Olivier Massot

Isolation and Characterization of an Endogenous Peptide from Rat Brain Interacting Specifically with the Serotonergic 1B Receptor Subtypes

The existence of endogenous compounds interacting with the serotonergic system was previously postulated. In the present work, rat brain tissues were extracted by acidic and organic procedures. The resulting extract was tested for its capacity to interact with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to 5-HT1 receptors. Compounds responsible for the observed inhibitory activities were isolated and purified by high pressure liquid chromatography. A tetrapeptide corresponding to a novel amino acid sequence Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of [3H]5-HT to 5-HT1 receptors at low concentration (IC50 = 10−10M). This effect corresponds to a specific interaction at 5-HT1B receptors since LSAL does not significantly affect other neurotransmitter bindings. LSAL appears heterogeneously distributed throughout the brain (hippocampus > cerebellum > striatum > brain stem) and in peripheral tissues (kidney > lung > stomach > blood > liver > spleen). Two other peptides, Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected [3H]5-HT binding compared with LSAL. They presumably represent degradation products of the functional peptide LSAL. The fact that LSAL interacts specifically with 5-HT1B receptors that inhibit the release of neurotransmitters and particularly that of 5-HT itself suggests that this peptide may be involved in mechanisms controlling 5-HT neurotransmission and, accordingly, may play an important role in pathophysiological functions related to 5-HT activity.

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K+-evoked overflow of [3H]dopamine was studied in rat striatal synaptosomes loaded with [3H]dopamine. The aim of the study was to investigate the participation of 5-HT1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca2+-dependent, tetrodotoxin-resistant K+-evoked overflow of [3H]dopamine was inhibited by CP93,129 (0.01–100 µM) in a concentration-dependent manner (IC50=1.8 µM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT1A receptor agonist, [+/–]DOI, a 5-HT2 receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT3 receptor agonist, at concentrations ranging from 0.01 µM to 100 µM did not show any significant effect. Neither ketanserin (1 µM and 5 µM), a selective 5-HT2/5-HT1D receptor antagonist, nor ondansetron (1 µM), a 5-HT3 receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT1B receptor antagonists, in contrast, used at a concentration of 1 µM, antagonized the inhibitory effect of CP93,129 (3 µM and 10 µM). SB224289, a selective 5-HT1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated Ki value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K+-evoked release of dopamine is regulated by the presynaptic 5-HT1B heteroreceptors.

Magnetic field desensitizes 5-HT1B 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-HT1B Receptors: A Novel Target for Lithium: Possible Involvement in Mood Disorders

Lithium ion is widely used to treat depressive patients, often as an initial helper for antidepressant drugs or as a mood stabilizer; however, the toxicity of the drug raises serious problems, because the toxic doses of lithium are quite close to the therapeutic ones. Thus, precise characterization of the target(s) involved in the therapeutic activity of lithium is of importance. The present work, carried out at molecular, cellular, and in vivo levels, demonstrates that 5-HT1B receptor constitutes a molecular target for lithium. Several reasons suggest that this interaction is more likely related to the therapeutic properties of lithium than to its undesirable effects. First, the observed biochemical and functional interaction occurs at concentrations that precisely correspond to effective therapeutic doses of lithium. Second, 5-HT1B receptors are well characterized as controlling the activity of the serotonergic system, which is known to be involved in affective disorders and the mechanism of action of various antidepressants. These findings represent progress in our knowledge of the mechanism of action of lithium that may facilitate clinical use of the ion and also open new directions in the research of antidepressant therapies.

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-HT-moduline, a 5-HT1B/1D receptor endogenous modulator, interacts with dopamine release measured in vivo by microdialysis

5-Hydroxytryptamine-moduline (5-HT-moduline) is an endogenous tetrapeptide (Leu-Ser-Ala-Leu) recently isolated and characterized from mammalian brain. This compound interacts with 5-HT1B receptors as a non-competitive, high-affinity antagonist and has the properties of an allosteric modulator. 5-HT-moduline could play an important role in the regulation of serotonergic transmission and also, through heteroreceptors, dopaminergic transmission. The aim of this work was to examine the potential ability of 5-HT-moduline to modify the basal extracellular concentration of dopamine and its metabolites (3-methoxytyramine, dihydroxyphenylacetic acid and homovanillic acid), in the rat striatum and to determine its potential interaction with the stimulating activity of a specific 5-HT1B receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl) pyrrolo [3,2-b] pyrid-5-one (CP-93,129), on the release of dopamine. The technique is based on in vivo microdialysis using probes implanted in the striatum of the conscious rat. Results showed that the perfusion of 5-HT-moduline directly into this structure (1.25 mM) increased the striatal level of dopamine by two-fold (104% of the absolute basal release values, P = 0.0015) and that of 3-methoxytyramine by 3-fold (293%, P = 0.0001) without any change in the terminal metabolite concentrations. The intrastriatal administration of CP-93,129 induced a statistically significant, dose-dependent increase of dopamine levels (P < 0.0001). Coperfusion of 5-HT-moduline did not significantly alter the effect of CP-93,129 at 0.1 and 0.5 mM, but appeared to have an additive effect on the lowest dose (P = 0.0406). The results obtained show that 5-HT-moduline directly administered into the striatum increases the release of dopamine in this area. Presumably, this effect results from the desensitization of 5-HT1B receptors located on dopamine terminals. However, the fact that a 5-HT1B receptor agonist (CP-93,129) also increased the release of dopamine in the striatum and that 5-HT-moduline exhibited a slight additive effect with that of a low concentration of CP-93,129 suggests that the two substances interact with different mechanisms.

Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells.

Abstract5-HT1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT1B or 5-HT1D receptors, it was shown that 5-HT-moduline prevents the binding of [3H]5-HT to 5-HT1B as well as to 5-HT1D receptors with similar biochemical characteristics: the IC50 of the peptide was 1.2×10–12 M for 5-HT1B and 9×10–13 M for 5-HT1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [3H]5-HT on 5-HT1B (–51.2±1%) as well as 5-HT1D binding (–47.2±7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the “scrambled” peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT1A or 5-ht6 receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [35S]GTPγS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC50=1.2±0.7×10–12 M for 5-HT1B and 9.8±4.0×10–12 M for 5-HT1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT1D as well as 5-HT1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT1B and 5-HT1D receptors.

A new peptide, 5-HT-moduline, isolated and purified from mammalian brain specifically interacts with 5-HT1B1D receptors

5-HT-Moduline (Leu-Ser-Ala-Leu) is a new endogenous peptide purified from rat brain which interacts specifically with 5-HT1B/1D receptors. The binding interaction of 5-HT-Moduline with 5-HT1B/1D receptors appeared to be a non-competitive process, since the Bmax value of [125I] cyanopindolol binding on rat brain cortical membranes was decreased without modification of the Kd. This interaction was conserved on NIH 3T3 cells expressing the 5-HT1B receptor (IC50 = 10(-11)M) suggesting that the binding site for 5-HT-Moduline is localized on the 5-HT1B receptor protein. The observed interaction may lead to functional alterations of 5-HT1B/1D receptors known to play an important role in regulating the release of 5-HT from serotonergic nerve terminals (autoreceptors) as well as the release of other neurotransmitters (heteroreceptors).

Interaction of lithium with 5-HT1B receptors in depressed unipolar patients treated with clomipramine and lithium versus clomipramine and placebo: preliminary results

Lithium is commonly used in combination with antidepressant drugs as a treatment for refractory depression; less often, it is used in non-resistant depression. The aim of this study was to examine the interaction of lithium with 5-HT(1B) receptors in 10 non-resistant unipolar depressed patients treated with clomipramine+lithium (C+L) vs. clomipramine+placebo (C+P). A mediation of the serotonergic system has been proposed in the literature to explain the clinical effect of lithium. Indeed, in a previous study of healthy human blood platelets, we demonstrated the interaction of lithium with adenylate cyclase activity coupled to 5-HT(1B) receptors. The functional activity of these receptors was measured by studying the inhibitory effect of L694,247, a 5-HT(1B) receptor agonist, on the adenylate cyclase activity determined by the production of cAMP. Using the same technique in the present study, we found that lithium significantly reduced the inhibition of adenylate cyclase activity induced by 5-HT(1B) receptor activation. This result confirms the specific interaction of lithium with 5-HT(1B) receptors. Moreover, a correlation between the percentage of 5-HT(1B) receptor-dependent adenylate cyclase inhibition and the clinical benefit of lithium was established, suggesting 5-HT(1B) receptors may be a target for the therapeutic effect of lithium.

Mapping of 5-HT-moduline binding sites in guinea-pig brain by film and digital autoradiography

5-HT-moduline is a cerebral tetrapeptide [Leu-Ser-Ala-Leu] that was recently isolated from bovine brain tissue and shown to interact specifically with 5-HT1B receptors, particularly in rodents. The pharmacological properties of 5-HT1B receptors in rodents are different from those in other species. In order to better understand the role of this peptide in non-rodent species, we determined the distribution of 5-HT-moduline binding sites in guinea-pig brain using both the film autoradiography and digital autoradiography with a newly developed high resolution beta-imaging techniques. We found that 5-HT-moduline binding sites were expressed in various brain regions. Quantitative analysis showed that densities of binding sites were similar to those observed previously in rat brain. Regions with the highest labelling included cortex, septum, hippocampus and some regions of basal ganglia. Our results extend previous data and show that 5-HT-moduline interacts with the two forms of 5-HT1B receptors that are distinct pharmacologically. By this interaction, 5-HT-moduline may play an important role in regulating the functional activity of 5-HT1B receptors, thereby contributes to the pathophysiology of serotonergic transmission.