Repeated exposure to aerosolized graphene oxide mediates autophagy inhibition and inflammation in a three-dimensional human airway model.

Hazard evaluation of engineered nanomaterials (ENMs) using real-world exposure scenario could provide better interpretation of toxicity end points for their use in the assessment of human safety and for their implications in many fields such as toxicology, nanomedicine, and so forth. However, most of the current studies, both in vivo and in vitro, do not reflect realistic conditions of human exposure to ENMs, due to the high doses implemented. Moreover, the use of cellular models cultured under submerged conditions limits their physiological relevance for lung exposure, where cells are primarily cultured at the air-liquid interface. Addressing such issues is even more challenging for emergent nanomaterials, such as graphene oxide (GO), for which little or no information on exposure is available. In this work, we studied the impact of repeated exposure of GO on a three-dimensional (3D) reconstruct of human bronchial tissue, using a nebulizer system focusing on short-term effects. The selected doses (reaching a maximum of ca. 20 â€‹µg/cm2 for a period of 4 weeks of exposure) were extrapolated from alveolar mass deposition values of a broader class of carbon-based nanomaterials, reflecting a full working lifetime of human exposure. Experimental results did not show strong toxic effects of GO in terms of viability and integrity of the lung tissue. However, since 2 weeks of treatment, repeated GO exposure elicited a proinflammatory response, moderate barrier impairment, and autophagosome accumulation, a process resulting from blockade of autophagy flux. Interestingly, the 3D airway model could recover such an effect by restoring autophagy flux at longer exposure (30 days). These findings indicate that prolonged exposure to GO produces a time window (during the 30 days of treatment set for this study) for which GO-mediated autophagy inhibition along with inflammation may potentially increase the susceptibility of exposed humans to pulmonary infections and/or lung diseases. This study also highlights the importance of using physiologically relevant in vitro models and doses derived from real-world exposure to obtain focused data for the assessment of human safety.

Dual inhibition of REV-ERBß and autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells.

REV-ERBα and REV-ERBß nuclear receptors regulate several physiological processes, including circadian rhythm and metabolism. A previous study reported the REV-ERBα gene to be co-overexpressed with ERBB2 in breast cancer cell lines. Surprisingly, we found that several tumor types, including a number of breast cancer cell lines, predominantly express the REV-ERBß variant. This pattern was independent of ERBB2 and ER status, and opposite to that of non-cancer mammary epithelial HMEC cells, in which REV-ERBα was the major variant. Consistent with this molecular profile, REV-ERB target genes in both circadian and metabolic pathways were derepressed upon silencing of REV-ERBß, but not REV-ERBα. Strikingly, we found that REV-ERBß is a determinant of sensitivity to chloroquine, a clinically relevant lysosomotropic agent that suppresses autophagy. The cytoprotective function of REV-ERBß appears to operate downstream of autophagy blockade. Through compound screening, we identified ARN5187, a novel lysosomotropic REV-ERBß ligand with a dual inhibitory activity toward REV-ERB-mediated transcriptional regulation and autophagy. Remarkably, although ARN5187 and chloroquine share similar lysosomotropic potency and have a similar effect on autophagy inhibition, ARN5187 is significantly more cytotoxic. Collectively, our results reveal that dual inhibition of REV-ERBß and autophagy is an effective strategy for eliciting cytotoxicity in cancer cells. Furthermore, our discovery of a novel inhibitor compound of both REV-ERB and autophagy may provide a scaffold for the discovery of new multifunctional anticancer agents.

Chromatin remodeling and circadian control: master regulator CLOCK is an enzyme.

The molecular machinery that governs circadian rhythmicity is based on clock gene products organized in regulatory feedback loops. Recently, we have shown that CLOCK, a master circadian regulator, has histone acetyltransferase activity essential for clock gene expression. The Lys-14 residue of histone H3 is a preferential target of CLOCK-mediated acetylation. As the role of chromatin remodeling in eukaryotic transcription is well recognized, this finding identified unforeseen links between histone acetylation and cellular physiology. Indeed, we have shown that the enzymatic function of CLOCK drives circadian control. We reasoned that CLOCK's acetyltransferase activity could also target nonhistone proteins, a feature displayed by other HATs. Indeed, CLOCK also acetylates a nonhistone substrate: its own partner, BMAL1. This protein undergoes rhythmic acetylation in the mouse liver, with a timing that parallels the down-regulation of circadian transcription of clock-controlled genes. BMAL1 is specifically acetylated on a unique, highly conserved Lys-537 residue. This acetylation facilitates recruitment of the repressor CRY1 to BMAL1, indicating that CLOCK may intervene in negative circadian regulation. Our findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

Photomorphogenesis in the hypogeous fungus Tuber borchii: isolation and characterization of Tbwc-1, the homologue of the blue-light photoreceptor of Neurospora crassa.

Truffles form a group of plant-symbiotic Ascomycetes whose hypogeous life cycle is poorly understood. Here we present initial evidence for the influence of light on Tuber borchii mycelial growth and the identification and cloning of a gene, Tbwc-1, homologous to a blue-light photoreceptor of Neurospora crassa. Blue-light irradiation of T. borchii colonies inhibits their apical growth. It also alters apical growth in N. crassa. In Neurospora, the response is controlled by a nuclear photoreceptor, NcWC-1 (White Collar-1), which consists of a sensor domain (LOV) and a transcriptional factor moiety. We isolated a gene (Tbwc-1) whose deduced amino acid sequence shows a high similarity and colinearity of domains with NcWC-1, except for the polyglutamine regions. As previously found in Neurospora, Tbwc-1 mRNA is under light control and its steady state level increases upon irradiation. In silico analysis of the TbWC-1 sensor domain (LOV) supports the hypothesis that TbWC-1 is a photoreceptor, while the absence of the two polyglutamine regions involved in transcriptional activation in Neurospora suggests that this function in Tuber could be lost.

The central role of magnesium deficiency in Tourette's syndrome: causal relationships between magnesium deficiency, altered biochemical pathways and symptoms relating to Tourette's syndrome and several reported comorbid conditions.

Prior studies have suggested a common etiology involved in Tourette's syndrome and several comorbid conditions and symptomatology. Reportedly, current medications used in Tourette's syndrome have intolerable side-effects or are ineffective for many patients. After thoroughly researching the literature, I hypothesize that magnesium deficiency may be the central precipitating event and common pathway for the subsequent biochemical effects on substance P, kynurenine, NMDA receptors, and vitamin B6 that may result in the symptomatology of Tourette's syndrome and several reported comorbid conditions. These comorbid conditions and symptomatology include allergy, asthma, autism, attention deficit hyperactivity disorder, obsessive compulsive disorder, coprolalia, copropraxia, anxiety, depression, restless leg syndrome, migraine, self-injurious behavior, autoimmunity, rage, bruxism, seizure, heart arrhythmia, heightened sensitivity to sensory stimuli, and an exaggerated startle response. Common possible environmental and genetic factors are discussed, as well as biochemical mechanisms. Clinical studies to determine the medical efficacy for a comprehensive magnesium treatment option for Tourette's syndrome need to be conducted to make this relatively safe, low side-effect treatment option available to doctors and their patients.

Endurance training effects on 5-HT(1B) receptors mRNA expression in cerebellum, striatum, frontal cortex and hippocampus of rats.

The 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. The present study investigated the effects of a 7 week period of physical training on the expression of cerebral 5-HT(1B) receptors by measuring corresponding mRNA levels in rat. Using RNase protection assay technique, we have observed no change in 5-HT(1B) receptor mRNA levels in the striatum and in the hippocampus after moderate as well as after intensive training. In contrast, a significant decrease in 5-HT(1B) receptor mRNA levels was observed in cerebellum of intensively trained rats. Moreover, in frontal cortex, a significant decrease in 5-HT(1B) receptors mRNA level occurred in both groups of trained rats. These data suggest the existence of regional differences in the effect of physical exercise on the expression of 5-HT(1B) receptors.

Effects of the inhibition of cyclo-oxygenase 1 or 2 or 5-lipoxygenase on the activation of the hypothalamic-pituitary-adrenal axis induced by interleukin-1beta in the male Rat.

The limited entry of interleukin-1beta (IL-1beta) into the central nervous system has led to the hypothesis that IL-1beta acts, through IL-1beta receptors located notably on endothelial cells, on the release of prostaglandins which in turn stimulate the hypothalamic-pituitary-adrenal (HPA) axis. We used cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors, before the injection of IL-1beta, to explore the role of arachidonic acid metabolic pathways on HPA axis activation. Adult male rats were i.m injected 20 min before i.p injection of IL-1beta, with (i): a COX-1/COX-2 inhibitor (ketoprofen); (ii) a COX-2 selective inhibitor (NS 398); or (iii) a 5-LOX inhibitor (BW A4C). Following this, rats were killed 90 min after i.p. IL-1beta injection and analysis for plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations and determination of anterior pituitary pro-opio melanocortin (POMC) gene transcription was conducted. Administration of the COX-1/COX-2 inhibitor led to a complete blockage of ACTH and corticosterone secretion and POMC gene transcription. The COX-2 inhibitor led to a complete blockade of ACTH secretion and POMC gene transcription but had no effect on corticosterone secretion. The 5-LOX inhibitor had no significant effect on any parameter. These results demonstrate the crucial role of eicosanoid pathways in mediating the stimulation of the HPA axis induced by IL-1beta. Moreover, we found a clear dissociation of the effect of the blockage of COXs upon ACTH and corticosterone secretion, suggesting that IL-1beta may act at the brain as well as at the adrenal cortex to stimulate the secretion of corticosterone.

Effects of physical training on functional activity of 5-HT1B receptors in rat central nervous system: role of 5-HT-moduline.

The effect of physical exercise was examined on the sensitivity of 5-HT1B receptors and on 5-HT-moduline tissue concentration in the central nervous system of rats. Rats were trained for 7 consecutive weeks to run on a treadmill. Three groups of animals were selected: group 1, sedentary rats (controls); group 2, animals running for 1 h at 18 m/min for 5 days per week (moderate training) and group 3, animals running for 2 h, at 30 m/min on a 7% grade for 5 days per week (intensive training). The animals were sacrificed 24 h after the last running. Rat brains were dissected out to obtain hippocampus and substantia nigra and kept at -80 degrees C until use. 5-HT1B receptor activity was determined by studying [35S]GTPgammaS binding in a substantia nigra membrane preparation from individual animals, after stimulation by a selective 5-HT1B receptor agonist (CP 93,129). 5-HT-moduline tissue content in hippocampus from individual animals was determined by ELISA using a polyclonal anti-5-HT-moduline antibody. In moderately trained animals (n=5), the CP 93,129-stimulated [35S]GTPgammaS binding curve was shifted to the right compared with controls (n=6), whereas the binding was totally suppressed in intensely trained animals (n=5). In parallel, 5-HT-moduline tissue concentration in the hippocampus was slightly increased in moderately trained animals (117.3 +/- 8.9%) (n=5), whereas it was significantly increased in intensely trained animals (182.6 +/- 29.5%) (n=5) compared to controls (100 +/- 6.11%) (n=6). These results show that 5-HT1B receptors are slightly desensitized in moderately trained animals and totally desensitized in intensely trained animals; moreover, they suggest that the observed desensitization is related to an increase of 5-HT-moduline tissue content; this mechanism may play a role in various pathophysiological conditions.

Magnetic field desensitizes 5-HT(1B) receptor in brain: pharmacological and functional studies.

It was previously suggested that exposure to magnetic fields (MFs) could generate dysfunction of the CNS. The physiological manifestations described lead us to postulate that these symptoms might be related to a dysfunction of the serotonergic system and particularly of the 5-HT(1B) receptors. Accordingly, MFs could modify the conformation of these receptors altering their functional activities. In rat brain membrane preparations, we showed that the affinity constant of 5-HT for 5-HT(1B) receptors was modified under exposure to MFs since K(d) varied from 4.7+/-0.5 to 12+/-3 nM in control and exposed (2.5 mT) membranes, respectively. This effect was intensity-dependent (the sigmoidal dose-response curve was characterized by an EI(50) of 662+/-69 microT and a maximal increase of 321+/-13% of the control K(d)), reversible, temperature-dependent and specific to the 5-HT(1B) receptors. Similar results have also been obtained with the human 5-HT(1B) receptors. In parallel assays, the functional activity of 5-HT(1B) receptors was investigated. The capacity of a 5-HT(1B) agonist to inhibit the cAMP production was reduced by 37% (53.7+/-3.5% to 33.7+/-4.1%) following exposure to MFs and the cellular activity of the receptors (inhibition of the synaptosomal release of 5-HT) also was markedly reduced (66.5+/-3.2% to 28.5+/-4.2%). These results clearly show that in in vitro assays, MF specifically interacts with 5-HT(1B) receptors, inducing structural changes of the protein that result in a functional desensitization of the receptors. Thus, in vivo, exposure to MFs may lead to physiological changes, particularly in the field of mood disorders where the 5-HT system is strongly involved.

5-HT-moduline controls serotonergic activity: implication in neuroimmune reciprocal regulation mechanisms.

The serotonergic neurotransmission is known as a neuromodulatory system exerting its activity in the central nervous system (CNS) as well as at the periphery. The anatomical and morphological organization of the system based on a marked centralization of the cellular bodies and the large, almost ubiquitary, presence of axonal projections of the neurons is in good agreement with this modulatory role. Furthermore, a very high number of varicosities located along the axonal branches are capable of releasing serotonin (5-HT). The amine stimulates a number of different specific receptor types which allows 5-HT to exert different activities on its various cellular targets. Among these receptors, the 5-HT1B subtypes play a particular role as they are autoreceptors located on 5-HT neurons terminals and heteroreceptors located on non-serotonergic terminals where they control the release of the neurotransmitter. 5-HT-moduline, an endogenous tetrapeptide, regulates the efficacy of these 5-HT1B receptors, hence, is able to control the serotonergic activity in a synchronous manner for the various varicosities from a single neuron and thus may favour the differential effect of that neuron on distinct cerebral functions. Accordingly, the peptide allows the 'fine tuning' of the cerebral activity by the serotonergic system to elaborate the response given by the brain to a particular stimulus, that is, stress situations. At the periphery, the serotonergic system also appears to possess a regulatory activity via 5-HT1B receptors. In particular, the receptors located on immunocompetent cells control their activity and are themselves regulated by 5-HT-moduline likely originating from adrenal medulla and released after acute stress. The serotonergic system appears to play a major role in the reciprocal signalling existing between the neuronal and the immune system. The participation of 5-HT-moduline is likely in physiological functions as well as in pathological disorders affecting central and peripheral activities.

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes: further evidence using 5-HT moduline, polyclonal 5-HT1B receptor antibodies and 5-HT1B receptor knock-out mice.

In previous paper based on classical pharmacological tools, we identified a Gi protein-coupled presynaptic 5-hydroxytryptamine (5-HT) 1B receptor causing inhibition of dopamine (DA) release in rat striatal synaptosomes. It was the aim of the present study to further explore this receptor, using 5-HT moduline, a polyclonal antibody directed against 5-HT1B receptors and 5-HT1B receptor knock-out mice. Preincubation of rat striatal synaptosomes with 5-HT moduline (0.1, 1, or 10 microM) significantly reduced the inhibitory effect of CP93,129, a selective rat 5-HT1B receptor agonist, on K+-evoked overflow of [3H]DA in a non-competitive manner: 5-HT moduline did not modify the IC50 of CP93,129, but concentration-dependently reduced the maximal inhibitory effect. Preincubation of rat striatal synaptosomes with a specific polyclonal 5-HT1B receptor antibody also resulted in a significant attenuation of the inhibitory effect of CP93,129 on K+-evoked overflow of [3H]DA. In female 129/Sv wild-type mice, CP93,129 and 5-carboxyamidotryptamine maleate (5-CT), a non-selective 5-HT1B receptor agonist, inhibited the K+-evoked [3H]DA overflow in a concentration-dependent manner. Sumatriptan, a selective rat 5-HT1D receptor agonist, did not modify the overflow of [3H]DA. SB224289, a selective 5-HT1B receptor antagonist, abolished the inhibitory effects of CP93,129 and 5-CT. The inhibitory effects of CP93,129 and 5-CT were absent in synaptosomes from 5-HT1B receptor knockout mice. No compensatory inhibition effect in mutant mice was observed using sumatriptan. In conclusion, the results show that a non-competitive antagonist of the 5-HT1B receptor concentration-dependently decreases the maximal inhibitory effect of a 5-HT1B receptor agonist on the synaptosomal K+-evoked release of [3H]DA in striatum. Moreover, a specific antibody raised against the receptor and particularly directed against a region of the receptor protein involved in signal transduction, namely the coupling with the G-protein, also antagonizes the inhibitory effect of the stimulation of 5-HT1B receptor on the release of [3H]DA. Ultimately the disruption of 5-HT1B receptor gene in 5-HT1B knock-out mice leads to a total suppression of the effect of 5-HT1B receptor agonists on [3H]DA release. These observations further support our previous observations using selective agonists/antagonists, indicating that 5-HT1B receptors control the release of neuronal DA as presynaptic heteroreceptors.

Application of the polymerase chain reaction to the RNase protection assay for 5-HT(1B) receptor mRNA levels measurement in rat brain tissues.

The RNase protection assay (RPA) is an extremely sensitive procedure for detection of messenger RNA (mRNA) in complex sample mixture of total RNA. However, its usefulness has been limited by the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction (PCR) to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for the 5-hydroxytryptamine(1B) (5-HT(1B)) receptor. Radiolabeled riboprobe was then synthesized using the PCR product as a template and used in RPA to detect mRNA for 5-HT(1B) receptor in rat brain. The internal control was the beta-Actin mRNA. Due to the simplicity of its design and the lack of need for subcloning, the DNA template synthesis by PCR facilitates the implementation of the RPA. Since the 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters and the protocol described here permits the determination of 5-HT(1B) receptor mRNA levels in the rat cerebellum, striatum, hippocampus and frontal cortex, this protocol is helpful in understanding the involvement of 5-HT(1B) receptors in various physiological phenomena.

5-Hydroxytryptamine-moduline: a novel endogenous peptide involved in the control of anxiety.

The serotonergic system is considered as a neuromodulatory system interacting with other neurotransmissions in the brain and participating in the elaboration of an adapted response of the central nervous system to external stimuli. Indeed, serotonin is involved in a large number of physiological events, such as temperature regulation, sleep, learning and memory, behaviour, sexual function, hormonal secretions and immune activity, and in parallel, it is also implicated in pathological disorders particularly in stress, anxiety, aggressivity and depression. At least 14 different types of serotonin receptors mediate serotonergic activity and among them, serotonin-1B receptors play an important role in the control of the serotonergic function. Serotonin-1B receptors are autoreceptors localized on serotonergic neuron terminals (varicosities) where they inhibit the evoked release of serotonin and its biosynthesis; they are also heteroreceptors located on non-serotonergic terminals, where they inhibit the release of the corresponding neurotransmitters (acetylcholine, GABA, noradrenaline, etc.). 5-Hydroxytryptamine-moduline, an endogenous tetrapeptide (Leu-Ser-Ala-Leu) recently isolated and characterized from rat and bovine brain extracts, was shown to specifically interact with serotonin1B receptors as an allosteric modulator having antagonistic properties in vitro and in vivo. Immuncytochemical studies using specific polyclonal anti-peptide antibodies have shown that this peptide is distributed heterogeneously in mouse brain and located in areas which also contain serotonin-1B receptors. Moreover, the content of these cerebral tissues in 5-hydroxytryptamine-moduline is affected by stress. In the present work, polyclonal anti-5-hydroxytryptamine-moduline antibodies were administered to mice via intracerebroventricular injections to study the in vivo effects of a lowering (or suppression) of this neuropeptide in the central nervous system. The inactivation of the peptide by the specific antibodies significantly modified the behaviour of the animals in two behavioural tests, the open-field and elevated plus-maze, known to be animal models related to anxiety behaviour. Treated mice displayed behaviour consistent with an anxiolytic effect of the antibody, suggesting a potential role of 5-hydroxytryptamine-moduline in the control of anxiety.

Acute stress induces a differential increase of 5-HT-moduline (LSAL) tissue content in various rat brain areas.

5-HT-moduline is an endogenous cerebral tetrapeptide (LSAL) which specifically interacts as an allosteric modulator with 5-HT1B receptors controlling serotonergic activity [O. Massot, J.C. Rousselle, M.P. Fillion, B. Grimaldi, I. Cloez-Tayarani, A. Fugelli, N. Prudhomme, L. Seguin, B. Rousseau, M. Plantefol, R. Hen, G. Fillion, 5-Hydroxytryptamine-moduline, a new endogenous cerebral peptide, controls the serotonergic activity via its specific interaction with 5-hydroxytryptamine1B/1D receptors, Mol. Pharmacol. 50 (1996) 752-762; J.C. Rousselle, O. Massot, M. Delepierre, E. Zifa, G. Fillion, Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic1B receptor subtypes, J. Biol. Chem. 271 (1996) 726-735; J.C. Rousselle, M. Plantefol, M.P. Fillion, O. Massot, P.J. Pauwels, G. Fillion, Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells, Naunyn-Schmiedeberg's Arch. Pharmacol. 358 (1998) 279-286]. Cerebral tissue contents of 5-HT-moduline were determined in various rat brain areas after an acute restraint stress, and after repetition of this stress, to examine whether or not mechanisms involving this peptide could be affected by stress situations. The measurement of the peptide was carried out using specific polyclonal antibodies [B. Grimaldi, M.P. Fillion, A. Bonnin, J.C. Rousselle, O. Massot, G. Fillion, Immunocytochemical localization of neurons expressing 5-HT-moduline in the mouse brain, Neuropharmacology 36 (1997) 1079-1087] in a dot-ELISA (enzyme-linked-immunosorbent assay) assay in cortex, hippocampus, hypothalamus, substantia nigra, striatum and in adrenal glands. Tissue contents of 5-HT-moduline progressively and transiently increased in most studied brain regions and reached a maximal value 20 min after the beginning of the restraint stress. The increase in 5-HT-moduline tissue contents represented 323% of the value observed in unstressed control animals in the cortex, 207% in the hippocampus, 149% in the hypothalamus and 156% in the substantia nigra. Thereafter, the peptide content of the latter tissues diminished during the last 20 min of restraint and returned to control values within 1 h after the end of the stress period. The striatum did not show any significant variation of 5-HT-moduline content during restraint stress. In adrenal glands, the 5-HT-moduline content rapidly decreased (60% of controls) after the beginning of the restraint stress, the effect of this stress being progressively less pronounced, still representing 80% of controls after 40 min. Repetition of the restraint stress daily for 3 weeks totally abolished the effect of the stress on variations of 5-HT-moduline tissue content in all the studied brain regions. These results show that an acute restraint stress induces a rapid and significant increase in the amount of 5-HT-moduline contained in various brain areas. This phenomenon is likely to be related to the stress-induced 5-HT1B receptor desensitization which was previously demonstrated.

Characterization of 5-ht6 receptor and expression of 5-ht6 mRNA in the rat brain during ontogenetic development.

We have determined the pharmacological characteristics of the rat 5-ht6 receptor stably expressed in CHO cells. Moreover, using RT-PCR experiments the in vivo expression of the gene encoding this receptor was studied in rat at various embryonic days (ED) starting from ED10 to birth (PN0) and at post-natal days (PN) up to PN36. The pharmacological analysis of the [3H]5-HT binding in stably transfected CHO cells expressing rat 5-ht6 receptors revealed the presence of a single class of high affinity saturable binding sites for 5-HT corresponding to an affinity constant: Kd=27.2+/-3.4 nM. This receptor also exhibited a high affinity for a number of typical and atypical antipsychotics, tricyclic antidepressant drugs and ergot alkaloïds. In stably transfected CHO cells, serotonin elicited a potent stimulation of adenylyl cyclase activity which was blocked by antipsychotic and antidepressant drugs. These results confirm the hypothesis that 5-ht6 receptors may correspond to an important target for atypical antipsychotics and reveal an original pharmacological profile for this receptor. The study of the ontogeny of the 5-ht6 mRNA in rat developing brain showed that 5-ht6 mRNA were first detectable with a high level on ED12, slighly decreased up to ED17 and then remained stable at high level until the adult age. The ontogenetic pattern of 5-ht6 mRNA expression appeared to correlate with the occurence of the first cell bodies of serotonergic neurons; the early expression of 5-ht6 mRNA and the fact that this receptor is positively coupled to the production of cAMP may suggest a role for 5-ht6 receptor in the early growth process involving the serotonergic system.

Molecular, cellular and physiological characteristics of 5-HT-moduline, a novel endogenous modulator of 5-HT1B receptor subtype.

The serotonergic transmission is considered as a neuromodulatory system in the Central Nervous System. 5-HT1B receptors play an important role in this modulatory activity. We have purified from mammalian brain an endogenous peptide, LSAL, we called 5-HT-moduline, interacting specifically with 5-HT1B receptors. This interaction is characterized by a high affinity (Ki = 10(-10) M) and a non-competitive mechanism. Direct [3H]5-HT-moduline binding revealed a single population of sites having an apparent affinity constant close to 10(-10) M. Autoradiographic studies showed a brain distribution of [3H]5-HT-moduline binding sites closely related to the 5-HT1B receptors. In functional studies, the peptide is able to reverse the activity of a 5-HT1B agonist in the nanomolar range. Furthermore, this antagonist effect is also observed in vivo on mice behavior. Immunocytochemistry revealed an heterogeneous distribution of 5-HT-moduline in mouse brain. The labeled structures correspond to cellular profiles with axon-like prolongations. Moreover, in vitro, LSAL is released in a Ca++, K(+)-dependent manner. Therefore, 5-HT-moduline behaves as a neurotransmitter. The fact that 5-HT-moduline induces the desensitization of 5-HT1B receptors reflects the existence of a novel and efficient mechanism able to rapidly modulate the serotonergic activity.

[Giant diverticulum of the duodenum. Case report]. / Diverticolo gigante del duodeno. Caso clinico.

The authors describe the case of a 49-year-old man with a giant diverticulum of the duodenum, with apparently scarce symptoms, pointed out by gastroscopy and X-ray of the upper gastrointestinal duct with barium meal. The removal of the lesion, obtained after surgical operation, showed a diverticulum with a 6 cm diameter, with signs of diverticulitis; the histological test did not point out is degenerative lesions. A survey of the recent literature on the matter is presented.

Immunocytochemical localization of neurons expressing 5-HT-moduline in the mouse brain.

The localization of 5-HT-moduline, an endogenous cerebral tetrapeptide (LSAL) which specifically interacts with 5-HT1B receptors (Rousselle et al., 1996; Massot et al., 1996) was examined in mouse brain using an immunocytochemistry technique with a polyclonal anti-peptide antibody highly specific for this tetrapeptide. Highest levels of 5-HT-moduline immunoreactivity were observed in the cerebral cortex including cingulate, retrosplenial, parietal and pyriform cortical areas and in the basal ganglia. Intense immunoreactivity also occurred in the hippocampus, subiculum, various hypothalamic and thalamic nuclei and in some midbrain regions such as the substantia nigra and the superior colliculi. Immunoreactive neurons generally showed intense and extensive labelling of the perikarya and dendritic arborizations with moderate to heavy characteristic deposits of reaction product along plasma membranes and within cytoplasm while the nuclei were devoid of reaction product. The results obtained indicated that 5-HT-moduline immunoreactivity was heterogenously distributed in neuronal structures of mouse brain. The distribution of 5-HT-moduline immunoreactivity closely correlated with that of 5-HT-moduline specific binding sites as visualized by autoradiography (Massot et al., 1996). Moreover, it seems to overlap with the distribution of serotonergic innervation and also with that of 5-HT1B receptors in mouse brain (Boschert et al., 1994; Bruinvels et al., 1994; Chopin et al., 1994; Langlois et al., 1995). These data provide evidence that 5-HT-moduline immunoreactivity is located in cells with the morphological appearance of neurones. Its localization in brain areas which also contain 5-HT1B receptors, is in good agreement with previous demonstrations that this peptide specifically interacts with 5-HT1B receptors to regulate their functional activity and accordingly controls the modulatory activity of the serotoninergic system on various CNS functions.

5-hydroxytryptamine-moduline, a new endogenous cerebral peptide, controls the serotonergic activity via its specific interaction with 5-hydroxytryptamine1B/1D receptors.

The serotonergic system controls the activity of neurotransmissions involved in numerous physiological functions. It is also thought to be crucially implicated in various pathologies, including psychiatric disorders such as depression, anxiety, and aggressiveness. The properties of 5-hydroxytryptamine (5-HT)-moduline, a novel endogenous peptide, have been tested in vitro and in vivo. Binding studies have shown that the peptide specifically interacts with 5-HT1B/1D receptors via a noncompetitive mechanism corresponding to a high apparent affinity (EC50 = 10(10) M). The interaction was shown in rat and guinea pig brain tissues and in cells transfected with either 5-HT1B or 5-HT1D beta receptor gene. [3H]5-HT-moduline binds to a single population of sites in mammalian brain (Kd = 0.4 nM in rat, Kd = 0.8 nM in guinea pig) as well as in transfected cells expressing the 5-HT1B or the 5-HT1D beta receptors (Kd = 0.2 and 0.6 nM, respectively). Furthermore, the binding is clearly specific of the LSAL sequence. Autoradiographic studies showed an heterogeneous brain distribution of this site. The interaction of 5-HT-moduline with the 5-HT1B/1D receptor corresponds to a decrease in the functional activity of the receptor (i.e., a decrease in the inhibitory effect of a 5-HT1B agonist on the evoked release of [3H]5-HT from synaptosomal preparation). It was also shown that 5-HT-moduline possess an in vivo effect in the social interaction test in mouse. Finally, it was demonstrated that 5-HT-moduline was released from brain synaptosomal preparation by a K+/Ca(2+)-dependent mechanism. In conclusion, 5-HT-moduline is a novel endogenous peptide regulating the serotonergic activity via a direct action at presynaptic 5-HT receptor. It may play an important role in the physiological mechanisms involving the serotonergic system, particularly in mechanisms corresponding to the elaboration of an appropriate response of the central nervous system to a given stimulus.