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.

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.

A new peptide, 5-HT-moduline, isolated and purified from mammalian brain specifically interacts with 5-HT1B/1D 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).

[3H]5-HT binding sites and 5-HT-sensitive adenylate cyclase in glial cell membrane fraction.

Glial cell membrane fractions were prepared using glial cells preparations isolated from horse brain striatum. [3H]5-HT binding was measured by the filtration technique and the adenylate cyclase activity determined by measuring the cAMP production using a radioimmunoassay. Serotonin binds to glial membrane fractions with an affinity corresponding to a dissociation constant Kd = nM. The corresponding site is serotoninergic specific: [3H]5-HT binding is inhibited by 5-HT agonists (5 OH NM-DMT, 5-MeOHT, 5-MeOH-DMT, NN-DMT) or antagonists (cinanserine, cyproheptadine, methysergide, LSD) and not (or poorly) inhibited by non-serotoninergic related drugs. The population of sites binding 5-HT, present in neuronal membrane preparations and determined in parallel assays is distinct from that observed in glial preparations. The glial membrane fractions contains an adenylate cyclase activated by 5-HT with an apparent affinity constant close to 1 microM. It is serotonin-specific and clearly distinct from the DA-stimulated adenylate cyclase present in the same preparation. The sites binding 5-HT and activating the adenylate cyclase with low affinities might be directly related. This system, clearly distinct from the postsynaptosomal serotoninergic receptor, represents presumably a glial serotoninergic receptor; however, it cannot be totally excluded that these sites may refer to presynaptic membranes.

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.

Multiple high affinity binding sites for 5-hydroxytryptamine: a new class of sites distinct from 5-HT1 and S2.

Two different classes of binding sites probably related to serotonergic receptors have already been reported: 5-HT1 binding sites recognize [3H]5-hydroxytryptamine with a high affinity (Kd = 3 nM) and S2 binding sites recognize [3H]spiroperidol and [3H]ketanserine. An additional population of sites has been observed in crude membrane preparations or fractions enriched with synaptosomal membranes obtained from rat brain cortex. This population was observed as a single class of sites in a synaptosomal fraction (L fraction--according to Laduron (1977)). It corresponded to a dissociation constant Kd = 13-15 nM, and Bmax = 0.80 +/- 0.15 pmol/mg protein. Displacement experiments showed that it recognized preferentially the 5-HT structure (bufotenin, 5-MeO-tryptamine). Tryptamine was a weak displacer and 5,7-dihydroxytryptamine totally inefficient. Neither 8-OH-DPAT, nor quipazine had any effect. Methiothepin, cinanserin and cyproheptadine displaced 5-HT from these sites whereas ergot derivatives did not. Contrary to 5-HT1 binding, this recently observed binding was not altered by GTP; alpha-MSH reduced the corresponding Bmax whereas Leu-enkephalin did not. The degenerative lesion of the serotonergic fibers led to a slight increase in the Bmax of the binding without altering the Kd which means that corresponding sites are not located on serotonergic fibers and might be postsynaptically located.

Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides.

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7-8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1 +/- 3.6 microM and a Vmax of 5.5 micromol min(-1) l(-1) per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726-735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

5-HT-moduline, a 5-HT(1B/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.

5-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 1.2x10(-12) M for 5-HT1B and 9x10(-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]GTPgammaS 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 +/- 0.7x10(-12) M for 5-HT1B and 9.8 +/- 4.0x10(-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.

5-Hydroxytryptamine receptors in neurones and glia.

Crude membranal fractions isolated from mammalian brain tissue contain two classes of recognition sites capable of binding [3H]5-HT with high affinity constants. These classes of sits are characteristics of the postsynaptosomal membrane fraction for the higher affinity and of a glial cell membrane fraction for the lowest. They are observed with similar properties in cultured neuronal and glial cell respectively. Two 5-HT stimulated adenylate cyclases are present in crude membrane fraction; they are also separable as neuronal and glial components. These observations correspond likely to the existence of two classes of receptors for 5-HT. Their mechanisms of regulation involve presumably structural conformation changes of the recognition site coupled to various states of the activity of the receptor.

5-Hydroxytryptamine stimulates two distinct adenylate cyclase activities in rat brain: high-affinity activation is related to a 5-HT1 subtype different from 5-HT1A, 5-HT1B, and 5-HT1C.

5-HT binding sites of the 5-HT1 type are heterogeneous and appear to comprise several subtypes (5-HT1A, 5-HT1B and 5-HT1C); their physiological role is as yet unclear. The stimulation of adenylate cyclase induced by 5-HT has been investigated in membrane fractions prepared from rat brain cortex. Enzymatic activity was determined by measuring cAMP production with an HPLC technique. It was shown that 5-HT stimulates adenylate cyclase activity with 2 activation constants (Kact): one shows a high apparent affinity (Kact = 0.8 nM) and the other a lower apparent affinity (Kact = 0.30 microM). The latter activity, induced by micromolar concentrations of 5-HT, was inhibited by spiperone at concentrations that block 5-HT1A binding. 5-Methoxytryptamine, bufotenin, and LSD also had a stimulatory biphasic effect on adenylate cyclase activity, whereas trifluoromethylphenylpiperazine, 5-carboxyamidotryptamine, 8-hydroxy-(2-di-n-propylamino)tetralin, RU 24969 had a monophasic effect. Enzyme activation by drugs acting in the micromolar range was inhibited by spiperone (1 microM), suggesting a link between this activation and 5-HT1A sites. On the other hand, the high-affinity activation of the enzyme induced by 5-HT, 5-methoxytryptamine, bufotenin, LSD, and the activation induced by TFMPP were not inhibited by spiperone (1 microM), by propranolol (3 microM), or by mesulergine (0.1 microM), which selectively block 5-HT1A, 5-HT1B, and 5-HT1C sites. Inhibition was produced by dihydroergotamine, methysergide, cinanserin, and mianserin, but not by naloxone, phenoxybenzamine, and phentolamine. Therefore, these activations seem related to 5-HT1 receptors but not to 5-HT1A, 5-HT1B, or 5-HT1C sites. Accordingly, binding of [3H]5-HT to 5-HT1-like sites was examined in the presence of spiperone (1 microM) and propranolol (3 microM); in these conditions, a high-affinity site (KD = 3.4 nM) was indeed revealed. The relative potencies of a series of drugs that stimulate or inhibit the activation of the adenylate cyclase with a high affinity and their ability to inhibit this binding of [3H]5-HT showed a positive correlation, strongly suggesting a direct relation between this recognition site for 5-HT and the production of a second messenger (cAMP). Moreover, this potential receptor is shown to be heterogeneously distributed within the brain, and was localized postsynaptically at serotonergic synapses.

New markers in Anopheles gambiae salivary glands after Plasmodium berghei infection.

In malaria, mosquito saliva and salivary glands play central roles in the multi-faceted interactions that occur among the parasite, its vector, and its host. Analyzing the processes involved in the survival and maintenance of the Plasmodium parasite in mosquito organs, and in its transmission into vertebrate hosts, may lead to the identification of new molecular targets for parasite control. We used comparative two-dimensional gel polyacrylamide electrophoresis (2D-PAGE), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), and high-performance liquid chromatography (HPLC), followed by Edman sequencing, to study saliva and salivary gland samples from Anopheles gambiae mosquitoes infected or not with Plasmodium berghei. Quantitative 2D-PAGE profile analysis showed that the intensities of seven spots were affected by the presence of the parasite in the salivary glands. Most of the proteins identified possessed a signal peptide. SELDI-TOF-MS revealed 32 proteins/peptides whose peak intensities differed between the Plasmodium-infected and non-infected control groups. Quantitative comparison of HPLC profiles of low-molecular-weight components from salivary gland extracts revealed several peptides and proteins with levels that were modulated by parasite infection. The results of these complementary approaches suggest that the infection of female A. gambiae mosquitoes by P. berghei alters the production levels of several salivary gland proteins and peptides, some of which (e.g., protein cE5, B3VDI9_ANOGA, and AGAP008216-PA) are known or predicted to be secreted in saliva and involved in blood feeding.

[Connection between 3H 5-HT and adenyl cyclase activation induced by 5-HT in preparations of cerebral glial membranes]. / Liaison de 3H 5-HT et activation adenylcyclasique induite par la 5-HT dans des préparations de membranes gliales cérébrales.

Purified glial membrane preparations have been isolated from horse brain striatum. Tritiated 5-HT bound to these membranes with a high affinity (KD = 10 nM); the corresponding binding is reversible and appears specific of the serotoninergic structure. In parallel, 5-HT activates an adenylate cyclase with a low affinity (KD = 1 microM). The sites involved in this binding and in this adenylate cyclase activation appear different from the serotoninergic sites reported in the neuronal membrane preparations.

[Increase in binding affinity of the 5-HT receptor and decrease in 5-HT sensitive adenylate cyclase activity following prolonged exposure to 5-HT (author's transl)]. / Augmentation de l'affinité du récepteur 5-HT et diminution de l'activité adénylate cyclase sensible à la 5-HT par exposition prolongée à la 5-HT.

Previously, a 5-hydroxytryptamine receptor has been described in neuronal membrane preparations. This receptor corresponds to a recognition site for 5-HT, and is seemingly related to an effector which consists of an adenylate cyclase activated by 5-HT and having a high apparent affinity constant (KD close to 1 nM). Moreover, this neuronal receptor is distinct of the glial serotoninergic receptor (Fillion et al., 1980). The method used to measure the binding of 3H-5-HT has been previously reported (FILLION et al., 1978). Adenylate cyclase activity was determined by measurement of cAMP using a radioimmunoassay (FILLION et al., 1979 a, b). Membranes were pretreated as follows: (1) membranes were preexposed to 5-HT (i.e.: incubated in the presence of 5-HT, generally 10 nM) at 37 degrees C, washed by centrifugation and resuspended in a medium free of 5-HT (or diluted in the same medium) or (2) membranes were incubated with a single concentration of 5-HT for a prolonged duration. Results were as follows: 1. The binding of 3H-5-HT, incubated for 10 min at 37 degrees C, corresponds to a KD close to 20 nM whereas a prolongation of the incubation duration to 15-25 induces the binding of 5-HT with a KD close to 2 nM. 2. Preexposure of the membranes to 5-HT leads to the same increase in affinity with, apparently, a reduced number of sites. 3. N-ethylmaleimide pretreatment of the membranes prior to their preexposure to 5-HT inhibits the change in affinity. 4. GTP, but not GDP, reverses the affinity change. 5. The adenylate cyclase activated by 5-HT is desensitized by preexposure of the membranes to 5-HT. 6. GTP reverses the process of desensitization. These results support the hypothesis of the existence of a regulatory mechanism for the 5-HT neuronal receptor. The regulatory mechanism would involve structural conformation changes of the recognition site corresponding to different affinities of the adenylate cyclase. A nucleotide binding protein is presumably involved in the coupling of these two subunits of the receptor.

Solubilization and characterization of [3H] 5HT high affinity binding sites (5HT1 and 5HT3).

The solubilization of the serotonergic 5HT1 and 5HT3 sites was performed with digitonin and sodium cholate at 1% (final concentration). Two binding sites for [3H]5HT were observed on rat or horse brain synaptosomal membranes solubilized with these detergents. The corresponding dissociation constants (KD) were 1-3 nM and 13-30 nM respectively. These values were closely similar to those corresponding to 5HT1 and 5HT3 sites located in intact membranes. The solubilized sites specifically bound 5HT. The effect of GTP decreasing the binding to 5HT1 sites was lost on solubilized 5HT1 sites; it was recovered, however, by addition of phospholipids (asolectin 0,2%). The apparent molecular weights of these sites were determined using the gel filtration method (438 and 235 K daltons). The photoactivation of [3H]5HT by U.V. light was used to label 5HT1 and 5HT3 sites irreversively in membranes. The binding of [3H]5HT following U.V. irradiation was not dissociated after dilution; it was saturable and prevented by serotonergic drugs and not by adrenergic or dopaminergic antagonists. Moreover, GTP added prior to the irradiation reduced it markedly thus showing that 5HT1 sites were labelled. Electrophoretic and fluorographic analyses of the labelled material evidenced a 60 K dalton-band specifically labelled with [3H]5HT (5 or 20 nM). These results tend to indicate that the 60 K dalton-proteic band might represent a proteic subunit constituting part of 5HT1 and 5HT3 sites.