pharmacological cross-reactivities

The biochemical characterization of a serotonin (5HT) receptor and the cloning of a dopamine (DA) receptor in the central nervous system (CNS) of the leech, Theromyzon tessulatum, is presented. Additionally, DA and 5HT binding sites were examined in the CNS by Scatchard analysis which showed a single, relatively high-affinity binding site with a Kd 1.1 nM and a Bmax 126+18 fmol/mg protein for [3H]DA and a Kd 2.1 nM and a Bmax 225 fmol/mg protein for [3H]5HT. The first 88 amino acids of the 5HT receptor, isolated by a 5HT-affinity column followed by anion exchange chromatography and C3 reverse-phase HPLC exhibited a 43% sequence homology with Lymnaea stagnalis 5HT-receptor. The isolated DA receptor revealed a single protein of 45 kDa with an anti-D1-R in Western blot. The first 80 N-terminal amino acid residues and a trypsin digested fragment of 31 residues were obtained, and based on these sequencing data, a molecular biology strategy using reverse transcriptase-polymerase chain reaction, was developed. An amplified 1-kb segment was obtained. The complete deduced sequence of 416 amino acid residues exhibited about 30.6% sequence homology with the vertebrate D1 receptor family. Moreover, we further demonstrate that the leech 5HT and DA receptors also exhibit 30% sequence identity with each other, explaining their pharmacological cross-reactivity. Finally, anti-D1-R immunocytochemistry revealed positive structures in the peripheral and central nervous system, e.g., neurons, sensory fibers and immune cells. This is the first biochemical and molecular characterization of a DA receptor in leeches.

Biochemical evidence of the sodium influx stimulating related peptide in the brain of the leech Theromyzon tessulatum

This paper reports the immunocytochemical and biochemical evidence of a sodium influx stimulating related peptide (SIS-like peptide) in the brain of the leech Theromyzon tessulatum. Cells immunoreactive to both polyclonal antisera raised against the N-terminal (fragment 10-19) or the C-terminal (fragment 67-76) sequences of the purified freshwater snail Lymnaea stagnalis sodium influx stimulating peptide (SISP) was detected in the brain of this leech. These immunocytochemical data were strengthened by biochemical results at the level of the protein and by in vitro translated RNA. By combined techniques i.e. high performance gel permeation chromatography, electrophoresis followed by immunoblot analysis with the two antisera and immunoprecipitation, we established the existence in leeches of a protein of ca 11 kDa and its precursor, a protein of ca 13 kDa related to the SISP. Moreover, results of injections of anti-SISP (10-19) preabsorbed or not with its homologous antigen to Theromyzon tessulatum confirmed the SIS-like substance involvement in osmoregulation by an anti-diuretic function.