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.

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.

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.

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 microM) in a concentration-dependent manner (IC50=1.8 microM; maximal inhibition by 35.5% of control). [+/-]8-OH-DPAT, a 5-HT(1A) receptor agonist, [+/-]DOI, a 5-HT2 receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT3 receptor agonist, at concentrations ranging from 0.01 microM to 100 microM did not show any significant effect. Neither ketanserin (1 microM and 5 microM), a selective 5-HT2/5-HT1D receptor antagonist, nor ondansetron (1 microM), 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 microM, antagonized the inhibitory effect of CP93,129 (3 microM and 10 microM). SB224289, a selective 5-HT1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K(i) 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.

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.

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.

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.

Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration.

Expression of seven serotonin or 5-hydroxytryptamine (5-HT) receptors (5-HT1D alpha, 5-HT1E, 5-HT2, 5-HT1A, 5-HT1C, 5-HT1D beta, and 5-HT6) was investigated in human normal fetal astrocytes and eight glioma cell lines by reverse transcription and polymerase chain reaction (RT-PCR). No expression of 5-HT1D beta and 5-HT6 was observed in any of the cell lines studied. The 5-HT1D alpha receptor was found to be expressed in two human glioma cell lines but not in normal astrocytes. In addition, only three glioma cell lines expressed the 5-HT1E receptor. The 5-HT1C receptor was expressed in six glioma cell lines but not in normal astrocytes while the 5-HT1A was found to be expressed in normal astrocytes from the left hemisphere and in six glioma cell lines but not in normal astrocytes from the cerebellum. Interestingly, the 5-HT2 receptor was expressed in all cells studied but very weakly in normal astrocytes. The effect of 5-HT on glioma cell proliferation, migration, and invasion was also investigated. Serotonin was found to positively modulate these three processes in vitro. These results suggest that 5-HT may play an important role in the control of the biological properties of human glioma cells.

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).

Production and characterization of an antibody directed against the mouse 5HT1B receptor.

We have developed a polyclonal antibody directed against a peptide located in the third intracellular loop of the 5HT1B receptor. Its characterization was carried out using NIH cells stably transfected with a eukaryotic expression vector containing the mouse 5HT1B receptor cDNA. The synthetic antigenic peptide had a unique sequence to the mouse 5HT1B receptor corresponding to amino acids 273-287 localized in the third intracellular loop. In dot blot analysis, antisera detected 2 ng to 2 micrograms of synthetic peptide at dilutions of 1/200-1/20,000 and bound antibody was visualized using an immunoperoxidase procedure. Preimmune serum showed no immunoreactivity to the synthetic peptide. NIH cells stably transfected expressing mouse 5HT1B receptor displayed an intense immunoreactivity with the antiserum. In contrast no immunoreactivity was seen in any of the control experiments. In the present study, we have produced a specific antibody which is an essential tool suitable for immunocytochemical applications such as regional distribution, anatomical localization and phenotypical characterization of the cells expressing the 5HT1B receptors in brain by double immunolabelling procedure.

Rabies virus selectively alters 5-HT1 receptor subtypes in rat brain.

Rabies virus infection in man induces a series of clinical symptoms, some suggesting involvement of the central serotonergic system. The results of the present study show that, 5 days after rabies virus infection in rat, the total reversible high-affinity binding of [3H]5-HT in the hippocampus is not affected, suggesting that 5-HT1A binding is not altered. 5-HT1B sites identified by [125I]cyanopindolol binding are not affected in the cortex 3 and 5 days after the infection. Accordingly, the cellular inhibitory effect of trifluoromethylphenylpiperazine (TFMPP) on the [3H]acetylcholine-evoked release, presumably related to 5-HT1B receptor activity, is not modified 3 days after infection. In contrast, [3H]5-HT binding determined in the presence of drugs masking 5-HT1A, 5-HT1B and 5-HT1C receptors, is markedly (50%) reduced 3 days after the viral infection. These results suggest that 5-HT1D-like receptor subtypes may be affected specifically and at an early stage after rabies viral infection.

Improved brain delivery of AZT using a glycosyl phosphotriester prodrug.

The concentration of AZT in mice plasma and brain was measured using HPLC after an ingestion of 20 mg/kg of AZT or the molar equivalent of hexadecyl 2-(alpha-D-mannopyranosidyl)ethyl 3'-azido-3'-deoxy-5'-thymidinyl phosphate 3. The results demonstrated the promising qualities of the prodrug 3 which gave AZT-5'-phosphate as the main metabolite: the total concentration of AZT derivatives detected in brain presented a peak of 156 nmol/g (5 nmol/g for AZT) at 1 h; the half-life was about 24 h (1 h for AZT) with an AUC of 4366 nmol h/g as compared to 4 nmol h/g for AZT. The lipophilic properties of 3 were confirmed by its in vitro transport of inside synaptosomes. The derivative 2-(alpha-D-mannopyranosidyl)ethyl 3'-azido-3'-deoxy-5'-thymidinyl phosphate (2) provided also a good delivery of AZT to the central nervous system, with values intermediate between those of AZT and 3.

Molecular cloning of two partial serotonin 5-HT1D receptor sequences in mouse and one in guinea pig.

The G protein coupled serotonin (5-HT) receptors, with seven membrane spanning domains, form a multigene family of which several members have been cloned and sequenced. The presence of 5-HT1D binding sites to our knowledge has not yet been reported in mouse. Here we describe the cloning and sequencing by the polymerase chain reaction (PCR) method of two 5-HT1D receptor sequences of the third cytoplasmic loop in mouse, strongly suggesting the existence of two 5-HT1D receptor genes, located on chromosome 4. A homologous sequence to one of them was cloned in guinea pig.