Membrane cholesterol oxidation inhibits ligand binding function of hippocampal serotonin(1A) receptors

We have monitored the ligand binding function of the bovine hippocampal 5-HT(1A) receptor following treatment of native membranes with cholesterol oxidase. Cholesterol oxidase is a water soluble enzyme that acts on the membrane interface to catalyze the conversion of cholesterol to cholestenone. Oxidation of membrane cholesterol significantly inhibits the specific binding of the agonist and antagonist to 5-HT(1A) receptors. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed no appreciable effect on membrane order due to the oxidation of cholesterol to cholestenone. These results therefore suggest that the ligand binding function of the 5-HT(1A) receptor is a cholesterol-dependent phenomenon that is not related to the ability of cholesterol to modulate membrane order. Importantly, these results represent the first report on the effect of a cholesterol-modifying agent on the ligand binding function of this important neurotransmitter receptor.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.     

Role of cholesterol in ligand binding and G-protein coupling of serotonin1A receptors solubilized from bovine hippocampus

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We report here that solubilization of the hippocampal 5-HT(1A) receptor by the zwitterionic detergent CHAPS is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity and extent of G-protein coupling. Importantly, replenishment of cholesterol to solubilized membranes using MbetaCD-cholesterol complex restores the cholesterol content of the membrane and significantly enhances the specific agonist binding activity and G-protein coupling. These novel results provide useful information on the role of cholesterol in solubilization of G-protein-coupled receptors, an important step for molecular characterization of these receptors.     

Effect of Mg2+ on guanine nucleotide sensitivity of ligand binding to serotonin1A receptors from bovine hippocampus

The serotonin1A (5-HT1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). We report here that guanine nucleotide sensitivity of agonist binding to hippocampal 5-HT1A receptors is dependent on the concentration of Mg2+. Our results show that agonist binding to 5-HT1A receptors is relatively insensitive to guanine nucleotides in the absence of Mg2+. In contrast to this, the specific antagonist binding is insensitive to guanine nucleotides, even in the presence of Mg2+. These results point out the requirement of an optimal concentration of Mg2+ which could be used in assays toward determining guanine nucleotide sensitivity of ligand binding to GPCRs such as the 5-HT1A receptor. Our results provide novel insight into the requirement and concentration dependence of Mg2+ in relation to guanine nucleotide sensitivity for the 5-HT1A receptor in particular, and GPCRs in general.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor

Insolubility in non-ionic detergents such as Triton X-100 is a widely used biochemical criterion for characterization of membrane domains. We report here a novel green fluorescent protein fluorescence-based approach to directly determine detergent insolubility of specific membrane proteins. We have applied this method to explore the detergent resistance of an important G-protein coupled receptor, the serotonin1A (5-HT1A) receptor. Our results show, for the first time, that a small yet significant fraction of the 5-HT1A receptor exhibits detergent insolubility. These results are validated by control experiments involving fluorescent lipid probes and protein markers. Our results assume relevance in the context of localization of the 5-HT1A receptor in membrane domains and its significance in receptor function and signaling.     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin 1A receptor

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT(1A) receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT(1A) receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT(1A) receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT(1A) receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT(1A) receptor function.     

Direct noncompetitive inhibition of 5-HT(3) receptor-mediated responses by forskolin and steroids

5-HT(3) receptors cloned from NCB-20 cells were expressed in Xenopus oocytes, and the effects of forskolin and steroids on the function of the receptors were investigated using the two-electrode voltage-clamp technique. Forskolin, 17-beta-estradiol, and progesterone inhibited the currents activated by 1 microM 5-HT in a reversible and concentration-dependent manner, with IC(50) values of 12, 33, and 89 microM, respectively. The inhibitory effects of forskolin and 17-beta-estradiol were independent of the membrane potential. Forskolin and 17-beta-estradiol significantly reduced the maximal amplitude of the 5-HT concentration-response curve (E(max)) without significantly affecting the EC(50), indicating that these compounds act as noncompetitive inhibitors of the 5-HT(3) receptor. The cAMP analogue, 8-Br-cAMP (0.2 mM), and the protein kinase A activator, Sp-cAMP (0.1 mM), did not affect the amplitude of 5-HT(3) receptor-mediated currents. The membrane-permeable protein kinase A inhibitor Rp-cAMP (0.1 mM) and the estrogen-receptor antagonist tamoxifen (1 microM) did not affect the inhibition of 5-HT-activated current. In addition, 5-HT(3) receptor-mediated currents were inhibited by both 1,9-dideoxy forskolin (30 microM), which does not activate adenylyl cyclase, and wForskolin (30 microM), a charged hydrophilic analogue of forskolin that is membrane impermeable. These results indicate that both forskolin and 17-beta-estradiol inhibit the function of the 5-HT(3) receptor in a noncompetitive manner and that this inhibition is independent of cAMP levels.     

Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT(1A) receptor by depleting cholesterol from native membranes using methyl-beta-cyclodextrin (MbetaCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MbetaCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT(1A) receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-gamma-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.     

Effect of sphingomyelinase treatment on ligand binding activity of human serotonin1A receptors

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. We have monitored the ligand binding of the human serotonin1A receptor stably expressed in CHO cells (termed CHO-5-HT1AR) following treatment with sphingomyelinase (SMase), an enzyme that specifically catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Our results show, for the first time, that the specific ligand binding activity of the serotonin1A receptor in membranes isolated from CHO-5-HT1AR cells is increased upon sphingomyelinase treatment. Saturation binding analysis reveals increase in binding affinity of the receptor under these conditions. This is accompanied by a reduction in membrane order, as monitored by fluorescence anisotropy of the membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in intact cells. These results represent the first report on the effect of sphingomyelinase treatment on the ligand binding activity of this important neurotransmitter receptor.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

Attenuation of ischemic efflux of endogenous amino acids by the novel 5-HT1A/5-HT2 receptor ligand adatanserin

Using sodium azide (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia in the hippocampal slice, we have evaluated the effects of the novel 5-HT(1A) partial agonist/5-HT(2) receptor antagonist adatanserin and the 5-HT(1A) receptor agonist BAYx3702 on the efflux of endogenous glutamate, aspartate and GABA. BAYx3702 (10-1000 nM) produced a significant (P<0.05) dose-related attenuation of ischemic efflux of both glutamate and GABA with maximum decrease being observed at 100 nM (73 and 69%, respectively). This attenuation was completely reversed by the addition of the 5-HT(1A) antagonist, WAY-100635 (100 nM). Similarly, adatanserin (10-1000 nM) produced a significant (P<0.05) dose-related attenuation in glutamate and GABA efflux with a maximum of 72 and 81% at 100 nM, respectively. This effect was completely reversed by the 5-HT(2A/C) receptor agonist, DOI but unaffected by WAY-100635. The 5-HT(2A) receptor antagonist MDL-100907 produced a comparable attenuation of glutamate when compared to adatanserin, while the 5-HT(2C) receptor antagonist, SB-206553, had no effect on ischemic efflux. None of these compounds significantly altered aspartate efflux from this preparation. In conclusion, the 5-HT(1A) receptor partial agonist 5-HT(2) receptor antagonist, adatanserin is able to attenuate ischemic amino acid efflux in a comparable manner to the full 5-HT(1A) agonist BAYx3702. However, in contrast to BAYx3702, adatanserin appears to produce it effects via blockade of the 5-HT(2A) receptor. This suggests that adatanserin may be an effective neuroprotectant, as has been previously demonstrated for full 5-HT(1A) receptor agonists such as BAYx3702.     

Circadian and seasonal rhythms of 5-HT receptor subtypes, membrane anisotropy and 5-HT release in hippocampus and cortex of the rat.

Specific serotonin binding (5-HT1, 5-HT1A, and 5-HT2 subtypes) and membrane anisotropy were measured at 2 h intervals over a 24 h period in the hippocampus and cortex of Wistar WU rats, housed under a 12 h light-dark cycle, with lights on at 07.00. All experiments were performed both in March and December. In the hippocampus significant circadian rhythms could be ascertained for 5-HT1 binding sites in March and December while for 5-HT1A (subtype of 5-HT1) binding sites the circadian rhythm was only significant in March. The membrane anisotropy also showed significant variations only in March. Circadian rhythms were also found in the cortex for 5-HT1 (December) and 5-HT2 (March and December) binding sites as well as for the membrane anisotropy (December). A correlation was found between membrane anisotropy and 5-HT1 and 5-HT2 binding sites in hippocampus and cortex, respectively. A circadian rhythmicity was also observed for serotonin release as measured by in vivo voltammetry in both brain areas. The results obtained on the diurnal variations of serotonin receptor subtypes and serotonin release and the probable inverse relationship of these two parameters may be relevant in understanding the coupling of pre- and postsynaptic activity.     

In vitro and in vivo pharmacological profile of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl] ethyl] piperidine (NRA0161)

Atypical antipsychotic properties of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl] piperidine (NRA0161) were investigated by in vitro receptor affinities, in vivo receptor occupancies and findings were compared with those of risperidone and haloperidol in rodent behavioral studies. In in vitro receptor binding studies, NRA0161 has a high affinity for human cloned dopamine D(4) and 5-HT(2A) receptor with Ki values of 1.00 and 2.52 nM, respectively. NRA0161 had a relatively high affinity for the alpha(1) adrenoceptor (Ki; 10.44 nM) and a low affinity for the dopamine D(2) receptor (Ki; 95.80 nM). In in vivo receptor binding studies, NRA0161 highly occupied the 5-HT(2A) receptor in rat frontal cortex. In contrast, NRA0161 did not occupy the striatal D(2) receptor. In behavioral studies, NRA0161, risperidone and haloperidol antagonized the locomotor hyperactivity in mice, as induced by methamphetamine (MAP). At a higher dosage, NRA0161, risperidone and haloperidol dose-dependently antagonized the MAP-induced stereotyped behavior in mice and NRA0161 dose-dependently and significantly induced catalepsy in rats. The ED(50) value in inhibiting the MAP-induced locomotor hyperactivity was 30 times lower than that inhibiting the MAP-induced stereotyped behavior and 50 times lower than that which induced catalepsy.These findings suggest that NRA0161 may have atypical antipsychotic activities yet without producing extrapyramidal side effects.     

Solubilization of Serotonin1A Receptors Heterologously Expressed in Chinese Hamster Ovary Cells

1. The serotonin1A (5-HT1A) 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. 2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration. 3. Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.     

Proterguride, a highly potent dopamine receptor agonist promising for transdermal administration in Parkinson's disease: interactions with alpha(1)-, 5-HT(2)- and H(1)-receptors

Dopamine receptor agonists play an important role in the treatment of Parkinson's disease and hyperprolactinemic conditions. Proterguride (n-propyldihydrolisuride) was already reported to be a highly potent dopamine receptor agonist, thus its action at different non-dopaminergic monoamine receptors, alpha(1A/1B/1D), 5-HT(2A/2B)- and histamine H(1), was investigated using different functional in vitro assays. The drug behaved as an antagonist at alpha(1)-adrenoceptors without the ability to discriminate between the subtypes (pA(2) values: alpha(1A) 7.31; alpha(1B) 7.37; alpha(1D) 7.35) and showed antagonistic properties at the histamine H(1) receptor. In contrast, at serotonergic receptors (5-HT(2A), 5-HT(2B)) proterguride acted as a partial agonist. The drug stimulated 5-HT(2A) receptors of rat tail artery in lower concentrations than 5-HT itself but failed to evoke comparable efficacy (proterguride: pEC(50) 8.34, E(max) 53% related to the maximum response to 5-HT; 5-HT: pEC(50) 7.03). Agonism at 5-HT(2B) receptors is presently considered to be involved in drug-induced valvular heart disease. Activation of 5-HT(2B) receptors in porcine pulmonary arteries by proterguride (pEC(50) 7.13, E(max) 49%; E(max) (5-HT) 69%), however, occurred at concentrations much higher than plasma concentrations achieving dopaminergic efficacy in humans. The results are discussed focussing on the relevance of action at 5-HT(2B) receptors as well as their significance for a transdermal administration of proterguride. Since it is well accepted that pulsatile dopaminergic stimulation is associated with treatment-related motor complications in the dopaminergic therapy of Parkinson's disease, the transdermal route of administration is of great clinical interest due to the possibility to achieve constant plasma concentrations.     

Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A(2) (sPLA(2)) and presynaptically active neurotoxin from venom of the long-nosed viper (Vipera ammodytes ammodytes). As a member of the M-type sPLA(2) receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional "propeptide" prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA(2)-binding C-type lectin-like domain 5 of the M-type sPLA(2) receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA(2)s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA(2) receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA(2) neurotoxicity.     

Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

Possible Role of Gangliosides in Regulating an Adenylate Cyclase-Linked 5-Hydroxytryptamine (5-HT1) Receptor

Cultured NCB-20 hybrid cells express adenylate cyclase-coupled receptors for 5-hydroxytryptamine (5-HT) that correspond biochemically and pharmacologically to 5-HT1 receptors in rodent brain membrane preparations, apart from a much-reduced affinity for 5-HT (160 nM compared to less than 5 nM in brain). Since NCB-20 cells also differ from rodent brain both qualitatively and quantitatively in their ganglioside composition, the effects of exogenously added gangliosides on the affinity of the 5-HT1 receptor for 5-HT were tested. Both GM1 ganglioside (the cholera toxin receptor) and tetrasialoganglioside GQ1b produced a 10-fold increase in receptor affinity for [3H]5-HT, measured by binding studies. All gangliosides, at submicromolar concentrations, resulted in significantly reduced EC50 values for 5-HT-mediated elevation of intracellular cyclic AMP levels. GQ1b had the capacity to most dramatically enhance the potency of 5-HT in mediating increases in cyclic AMP levels. Gangliosides had no effect on the potency of DADLE or 3,4-dihydroxyphenylethylamine (dopamine)-mediated depression of cyclic AMP levels, suggesting some specificity for 5-HT. Our data are interpreted as implying a specific role for polysialogangliosides in modulating the affinity of the 5-HT1 receptor and the coupling of the 5-HT1 receptor-guanine nucleotide binding protein adenylate cyclase complex.