Dopamine D2 and 5-hydroxytryptamine 5-HT(₂A) receptors assemble into functionally interacting heteromers

In view of the co-distribution of dopamine D_2LR and 5-hydroxytryptamine 5-HT_2A receptors (D_2LR and 5-HT_2AR, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D_2LR-5-HT_2AR heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D_2LR and the 5-HT_2AR form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D_2LR-5-HT_2AR heteromeric signaling was analyzed we found that the 5-HT_2AR-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D_2LR as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D_2LR-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D_2LR and 5-HT_2AR within the heteromer led to inhibition of the D_2LR functioning, thus suggesting the existence of a 5-HT_2AR-mediated D_2LR trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D₂R-5-HT_2AR may be involved in the receptor interface. Overall, the presence of the D_2LR-5-HT_2AR heteromer in discrete brain regions is postulated based on the existence of D_2LR-5-HT_2A receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.     

Interactions of the Melanocortin-4 Receptor with the Peptide Agonist NDP-MSH

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His₆-Phe₇-Arg₈-Trp₉, which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle₄,d-Phe₇]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R^?), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level.The mutation of residues in the human MC4R—such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)—to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between d-Phe₇ on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp₉.For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the β₂-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the β₂-adrenergic receptor.     

Role of Disulfides and Sulfhydryl Groups in Agonist and Antagonist Binding in Serotonin1A Receptors from Bovine Hippocampus

SUMMARY 1. The serotonin_1A (5-HT_1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT_1A receptors from bovine hippocampus.2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT_1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity.3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites.4. In addition, ligand-binding studies in presence of GTP--S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT_1A receptor and the G-protein.5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT_1A receptors.     

Three "hotspots" important for adenosine A(2B) receptor activation: a mutational analysis of transmembrane domains 4 and 5 and the second extracellular loop

G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Despite the progress made in the last decade in molecular and structural biology, their exact activation mechanism is still unknown. Here we describe new insights in specific regions essential in adenosine A_2B receptor activation (A_2BR), a typical class A GPCR. We applied unbiased random mutagenesis on the middle part of the human adenosine A_2BR, consisting of transmembrane domains 4 and 5 (TM4 and TM5) linked by extracellular loop 2 (EL2), and subsequently screened in a medium-throughput manner for gain-of-function and constitutively active mutants. For that purpose, we used a genetically engineered yeast strain (Saccharomyces cerevisiae MMY24) with growth as a read-out parameter. From the random mutagenesis screen, 12 different mutant receptors were identified that form three distinct clusters; at the top of TM4, in a cysteine-rich region in EL2, and at the intracellular side of TM5. All mutant receptors show a vast increase in agonist potency and most also displayed a significant increase in constitutive activity. None of these residues are supposedly involved in ligand binding directly. As a consequence, it appears that disrupting the relatively “silent” configuration of the wild-type receptor in each of the three clusters readily causes spontaneous receptor activity.     

Transmembrane helix 6 observed at the interface of β2AR homodimers in blind docking studies

Peptide– and protein–protein dockings were carried out on β₂-adrenergic receptor (β₂AR) to confirm the presence of transmembrane helix 6 (TM6) at the interface region between two β₂AR monomers, thereby its possible role in dimerization as suggested in numerous experimental and computational studies. Initially, a portion of TM6 was modeled as a peptide consisting of 23 residues and blindly docked to β₂AR monomer using a rigid body approach. Interestingly, all highest score conformations preferred to be near TM5 and TM6 regions of the receptor. Furthermore, longer peptides generated from a whole TM region were blindly docked to β₂AR using the same rigid body approach. This yielded a total of seven docked peptides, each derived from one TM helix. Most interestingly, for each peptide, TM6 was among the most preferred binding site region in the receptor. Besides the peptide dockings, two β₂AR monomers were blindly docked to each other using a full rigid-body search of docking orientations, which yielded a total of 16,000 dimer conformations. Each dimer was then filtered according to a fitness value based on the membrane topology. Among 149 complexes that met the topology requirements, 102 conformers were composed of two monomers oriented in opposite directions, whereas in the remaining 47, the monomers were arranged in parallel. Lastly, all 149 conformers were clustered based on a root mean-squared distance value of 6 Å. In agreement with the peptide results, the clustering yielded the largest population of conformers with the highest Z-score value having TM6 at the interface region.     

Dopamine D1 receptor and serotonin 5-HT1A receptor agonist effects of the natural product (–)-stepholidine: molecular modelling and dynamics simulations

In this study, by homology modelling and molecular dynamics (MD) simulation, models of l-stepholidine (l-SPD) activating the 5-HT_1A and D₁ receptors were constructed. In 100-ns MD simulations, the D₁ and 5-HT_1A receptors were activated by the partial agonist l-SPD, conforming with the global toggle switch activation model and the sequential activation model. The residues Y^7.53 and Y^5.58 swing significantly between different transmembrane (TM) domains after activation. Similarities between D₁ and 5-HT_1A included (1) the outward motion of TM-5; (2) the ionic lock was independent of the tilt of TM-6 and (3) there was an apparent bending of TM-6, and the ring of l-SPD formed strong π–π interactions with residue W^6.48. Differences between the two included the following: (1) in 5-HT_1A, l-SPD formed a hydrogen bond with Ala172^5.46 of TM-5, and the intracellular end of TM-5 moved outward slowly; that hydrogen bond did not form with the D₁ receptor; (2) l-SPD formed stronger interactions with D^3.32 and W^6.48 in the D₁ receptor than in the 5-HT_1A receptor and (3) the hydrogen bonding network was somewhat different in SPD-5-HT_1A and SPD-D₁ receptors. We propose the interaction between l-SPD and D^3.32 or/and W^6.48 is the original driving force during the whole activation process.     

Long-term Stress with Hyperglucocorticoidemia-induced Hepatic Steatosis with VLDL Overproduction Is Dependent on both 5-HT2 Receptor and 5-HT Synthesis in Liver

Hepatic triglycerides production and adipose lipolysis are pivotal for long-term stress (LTS) or hyperglucocorticoidemia-induced insulin resistance. 5-hydroxytryptamine (5-HT) has been demonstrated to induce hepatic lipid metabolic abnormality by activating mammalian target of rapamycin (mTOR). In present study, we explored whether 5-HT is involved in LTS effects in liver using restraint stress-exposed rats and cultured primary rat hepatocytes and HepG2 cells. LTS with hyperglucocorticoidemia induced hepatic 5-HT synthetic increase with tryptophan hydroxylase 1 (Tph1) up-regulation, and 5-HT2 receptor (5-HT₂R, including 5-HT2A, 2B receptor) up-regulation in liver and visceral adipose, as well as hepatic mTOR activation with triglycerides and VLDL overproduction with steatosis, and visceral adipose lipolytic increase with high blood free fatty acids (FFAs) level. 5-HT exposure exhibited LTS-like effects in both tissues, and both LTS and 5-HT effects could be abolished significantly by blocking 5-HT₂R. In HepG2 cells dexamethasone or palmitate-induced mTOR activation with triglycerides and VLDL overproduction were accompanied by up-regulations of 5-HT synthesis and 5-HT₂R, which were significantly abolished by gene silencing Tph1 or 5-HT₂R and were almost fully abolished by co-silencing of both, especially on VLDL overproduction. Chemical inhibition of Tph1 or/and 5-HT₂R in both hepatocytes exhibited similar abolishment with genetic inhibition on dexamethason-induced effects. 5-HT-stimulated effects in both hepatocytes were fully abolished by blocking 5-HT₂R, while 5-HT itself also up-regulated 5-HT₂R. In conclusion, up-regulated hepatic 5-HT synthesis and 5-HT₂R induced by both glucocorticoid and FFAs are crucial for LTS-induced hepatic steatosis with VLDL overproduction, while 5-HT by acting on 5-HT₂R mediates mTOR activation in liver.     

Fighting experience alters brain androgen receptor expression dependent on testosterone status

Contest decisions are influenced by the outcomes of recent fights (winner–loser effects). Steroid hormones and serotonin are closely associated with aggression and therefore probably also play important roles in mediating winner–loser effects. In mangrove rivulus fish, Kryptolebias marmoratus, individuals with higher testosterone (T), 11-ketotestosterone and cortisol levels are more capable of winning, but titres of these hormones do not directly mediate winner–loser effects. In this study, we investigated the effects of winning/losing experiences on brain expression levels of the receptor genes for androgen (AR), oestrogen α/β (ER_α/β), glucocorticoid (GR) and serotonin (5-HT_1AR). The effect of contest experience on AR gene expression depended on T levels: repeated losses decreased, whereas repeated wins increased AR gene expression in individuals with low T but not in individuals with medium or high T levels. These results lend strong support for AR being involved in mediating winner–loser effects, which, in previous studies, were more detectable in individuals with lower T. Furthermore, the expression levels of ER_α/β, 5-HT_1AR and GR genes were higher in individuals that initiated contests against larger opponents than in those that did not. Overall, contest experience, underlying endocrine state and hormone and serotonin receptor expression patterns interacted to modulate contest decisions jointly.     

Inhibitory effects of tryptamine on tolbutamide-induced hypoglycemia in mice: Mediation by 5-HT receptors

Effects of tryptamine on tolbutamide-induced hypoglycemia were investigated in mice. Tryptamine significantly inhibited hypoglycemia elicited by tolbutamide. The inhibitory effects of tryptamine were strongly blocked by the 5-HT₁ and 5-HT₂ receptor antagonist methysergide and the 5-HT₂ receptor antagonist ketanserin, while the 5-HT₃ receptor antagonist ICS 205–930 was without effect. Tryptamine induced hyperglucagonemia in tolbutamide-treated mice, and this effect elicited by tryptamine was strongly inhibited by the 5-HT₂ receptor antagonist ketanserin. These results suggest that the inhibitory effects of tryptamine on tolbutamide-induced hypoglycemia are mediated by 5-HT₂ receptors and that tryptamine is involved in glucagon release.     

Internalization of Human 5-HT<Subscript>4a</Subscript> and 5-HT<Subscript>4b</Subscript> Receptors is Splice Variant Dependent

The family of 5-HT₄ receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT_4a and h5-HT_4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT_4a-GFP and h5-HT_4b-GFP were generated by fusing the coding sequence of the 5-HT₄ receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT_4b-GFP receptor, but not of the h5-HT_4a-GFP receptor. The h5-HT_4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT_4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT_4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT_4a and 5-HT_4b receptors.     

Quinoxalin-2-carboxamides: synthesis and pharmacological evaluation as serotonin type-3 (5-HT3) receptor antagonists

A series of quinoxalin-2-carboxamides were designed as per the pharmacophoric requirements of 5-HT₃ receptor antagonists and synthesized by condensing the carboxylic group of quinoxalin-2-carboxylic acid with various amines in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The structures of the synthesized compounds were confirmed by physical and spectroscopic data. The carboxamides were evaluated for their 5-HT₃ receptor antagonisms in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-HT₃ agonist, 2-methy-5-HT. All the synthesized compounds showed 5-HT₃ receptor antagonism, (4-benzylpiperazin-1-yl)(quinoxalin-2-yl)methanone was the most potent compound among this series.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism,antagonism, or inverse agonism?

1. The 5-HT₂ receptors subdivision into the 5-HT_2A/2B/2C subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT₂ receptors role on memory consolidation.2. The SB-200646 (a selective 5-HT_2B/2C receptor antagonist) and LY215840 (a nonselective 5-HT_2/7 receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP).3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT_2A receptor antagonist) and to a LY215840 high dose.4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine; while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs.5. It is suggested that 5-HT_2B/2C receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT₂ receptors remains unclear at this time.6. Notably, the 5-HT₂ receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreased cholinergic, glutamatergic, and/or serotonergic neurotransmission.     

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT_2A receptor. Active state of 5-HT_2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT_2A receptor active state model was constructed by homology modelling using active state β₂-adrenergic receptor (β₂-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.     

Rigidized 1-aryl sulfonyl tryptamines: synthesis and pharmacological evaluation as 5-HT6 receptor ligands

A series of N₁-arylsulfonyl-3-(pyrrolidin-3-yl)-1H-indole and N₁-arylsulfonyl-3-(4-chloro-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole derivatives (tryptamine derivatives with rigidized side chain) have been prepared and tested for their binding affinity to 5-HT₆ receptor. Several compounds displayed potent binding affinity for the 5-HT₆ receptor when tested in in vitro binding assay. The primary SAR indicates that rigidification of dimethylamino alkyl chain at C₃ of indole carbon maintains the binding affinity to 5-HT₆R. The lead compound N₁-benzenesulfonyl-3-(4-chloro-1-methyl-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole, 10a (K_b = 0.1 nM) has shown excellent in vitro affinity and was active in animal models of cognition like NORT and water maze.     

The Central 5-HT3 Receptor in CNS Disorders

Among the characterized 5-HT receptors of the central nervous system, the type 3 receptor subtype (5-HT₃R) is the only one known to be a ligand-gated ion channel. Its early pharmacological characterization and mapping by radioligand binding autoradiography suggested that this receptor may, among other actions, regulate dopamine release in the nigro-striatal pathway and reduce alcohol consumption in experimental animals while antagonists of this receptor have been reported to treat anxiety disorders. Following the cloning of this receptor in 1991, direct cellular localization was made possible by in situ hybridization and immunohistochemical analysis. Here we summarize our recent efforts showing that 5-HT₃R-expressing neurons are mainly GABA containing cells in the rat neocortex, olfactory cortex, hippocampus, and amygdala which also often contain chole-cystokinin (CCK) immunoreactivity. These results provide a means to unify some of the initial pharmacological observations.     

Cysteine residues are critical for chemokine receptor CXCR2 functional properties

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.     

Mechanism of dimerization of the human melanocortin 1 receptor

The melanocortin 1 receptor (MC1R) is a dimeric G protein-coupled receptor expressed in melanocytes, where it regulates the amount and type of melanins produced and determines the tanning response to ultraviolet radiation. We have studied the mechanisms of MC1R dimerization. Normal dimerization of a deleted mutant lacking the seventh transmembrane fragment and the C-terminal cytosolic extension excluded coiled-coil interactions as the basis of dimerization. Conversely, the electrophoretic pattern of wild type receptor and several Cys → Ala mutants showed that four disulfide bonds are established between the monomers. Disruption of any of these bonds abolished MC1R function, but only the one involving Cys35 was essential for traffic to the plasma membrane. A quadruple Cys35-267-273-275Ala mutant migrating as a monomer in SDS-PAGE in the absence of reducing agents was able to dimerize with WT, suggesting that in addition to disulfide bond formation, dimerization involves non-covalent interactions, likely of domain swap type.     

5-HT<Subscript>6/7</Subscript> Receptor Antagonists Facilitate Dopamine Release in the Cochlea via a GABAergic Disinhibitory Mechanism

In humans, serotonin (5-HT) has been implicated in numerous physiological and pathological processes in the peripheral auditory system. Dopamine (DA), another transmitter of the lateral olivocochlear (LOC) efferents making synapses on cochlear nerve dendrites, controls auditory nerve activation and protects the sensory nerve against overactivation. Using in vitro microvolume superfusion techniques we tested 5-HT₆ and 5-HT₇ receptor antagonists whether they can influence dopamine (DA) release from the guinea-pig cochlea in control and in ischemic conditions using currently available and new 5-HT₆ and 5-HT₇ antagonists and mixed antagonists, which were synthesized and characterized for the current study. While the 5-HT₇ antagonist SB-258719 was ineffective, SB-271046, which blocks the 5-HT₆ receptor, caused a significant increase in cochlear DA release what is contradictory with the excitatory nature of this type of receptor. Moreover, the mixed 5-HT_6/7 antagonist EGIS-12233 induced an even more pronounced increase in the resting DA release. To understand why the block of an excitatory receptor results in an increase instead of a decrease in function, we investigated the possible involvement of an indirect neural mechanism through an inhibitory system. In the presence of the GABA_A receptor blocker bicuculline, EGIS-12233 failed to increase the release of DA, suggesting that the serotonin receptor modulation of DA release from the lateral olivocochlear efferents in the cochlea was produced indirectly by decreasing the GABAergic inhibitory tone on dopaminergic nerve endings. The mixed 5-HT₇/D₄ receptor antagonist EGIS-11983 significantly increased both the stimulation-evoked and the resting DA release, while the selective D4 blocker L-741,741 alone had no significant effect. Ischemia, simulated by oxygen and glucose deprivation from the perfusion solution had no action on the effect of the drugs. Drugs that can increase the release of DA from LOC terminals in the cochlea may have a role in the treatment of sensorineural hearing loss.