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

Abstract

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-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

“…1). The fact that ALLS, a peptide having the same structure as LSAL in a different sequence, was inactive supports the view that the effect is specific for LSAL (5-HT moduline) as already reported in previous studies (Massot et al 1996;Rousselle et al 1998;Seguin et al 1997). The antagonistic effect of 5-HT moduline was shown at concentrations (100 nM) higher than those corresponding to the apparent binding affinity (<1 nM; Massot et al 1996); this could be related to its rapid degradation under the experimental conditions used whereas binding experiments of 5-HT moduline to brain homogenate were performed at 4°C, which decreased the degradation of the peptide (Massot et al 1996).…”

“…The endogenous peptide, 5-HT moduline (LSAL), has been recently demonstrated to be a selective non-competitive 5-HT 1B/1D receptor antagonist, probably acting as an allosteric modulator of the 5-HT 1B/1D receptor protein (Massot et al 1996;Rousselle et al 1998). Preincubation of the rat striatal synaptosomes with various concentrations of 5-HT moduline significantly reduced the effect of CP93,129 on the K + -overflow of [ 3 H]DA in a non-competitive manner (Fig.…”

High-resolution imaging of brain 5-HT1B receptors in the rhesus monkey using [11C]P943

“…The in vitro pharmacology profile for unlabeled P943 is shown in Table 1, demonstrating its potency and selectivity towards 5-HT 1B ; however, no assay was performed for its selectivity towards 5-HT 1B vs. 5-HT 1D as there is no drug selective only for 5-HT 1B or 5-HT 1D. GR 127,935 is a potent and effective antagonist for human 5-HT 1D and 5-HT 1B with a pKi value of 9.0 and 8.6 for 5HT 1B and 5HT 1D , respectively [27,28,29]. In our blocking study with GR 127,935, a near complete blockade of [ 11 C]P943 binding was observed after pretreatment with GR 127,935.…”

5-HT Receptor Regulation of Neurotransmitter Release

“…Furthermore, the CP-93129-induced inhibition was reduced 1) by pretreatment with a specific polyclonal 5-HT 1B receptor antibody directed against a region of the receptor protein involved in signal transduction and 2) by pretreatment with 5-HT moduline, a tetrapeptide with the amino acid sequence LSAL (Sarhan et al, 2000). This endogenous peptide was shown to be a selective noncompetitive 5-HT 1B/1D antagonist, probably acting as an allosteric modulator of the 5-HT 1B receptor protein (Massot et al, 1996;Rousselle et al, 1998). It is also compatible with the 5-HT 1B character of the presynaptic 5-HT heteroreceptor that G i /G o proteins appear to be involved in signal transduction, as suggested by the attenuation of the inhibitory effects of 5-HT 1B receptor agonists by pertussis toxin (Sarhan and Fillion, 1999), Inhibitory presynaptic 5-HT 1B receptors on dopaminergic nerve terminals have also been shown in the mouse striatum by application of the same elegant approach as in the rat striatum: the K ϩ -evoked [ 3 H]DA release was inhibited by CP-93129 from mouse striatal synaptosomes and this effect was sensitive to blockade by SB 224289 (Sarhan et al, 2000).…”