Virginie Laurent

Endogenous melatonin provides an effective circadian message to both the suprachiasmatic nuclei and the pars tuberalis of the rat

Abstract:  The suprachiasmatic nuclei (SCN) distribute the circadian neural message to the pineal gland which transforms it into a humoral circadian message, the nocturnal melatonin synthesis, which in turn modulates tissues expressing melatonin receptors such as the SCN or the pars tuberalis (PT). Nuclear orphan receptors (NOR), including rorβ and rev‐erbα, have been presented as functional links between the positive and negative loops of the molecular clock. Recent findings suggest that these NOR could be the initial targets of melatonin’s chronobiotic message within the SCN. We investigated the role of these NOR in the physiological effect of endogenous melatonin on these tissues. We monitored rorβ and rev‐erbα mRNA expression levels by quantitative in situ hybridization after pinealectomy. Pinealectomy had no effect on NOR circadian expression rhythms in the SCN in 8‐day pinealectomized (PX) animals. However in animals PX for 3 months, significant desynchronization between per1 and per2 transcription patterns appeared. These results suggest that endogenous melatonin could sustain the circadian rhythmicity and the phase relationship between the molecular partners of the SCN circadian system on a long‐term basis. On the other hand, pinealectomy decreased the level and abolished the rhythmicity of NOR mRNA expression in the PT. These effects were partially prevented by daily melatonin administration in the drinking water. These results show that NOR can be regulated by the melatonin circadian rhythm in the PT and could be the link between the physiological action of melatonin and the core of the molecular circadian clock in this tissue.

Melatonin affects nuclear orphan receptors mrna in the rat suprachiasmatic nuclei

The pineal hormone melatonin nocturnal synthesis feeds back on the suprachiasmatic nuclei (SCN), the central circadian clock. Indeed, daily melatonin injections in free-running rats resynchronize their locomotor activity to 24 h. However, the molecular mechanisms underlying this chronobiotic effect of the hormone are poorly understood. The endogenous circadian machinery involves positive and negative transcriptional feedback loops implicating different genes (particularly period (Per) 1-3, Clock, Bmal1, cryptochrome (Cry) 1-2). While CLOCK:BMAL1 heterodimer activates the rhythmic transcription of per and cry genes, the PER and CRY proteins inhibit the CLOCK:BMAL1 complex. In previous studies, we observed that the immediate resetting effect of a melatonin injection at the end of the subjective day on the SCN circadian activity did not directly involve the above-mentioned clock genes. Recently, nuclear orphan receptors (NORs) have been presented as functional links between the regulatory loops of the molecular clock. These NORs bind to a retinoic acid receptor-related orphan receptor response element (RORE) domain and activate (RORalpha) or repress (REV-ERBalpha) bmal1 expression. In this study, we investigated whether melatonin exerts its chronobiotic effects through transcriptional regulation of these transcription factors. We monitored roralpha, rorbeta and rev-erbalpha messenger RNA (mRNA) expression levels by quantitative in situ hybridization, up to 36 h following a melatonin injection at circadian time (CT) 11.5. Results clearly showed that, while roralpha was not affected by melatonin, the hormone partially prevented the decrease of the rorbeta mRNA expression observed in control animals during the first hours following the injection. The major result is that the rev-erbalpha mRNA expression rhythm was 1.3+/-0.8-h phase-advanced in melatonin-treated animals during the first subjective night following the melatonin administration. Moreover, the bmal1 mRNA expression was 1.9+/-0.9-h phase-shifted in the second subjective night following the melatonin injection. These results clearly suggest that the NOR genes could be the link between the chronobiotic action of melatonin and the core of the molecular circadian clock.

A comparison of the leech Theromyzon tessulatum angiotensin I-like molecule with forms of vertebrate angiotensinogens: a hormonal system conserved in the course of evolution.

After five steps of purification including gel permeation, anti-angiotensin I affinity column chromatography followed by reverse-phase HPLC, a peptide immunoreactive to two different antisera (anti-angiotensin II and anti-angiotensin I) was purified to homogeneity from extracts of the leech Theromyzon tessulatum. The first 14 amino acid residues of the purified peptide (DRVYIHPFHLLXWG) established by automated Edman degradation, reveal the existence in leeches of an angiotensin I-like molecule close to human angiotensin I. The sequence of the purified peptide presents 78.5% of homology with the N-terminal part of human angiotensinogen. Moreover, in its sequence, this peptide presents the cleavage sites of vertebrate angiotensin metabolic enzymes, i.e. the renin and the angiotensin-converting enzyme. This finding constitutes the first biochemical characterization of an angiotensin I in Invertebrates. It also reflects the high conservation of angiotensins in the course of evolution, suggesting a fundamental role of this family in fluid homeostasis.

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37 degrees C, the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu11-Val12-Tyr13-Ser14 peptide as products, with a specific activity of 1.35 pmol AI/min/mg (Km 22 microM; Kcat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC50 = 4.6 microM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.

Biochemical Properties of the Angiotensin- Converting-Like Enzyme From the Leech Theromyzon tessulatum

This article reports the evidence and the biochemical properties of an angiotensin-converting (ACE)-like enzyme from head parts of the leech Theromyzon tessulatum. After solubilization from membranes with Triton X-114, the ACE-like enzyme was purified from the detergent-poor fraction. Four steps of purification including gel permeation and anion exchange chromatographies followed by a reversed-phase HPLC were needed. This poor glycosylated peptidyl dipeptidase (of ca. 120 kDa) hydrolyzes, at pH 8.4 and at 37 degrees C, the Phe8-His9 bond of angiotensin I with a high catalytic activity (i.e., K(m): 830 microM and Kcat/K(m): 153 s-1 mM-1). The hydrolysis of angiotensin I is inhibitable at 80% by captopril (IC50 = 175 nM) and lisinopril (IC50 = 35 nM). This activity is strictly dependent on the presence of NaCl and is increased by Zn2+. This zinc metallopeptidase also attacks peptides that have in their sequence either Gly-His, Gly-Phe, or Phe-His bond [e.g., enkephalins (Kcat/K(m): 12 s-1 mM-1) or bradykinin (Kcat/K(m): 2200 s-1 mM-1]. Taken together, these arguments are consistent with an ACE-like activity implicated in metabolism of angiotensins and bradykinin in leeches.

A comparison of the N-terminal sequence of the leech Theromyzon tessulatum angiotensin converting-like enzyme with forms of vertebrate angiotensin converting enzymes

This paper reports the purification of an angiotensing-converting like enzyme (ACE) of ca. 120 kDa from extracts of head membranes of the leech Theromyzon tessulatum. After solubilization with Triton X-114, the ACE-like enzyme contained in the detergent-poor fraction was separated using five steps of purification including gel permeation and anion exchange chromatographies followed by reverse-phase HPLC. The first 23 amino acid residues of the N-terminal part (GLDPELSPGCFSADEAGAQLFAE) of the purified S-pyridylethylated leech ACE established by automated Edman degradation revealed ca. 87% sequence identity with the N-terminal sequence of the guinea pig ACE. This enzyme cleaves the hyppuryl-His-Leu substrate with a specific activity of 5600 nmol hyppurate min-1 mg protein-1. Hydrolysis of this substrate by ACE-like enzyme is inhibited at 80% by 10 microM captopril or 10 microM lisinopril (IC50 of 200 nM and 50 nM, respectively). This enzyme is close in sequence and in activity to single domain vertebrate ACE. This is the first N-terminal sequence of an ACE-like enzyme determined in invertebrates.

Presence and biochemical properties of a molluscan invertebrate angiotensin-converting enzyme

A soluble 65582.9 Da (in MALDI-TOF) angiotensin converting (ACE)-like enzyme has been purified by a captopril-Sepharose affinity column chromatography from the mollusk Mytilus edulis. This glycosylated peptidyl dipeptidase, with an N-terminal sequence of LDPELSPGCFVANQAGGQLF, hydrolyses the Phe8-His9 bond (at pH 8.4 and 37 degrees C) of angiotensin I with a high catalytic activity i.e. Km: 168 microM and Kcat/Km: 262 s-1 mM-1. The hydrolysis of angiotensin I is inhibited by the specific ACE inhibitors captopril and lisonopril (Ic50 = 50 nM). This activity is increased by Cl- (optimal Cl- concentration 400 mM) and by Zn2+. This zinc metallopeptidase also attacks peptides having a Gly-His, Gly-Phe or a Phe-His bond in their sequence e.g. leucine-enkephalin (Kcat/Km: 1200 s-1 mM-1 or bradykinin (Kcat/Km: 2500 s-1 mM-1). Mytilus ACE displays properties of the C-domain of human ACE, indicating a high degree of conservation during evolution. These results are consistent with an ACE activity implicated in metabolism of several neuropeptides in mollusks.

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.

METABOLISM OF ANGIOTENSINS BY HEAD MEMBRANES OF THE LEECH THEROMYZON TESSULATUM

Angiotensins (angiotensin I, angiotensin II, angiotensin II‐amide) have been isolated in leeches and such peptides are involved in diuresis in these animals. To explore possible inactivation mechanisms of these peptides, angiotensins were incubated with head membranes of the leech T. tessulatum. Membranes derived from head parts of this leech are very rich in peptidases. They contain endopeptidase‐24.11‐like enzyme (NEP‐like) associated with a battery of exopeptidase. The way that angiotensins are degraded by the combined attack of these membrane peptidases has been investigated. The contribution of individual peptidases was assessed by adding inhibitors (phosphoramidon, captopril and amastatin) to the membrane fractions, when they were incubated with the peptides. In the case of angiotensin I, the primary attack was performed by a combined action of the NEP‐like and the ACE‐like enzymes, followed by aminopeptidase attacks. Angiotensin II and III were hydrolyzed by NEP‐like enzyme at the same Tyr‐Ile bond, whereas the N‐terminal arginine residue of angiotensin III was removed by an arginyl aminopeptidase. These results show that angiotensins are efficiently degraded by membranes and that NEP‐like enzyme plays a key role in this process.

Cone Viability Is Affected by Disruption of Melatonin Receptors Signaling.

Purpose Previous studies have demonstrated that melatonin has an important role in the modulation of photoreceptor viability during aging and may be involved in the pathogenesis of age-related macular degeneration.This hormone exerts its influence by binding to G-protein coupled receptors named melatonin receptor 1 (MT1) and 2 (MT2). Melatonin receptors 1 and 2 activate a wide variety of signaling pathways. Methods Melatonin-proficient mice (C3H/f+/+) and melatonin-proficient mice lacking MT1 or MT2 receptors (MT1−/− and MT2−/−) were used in this study. Mice were killed at the ages of 3 and 18 months, and photoreceptor viability was determined by counting nuclei number in the outer nuclear layer (ONL). Cones were identified by immunohistochemistry using peanut agglutinin (PNA) and green/red and blue opsin antibodies. Protein kinase B (AKT) and forkhead box O (FOXO1) were assessed by Western blotting and immunohistochemistry. Results The number of nuclei in the ONL was significantly reduced in C3Hf+/+, MT1−/−, and MT2−/− mice at 18 months of age with respect to 3-month-old animals. In 18-month-old MT1−/− and MT2−/− mice, but not in C3H/f+/+, the number of cones was significantly reduced with respect to young MT1−/− and MT2−/− mice or age-matched C3H/f+/+. In C3H/f+/+, activation of the AKT-FOXO1 pathway in the photoreceptors showed a significant difference between night and day. Conclusions Our data indicate that disruption of MT1/MT2 heteromer signaling induces a reduction in the number of photoreceptors during aging and also suggest that the AKT-FOXO1 survival pathway may be involved in the mechanism by which melatonin protects photoreceptors.