The effect of 5-HT receptor ligands on the uptake of [3H]5-hydroxytryptamine into rat cortical synaptosomes

doi:10.1016/0014-2999(93)90996-U

European Journal of Pharmacology

Volume 239, Issues 1–3, 3 August 1993, Pages 211-214

https://doi.org/10.1016/0014-2999(93)90996-U Get rights and content

Abstract

The effect of 5-HT receptor agonists and antagonists to inhibit [³H]5-HT uptake was investigated in rat cortical synaptosomes. The 5-HT (5-hydroxytryptamine) uptake inhibitors paroxetine and fluoxetine yielded pK_i values of 8.41 ± 0.12 and 7.43 ± 0.06 respectively. The 5-HT₃/5-HT₄ receptor antagonist tropisetron and the 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) had similar inhibitory potencies to cocaine (pK_i values of 6.58 ± 0.04, 6.47 ± 0.14 and 6.45 ± 0.12 respectively). The dopamine and noradrenaline uptake inhibitors GBR12909 and desipramine had comparable values of 6.5 ± 0.05 and 6.13 ± 0.07. Other 5-HT receptor ligands had pK_i values less than 6.0 (R(+)-zacopride, MDL72222, R(+)/S(−)-zacopride) or 5.0 (5-methoxytryptamine, m-chlorophenylbiguanide, S(−)-zacopride, SDZ205–557, ondansetron and renzapride). It is concluded, with the possible exception of tropisetron and 8-OH-DPAT, that it is unlikely that the effects of the 5-HT receptor ligands to inhibit 5-HT uptake contribute to their effects in vivo.

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Phenyl group of indole ring in Lipid B was shown to have π-π stacking interactions with Phe 330 and fused tetrahydropyran with Tyr 109 and 5-hydroxyl group of indole and hydroxyl group in linker was shown to have hydrogen bond interactions with Asp129 (Fig. 13C). The involvement of hydroxyl group of serotonin moiety may favor hydrogen bonding interactions with the receptor, in accordance with the reported mutational and modeling studies (Kitson, 2007; Cheng et al., 1993) where serotonin1A receptor prefers ligands with hydrogen bond acceptor at a position corresponding to the hydroxyl group in serotonin. The docking studies similarly show that Lipid B with the free hydroxyl group is available for interaction with serotonin1A receptor, (Fig. 13).
Tocopherol-based lipids are widely used for nucleic acid delivery. Using tocopherol molecules, we designed and synthesized 5-HT functionalized lipids by tethering 5-hydroxytryptamine (5-HT), a small molecule ligand as the head group to a natural amphiphilic molecule namely α-tocopherol (Vitamin E). This is with the aim of delivering nucleic acids specifically into cells expressing the serotonin receptors (5-hydroxytryptamine[5-HT]) which are abundant in the central nervous system. In order to achieve target recognition, we adopted an approach wherein two structurally different lipid molecules having serotonin as the head group was conjugated to tocopherol via different linkers thus generating lipids with either free –NH₂ or –OH moiety. The corresponding lipids designated as Lipid A (Tocopheryl carbonate serotonin-NH₂) and Lipid B (Tocopheryl 2-hydroxy propyl ammonium serotonin-OH), were formulated with co-lipids 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC) and evaluated for their ability to deliver plasmid DNA through reporter gene expression assays in vitro. Furthermore, the physicochemical characteristics and cellular interactions of the formulations were examined using serotonin-receptor enriched cells in order to distinguish the structural and functional attributes of both lipids. Cell-based gene expression studies reveal that in comparison to Lipid A, a formulation of Lipid B prepared with DOPE as the co-lipid, contributes to efficient uptake leading to significant enhancement in transfection. Specific interactions explored by molecular docking studies suggests the role of the hydroxyl moiety and the enantiospecific significance of serotonin- conjugated tocopherol lipids in recognizing these receptors thus signifying a promising lipid-based approach to target the serotonin receptors in the central nervous system.
Antidepressant-like activity of VN2222, a serotonin reuptake inhibitor with high affinity at 5-HT<inf>1A</inf> receptors
2002, European Journal of Pharmacology
It has been suggested that drugs combining serotonin (5-hydroxytryptamine, 5-HT) transporter blockade and 5-HT_1A autoreceptor antagonism could be a novel strategy for a shorter onset of action and higher therapeutic efficacy of antidepressants. The present study was aimed at characterizing the pharmacology of 1-(3-benzo[b]tiophenyl)-3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propanol (VN2222) a new synthetic compound with high affinity at both the 5-HT transporter and 5-HT_1A receptors and devoid of high affinity at other receptors studied, with the only exception of α₁-adrenoceptors. In keeping with the binding affinity at the 5-HT transporter, VN2222 inhibited 5-HT uptake in vitro both in rat cortical synaptosomes and in mesencephalic cultures and also in vivo when administered locally into the rat ventral hippocampus. After systemic administration, VN2222 exhibited an inverted U-shape effect so the inhibition of [³H]5-HT uptake ex vivo and the increase in 5-HT extracellular levels in microdialysis experiments was observed at low doses of 0.01–0.1 mg/kg whereas higher doses were ineffective. In studies related to 5-HT_1A receptor function, 0.01–0.1 μM VN2222 produced a partial inhibition of forskolin-stimulated cAMP formation behaving as a weak agonist of 5-HT_1A receptors. In body temperature studies, 5 mg/kg VN2222 produced a mild hypothermic effect in mice, suggesting a weak agonist activity at presynaptic 5-HT_1A receptors; much lower doses (0.01–0.5 mg/kg) partially antagonized the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) possibly through 5-HT transporter blockade. In the learned helplessness test in rats, an animal model for antidepressants, 1–5 mg/kg VN2222 reduced significantly the number of escape failures. Consequently, VN2222 is a new compound with a dual effect on the serotonergic system, as 5-HT uptake blocker and 5-HT_1A receptor partial agonist, and with a remarkable activity in an animal model of depression with high predictive validity.
The effects of ibogaine on dopamine and serotonin transport in rat brain synaptosomes
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Ibogaine has been shown to affect biogenic amine levels in selected brain regions. Because of the involvement of these neurotransmitters in drug addiction, the effects of ibogaine on biogenic amine transport may contribute to the potential anti-addictive properties of ibogaine in vivo. With rat brain synaptosomes as our experimental system, we measured the effects of ibogaine on the uptake and release of dopamine (DA) and serotonin (5-HT). Ibogaine competitively blocked both DA and 5-HT uptake with IC₅₀ values of 20 μM at 75 nM ³H-DA and 2.6 μM at 10 nM ³H-5-HT. Ibogaine had no effect on K⁺-induced release of ³H-DA from preloaded synaptosomes, but 20 μM and 50 μM ibogaine inhibited roughly 40% and 60%, respectively, of the K⁺-induced release of ³H-5-HT from preloaded synaptosomes. In the absence of a depolarizing stimulus, ibogaine evoked a small release of ³H-DA but not ³H-5-HT. These relatively low-potency effects of ibogaine on DA and 5-HT uptake in synaptosomes are consistent with the low binding affinity of ibogaine that has been previously reported for DA and 5-HT transporters. Our results show that if ibogaine modulates DA and 5-HT levels in the brain by directly blocking their uptake, then a concentration of ibogaine in the micromolar range is required. Furthermore, if the anti-addictive effects of ibogaine require this concentration, then ibogaine likely exerts these effects through a combination of neurotransmitter pathways, because binding affinities and functional potencies of ibogaine in the micromolar range have been reported for a variety of neuronal receptors and transporters.
Serotonin transporters in adult rat brain astrocytes revealed by [<sup>3</sup>H]5-HT uptake into glial plasmalemmal vesicles
1998, Neurochemistry International
Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na⁺-dependent, paroxetine and fluoxetine sensitive [³H] 5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a K_m value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [³H]-glutamate and [³H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [³H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (K_m approximately 40 nM), Na ⁺ dependent, paroxetine-sensitive manner. The [³H]5-HT uptake capacity (V_max ) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised ‘neuronal’ serotonin transporter.
Further characterization of forebrain serotonin receptors mediating tachycardia in conscious rats
1998, Brain Research Bulletin
It has been shown recently that activation of forebrain serotonin_1A (5-HT_1A) receptors, likely within the preoptic area, elicits a slight increase in blood pressure and a substantial tachycardia. The present studies were designed to characterize: (1) the requirement of the 5-HT_1A receptor agonist R(+)-8-hydroxy-2-(di-n-propylamino) tetralin [R(+)8-OH-DPAT]-induced tachycardia on the integrity of serotonergic innervation of the preoptic area, (2) the ability of the 5-HT_1A receptor partial agonist buspirone to elicit cardiovascular responses when microinjected into the preoptic area, (3) the role of 5-HT₂ and 5-HT₃ receptors in the preoptic area in cardiovascular regulation, and (4) the site specificity of the tachycardia produced by R(+)8-OH-DPAT. The data suggest that activation of 5-HT_1A receptors, but not 5-HT₂ or 5-HT₃ receptors, within or very near the preoptic area increases blood pressure and heart rate in conscious rats. Furthermore, the full response is dependent on afferent serotonergic innervation, suggesting a presynaptic modulatory role for 5-HT in the preoptic area.
Quantitative microdialysis of serotonin and norepinephrine: Pharmacological influences on in vivo extraction fraction
1996, Journal of Neuroscience Methods
The present study was designed to investigate the potential influence of various neuronal processes including uptake, release and metabolism, on the in vivo microdialysis extraction fraction (E_d) of serotonin (5HT) and norepinephrine (NE). Paroxetine administration decreased the E_d of 5HT in the nucleus accumbens from 24 ± 3 to 18 ± 0.2% (p < 0.05). Similarly, desipramine infusion reduced the NE E_d from 35 ± 2 to 26 ± 1% (p < 0.05). However, perfusion with pargyline or tetrodotoxin had no effect on the E_d of either 5HT or NE. Perfusion with agonists for the 5HT, α-adrenergic, D₂ and histamine receptors had no effect on the E_d of 5HT. In the same manner, perfusion with the α-adrenergic agonists, methoxamine or clonidine, did not affect the E_d of NE. These data are in agreement with experimental results obtained for dopamine in the nucleus accumbens and the theory of quantitative microdialysis which predicts that only changes in the rate of clearance will change E_d of monoamines. These results suggest that, like DA, changes in the E_d for 5HT or NE are indicative of changes in the reuptake of these neurotransmitters. The results also indicate that pharmacological agents which do not affect uptake have no effect on the extraction fraction.

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The effect of 5-HT receptor ligands on the uptake of [3H]5-hydroxytryptamine into rat cortical synaptosomes

Abstract

Pharmacol. Biochem. Behav.

Eur. J. Pharmacol.

Pharmacol. Biochem. Behav.

Pharmacol. Ther.

Pharmacol. Biochem. Behav.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

TIPS

A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein-dye binding

Anal. Biochem.

Pharmacological properties of GR38032F, a novel antagonist at 5-HT3 receptors

Br. J. Pharmacol.

Pharmacological characterisation of a neuronal receptor for 5-hydroxytryptamine in guinea pig ileum with properties similar to the 5-HT4 receptor

J. Pharmacol. Exp. Ther.

Screening for new antidepressant compounds

The effect of 5-HT receptor ligands on the uptake of [³H]5-hydroxytryptamine into rat cortical synaptosomes

Pharmacological properties of GR38032F, a novel antagonist at 5-HT₃ receptors

Pharmacological characterisation of a neuronal receptor for 5-hydroxytryptamine in guinea pig ileum with properties similar to the 5-HT₄ receptor