Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT_1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT_1A autoreceptor-activated inwardly rectifying K⁺ conductance in rat dorsal raphe 5-HT neurons. 5-HT_1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba²⁺ (EC₅₀ = 9.4 μM, full block with 100 μM) and by SCH23390 (EC₅₀ = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT_1A autoreceptor-activated GIRK conductance with high potency (EC₅₀ = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC₅₀s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K⁺ channels coupled to 5-HT_1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.     

Binding of STW 5 (Iberogast®) and its components to intestinal 5-HT,muscarinic M3, and opioid receptors

Clinical studies with the fixed herbal combination product STW 5 (Iberogast^®) have indicated an efficacy comparable to metoclopramide (5-HT₃ antagonist) and cisapride (5-HT₄ agonist) in functional gastro-intestinal diseases like functional dyspepsia (FD) and irritable bowel syndrome (IBS). Since serotonin (5-HT₃ and 5-HT₄) and muscarinic M₃ receptors are known to play a central role in the etiology of FD and IBS, the extracts contained in STW 5 and several of their phytochemical components were studied in vitro for binding affinities to these receptors of the intestine. STW 5 inhibited the binding of ³H-GR113808 and ³H-4-DAMP to 5-HT₄ and M₃ receptors, respectively, about 10 times more potently than the binding of ³H-GR65630 to 5-HT₃ receptors. IC₅₀ values for STW 5 did correspond to extract dilutions of 1:1000 (M₃ binding) and 1:2000 (5-HT₄ binding). In addition, STW 5 also potently inhibited the binding to opioid receptors with an IC₅₀ value of 1:2000. Of the nine herbal extracts contained in STW 5, the fresh plant extract of bitter candy tuft (Iberis amara) selectively inhibited binding to M₃ receptors, while ethanolic extracts of celandine herb and chamomile flower were selective to 5-HT₄, and liquorice root to 5-HT₃ receptors. Binding affinities to human recombinant 5-HT₃, 5-HT₄ and M₃ receptors were qualitatively similar to those of the corresponding intestinal receptors. The benzylisoquinoline alkaloid berberine had significant inhibitory action on 5-HT₄ and M₃ binding, showing IC₅₀ values of 40 ng/ml (100 nM) and 200 ng/ml (500 nM), respectively, but is present in the extract of celandine herb only in traces, so that also for the celandine extract a cooperative effect of several phytochemical constituents can be assumed. These in vitro data indicate that 5-HT₄ (to a lesser degree 5-HT₃), muscarinic M₃, and opioid receptors represent target sites for the treatment of FD and IBS with STW 5 (Iberogast^®).     

Atypical SCH23390 Binding Sites Are Present on Bovine Adrenal Medullary Membranes

D1-selective dopamine receptor agonists inhibit secretagogue-stimulated catecholamine secretion from bovine adrenal chromaffin cells. The purpose of the studies reported here was to use the radiolabeled D1-selective dopamine receptor antagonist, SCH23390, to characterize putative D1-like dopamine receptors responsible for this effect. Characterization of SCH23390 binding sites demonstrated an unusual pharmacological profile inconsistent with classical D1-like receptors. [¹²⁵I]SCH23390 bound to adrenal medullary membranes was competed for by non-radioactive iodo-SCH23390 (Kd = 490 ± 50 nM), but not by (+)butaclamol. Other classical D1 antagonists had little, if any, effect. Competition with dopamine receptor agonists demonstrated a relative rank order of potency profile characteristic of D1-like dopamine receptors, however, K_is were higher than those found in other tissues. The K_is for competition of [¹²⁵I]SCH23390 binding by C1-APB and SKF38393 (16 and 118 M, respectively) are nearly identical to the IC₅₀s previously observed for inhibition of secretion (9 and 100 M, respectively). Combined these data suggest that adrenal medullary membranes contain a novel SCH23390 binding site involved in the inhibition of secretion by D1-selective agonists.     

The relaxing effect of SCH 23390 in the molluscan smooth muscle

1. SCH 23390 (SCH, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) produced the relaxation of ACh-induced contraction in the anterior byssus retractor muscle of Mytilus edulis (ABRM) in a dose-dependent manner between 10⁻⁹-10⁻⁶M.2. The dose-response curve of SCH was shifted in parallel to the right by ketanserin (KET) with pA₂ value of 5.14 ± 0.08 and by 1-(1-naphthyl)piperazine (NAP) with that of 5.06 ± 0.01, but not by cyproheptadine (CYP), mianserin (MIA), butaclamol (BUTA), ICS 205–930 (ICS) and MDL 72222 (MDL) at 3 × 10⁻⁵ M.3. α-Methyl-serotonin (α-Me-5-HT), a selective 5-HT₂ receptor agonist dose-dependently relaxed the ACh-induced contraction of ABRM. The dose-response curve of α-Me-5-HT was shifted in parallel to the right by KET with pA₂ value of 5.01 ± 0.02, but not by BUTA, CYP, MIA, ICS and MDL at 3 × 10⁻⁵ M.4. These findings suggest that 5-HT₂-like receptors exist in the ABRM, and that the relaxation induced by SCH is mediated through these receptors.     

Photoaffinity Labelling and Solubilization of the Central 5-HT1A Receptor Binding Site

Abstract

Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT_1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT_1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [³H]8-OH-DPAT from 5-HT_1A sites in rat hippocampal membranes with an IC₅₀ of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4°C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT_1A binding sites. This blockade was specific of 5-HT_1A sites since the other serotoninergic sites, 5-HT_1B, 5-HT₂ and also the presynaptic 5-HT₃ sites were not affected by the treatment. In addition, the binding of [³H]Spiperone and [³H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM ³H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, ³H-incorporation into the 63 kD band could be prevented by uM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT_1A ligands such as isapirone. In contrast, the 5-HT₂ antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT_1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50 % recovery of 5-HT_1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT_1A sites.     

5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release

The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT₃) receptors. We report that recombinantly expressed 5-HT₃ receptor binding sites are reduced by chronic exposure to 5-HT (IC₅₀ of 154.0 ± 45.7 μm, t_½ = 28.6 min). This is confirmed for 5-HT₃ receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC₅₀ of 2.3 ± 1.0 μm, t_½ = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.     

The binding of SCH 39166 and SCH 23390 to 5-HT1C receptors in porcine choroid plexus

SCH 39166 is a novel benzonaphthazepine, which has been characterized as a potent and selective D1 antagonist. Recently, its D1 selective benzazepine predecessor, SCH 23390, has been shown to bind to 5-HT1C binding sites in the choroid plexus. Therefore, the present studies were undertaken to determine if SCH 39166 has any measurable affinity for 5-HT1C binding sites. Our results indicate that SCH 39166 exhibited poor affinity for the 5-HT1C receptor, with a Ki of 1327 nM. In contrast, SCH 23390 inhibited [3H]-mesulergine binding to 5-HT1C receptors with a Ki of 30 nM. The non-selective 5-HT antagonist, methysergide, inhibited binding with a Ki of 2.4 nM. Finally, studies with the stereoisomers of SCH 39166 and SCH 23390 demonstrated that stereoselectivity at the 5-HT1C site is significantly less than for the D1 site.     

Dissociation of the plasticity of 5-HT1A sites and 5-HT transporter sites

To study the early effects of neonatal 5,7-dihydroxytryptamine lesions on 5-hydroxytryptamine_1A (5-HT_1A) receptors, we measured regional [³H]8-OH-DPAT-labeled 5-HT_1A sites in binding assays and compared them to our previous studies of [³H]paroxetine-labeled 5-HT transporter sites during the first month in the same rats. While there were significant time- and dose-dependent effects of 5,7-DHT on 5-HT transporter sites, there were no significant changes in 5-HT_1A sites in cortex, hippocampus, diencephalon, brainstem, cerebellum, or spinal cord. 5,7-DHT lesions also did not alter the K_i of Gpp(NH)p at brainstem 5-HT_1A sites or the K_i of 5-HT in cortex or brainstem in the presence or absence of GTPS or Gpp(NH)p. There were significant regional differences between the density of 5-HT_1A sites and 5-HT transporter sites. The ontogeny of brainstem 5-HT_1A sites was a pattern of increases until three weeks postnatal, and 5,7-DHT lesions did not alter the ontogeny of 5-HT_1A sites. These data suggest differential plasticity of 5-HT_1A and 5-HT transporter binding sites during the first month after neonatal 5,7-DHT lesions.     

[3H]Spiroxatrine labels a serotonin1A-like site in the rat hippocampus

[³H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT_1A sites in the rat hippocampus. Analysis of the binding of [³H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC₅₀= 20.7 nM for the [³H]spiroxatrine binding sites, consistent with the labeling of 5-HT₁ sites, while dopamine and norepinephrine had very low affinity (IC₅₀=57600 nM and >10⁻⁴ M respectively). Saturation studies of the binding of [³H]spiroxatrine revealed a single population of sites with a K_d=2.21 nM. Further pharmacologic characterization with the 5-HT_1A ligands 8-hydroxy-2-(di-n-propylamino) tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [³H]spiroxatrine labeling 5-HT_1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [³H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT_1A receptor model) in the rat hippocampus. Since [³H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT_1A site, it should provide new information about the properties of this putative serotonergic receptor.     

Synthesis and antidepressant activity of a series of arylalkanol and aralkyl piperazine derivatives targeting SSRI/5-HT1A/5-HT7

A series of arylalkanol and aralkyl piperazine derivatives have been synthesized and evaluated for 5-HT reuptake inhibitory abilities and binding affinities at the 5-HT_1A/5-HT₇ receptors. Antidepressant activities of the compounds in vivo were screened using the forced swimming test (FST). The results indicated that the compound 8j exhibited high affinities for the 5-HT_1A/5-HT₇ receptors (5-HT_1A, k_i?=?0.84?nM; 5-HT₇, k_i?=?12?nM) coupling with moderate 5-HT reuptake inhibitory activity (RUI, IC₅₀?=?100?nM) and showed a marked antidepressant-like activity in the FST model.     

Cortical 5-HT1A receptor downregulation by antidepressants in rat brain

Total 5-HT binding sites and 5-HT_1A receptor density was measured in brain regions of rats treated with imipramine (5 mg/kg body wt), desipramine (10 mg/kg body wt) and clomipramine (10 mg/kg body wt), for 40 days, using [³H]5-HT and [³H]8-OH-DPAT, respectively. It was observed that chronic exposure to tricyclic antidepressants (TCAs) results in significant downregulation of total [³H]5-HT binding sites in cortex (42–76%) and hippocampus (35–67%). The 5-HT_1A receptor density was, however, decreased significantly (32–60%) only in cortex with all the three drugs. Interestingly, in hippocampus imipramine treatment increased the 5-HT_1A receptor density (14%). The affinity of [³H]8-OH-DPAT was increased only with imipramine treatment both in cortex and hippocampus. The affinity of [³H]5-HT to 5-HT binding sites in cortex was increased with imipramine treatment and decreased with desipramine and clomipramine treatment. 5-HT sensitive adenylyl cyclase (AC) activity was significantly increased in cortex with imipramine (72%) and clomipramine (17%) treatment, whereas in hippocampus only imipramine treatment significantly increased AC activity (50%). In conclusion, chronic treatment with TCAs results in downregulation of cortical 5-HT_1A receptors along with concomitant increase in 5-HT stimulated AC activity suggesting the involvement of cortical 5-HT_1A receptors in the mechanism of action of TCAs.     

Agonists and Antagonists Bind to an A-A Interface in the Heteromeric 5-HT3AB Receptor

The 5-HT₃ receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT₃AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT₃AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [³H]granisetron binding affinity (K_d) and 5-HT EC₅₀ were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC₅₀ values were increased. In B-to-A mutant receptors there were no changes in K_d, although EC₅₀ values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT₃A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT₃AB receptor.     

Effects of Geissoschizine Methyl Ether, an Indole Alkaloid in Uncaria Hook, a Constituent of Yokukansan, on Human Recombinant Serotonin7 Receptor

Effects of seven alkaloids, geissoschizine methyl ether (GM), hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine, in Uncaria hook, a constituent of the kampo medicine yokukansan, on serotonin₇ (5-HT₇) receptor were investigated using Chinese hamster ovary (CHO) cell membranes and human embryonic kidney 293 (HEK293) cells stably expressing the human recombinant 5-HT₇ receptor. A competitive binding assay using CHO membranes showed that GM (IC₅₀ = 0.034 μM) more strongly inhibited the binding of the radioligand [³H] LSD to 5-HT₇ receptor than the other alkaloids, suggesting that GM is bound to 5-HT₇ receptor. Agonistic/antagonistic effects of GM (1–50 μM) on the receptor were evaluated by measuring intracellular cAMP levels in HEK239 cells. GM (IC₅₀ = 6.0 μM) inhibited 5-HT-induced cAMP production in a concentration-dependent manner, as well as the specific 5-HT₇ receptor antagonist SB-269970 (0.1–1 μM). However, GM did not induce intracellular cAMP production as 5-HT did. These results suggest that GM has an antagonistic effect on 5-HT₇ receptor.     

Identification of 5HT2-Receptors on Longitudinal Muscle of the Guinea Pig Ileum

Abstract

In binding experiments with the radioligands [³H]Ketanserin (HKet) and [¹²⁵I]LSD (ILSD) 21 compounds were investigated using rat brain cortex membranes. The pK_D-values of the compounds were virtually independent of the radioligand used and their rank order was consistent with classification of the binding sites as being of the 5-HT₂-type. In contrast, in the longitudinal muscle of the guinea pig ileum in the presence of 0.3 μM cinanserin, ILSD labelled sites which were quite different to those in the cortex. In a functional test antagonism of the 5HT induced contraction of the guinea-pig ileum was measured in the presence of 1 μM atropine. The pharmacological inhibition constants (IC₅₀-values) of 8 compounds correlated well with the dissociation constants for HKet binding in the cortex and did not correlate with the data from ILSD binding in the guinea pig ileum. It is concluded that the ileum contains postjunctional 5HT₂-receptors which mediate contraction. The nature of the ILSD binding sites in the ileum remains to be elucidated.     

The Serotonin 5-HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin-Mediated Phosphoinositide Hydrolysis

Abstract: Serotonin (5-hydroxytryptamine; 5-HT) 5-HT_2A and 5-HT_2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT_2A-linked and 5-HT_2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca²⁺ (300 nM) and GTPγS (1 µM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT_2A antagonist, spiperone, and the nonselective 5-HT_2A/2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT_2C receptors as opposed to 5-HT_2A receptors. Radioligand binding assays with [³H]RP 62203 and [³H]-mesulergine were used to quantify 5-HT_2A and 5-HT_2C sites, respectively, in caudate. From these data, the B_max for caudate 5-HT_2A sites and 5-HT_2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT_2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT_2A- and 5-HT_2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT_2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT_2C sites. The significance of these observations with respect to the physiological function of 5-HT_2C receptors is discussed.     

Heterogeneity of Cortical and Hippocampal 5-HT1A Receptors: A Reappraisal of Homogenate Binding with 8-[3H]Hydroxydipropylaminotetralin

Abstract: The selective serotonin (5-HT) agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5-HT_1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [³H]8- OH-DPAT (free ligand from 200 pM to 160 nM) revealed high-affinity 5-HT_1A receptors (K_H= 0.7–0.8 nM) and lowaffinity (K_L= 22–36 nM) binding sites. The kinetics of [³H]8-OH-DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high- and low-affinity binding sites. When the high-affinity sites were labeled with a 1 nM concentration of [³H]8- OH-DPAT, the competition curves of agonist and antagonist drugs were best fit to a two-site model, indicating the presence of two different 5-HT_1A binding sites or, alternatively, two affinity states, tentatively designated as 5-HT_1AHIGH and 5-HT_1A^LOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5-HT_1A sites are modulated by 5′-guanylylimidodiphosphate, inhibition experiments with 5-HT were performed in the presence or in the absence of 100 μM 5′-guanylylimidodiphosphate. The binding of 1 nM [³H]8-OH-DPAT to the 5-HT_1A^HIGH site was dramatically (80%) reduced by 5′-guanylylimidodiphosphate; in contrast, the low-affinity site, or 5-HT_1A^LOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high-affinity 5-HT_1A^HIGH site corresponds to the classic 5-HT_1A receptor, whereas the novel 5-HT_1A^LOW binding site, labeled by 1 nM [³H]8-OH-DPAT and having a micromolar affinity for 5-HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5-HT_1A sites and the 5-HT innervation, rats were treated with p-chlorophenylalanine or with the neurotoxin p-chloroamphetamine. The inhibition of 5-HT synthesis by p-chlorophenylalanine did not alter either of the two 5-HT_1A sites, but deafferentation by p-chloroamphetamine caused a loss of the low-affinity [³H]8-OH- DPAT binding sites, indicating-that these novel binding sites may be located presynaptically on 5-HT fibers and/or nerve terminals.