Magnetic properties and the crystal field problem for tetragonal HoBa(2)Cu(3)O(x).

Magnetic susceptibilities of the first and third orders as well as the magnetization of the singlet paramagnet HoBa(2)Cu(3)O(x) (x≈6.0) at low temperatures are studied experimentally using a SQUID magnetometer and compared with theoretical calculations. The magnetic behaviour of single-crystal HoBa(2)Cu(3)O(x) at low temperature is found not to follow the one calculated on the basis of the known crystal field parameters. Different effects which may change the magnetic properties are analysed and discussed: low symmetry components of the crystal field due to some disorder in the oxygen subsystem, a noncompensated effective field from the ordered Cu subsystem acting on the Ho(3+) ions, and others.

High field magnetic transitions in the mixed holmium-yttrium iron garnet Ho(0.43)Y(2.57)Fe(5)O(12).

High static magnetic field magnetization measurements have been performed up to 23 T on Ho(0.43)Y(2.57)Fe(5)O(12) single crystals at helium temperature (T = 4.2 K) with fields applied along the three main cubic axes: [Formula: see text], [Formula: see text] and [Formula: see text]. The change from the spontaneous ferrimagnetic structure in zero magnetic field to the fully ferromagnetic one in high field takes place through several intermediate phases separated by transitions with step-like magnetization behaviour, but without any observed hysteresis. Using the effective spin Hamiltonian approximation, we show that the general features of these transitions can be accounted for by a large magnetocristalline anisotropy of the Ho(3+) moments of the uniaxial type along the local z axis of each rare-earth site. The model is in better agreement with the experiments than its Ising limit, widely used before, but is still unsuccessful in predicting the 'umbrella' magnetic structures found by previous neutron and NMR experiments.

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 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.

Potential of 5-HT-moduline as a drug target for affective disorders.

The serotonergic system is one of the aminergic neurotransmitter systems participating in the maintenance of homeostasis of the organism in mammals; accordingly, it regulates the activity of various cerebral functions to organize adapted responses of the brain to environmental stimuli. The regulatory activity of the serotonergic system itself is modulated by an endogenous mechanism based on an allosteric interaction involving a newly discovered peptide, 5-HT-moduline, and the 5-HT1B receptor which, as an autoreceptor, controls the release of 5-HT from serotonergic neuron terminals. 5-HT-moduline specifically interacts with 5-HT1B receptors at nanomolar concentrations resulting in the desensitization of the receptor. As 5-HT1B autoreceptors have an inhibitory effect on the release of 5-HT, 5-HT-moduline ultimately increases its release. The peptide is characterized by several criteria which correspond to those of a neurotransmitter, strongly suggesting that 5-HT-moduline is a novel neuropeptide locally controlling serotonergic activity. 5-HT-moduline is released in various parts of the brain, particularly under conditions of stress; moreover, its deactivation by specific antibodies in mice induces changes in the behavior of the animal. These results strongly suggest that the peptide may play a role in the physiopathology of central nervous system disorders in mammals, particularly in conditions related to stress and anxiety. Furthermore, the fact that 5-HT-moduline increases the release of 5-HT suggests that synthetic drugs which recognize the 5-HT-moduline binding site on 5-HT1B receptors and mimic the effect of the peptide, may have antidepressant properties by increasing the release of 5-HT.

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.

Structural requirements of 5-hydroxytryptamine-moduline analogues to interact with the 5-hydroxytryptamine1B receptor.

5-Hydroxytryptamine-moduline is an endogenous cerebral tetrapeptide that regulates the activity of 5-hydroxytryptamine1B receptors. Direct binding of 5-[3H]hydroxytryptamine-moduline on rat brain homogenate evidenced the existence of two interacting sites for the peptide, very likely corresponding to different conformations of the 5-hydroxytryptamine1B receptor: The peptide first binds to a low-affinity state of the receptor (pIC50 = 7.68+/-0.14) and then induces (or stabilizes) a high-affinity complex (pIC50 = 11.62+/-0.18). This work focuses on the ability of 5-hydroxytryptamine-moduline analogues to recognize the high- and low-affinity sites for 5-hydroxytryptamine-moduline. The results obtained show that the two conformers of the 5-hydroxytryptamine1B receptor have similar but not identical binding pockets for 5-hydroxytryptamine-moduline. These two sites proved to be stereoselective and selective for tetrapeptides and favored the binding of peptides with hydrophobic amino acids in positions 1 and 4, serine in position 2, and a short amino acid in position 3. However, the serine in position 2 seems to be more important for the interaction of the peptide with the low-affinity site than the high-affinity one, which only needs a short hydrophobic amino acid in position 2.

Differential sensitivity of 5-HT1B auto and heteroreceptors.

The effect of the native and rodent-selective 5-HT1B receptor agonists (5-hydroxytryptamine (5-HT) and CP93,129) on the K+-evoked overflows of [3H]5-HT, [3H]dopamine (DA) and [3H]acetylcholine (ACh) was studied in synaptosome preparations obtained from rat brain striatum or hippocampus loaded with radiolabeled neurotransmitter. The aim of the study was to compare the different potencies of the specific 5-HT1B receptor agonists to stimulate the auto and heteroreceptors and to modulate the different neurotransmitter release. Results show that under the same experimental conditions, 5-HT and CP93,129 exhibited significantly higher potencies in inhibiting the K+-evoked overflow of [3H]5-HT from synaptosomes of rat striatum (IC50=2.0+/-1.8 nM and 20.5+/-3.1 nM, respectively) than in inhibiting the K+-evoked overflow of [3H]DA from synaptosomes of the same cerebral region (IC50= 0.8+/-0.2 microM and 1.8+/-0.4 microM, respectively), or [3H]ACh from synaptosomes of hippocampus (IC50=1.7+/-0.8 microM for CP93,129). The inhibitory effects of the 5-HT1B receptor agonists on [3H] K+-overflows were antagonized by the selective 5-HT1B receptor antagonist (SB224289), further indicating that the observed effects were 5-HT1B receptor specific. Sumatriptan, a selective r5-HT1D receptor agonist, did not show any significant effect on the K+-overflow of [3H]5-HT in the range of concentrations (10(-10) to 10(-6) M), and did not affect the K+ overflow of [3H]DA or [3H]ACh at concentrations (10(-9) to 10(-4) M), which exclude the involvement of 5-HT1D receptors. These inhibitory effects of the 5-HT1B receptor agonists were highly attenuated by pertussis toxin in the three systems studied, suggesting the involvement of Gi/Go-proteins in the transduction mechanism pathway of the receptor generated signal. In conclusion, these results suggest that 5-HT1B heteroreceptors located on dopaminergic and cholinergic terminals exhibit a lower sensitivity to 5-HT1B receptor agonist and antagonist than do 5-HT1B autoreceptors. The observed difference in functional sensitivities of 5-HT1B auto- and heteroreceptors may represent important consequences in the physiological control of the release of serotonin versus that of other neurotransmitters.

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.

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.

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.

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.

[5HT-moduline an new peptide specifically interactive with the serotonergic system: physiopathologic implications]. / La 5HT-moduline, un nouveau peptide endogène interagissant spécifiquement avec le système sérotonergique: implications physiopathologiques.

5-HT1B receptors play a specific role as one of the many receptors regulating serotonergic activity. This endogenous peptide was recently isolated and characterized. Specific interaction has been demonstrated. 5-HT-moduline appears to be implicated in central nervous system response to various stimuli, particularly stress. It might play an important role in adaptation/inadaptation mechanisms (stress, anxiety, depression).

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.