Michel Salzet

Neuropeptide-derived antimicrobial peptides from invertebrates for biomedical applications

Neuropeptide-derived antimicrobial peptides from invertebrates for biomedical applications

M.

Introduction

Hospitals worldwide have become literal breeding grounds for some of the most deadly and powerful bacteria. With the increased use of antibiotics, resistance has become more frequent, leaving healthcare professionals with ineffective therapies for bacterial infections. This conducts to the identification of new pathogenic micro-organisms ledding to huge discoveries, as for example many arthritogenic bacteria (Chlamidia, Yersinia, Salmonella and Shigella), which are implicated in chronical rhumatismalpathogenic affections [2][3][4][5][6]. The discovering of such new pathogenic microorganisms stimulate since a long time researches. The fascinating discovering has changed the classical conception of the simple relationship between host and bacteria in the direct pathogenesis, suggesting that some micro organisms can have an unknown role in some of affections presumably aseptic.

This results that patients may enter a hospital for heart surgery and develop an infection of the surgical site. Many of these hospital-acquired infections are caused by Staphylococcus aureus (S. aureus), a potentially fatal bacterium that can live for extended periods on medical devices such as intravenous (IV) lines and catheter tubes. Staphylococcal infections range from local skin infections to endocarditis (heart valve infection), osteomyelitis (bone infection), sepsis (bloodstream infection), pneumonia and surgical-site infections in hospitalized patients [2][3][4][5][6]. By 1982 only 10% of S. aureus strains were susceptible to penicillin. In U.S. hospitals from 2% in 1975 to 35% in 1996 are become resistant to methicillin. The proportion of methicillin-resistant S. aureus (MRSA) has increased significantly. It is now estimated that about half of all S. aureus strains at many medical institutions are resistant to methicillin.

In the same bacterial family, methicillin-resistant S. epidermidis (MRSE) also compromises patient health. This organism, found primarily on skin tissue, was once considered a non-threatening contaminant. Now, it has been established as a leading cause of hospital-acquired bloodstream infections. More than 80% of S. epidermidis isolates in U.S. hospitals are methicillin resistant, and recent studies have found resistance to quinolones, cephalosporins and vancomycin. The emergence of S. epidermidis as a pathogen has been fuelled by the widespread use of catheters, prosthetic joints, valves and other invasive medical devices, and is a growing concern, particularly for immuno-compromised cancer patients.

Vancomycin is often used as the antibiotic of last resort for treating S. aureus and other bacterial infections, but not for long. This powerful antibiotic is now ineffective against many enterococcal infections. Vancomycin-resistant enterococci (VRE), for example, have developed resistance to all known antibiotics as a result of both successful mutations and the receipt of DNA from other drug-resistant bacteria. The US Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, reported a twenty-fold increase in the percentage of hospital-acquired enterococci found to be resistant to the drug vancomycin from January 1989 to March 1993. In fact, the mortality rate associated with VRE bloodstream infections has been estimated at 55%.

Vancomycin is frequently used to treat bacteria that are resistant to all other antibiotics. Consequently, the emergence of vancomycin resistance among enterococci may accelerate the spread of vancomycin-resistant genes among other bacteria, eventually limiting the usefulness of this drug and leaving healthcare professionals without options for fighting bacterial diseases. In April 1997, the CDC reported the alarming emergence of S. aureus strains that show intermediate levels of resistance to vancomycin. The recent appearance of these strains in Japan and the U.S. suggests the high probability that strains of S. aureus completely resistant to vancomycin may soon emerge.

Thus, the discovery of new strain of bacteria implicated in chronically affections, justify our pathogenic and therapeutic conceptions that require natural antibacterial defences.

The development of new antibiotic compounds, originating from natural resources for drug development is a promising research territory. With their high number of species and representatives in all ecological niches, insects are the largest (80% of all fauna) and widespread group within the Animal Kingdom. Invertebrates such like insects have developed a battery of natural antibacterial peptides e.g. defensins, cecropins [7,8] as innate response to bacteria. Most of these peptides are conserved in course of evolutions such like defensins or cryptidins, peptides found in human intestine [9][10][11][12][13]. Moreover, new antibacterial peptides have recently been identified in glandular cells of amphibian skin (magainins, dermaseptins..), fishes, most classes of invertebrates and plants reflecting the universal characters of such natural defences [14][15][16][17]. The used of these informations coming from invertebrates can be an useful tools for caring septic diseases like Heart failure, the Crohn disease or reactional arthritis [1][2][3][4][5][6]. Moreover, since these last 15 years, several antibacterial peptides have also been identified in mammals particularly in secretions of immune cells or genital, digestive and intestinal epithelia tracts and skin of human [19][20][21] Secretory granules from adrenal medullary chromaffin cells contain a complex mixture of low molecular mass constituents, such as catecholamines, ascorbate, nucleotides, calcium and several water-soluble peptides and proteins [22][23][24][25]. These components are released into the circulation in response to splanchnic nerve stimulation. Relatively large amounts of pro-enkephalin-A and chromogranin-derived peptides are also found. Metz-Boutique's group has shown in bovine that antibacterial activity is present within the intragranular chromaffin granule matrix and the extracellular medium following exocytosis [22][23][24][25]. These peptides inhibit the growth of Gram-positive bacteria (Micrococcus luteus and Bacillus megaterium) at micromolar concentrations. In addition, antibacterial assays on soluble chromaffin granule material, recovered from HPLC, indicate the presence of several other endogenous peptides with potent antibacterial activity against Gram-positive and -negative bacteria. These new antibacterial peptides, derived from chromogranin-and pro-enkephalin-A precursors, are stored with catecholamines and released during stress [22][23][24][25]. Bactericidal activities of the chromogranin-or pro-enkephalin-A-derived peptides are modulated by the degree of maturation of the precursor and by the presence of post-translational modifications (phosphorylations, O-glycosylation). Natural processing of these precursors at the N-and Cterminals generates most of these peptides [22][23][24][25].

Recently, since the discovering of panoply of new antibacterial peptides in invertebrates [26] (Table 1), the use of such animals in this research field is in constant progress. In fact, invertebrates had developed an adapted system of defence to fight and destroy efficiently pathogen agents. Invertebrates fight against micro organisms in synthesizing and in releasing in same time a battery of antibacterial peptides during septic infections. Thus, the repeated discovery of antibiotic molecules present in both Vertebrates and Invertebrates reflect the presence of a rapid chemical mechanism of defence against bacteria, the innate immune response co-existing with the specific one [26]. Moreover, knowing the fact that a bacteria is multiplied 50 fold faster than a B cell and that these antimicrobial peptides diffuse quicker and easier than antibodies, this shows that this type of economic molecules with a broad spectrum allow a fast defensive response superior to microorganisms multiplication. These peptides appear perfectly adapted to offer such efficacious defensive line against infectious agents.

Maggots Therapy: small antimicrobial peptides peptides : the neurodocrine link

When we think about saprophytic insects, which live during their complete larval development in faces or decomposing organic material the question raises how these animals can live in an environment, which is so rich in pathogen micro-organisms. Insects mount a very effective humoral and cellular response to invasive microorganisms, which differs from the defense mechanisms, known from in vertebrates. A specific cell mediated and acquired immunity does not appear to exist in insects. Phagocytosis of bacteria or a melanotic encapsulation response to metazoan parasites is considered to be an important defense mechanism [26]. Insects also respond to bacterial invasion with the synthesis of an array of antibacterial peptides. In recent years these compounds have been studies extensively and classified into distinct families (for reviews on the cecropins, attacins, diptericins, defensins and proline and glycine rich compounds, [START_REF] Hétru | Molecular Mechanisms of Immune Respones in Insects[END_REF]).

The beneficial effect of fly larvae for wounds has been known for a very long time. In the 19 th century, Baron Larrey (physician-in-chief in Napoleon's army) and Dr. Joseph Jones (medical officer during the American Civil War) reported that soldiers, who remained wounded for several days in the battle field, did not die thanks to the healing effects of maggots (larvae of dipterans) which had intruded the open wounds [START_REF] Chernin | [END_REF]. Maggot therapy was extensively used in the 1930's and 1940's in more than 300 hospitals in the USA alone for the treatment of suppurative skin infections, but was later abandoned with the introduction of antibiotics and the use of aggressive surgical debridements. Since the mid 1990's the method was re-introduced not only in the USA, but also in Europe, i.e. University Hospital Antwerp [29]. Maggot therapy [Fig. 1] is the salvage therapy for cases where the poor blood supply to the deep wounds and subsequent inability of immunological mediators and systemic antibiotics to reach the infected areas, prevent the healing process (patients with diabetic feet and pressure ulcers, [30]). In such cases, antibiotic treatment, surgical debridement and drainage fail to stop the progressive tissue destruction [31][32][33][34].

Maggots are naturally exposed to an infected environment. Such ongoing exposure causes evolutionary pressure to develop a unique antibacterial mechanism, including the secretion of substances that protect them against highly pathogenic bacteria. As mentioned above several antibacterial peptides have been identified in the animal kingdom [26]. These antibacterial peptides usually exceed 3 kDa and some of them are at developed by companies i.e. Intrabiotics, Magainin Pharmaceutical Inc, Micrologix Biotech Inc, Entomed [26]. Most of the known high molecular weight peptides are their overall cationic charge, which facilitates membrane binding, upon which they organize into -helix and form pores in the cell membrane. However, these characteristics also make these antibacterial agents potentially cytotoxic for eukaryotic cells as well [Table 1].

It is necessary to focus on low-molecular weight compounds, since these are generally more stable and effective. Interest in low-molecular antibacterial compounds has increased during recent years because of the major health problems caused by antibiotic resistance in the last few years in Europe. Two small molecular weight antibacterial compounds have recently been identified by Leem et al., [35,36]: the 573 Da N-ß-Alanyl-5-S-glutathionyl-3,4dihydroxye-Phe has been identified in adult flies (Sarcophaga peregrina) and the 148 Da phydroxycinnamaldehyde (MW: 148) from larva of the saw fly, Acantholyda parki S. Schoofs and colleagues have recently demonstrated that -Ala-Tyr (MW:252; [Fig. 2]), which was identified in larvae of the gray flesh fly [36][37][38][39][40] displays potent antibacterial activity in maggots. From the same species, other antibacterial compounds were purified [38]. They have a molecular weight of 224 and 243 Da, respectively [38]. Their structures were identified by ESI-Qq-TOF mass spectrometry (MS-MS-mode) and correspond to 3-hydroxykynurenine and dehydro-Ala-Arg, respectively [START_REF] Chernin | [END_REF]. All these peptides have in common to exhibited paralytic activity when injected into adult insects [38]. No cytotoxic effect could be observed in vitro on insect neuronal cells [38]. When applied to rat C6 glioma cells, rat primary neurons or human neuroblastoma cell lines IMR32 and SHSY5Y, no cytotoxic effect of -AY was detected up to 10 mM concentrations 38. Intraspinal injection in rats gave no significant effect at concentrations as high as 198 mM 38. However, the effects of -AY application in vertebrates must be further examined. Besides for clinical applications, -AY may be used as antimicrobial additive to cell culture growth media, or as preservative in foods, pharmaceuticals, cosmetics and other products susceptible to microbial deterioration.

The exact mode of action of p-hydroxycinnamaldehyde and -AY has not been elucidated as yet. However, the cytotoxicity of hydroxylated phenolic compounds and their oxidation products has been long recognised and is thought to be due to the production of free radicals 38 and to their inhibitory interactions with proteins in general. The inhibitory effect of such compounds on micro-organisms is, among other things, dependent on the rate of hydroxylation 38. The main effectors of activity are quinones, which are aromatic rings with two ketone substitutions. Hydroxylated amino acids can be converted into quinones by phenoloxidases such as tyrosinase [Fig. 3]. The bactericidal effect of -AY at concentrations higher than the MIC value, was rapid for bacterial cells that are suspended in microbial growth medium but not for bacterial cells in phosphate buffer. The most likely explanation is that only actively metabolizing cells are sensitive to the bactericidal effect of -AY. Rather, the observation that growing cells are more sensitive, suggests that -AY may interact with a vital metabolic process.

The biochemical characterization of low molecular weight antibacterial compounds in insect larvae will reveal a new class of antibacterial substances, with potential to serve as lead compounds for a new brand of antibiotics. In fact, although several interesting antimicrobial compounds have meanwhile reached the stage of clinical trials www.phrma.org, possible applications are often limited to topical treatments. Being mostly peptidergic in nature, these natural antimicrobial compounds are susceptible to proteolytic breakdown by proteases, encountered during internal digestion in the gastro-intestinal tract or transport in the bloodstream, and consequently have too limited half-life upon systemic application. Moreover, most antimicrobial peptides have a high content of Lys and Arg, which are the main target of trypsine-like proteases. As such, it is important to identify smaller, perhaps even non-peptidergic substances with antimicrobial activity, as these can be expected to be more stable. Being small, they are also more likely to be suitable for production by chemical synthesis, thus bypassing the need for more expensive and labour intensive recombinant expression systems.

Leeches : antibacterial peptides for Cardiovascular diseases diagnosis

Haematophagous leeches are ecto-parasites and have a long life cycle e.g. 10 years for Hirudo medicinalis or 30 years for Heamentheria officinalis [START_REF] Sawyer | Leech Biology and behavior[END_REF] which defend themselves against bacteria attacks after injury or after a blood meal. Regarding their life cycle they appear to have an immune system very powerful. We recently demonstrate that these animals have a high anti-microbial panoply, some are bore on neuropeptide precursors [START_REF] Tasiemki | [END_REF]. These precursors are processed into active neuro-and antibacterial peptides. We found in leeches 15 min after bacteria infection enkephalins and panoply of antibacterial peptides such like enkelytin-like, peptide B-like [START_REF] Tasiemki | [END_REF] and chromacin-like peptides [43] [Fig. 4]. These molecules implicated in innate immune defense initiate and reinforce the inflammation immune response [44]. Moreover, they present sequence identity with vertebrate antibacterial molecules, we recently found in human plasma after coronary cardiology by-pass [45]. In fact, during coronary cardiology by-pass, time-course experiments showed the presence of a new family of antibacterial peptides i.e. serine rich, bi-phosphorylated and related to enkelytin, peptide B, fribinopeptide A and the apolipoprotein CIII [Table 2] and opioids (Methionine-Enkephalin and Methionine-Enkephalin Arg Phe) in plasma after surgery [44,45]. This suggests a new processing of these neuropeptide precursors in immunocytes or endothelial cells, not yet known.

Recent data in human have shown that that an injury to the endothelium might initiates and promotes the atherosclerotic process and this 'response to injury' concept is now widely accepted [46]. Information obtained in the past few years concerning the various atheromatous cellular components in spontaneous atherogenesis pointed out, at earlier stage, an accumulation of monocyte-derived macrophages, foam cell formation and interactions among lipoproteins, lymphocytes and other immune mediators, the process ultimately involving an inflammatory state in which macrophages and T lymphocytes play a major role [47][48][49]. In addition, it has been suggested that 'plaque activity' and the function of the cellular components can be a more important determinant of the clinical manifestations of atherosclerosis than is the percentage of stenosis of arterial lesions [50]. Antigen within the plaque may enhance the immunological activity and therefore the atherogenic inflammation. Suggested antigenic stimuli include modified cholesterol-rich lipoproteins [51], but also exogenous pathogens as bacteria. Indeed, over the past decade, increasing evidence obtained from epidemiological studies has accumulated suggesting a role for infectious agents in the genesis of atherosclerosis: Helicobacter pylori [52,53], Chlamydia pneumoniae [54], Pneumococcal endocartis [55], coagulase-negative staphylococci [56], Streptococcus pneumoniae [57]. These data even led to the development of adequate clinical trials to evaluate the putative protective effect of antibiotics in patients suffering a coronary artery disease [58,59]. In contrast, two prospective studies, involving a large cohort of white male physicians, did not confirm any association between the risk of myocardial infarction and prior infection with cytomegalovirus, herpes simplex virus, or Chlamydia pneumoniae [60,61].

As recently reviewed by Epstein and Zhu [62], these conflicting results may be largely due to the design of the studies. Most of the significant associations between bacterial infection and cardiovascular diseases were reported in retrospective studies in which information of a prior exposure to the suspected risk factor is obtained after the occurrence of the disease. Such a design does not allow taking into account for time-delay between exposure and disease onset, a notion that could be determinant in some cases. From this point of view, prospective studies offer additional information concerning the physiopathological process. Another criticism concerns the representativeness of the population samples. Indeed, in the prospective study, the absence of association between prior infection and MI was shown in a large, but extremely homogeneous, cohort of physicians. Compared to the general population, this cohort strongly differs in terms of socio-economic status and knowledge of other risk factors, a significant bias reducing the possible generalisation of the conclusions. The third important aspect that may explain conflicting results may be the end points. Significant associations between prior infection and the presence of atherosclerotic lesions do not imply similar associations with acute ischemic phase of the disease, such as MI or stroke. Indeed, the disruption of the atherosclerotic plaque, the most frequent critical step leading to ischemia, essentially depends on the structure of the lesion at the time of onset, in which initial bacterial infection probably poorly interferes. The final argument should be that, in most of these studies, prior infection was assessed by an immunological diagnosis implicating the activation of an advanced but late mechanism of defence. It may be postulated that other molecules, such as antibacterial peptides, should be more sensitive and earlier markers of the risk of CAD related to prior infection than antibodies. Indeed, data obtained from Invertebrates demonstrated the existence of an innate immune response based on short peptides developing an antibacterial activity. This process is suspected to constitute a rapid chemical mechanism of defence against infection, also characterised in humans.

Figure 1 :Figure 2 :

 12 Figure 1 : Maggot Therapy for diabetical foot woods (Acker, 1999)

Figure 3 :

 3 Figure3: Conversion of tyrosine to quinone by tyrosinase (from[38]).

Figure 4 :

 4 Figure 4: Antimicrobial (theromacine, theromyzine, enkelytine) peptides found in leech coccons [63] inset photograph represents a Theromyzon tessulatum with cocoons.

Table 1 -antimicrobial peptides from invertebrates (up date from [26])

 1

	Structure	Representative peptides	Organism	Antimicrobial activity
	Cationic peptides
	Linear alpha-helix peptides
		cecropins	insects,crustaceans	bacteria, fungi, virus, protozoa,
	metazoa
	Linear peptides rich in certain amino-acid
	Pro-rich drosocin, metchnikowins	fruit fly	bacteria
		pyrrhocoricin, metalnikowins	bugs	bacteria, fungi
		lumbricins	eartworm, leech	bacteria
	Gly-rich	diptericins, attacins	dipteran	bacteria
		Armadillins	crustacean	bacteria
	Arg-rich	perinerin	polychaeta	fungi
	His-rich	theromyzin	leech	bacteria,
	Single disulfide bridge
		thanatin	bug	bacteria, fungi
		Aranacins	polychaeta	bacteria
	Two disulfide bridges
		tachyplesin II	horseshoe crab	bacteria, fungi, virus
		androctonin	scorpion	bacteria, fungi
	Three disulfide bridges
		defensins	insects	bacteria, fungi, protozoa
		penaeidins	shrimps	bacteria, fungi
	More than three disulfide bridges
		tachycitin	horseshoe crab	bacteria, fungi
		drosomycin	fruit fly	fungi
		gambicin	mosquitoes	bacteria, fungi, protozoa
		helyomicins	lepidopteran	bacteria, fungi
		theromacin	leeches	bacteria
		ASBF	nematodes	bacteria
	Cyclic peptides
		Jasplakinolide,	sponges	bacteria, fungi
		cyclo-(glycyl-L-prolyl-L-glutamyl),
		cyclo-(glycyl-L-seryl-L-prolyl-L-glutamyl)
	Others
		Granulysin, discodermin A	sponges	bacteria

Table 2 :

 2 New antibacterial peptides isolated in human plasma from people suffering of cardiovascular disease

	Peptides	Mass (kDa)	pHi	Sequences
	Fibrinopeptide 20-35	3116,3	4,53 DSGEGDFLAEGGGVRGPRVVERHQSACKDS
	Apolipoprotein 21-57	3810,5	4,49 SEAEDASLLSFMQGYMKHATKTAKDFTALSSVQES
	Peptide B	3616,9	4,18 FAEALPSDEEGESYSKEVPEMEKRYGGFMRF
	Enkelytin	3313,6	3,98 FAEALPSDEEGESYSKEVPEMEKRYGGFM

ACKNOWLEDGEMENTS

This work was supported in part by the MNERT, the CNRS, the FEDER and the Conseil Régional de la région Nord-Pas De Calais and génopole of lille. The author would like to thank the referees for their recommendations.


Michel Salzet
email: michel.salzet@univ-lille1.fr

Molecular Aspect of Annelid Neuroendocrine system

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Table of Contents Introduction

Annelids neural tissues do not contain anatomical correlates of hypothalamus or pituitary. However, they possess localized ganglionic regions rich in mammalian-like neuroendocrine signaling molecules [1]. These molecules appear free in the animals' hemolymph, demonstrating distant signaling via several target tissues, including immune cells [1,2]. Thus, the fact that a classical closed conduit system to carry signaling molecules does not exist in Annelids should not detract from an endocrine presence because the baseline functioning of the system depends on distance between the origin of a signal molecule and its target tissue/receptor.

Neuroendocrine peptide families in Annelids similar to Vertebrates ones

Among the 40 neuropeptides isolated, so far, in annelids (Tables 1 and2), the most part already sequenced are related to the ones previously isolated in vertebrates [1][2][3][4][5]. More recently some EST databanks of annelids have been carried out by several groups and have opened the door of the discovering of novel neuropeptide families like the angiotensins, the oxytocin/vasopressin, the myotropic peptides, and the opioid families. With the exception to the myotropic peptides, which are more specific to the annelids, the other molecules families have also been identified in vertebrates in which they play crucial roles as neurohormones. These data further strengthen the existence of a neuroendocrine system in the annelids.

Angiotensin-like peptides

Biochemical identification of a "central" angiotensin II (AII)-like peptide in the leech Erpobdella octoculata was demonstrated and found to be amidated. [6][7][8][9]. This constituted the first characterization of an angiotensin-like peptide in an invertebrate, demonstrating its conservation during the course of evolution. An identification of the proteins immunoreactive to anti-AII was found both in brain extracts and in vitro translated brain RNA products [6]. The pro-AII precursor detected in the brain extracts possesses a ca 19 kDa molecular mass and is also a "multiple hormone precursor" as it is also recognized by two other antisera : a polyclonal γ-MSH and a specific monoclonal antibody directed against leech neurons, Tt159 [10]. Furthermore, we found in leeches a ca. 11-kDa peptide with a sequence of DRVYIHPFHLLXWG, which exhibits a 78.5% sequence identity to the N-terminus of the angiotensinogen and a 100% sequence identity to AI [7].

Biosynthesis study of leech AII revealed the existence of a renin- [11] and angiotensinconverting like enzymes (ACE)-like enzymes implicated in its catabolism [13]. Leech ACE has been cloned and an approximative 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE) [14]. Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated Nacetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE [14]. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells [14]. EST screening reveals that both membrane and soluble somatic forms of ACE are present in leeches. In the polychaete Neanthes virens a Dipeptidyl carboxypeptidase resembling mammalian ACE has also been demonstrated [15,16].The purified enzyme was homogeneous by SDS-PAGE, with a molecular mass of 71 kDa by SDS-PAGE and 69 kDa by gel filtration, indicating that it is monomeric like the leech TtACE [14]. The isoelectric point was 4.5 and optimum pH for the activity was 8.0. It showed a specific activity of 466.8 U/mg [16].

Experiments conducted on the biological activity of the AIIamide established that this peptide is involved in the control of leeches' hydric balance exerting a diuretic effect [17]. Similarly, in the Polychaeta Nereis diversicolor, injections of polyclonal antisera against AII provoked a partial inhibition of the increase in body weight in animals exposed to hypo-osmotic medium. In a subsequent test, injections of synthetic AII-amide and, to a lesser extent AII enhanced the increase in body weight and, therefore, strengthened the importance of these peptides in the neuroendocrine control of Nereis osmoregulation [18]. In clam worm Perinereis sp, angiotensin III as well as angiotensin II enhanced an increase in body weight under a hypoosmotic condition and suppressed a decrease in body weight under a hyper-osmotic condition. When clam worms were treated with tetrachloroaurate (III) after angiotensin-treatment, these enhancing and suppressive effects of the angiotensins under hypo-and hyper-osmotic conditions were inhibited. In contrast, when clam worms were pretreated with tetrachloroaurate (III) before angiotensin-treatment, these effects of angiotensins were not inhibited. Since tetrachloroaurate (III) is a representative blocker of aquaporins, these results indicate that angiotensin III as well as angiotensin II regulates water flow through aquaporins in clam worms [19,20] In leeches, the fact that AIIamide injections at different doses in T. tessulatum suggests the existence of two different types of receptors, one at high and the other one at low affinities towards AIIamide, we focused our interest on the identification of the leech AIIamide receptors. Binding experiments on T. tessulatum brain membranes with mono [ 125 I]AIIamide reveals 70% specific binding and a IC 50 of 10 nM [21]. In addition, biochemical studies using commercial anti-AT1 receptor reveal the existence of a specific protein at a molecular weight of 140 kDa [21] which is confirmed by EST analysis where AT1 and AT2 like receptors have been detected with 27 and 32% sequence identity, respectively. Immunocytochemical studies performed at the level of the brain confirmed the presence of labeling in neurons and glial cells to anti-AII, antileech renin, anti-leech ACE and anti-AT1 [21]. Leech coelomocytes are also immunoreactive to polyclonal antisera raised against the T. tessulatum 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 [21]. Moreover, 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). Immunocytochemical studies 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. These data report a coaction between cytokines like substances and neuropeptides in an immune process and the involvement of the RAS in modulation of the immune response [21].

Complementary to these findings, evidences of natural ACE inhibitors have been demonstrated in leeches [22]. In fact, the leech osmoregulator Factor (IPEPYVWD, see the LORF section), a neuropeptide found in both central nervous system and sex ganglia of leeches is involved in water retention control through inhibition of ACE with an IC 50 of 19.8 µM for rabbit ACE. Its cleavage product the IPEP is a better competitive inhibitor with an IC 50 of 11.5µM. Competition assay using p-[ 3 H]benzoylglycylglycylglycine and insect ACE established that LORF and IPEP are natural inhibitors for invertebrate ACE. 54% of insect ACE activity is inhibited with 50 µM d'IPEP and 35% with 25 µM de LORF like [22].

Taken together, these data strongly suggest the existence of a complete RAS in annelids which was conserved in course of evolution.

Oxytocin/vasopressin peptides

The peptides of the vasopressin/oxytocin family have been discovered throughout the animal kingdom. They are alike, sharing at least five of nine residues and a disulfide-linked ring structure, which puts severe constraints on conformational flexibility. In vertebrates, gene duplication gave rise to two distinct families i.e. the vasopressin (VP) one and the oxytocin (OT) one (Table 3). The differential binding of VP and OT to their respective receptors is largely due to the amino acid residue in position 8 i.e. a basic one in VP-related peptides and a neutral one in OT-related peptides. In the leech Erpobdella octoculata, a VP related peptide; the lysineconopressin (CFIRNCPKG) has been isolated [23]. However, EST screening in Hirudo medicnalis has revealed a peptide related to lysine-conopressin but belonging to the OT family (CFIRNCPLG), named Hirudotocin (Salzet, Unpublished data). In Oligochaeta, a peptide related to this family (CFVRNCPTG), the annnetocin has been discovered [24]. The proannetocin showed 37.4-45.8% amino acid homology to other prohormones. In the neurophysin domain, 14 cysteines and amino acid residues essential for association of a neurophysin with a vasopressin/oxytocin superfamily peptide were conserved, suggesting that the Eisenia neurophysin can bind to annetocin [25]. Furthermore, in situ hybridization experiments demonstrated that the annetocin gene is expressed exclusively in neurons of the central nervous system predicted to be involved in regulation of reproductive behavior [25]. Similar results are obtained for the Hirudotocin (Salzet, unpublished data).

These peptides act on osmoregulation via nephridia [23][24][25]. Lysine-conopressin inhibits the Na + amiloride dependent transitory current before to highly stimulate it on Hirudo medicinalis stomach or tegument preparation [17]. Moreover, lysine-conopressin induces egg laying in earthworm like OT does in vertebrates [26]. This confirms that through the course of evolution the OT/VP peptides family has conserved its function on both osmoregulation and reproduction Annetocin receptor (AnR) has been cloned [4]. The deduced precursor displays high sequence similarity with OT/VP receptors. Genomic analysis of the AnR gene revealed that the intron-inserted position is conserved between the AnR gene and the mammalian OT/VP receptor genes (Fig. 1). These results indicate that AnR and mammalian OT/VP receptors share a common ancestor gene. Administration of annetocin to the AnR expressed in Xenopus oocytes induced a calcium-dependent signal transduction. Reverse transcriptase-PCR analysis and in situ hybridization showed that the AnR gene is expressed specifically in the nephridia located in the clitellum region, although the nephridia are distributed throughout the worm body. This result suggests that annetocin induces egg-laying behaviour through its action on the nephridia. This is the first description concerning the functional correlation between an invertebrate OT/VP-related peptide and egg-laying behaviour [4]. In leeches, a 1.7 kb cDNA for an AVP-related receptor has been cloned from the leech Theromyzon tessulatum [START_REF] Levoye | Theromyzon tessulatum[END_REF]. The open reading frame encodes a 435amino acid transmembrane protein that displays seven segments of hydrophobic amino acids, typical of G-protein-coupled receptors. The overall predicted protein exhibits about 30% aminoacid identities to other invertebrate, as well as vertebrate, AVP/OT receptor family members, and displays conserved characteristic features belonging to the AVP/OT receptor superfamily. RT-PCR expression experiments showed that mRNA is expressed in the genital tract, the ovary and the brain. The receptor expression is stage specific, showing a weak expression after the two first blood meals, increasing dramatically after the last blood meal during the period of sexual maturation and disappearing after egg laying. Thus, the leech AVP-related receptor may mediate reproductive functions. When expressed in COS-7 cells, the receptor binds ligands with the following rank order of potency: AVP= Arg-vasotocin >Arg-conopressin >mesotocin = OT = Lys-conopressin=isotocin>annetocin. This shows an AVP-like pharmacological profile. The transfected receptor mediates AVP-induced accumulation of inositol phosphates, indicating that the leech AVP-related receptor is functional [START_REF] Levoye | Theromyzon tessulatum[END_REF].

Taken together, these data demonstrate the characterization of AVP/OT superfamily receptor in annelids, which are considered the most distant group of coelomate metazoans possessing a functional AVP/OT-related endocrine system [4 ,27].

Opioids

Enkephalins have been isolated in Annelids [28] and its precursor the proenkephalin shows sequence similarity with amphibian proenkephalin (26.2 %) (29,30). The proenkephalin contains Met-and Leu-enkephalins in a ratio of ½ and Met-enkephalin-Arg-Gly-Leu and Metenkephalin-Arg-Phe flanked by dibasic amino acid residues, which are targets for proteolytic enzymes such like prohormone convertases present in leeches ( [29], Fig. 1). Specific receptors for these peptides have been characterized in neural and in immune systems [29]. Enkephalin derived peptides seem to be implicated in innate immune response (31). In fact, 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 [32]. 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 bovine's 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 [32]. 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 [30,[32][33][34].

Besides enkephalins, two other opioids derived from two other opioid precursors have been characterized, α-Neo-endorphin [35) and γ-MSH [36]. Characterization of the entire prodynorphin opioid precursor revealed that it exhibits a 28.8% sequence identity to rat, and 22% to the human and pig [37]. Although the α Neo endorphin is identical to the one found in vertebrates, the dynorphins are slightly shorter. A POMC-like molecule has also been demonstrated in leeches [38]. Of the six peptides, three showed high sequence similarity to their vertebrate counterparts, namely, met-enkephalin, α-MSH and ACTH (100, 84.6 and 70% respectively) whereas γ-MSH, β-endorphin and γ-LPH exhibited only 45, 20 and 10% sequence identity. No dibasic amino acid residues were found at the C-terminus of the γand β-MSH peptides suggesting that they are not produced in the leech or that they could be synthesized via an alternate biosynthetic pathway. In contrast, the leech α-MSH was flanked at its C-terminus by the Gly-Arg-Lys amidation signal. Circulating levels of ACTH and MSH were 10 and 1 fmol/microl hemolymph, respectively. Morphine, in a dose-dependent manner, increased the levels of both peptides threefold; this effect was blocked by naloxone treatment. Similar results were found with the anandamide. Leech ACTH was processed to MSH by the enzymes neutral endopeptidase (24.11) and angiotensin-converting enzyme. Leech α-MSH had the same activity as authentic alpha-MSH in two bioasssay systems [38].

Taken together, the results from leeches now demonstrate, those opioid precursors and many of the derived bioactive peptides, i.e., α-MSH and ACTH, which are important in mammalian neuroendocrine signaling, are present in invertebrates. This adds to the growing body of evidence that a neuroendocrine apparatus is also present in simple animals.

Neuroendocrine peptide families in Annelids similar to Molluscs ones

Annelids and Molluscs are both lophochotrozoans and in this context several families of peptides have been well conserved in these two groups. The most represented one is the Rxamides family. However other molecules families have been detected in leech and lymnaea or Aplysia but are also now discovered in vertebrates like the RFamide peptide family [7].

RXamide Peptides

Several related RXmides have been found in annelids [39][40][41][42][43][44][45][46]. A myomodulin-like peptide, GMGALRLamide, has been purified and sequenced from the medicinal leech nerve cords (45). Myomodulin-like immunoreactivity has recently been found to be present in a set of leech neurons, including Leydig neurons [45,46]. The glial responses to Leydig neuron stimulation persisted in high-divalent cation saline, when polysynaptic pathways are suppressed, indicating that the effects on the glial cell were direct [42]. The glial responses to myomodulin A application persisted in high-Mg 2+ /low-Ca 2+ saline, when chemical synaptic transmission is suppressed, indicating a direct effect of myomodulin A on the glial membrane (42). The glial hyperpolarization evoked by myomodulin A was dose dependent (EC 50 = 50 nM) and accompanied by a membrane conductance increase of approximately 25%. Ion substitution experiments indicated that myomodulin A triggered a Ca 2+ -independent K + conductance (42). Moreover, synthetic leech myomodulin-like peptide [46] showed identical neuronal modulation effect on the giant leech Retzius cell comparable to that by the synthetic Aplysia myomodulin APMGMLRLamide [47][48][49]. This neural and muscular modulation has been shown to be important for shaping and modifying behavior. Experiments focused on the Retzius cell (R) revealed that the myomodulin-like peptide increased the excitability of the R cell such that the cell fires more action potentials with a shorter latency to the first action potential. This effect is mediated by the activation of a Na+-mediated inward current near the cell resting membrane potential [44]. EST library scanning reveals the presence of a myomodulin precursor sharing several peptides of this family bracketed by di-basic residues like GVSFLRMG, SLDMLRMG, AVSMLRMG, AVSLLRMG (Salzet, unpublished data) and presenting 57% sequence homology with Lymnaea or Aplysia myomodulin precursor (49, 50) (Fig. 2).

In polychaete annelids a heptapeptide, AMGMLRMamide, termed Pev-myomodulin, was isolated from Perinereis vancaurica using the esophagus of the animal as the bioassay system. The sequence of the annelid peptide is highly homologous with those of the myomodulin-CARP-family peptides found in molluscs. The annelid peptide is regarded as a member of the myomodulin-CARP family, though all the molluscan peptides have a Leu-NH 2 at their C-termini. The annelid peptide showed a potent contractile action on the esophagus of the annelid. The peptide may be an excitatory neuromediator involved in the regulation of the esophagus. Among various myomodulin-CARP-family peptides and their analogues, the annelid peptide showed the most potent contractile action on the esophagus [66]. Replacement of the C-terminal Met-NH2 of the annelid peptide with a Leu-NH 2 decreased its contractile potency, while replacement of the Cterminal Leu-NH 2 of myomodulin and CARP with a Met-NH2 increased their potency. The Cterminal Met-NH 2 of the annelid peptide seems to be important, but not essential, for exhibiting its contractile activity on the esophagus [66].

RF-amide peptides

FRaP family is present in the polychaetes Nereis virens and Nereis diversicolor [51][START_REF] Baratte | Isolation and characterization of authentic Phe-Met-Arg-Phe-NH2 and the novel Phe-Thr-Arg-Phe-NH2 peptide from Nereis diversicolor[END_REF][START_REF] Krajniak | Authentic FMRFamide is present in the polychaete Nereis virens[END_REF]. In this last species, two other RFamide peptides (FM(O)RFamide and FTRFamide) have been isolated [51][START_REF] Baratte | Isolation and characterization of authentic Phe-Met-Arg-Phe-NH2 and the novel Phe-Thr-Arg-Phe-NH2 peptide from Nereis diversicolor[END_REF][START_REF] Krajniak | Authentic FMRFamide is present in the polychaete Nereis virens[END_REF]. Pharmacological data suggest that RFamide peptides are involved in the control of heartbeat and body wall tone in the polychaete Sabellastarte magnifica [54,55] and in the oligochaete Eisenia foetida [56].

In the eathworm Eisenia fetida, FMRF-like peptides are co-localized with serotonin, suggesting a role as neuromodulators influencing serotoninergic neurons [56,57]. In Lumbricus terrestris, FMRF-like peptides seem to be involved in both central integratory processes, neuromuscular regulation and sensory processes [56,57].

In Hirudinae, anti-FMRFamide immunoreactivity is found in cell bodies and neuronal processes of the central nervous system [58][59][60][61][62][63]. In the segmental ganglia of the ventral nerve cord, this immunoreactivity is localized in heart excitatory (HE) motor neurons, heart accessory (HA) modulatory neurons and several motor neurons innervating the longitudinal and mediodorso ventral muscles [64][65][66]. Among the 21 segmental ganglia (SG1-SG21) of the ventral nerve cord of leeches, SG5 and SG6 that innervate the sexual organs are designated as sex SG. These sex ganglia contain, as compared to the non-sex ganglia, an additional population of neurons immunostained with anti-FMRFamide in Hirudo medicinalis [67]. Furthermore, two RFamide peptides (FMRFamide and FLRFamide) were identified in Hirudo medicinalis [60,68].

These peptides increase the strength and accelerate the rate of myogenic contractions as well as inducing myogenic contractions in quiescent hearts [65,66]. Besides these tetrapeptides, we characterized an extended form of FLRFamide, the GDPFLRFamide from sex ganglia extracts of E. octoculata [60]. In T. tessulatum presence of RFamide peptides in neurosecretory granules in fibers of the neurohaemal area suggests that at least one of the characterized peptides is secreted into the dorsal vessel. The brain could exert a neuroendocrine control of certain functions via RFamide peptides. Taking into account a previous study showing a loss of weight of T tessulatum after a GDPFLRFamide injection and an increase of weight after a FMRFamide injection [60], we surmise that GDPFLRFamide may act as a diuretic hormone and FMRFamide as an anti-diuretic hormone. Electrophysiological experiments confirmed our speculation [17]. The anti-diuretic effect of FMRFamide seemed not due to a direct action on the caecal epithelium. Nevertheless, the control of the hydric balance might be also exerted directly on the nephridia. Indeed, Wenning et al. demonstrated that the nephridial nerve cells, which innervate the nephridia and contact the urine forming cells, contain RFamide peptide(s) in H .medicinalis [61,69]. Furthermore, these authors showed that FMRFamide leads the hyperpolarization and decreases the rate of firing of the nephridial nerve cells, suggesting autoregulation of peptide release.

Leech egg-laying hormone

In leeches, egg-laying may be under the control of a leech egg laying hormone (L-ELH) [70]. In Eisenia fetida, although that the OT-VP related peptide, annetocin, is known to potentate the pulsatory contractions in bladder-shaking movement of the nephridia, indicating an involved of osmoregulation though nephridial function, this peptide is also implicated in egg laying behaviors [26]. In fact, annetocin, induced a series of egg-laying-related behaviors in the earthworms. These stereotyped behaviors consisted of well-defined rotatory movements, characteristic body-shape changes, and mucous secretion from the clitellum [26]. Each of these behaviors is known to be associated with formation of the cocoon in which eggs are deposited. In fact, some of the earthworms injected with annetocin (> 5 nmol) laid eggs. Such egg-laying-related behaviors except for oviposition were also induced by oxytocin, but not by Arg-vasopressin. Furthermore, annetocin also induced these egg-laying-like behaviors in the leech Whitmania pingra, but not in the polychaete Perinereis vancaurica. These results suggest that annetocin plays some key role in triggering stereotyped egg-laying behaviors in terrestrial or fresh-water annelids that have the clitella [26].

Are some Neuroendocrine Signaling Molecules specific to Annelids?

The history of neurobiology demonstrates the significance of the invertebrate nervous system as a valuable model. The giant axon of the squid and crayfish neuromuscular junction stands out in this regard. This field of scientific endeavor also stands out for its demonstration of the conservation of signaling molecules and their functions during evolution. Probably the first peptide found in invertebrates that later was also found in mammals is the Hydra head activator peptide [71][72][73]. In Hydra this peptide modulates morphogenesis, cellular growth and differentiation. In mammals, this peptide sequence proved to be unique in that it differed from that of any known peptide. Furthermore, the investigators surmise that this peptide may have the same functions in mammals. We propose here to extend this several families specifically found in annelids and not yet found in mammals.

Leech osmoregulator Factor

In early reports we found that LORF (IPEPYVWD) is a peptidergic-signaling molecule involved in osmoregulation [74]. Furthermore, electrophysiological experiments conducted in the leech H. medicinalis revealed an inhibition of the efficacy of Na + conductance in leech skin [17]. Immunocytochemical studies with an antiserum against synthetic LORF found a great amount of positive immunoreactive neurons in all ganglia. This material was present in a single type of electron-dense secretory granules of a size of 80-100 nm [32]. However, we recently showed its presence in rat tissues, including discrete brain areas (75), which demonstrate that this peptide, originally isolated from the leech, is also present in mammals. Furthermore, in both the leech central nervous system (CNS) and the rat brain, e.g., hypothalamus, LORF is coupled to nitric oxide (NO) release [75]. It is also capable of stimulating NO release from human saphenous vein fragments [75]. These results showed that LORF is not specific to annelids but they also demonstrated that Leech represents a suitable model to isolate novel peptides also conserved in vertebrates.

GGNG Peptides

The most specific peptides presently isolated in annelids are the ones related to the GGNG, excitatory peptides [76][77][78][79][80] (Fig. 3). In leeches, these peptides elicit muscular contractions of isolated preparation of penis and intestine, suggesting that they may play a role in reproduction behavior like the PLGWamide in mollusks [76][77][78][79][80]. In earthworm, they elicit gut and esophagus muscular contractions. No peptides homologous to GGNG peptides have been isolated so far in any living organisms.

Are Neuropeptide Processing Enzymes Present in Invertebrates?

The biologically active neuropeptides are produced via the proteolytic processing of precursor molecules. Among the enzymes involved in neuropeptide precursors processing, the serine proteases belonging to the subtilisin/kexin family and known as the proprotein convertases (PCs) are playing, at least in mammals, a crucial role. In mammals, this family includes, so far, seven members (PC1, PC2, furin, PC4, PACE4, PC5 and PC7) which are differentially involved in the processing, generally at single or dibasic residues, of a large number of protein precursors including neuropeptides, growth factors, receptors, enzymes and viral envelope glycoproteins [81]. Between these enzymes, only PC1 and PC2, which are abundantly expressed in neurons and endocrine cells, play an important role in the neuropeptide precursors processing. Although these latter enzymes have been cloned in numerous species including mammals, amphibians, molluscans, insects and nematods, they have not yet been characterized in annelids. EST screening confirms the presence of prohormone convertases in leech brain sharing at least 65% sequence identity.

Conclusion

In conclusion, given the wealth of information now emerging on these mammalian-like neuroendocrine processes found in invertebrates, it would appear that this system, in all probability, originated in "simple" animals. Therefore, aside from its historical origin, it may be more appropriate to speak of the mammalian neuroendocrine system as Annelid-like.

Figure 1 :

 1 Figure1: Sequence alignment of OT/VP receptor in Invertebrates using Mulatlin software (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_multalin.html): cephalotocin R and octopressin R[82,83], lysine and arginine-conopressin R[START_REF] Van Kesteren | A novel G proteincoupled receptor mediating both vasopressin-and oxytocin-like functions of Lysconopressin in Lymnaea stagnalis[END_REF], annetocin R[4], leech OT/VP R[START_REF] Levoye | Theromyzon tessulatum[END_REF]and Hirudotocin R sequence are from leech EST (Salzet, unpublished data). Inset a phylogenetic tree of the Invertebrate receptor using genebee software (http://www.genebee.msu.su/services/phtree_reduced.html).

Figure 2 :

 2 Figure 2 : Sequence alignment of Myomodulin precursor from Mollusks [49,50] and from leech EST (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_multalin.html):

Figure 3 :

 3 Figure 3 : Sequence alignment of Annelids Excitatory peptide Precursor [76-79] (http://npsapbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_multalin.html):

Table 1 : Characterized Annelid Neuropeptides from leeches

 1

	species	Sequence	Name
	Theromyzon
	tessulatum

Table 2 : Characterized Annelids Neuropeptides other Annelids

 2

	Species	Sequence	Name
	Perinereis Vancaurica	AMGMLRMamide	Myomodulin-CARP
		WVGDVQ	Esophagus regulation peptides
		ATWLDT
		WMVGDVQ
		FYEGDVPY
	Nereis diversicolor	FMRF-amide, FM(O)RF-amide	FMRF-amide
		FTRF-amide	FTFR-amide
	Nereis virens	FMRF-amide	FMRF-amide
		FLRF-amide	FLRF-amide
	Eisenia Foetida	CFVRNCPTGamide	Annetocin
		APKCSGRWAIHSCGGNG	GGNG1
		GKCAGQWAIHACAGGNG	GGNG2
		RPKCAGRWAIHSCGGGNG	GGNG3

Table 3 : Oxytocin/vasopressin peptides discovered in invertebrates

 3

	Species	Sequence	Name
	Erpobdella octoculata	CFIRNCPKG-amide	Lysine-Conopressin
	Hirudo medicinalis	CFIRNCPLGamide	Hirudotocin
	Eisenia Foetida	CFVRNCPTGamide	Annetocin
	Conus Georgaphus	CFIRNCPKG-amide	Lysine-Conopressin
	Conus Striatis	CIIRNCPRG-amide	Arginine-Conopressin
	Octopus	CYFRNCPIG-amide	Cephalotocin
	Octopus	CFWTSCPIG-amide	Octopressin
	Locusta migratoria	CLITNCPRG-amide	Diuretic peptide

Acknowledgments

This work was in part supported by the Centre National de Recherche Scientifique (CNRS) and the MNERT.


Isabelle Fournier

Robert Day

Michel Salzet

Direct Analyses of neuropeptides by in situ MALDI-TOF mass spectrometry in the rat brain

The measure of neuropeptides is a important tool in biology to better define endocrine and neuroendocrine function. Traditionally most methods have relied on the development of specific antibodies. Newer molecular methodologies have used measures of gene expression of neuropeptide precursors, such as Northern blot, PCR or in situ hybridization analysis. Matrix-assisted laser desorption/ionization (MALDI) mass analysis is a novel powerful technique for investigation of neuropeptides. Multiple peptides and peptide forms can be detected simultaneously and with great sensitivity in tissue extracts or partially purified samples. We have now adapted a MALDI methodology for the direct measurement of neuropeptides on fresh rat brain tissue sections. We have validated the method by examining peptidergic mass profiles of the supraoptic nucleus (SON) and caudate putamen hypothalamic regions. Interestingly, mass profiles shown that vasopressin which is specifically present in the SON is modulated when animals are treated with lipopolysaccharides. MALDI-MS on brain slides is a novel complementary technique for neurobiologists and endocrinologists in order to investigate the dynamic and regional repartition of neuropeptides during physiological events.

INTRODUCTION

Neuropeptides serve important functions in the brain and in the periphery in cell to cell communication. The ability to rapidily and effectively establish peptides levels and forms is a critical aspect of neuroendocrinology. Changes in peptide levels can reflect there importance in specific events and can lead to establish their direct functions. The measure of various neuropeptides can be a long and complicated process which often involves the measure of only one peptide at a time, after sometimes lengthy and arduous purification procedures. Mass spectrometric techniques have offered a novel rapid and sensitive methodology for the detection and measure of multiple peptides within one sample. Research employing mass spectrometry for proteomic studies i.e. to characterize a protein pattern in normal or in pathological tissues has been intense. Of particular interest has been the development of direct analyses on fresh tissues using matrix assisted laser desorption-ionisation (MALDI) mass spectrometry. Since the last five years the use of invertebrate models for direct analyses of neuropeptides secreted by neurons using fresh tissues dissected and deposited in vial with matrix has been well developed (Critchey, [START_REF] Critchley | Identification of peptides by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and direct analysis of the laterobuccal nerve from the pond snail Lymnaea stagnalis[END_REF]Jimenez et al., 1998;Hseich et al., 1998;Jimenez and Burlingame, 1998;[START_REF] Worster | Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene[END_REF][START_REF] Henry | HPLC and electrospray ionization mass spectrometry as tools for the identification of APGWamide-related peptides in gastropod and bivalve mollusks: comparative activities on Mytilus muscles[END_REF]. Sweedler et al have recently studied the mollusc Aplysia californica using the direct profiling of neuropeptide precursor processing in a single cell [START_REF] Li | In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysisAnal[END_REF]. This technique allows to perform in situ sequencing using post-source decay, fragmentation technique (Kaufman et al., 1996;[START_REF] Chaurand | Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry[END_REF][START_REF] Fournier | Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[END_REF]. This is a strategy for peptide identification and characterization of post-translational modifications. However a completely novel approach was examined by Caprioli and collaborators. They performed the direct spatial mapping of peptides in cells using mass spectrometry technique [START_REF] Stoeckli | Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues[END_REF]. This group developed the used of MALDI-MS to image fresh tissues slices after coating the sample with matrix. Molecular ion images are then successfully generated from area of rat pituitary where over 50 different peptides are observed as well as their precursors, isoforms and metabolic fragments [START_REF] Chaurand | Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry[END_REF]. The development of new imaging computer software allowing both instrument control and data imaging acquisition and processing for MALDI-MS of thin tissue sections has now open the door of a new world for mass spectrometry application on biological tissue (Stoecki et al., 1999;[START_REF] Stoeckli | Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues[END_REF].

In the present study, we performed for the first time a comparative study from rat subjected to a bacteria challenge or not on fresh tissues slices using MALDI-MS in order to investigate neuropeptides by in situ MALDI-TOF mass spectroscopy. We focus our interest on peptides in the supraoptic nucleus, especially the vasopressin and demonstrated that mass spectrometry can also be a tool for in situ and dynamic studies.

MATERIAL and METHODS

Laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometric analysis was performed with a Applied Biosystems (Framingham, MA, USA) voyager-DE STR time of flight mass spectrometer with delayer extraction, operating with a pulsed nitrogen laser at 337 nm.S Positive-ion mass spectra were acquired in linear, delayed extraction mode with an accelerating potential of 20 kV, a 96% grid potential and a delay time of 150-1000 ns, depending on the mass range. Individual calibrations were performed on each slide. Each spectrum is the results of 200 averaged laser pulses.

Tissue preparation

Adult female Wistar rats (animal use accreditation by the French Ministry of the Agriculture N° 04860) were used in this study and maintained under standard care. Female rats weighing 300-350g were injected i.p. with lipopolysaccharides ( ) and scarified 12hours later Brain were dissected from the animals and fresh frozen sections (12 µm) were obtained on cryostat. Sections were then transferred to the MALDI-MS target before embedded with alpha-cyano-4-hydroxycinnamic acid matrix. In order to localize the supraoptic nucleus as well alternative sections were transferred to glass slides and stained with toluidine blue. Several matrix were used in this study in order to find the one the more efficient i.e. sinapinic acid, alpha-cyano-4-hydroxycinnamic acid.

RESULTS and DISCUSSION

As seen in figure 1, mass spectrum profiles of supraoptic nucleus or caudate putamen revealed a larger number of masses reflecting the high sensitivity of the technique in order to detect in situ neuropeptides. The range of peptide detection depends of the nature of the matrix used. Alpha-cyano-4-hydroxycinnamic acid allows to detect peptides with a molecular mass ranged between 500 to 3000 Da with a 0.1 Da precision. By contrast (Fig. 2), sinapinic acid matrix is more useful for larger peptides (> at 3 Da to 20 kDa).

Comparison of mass profiles obtained in supraoptic nucleus and in caudate putamen revealed common masses between both brain region corresponding to dynorphin A (1-8) (981.75 Da), dynorphin B (1570.27 Da), CCK (10-20) (1248.98 Da), CCK (1-8) sulfoxyde (1079.86) or unknown masses 2603.6 Da, 2830.4 Da, 3534 Da. By contrast, Vasopressin (1084.4 Da) which is known to be specific of the supraoptic nucleus has only been found in this brain region while it is absent in caudate putamen (Fig. 3a).

Previous treatment of rats with lipopolysaccharides (LPS) in order to mimic bacteria challenge have showed shown the extensive release of the arginine-vasopressin (AVP) from the supraoptic nucleus cells (Fig. 3c). No mass corresponding to this peptide can be detected 12 hours post-injection although CCK [START_REF] Kaufmann | Post-source decay and delayed extraction in matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. Are there trade-offs?[END_REF][START_REF] Laiko | Desorption/ionization of biomolecules from aqueous solutions at atmospheric pressure using an infrared laser at 3 microm[END_REF][START_REF] Li | In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysisAnal[END_REF][START_REF] Li | Single-cell MALDI: a new tool for direct peptide profiling[END_REF][START_REF] Matsunaga | LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus[END_REF][START_REF] Nava | Lipopolysaccharide increases arginine-vasopressin release from rat suprachiasmatic nucleus slice cultures[END_REF][START_REF] Salzet | FMRFamide-related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata. Identification and involvement in the control of hydric balance[END_REF][START_REF] Schleuder | IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching[END_REF][START_REF] Stoeckli | Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues[END_REF][START_REF] Stoeckli | Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument[END_REF][START_REF] Worster | Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene[END_REF] and other peptides are still present. Rats injected with a saline solution did not provoke vasopressin release (Fig. 3b). These data are in agreement with those of by [START_REF] Nava | Lipopolysaccharide increases arginine-vasopressin release from rat suprachiasmatic nucleus slice cultures[END_REF]. In fact, these authors demonstrated that LPS administered in the medium for 3h increased significantly the arginine-vasopressin release lasting up to 6h after treatment. These results provide the evidence that LPS influences AVP secretion in SON neurons. Moreover, [START_REF] Matsunaga | LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus[END_REF] showed that administration of low dose (5 g/kg) and high dose (125 g/kg) of LPS induced intense nuclear c-fos Fos immunoreactivity in many oxytocin (OXT) and AVP neurons in all the observed hypothalamic regions. The percentage of c-fos positive nuclei in OXT magnocellular neurons was higher than that of AVP magnocellular neurons in the supraoptic nucleus (SON), the magnocellular neurons in the paraventricular nucleus (magPVN), rostral SON (rSON), and nucleus circularis (NC), whose axons terminate at the posterior pituitary for peripheral release. The percentage of c-fos positive nuclei in AVP parvocellular neurons in the paraventricular nucleus (parPVN) was higher than that of OXT parvocellular neurons, whose axons terminate within the brain for central release. Moreover, the percentage of c-fos-positive nuclei in AVP magnocellular neurons of the SON and rSON was significantly higher than that of the magPVN and NC when animals were given LPS via intraperitoneal (i.p.)-injection. This regional heterogeneity was not observed in OXT magnocellular neurons of i.p.-injected rats or in either OXT or AVP magnocellular neurons of intravenous (i.v. )-injected rats. This suggest that LPS-induced peripheral release of AVP and OXT is due to the activation of the magnocellular neurons in the SON, magPVN, NC, and rSON, and the central release of thesehormones is in part derived from the activation of parvocellular neurons in the PVN. The sum of these data may explain some central effects observed in vivo after lipopolysaccharides administration.

Interestingly, most peptides identified by their mass like vasopressin (1084.4 Da) also contained an analogue which is associated with potassium (MK+ : 1122.6) but not with sodium (MNa+) (Fig. 3a). This reflects the fact that in situ neuropeptides are complexed with potassium and less with sodium. Also, some peptides are oxidized like the CCK [START_REF] Bennett | Characterization of eight forms of corticotropin-like intermediary lobe peptide from the rat intermediary pituitary[END_REF][START_REF] Caprioli | Molecular imaging of biological samples : localization of peptides and proteins using MALDI-TOF MS[END_REF][START_REF] Chaurand | Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry[END_REF][START_REF] Critchley | Identification of peptides by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and direct analysis of the laterobuccal nerve from the pond snail Lymnaea stagnalis[END_REF][START_REF] Fournier | Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[END_REF][START_REF] Henry | HPLC and electrospray ionization mass spectrometry as tools for the identification of APGWamide-related peptides in gastropod and bivalve mollusks: comparative activities on Mytilus muscles[END_REF][START_REF] Hsieh | Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis[END_REF][START_REF] Jimenez | Ultramicroanalysis of peptide profiles in biological samples using MALDI mass spectrometry[END_REF]. This could be explained by the fact that although the peptides are complexed with the MALDI matrix, oxidation can occurred or as we have already found during FMRFamide purification by HPLC, oxidation can be considered as an inactivation process of active peptide [START_REF] Salzet | FMRFamide-related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata. Identification and involvement in the control of hydric balance[END_REF].

Taken together results reflect the high sensitivity and discrimination power of the MALDI-MS technique. These data are also in agreement with those of [START_REF] Caprioli | Molecular imaging of biological samples : localization of peptides and proteins using MALDI-TOF MS[END_REF]. In the rat pituitary, using MALDI-MS, they detected all the pro-opiomelanocortin derived peptides as well as other neuropeptides which are known to be absent in supraoptic nucleus and caudate putamen and confirmed by MALDI-MS profiles obtained in the present study (Figs. 1 and2). Thus over 50 neuropeptides have been detected in brain slides based on their molecular masses measurement. As suggested by [START_REF] Chaurand | Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry[END_REF] truncated analogs with either N-terminal or C-terminal residues, partially acetylated and unacetylated forms and in several cases possible phosphorylated and sulphated forms have been detected as previously by HPLC, immunoassays, microsequencing, electrospray mass spectrometry [START_REF] Bennett | Characterization of eight forms of corticotropin-like intermediary lobe peptide from the rat intermediary pituitary[END_REF]. Thus, in situ MALDI-MS allows us to investigate the distribution pattern of neuropeptides in brain and can also be used for dynamic studies.

In terms of present limitations, at the present time it is difficult to perform mass spectrometry peptide sequencing employing MALDI combine with post-source decay (PSD) fragment ion mass analysis directly on the tissue. The ability to rapidly identify structurally peptides in situ would permit the determination of the many unknown peptide fragments observed. The low level of peptides or the energy necessary to breakdown in situ peptides could be one explanation for the present limitations. However, the use of infrared laser in such experimental conditions may provide the required alternative solution. [START_REF] Schleuder | IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching[END_REF][START_REF] Laiko | Desorption/ionization of biomolecules from aqueous solutions at atmospheric pressure using an infrared laser at 3 microm[END_REF].

Legend Figures

Figure 1

 1 Figure 1Rat brain sections (12µm) obtained with a cryostat. a: MALDI-TOF mass spectrum in the positive mode recorded directly from the supraoptical nucleus (matrix: CHCA).b: MALDI-TOF mass spectrum in the positive mode recorded directly from the Caudate Putamen region (matrix CHCA).

Figure 2 a

 2 Figure 2 a: MALDI-TOF mass spectrum in the positive mode recorded directly from the supraoptical nucleus (matrix: sinapinic acid).b : MALDI-TOF mass spectrum in the positive mode recorded directly from the supraoptical nucleus (matrix: sinapinic acid).

Figure 3 a

 3 Figure 3 a: MALDI-TOF mass spectrum in the positive mode recorded directly from the supraoptical nucleus (matrix: CHCA). Zoom on Vasopressin ions. (b,c) Comparison of MALDI-TOF mass spectra recorded in the supraoptical nucleus (matrix CHCA): control (saline injection : b) vs. challenged animals (c)

Acknowledgements

The Centre National de la Recherche Scientifique, the MNERT, the Genopole of Lille, the FEDER, the Conseil Regional Nord-Pas de Calais and the Canadian Institutes of Health Research (CIHR) supported this work.