Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

Antisense Oligonucleotide-Induced Reduction in 5-Hydroxytryptamine7 Receptors in the Rat Hypothalamus Without Alteration in Exploratory Behaviour or Neuroendocrine Function

Abstract: The effect of a 5-hydroxytryptamine₇ (5-HT₇) receptor-directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5-HT₇ antisense oligonucleotide treatment significantly reduced [³H]5-HT binding to hypothalamic 5-HT₇ receptors, whereas cortical 5-HT_2C density remained unchanged. In rats on a food-restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle-treated controls by day 4 of administration. 5-HT₇ antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus-maze behaviour on day 6 of intracerebroventricular treatment. 5-HT₇ antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5-HT turnover in either 5-HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5-HT₇ antisense oligonucleotide administration selectively reduced rat hypothalamic 5-HT₇ receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5-HT₇ receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Allosteric Interactions Among Agonists and Antagonists at 5-Hydroxytryptamine3 Receptors

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine₃ (5-HT₃) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT₃ receptors. The affinities of competitive 5-HT₃ receptor antagonists were similar regardless of whether the receptors were labeled with [³H]RS-42358, [³H]granisetron, or 1-( m -[³H]chlorophenyl)biguanide ([³H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [³H]mCPG. The dissociation of [³H]mCPG, [³H]RS-42358, and [³H]RS-25259, but not [³H]granisetron, from both cloned and native 5-HT₃ receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT₃ receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

Immunological Characterization and Transmembrane Topology of 5-Hydroxytryptamine3 Receptors by Functional Epitope Tagging

Abstract: 5-Hydroxytryptamine₃ (5-HT₃) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABA_A, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5-HT₃ receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5-HT₃ receptors in transfected COS-7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti-fusion protein antibodies directed against a wild-type receptor intracellular domain. The extracellular location of the distal C-terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5-HT₃ serotonin-gated channels. In this region, the significant homology between members of this class of neurotransmitter-gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate-gated and ATP-gated channels.     

Ultrastructure of the 5-Hydroxytryptamine3 Receptor

Abstract: We have determined the ultrastructure of 5-hydroxytryptamine₃ (5-HT₃) serotonin receptors purified from NG108-15 mouse neuroblastoma × rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT₃ receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT₃ receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.     

5-Hydroxytryptamine3 Receptors Sited on Cholinergic Axon Terminals of Human Cerebral Cortex Mediate Inhibition of Acetylcholine Release

Synaptosomes prepared from freshly obtained human cerebral cortex and labeled with [3H]choline have been used to investigate the modulation of [3H]acetylcholine ([3H]ACh) release by 5-hydroxytryptamine (5-HT). The Ca(2+)-dependent release of [3H]-ACh occurring when synaptosomes were exposed in superfusion to 15 mM KCl was inhibited by 5-HT (0.01-1 microM) in a concentration-dependent manner. The effect of 5-HT was mimicked by 1-phenylbiguanide, a 5-HT3 receptor agonist, but not by 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT1A receptor agonist. The 5-HT3 receptor antagonists tropisetron and ondansetron blocked the effect of 5-HT, whereas spiperone and ketanserin were ineffective. It is suggested that cholinergic axon terminals in the human cerebral cortex possess 5-HT receptors that mediate inhibition of ACh release and appear to belong to the 5-HT3 type.     

Pertussis Toxin Attenuates 5-Hydroxytryptamine1A Receptor-Mediated Inhibition of Forskolin-Stimulated Adenylate Cyclase Activity in Rat Hippocampal Membranes

The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in rat hippocampal membranes isolated from animals treated with vehicle or islet-activating protein (IAP; pertussis toxin). In vehicle-treated animals, 5-HT, 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, and gepirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity with EC50 values of 60, 76, 376, and 530 nM, respectively. IAP treatment reduced by 30-55% the 5-HT1A agonist inhibition of adenylate cyclase activity via 5-HT1A receptors. The data indicate that the inhibitory guanine nucleotide-binding protein or Go (a similar GTP-binding protein of unknown function purified from brain) mediates the 5-HT1A agonist inhibition of hippocampal adenylate cyclase.     

Mianserin-Induced Down-Regulation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Stably Expressed in the Human Neuroblastoma Cell Line SH-SY5Y

Abstract: We have assessed the ability of the serotonergic antagonist mianserin to modulate the number and functional activity of human 5-hydroxytryptamine_2A (5-HT_2A) and 5-HT_2C receptors stably expressed in the human neuroblastoma cell line SH-SY5Y. Incubation of cells expressing the 5-HT_2A receptor with mianserin (100 n M ) for 24 h caused a significant decrease (48%) in the binding capacity of [³H]ketanserin. This receptor down-regulation was associated with a corresponding decrease in the maximal production of inositol phosphates induced by 5-HT but not by carbachol. Exposure of cells expressing the 5-HT_2C receptor to mianserin (100 n M ) for 72 h but not for 24 h similarly resulted in a significant reduction (44%) in [³H]mesulergine binding. Corresponding analysis of inositol phosphate production by 5-HT at the 5-HT_2C receptor after incubation with mianserin showed no change in maximal response after 24 h. No change in the binding capacity of either radioligand was seen after incubation with mianserin for 1 h. A decrease in the binding affinity of both radioligands was also observed after mianserin treatment, but this decrease was similar after 1 h of incubation to that seen after 24 or 72 h, and was probably due to the retention of mianserin within the tissue. We conclude that antagonist down-regulation is evident at human 5-HT_2A and 5-HT_2C receptors stably expressed in a human neuroblastoma cell line and is probably mediated by a direct action of mianserin at the receptor.     

A Single Amino Acid Difference Accounts for the Pharmacological Distinctions Between the Rat and Human 5-Hydroxytryptamine1B Receptors

Abstract: Molecular cloning of the rat and human 5-hydroxytryptamine_1B (5-HT_1B) receptors has revealed that the primary amino acid sequence of these two receptors is >90% identical. Despite this high degree of primary sequence homology, these two receptors have significantly different pharmacological properties. A mutant human 5-HT_1B receptor was constructed in which Thr³⁵⁵ was replaced by Asn, the corresponding residue at this position in the rat 5-HT_1B receptor. The pharmacology of the mutant human 5-HT_1B receptor was very similar to that of the rat 5-HT_1B receptor. Specifically, the mutant receptor had much higher affinity for pindolol, [¹²⁵I]-iodocyanopindolol, propranolol, and CP-93,129 than the wild-type receptor. In contrast, the mutant had significantly lower affinity for sumatriptan, N,N -dipropyl-5-carboxamidotryptamine, 5-methoxy- N,N -dimethyltryptamine, methysergide, metergoline, and rauwolscine. These data suggest that a single amino acid difference at position 355 is responsible for the pharmacological differences between the rat and human 5-HT_1B receptors.     

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5-hydroxytryptamine (5-HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5-HT receptors. The aim of this study was to subclassify the 5-HT₂ receptor mediating inhibition of the cyclic GMP response elicited by N -methyl- d -aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5-HT_2A, 5-HT_2B, and 5-HT_2C subtypes, differentially affected the inhibition by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5-HT_2C subtype.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Identification and Characterisation of 5-Hydroxytryptamine3 Recognition Sites in Human Brain Tissue

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.     

A Method for the In Vivo Investigation of the Serotonergic 5-HT2 Receptors in the Human Cerebral Cortex Using Positron Emission Tomography and 18F-Labeled Setoperone

Following previous validation in baboons, we have studied the characteristics of [18F]setoperone as a radioligand for investigating serotonergic 5-hydroxytryptamine2 (5-HT2) receptors in the normal, unmedicated human brain with positron emission tomography (PET); subjects orally pretreated with therapeutic amounts of ketanserin, sulpiride, or prazosin were also studied to evaluate the specificity and sensitivity of [18F]setoperone brain specific binding. In controls (n = 10), the tracer showed a clear-cut retention in both frontal cortex and striatum (known to contain a high density of 5-HT2 receptors) relative to cerebellum (known to be devoid of 5-HT2 receptors). In the seven young controls (20-39 years old), the frontal cortex/cerebellum and striatum/cerebellum ratios increased during the first hour to reach similar values of 2.53 +/- 0.12 and 2.38 +/- 0.11 (mean +/- SEM), respectively, and were essentially stable during the second hour. Pretreatment with ketanserin (a 5-HT2 blocker) significantly reduced the frontal cortex/cerebellum ratio to 0.7-1.0 at 65 min, whereas the striatum/cerebellum ratio was significantly, but only partially, reduced. During sulpiride treatment (a D2 blocker), the frontal cortex/cerebellum ratio was not altered, whereas the striatum/cerebellum ratio was significantly, but only partially, reduced. With prazosin pretreatment (an alpha 1-adrenergic blocker), neither the frontal cortex/cerebellum nor the striatum/cerebellum ratio was modified. These data in humans with PET demonstrate that [18F]setoperone labels with high sensitivity and selectivity 5-HT2 receptors in the frontal cortex; in striata, however, binding is to both 5-HT2 and D2 receptors. The deproteinated-to-whole plasma radio-activity concentration ratio increased with time following injection. The mean percentage of intact [18F]setoperone, in deproteinated plasma, was 82, 74, 53, 45, 30, and 22% at 5, 10, 20, 30, 60, and 110 min following injection, respectively. These data indicate that [18F]setoperone (a) is significantly bound to plasma proteins and (b) is significantly metabolized into several labeled metabolites that are much more hydrophilic than setoperone and, hence, presumably do not cross the blood-brain barrier. These results suggest the suitability of [18F]setoperone data for modeling of 5-HT2 receptor binding in brain.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

Chimeric D2/D3 Dopamine Receptors Efficiently Inhibit Adenylyl Cyclase in HEK 293 Cells

Abstract: Despite a high degree of sequence homology, the dopamine D₂ and D₃ receptors have substantially different second messenger coupling properties. We have used chimeric D₂/D₃ receptors to investigate the contribution of the intracellular loops to the signaling properties of these receptors. In HEK 293 cells, D₂ receptors inhibit prostaglandin E₁-stimulated cyclic AMP levels by >90%, whereas D₃ receptors inhibit cyclic AMP accumulation by only 20%. In chimeras that have the second or third intracellular loop, or both loops simultaneously, switched between the D₂ and D₃ receptors, the maximal inhibition of adenylyl cyclase is 60–90%. In addition, the potency of quinpirole to inhibit adenylyl cyclase activity at some of the chimeras is altered compared with the wild-type receptors. It appears that the intracellular loops of the D₃ receptor are capable of interacting with G proteins, as when these loops are expressed in the D₂ receptor, the chimeras inhibit adenylyl cyclase similarly to the wild-type D₂ receptor. Our data suggest that the overall conformation of the D₃ receptor may be such that it interacts with G proteins only weakly, but when the intracellular loops are expressed in another context or the D₃ receptor structure is altered by the introduction of D₂ receptor sequence, this constraint may be lifted.     

Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. I. Modulation of Central 5-Hydroxytryptamine Receptors in the Rat

Abstract: The present study indicates that central 5-hydroxytryptamine (5-HT; serotonin) receptors can be modulated in opposite directions by Ca²⁺ and guanine nucleotides [guanosine triphosphate (GTP), β, γ-imidoguanosine 5′-triphosphate (GppNHp)]. Thus CaCl₂ (≥0.5 mm ) inhibited whereas GTP and GppNHp (10 μm ) stimulated the 5-HT-sensitive adenylate cyclase in the hippocampus of newborn rats. Both the affinity (K_d ^?1) and the number (B_max) of [³H]5-HT binding sites in hippocampal membranes from adult rats were increased in the presence of Ca²⁺ (≥0.25 mm ); GTP (≥0.1 mm ) and GppNHp (≥0.3; μm ) produced reverse effects. The efficacy of guanine nucleotides in inhibiting specific [³H]5-HT binding was counteracted by Ca²⁺: the addition of this cation (5mm -CaCl₂) to the assay mixture resulted in a 40-fold increase in the IC₅₀ for GTP; the IC₅₀ for GppNHp increased five-fold under the same condition. The examination of the respective effects of Ca²⁺ and of GTP on the specific binding of [³H]5-HT to various hippocampal membrane preparations (from developing rats, from subcellular fractions of adult tissues, and from adult rats after the selective degeneration of serotoninergic innervation in the forebrain) indicated that the amplitudes of the Ca²⁺-induced increase and of the GTP-induced decrease were generally correlated. This conclusion did not apply to striatal membranes of kainic acid-treated rats because [³H]5-HT binding sites persisting after the intrastriatal injection of kainic acid (i.e., half of the total number in striatal membranes from control rats) were markedly less affected by GTP but at least as responsive as control membranes to the Ca²⁺-induced increase. These data are compatible with the hypothesis of a possible coupling of some–but not all–[³H]5-HT binding sites to adenylate cyclase in the rat brain.     

Characterization and Location of Met5-Enkephalin-Arg6-Phe7 Stored in Various Rat Brain Regions

A specific antiserum against met⁵-enkepha-lin-arg⁶-phe⁷ was raised and used to study the distribution and characterization of met⁵-enkephalin-arg⁶-phe⁷-like immunoreactive material in rat brains by radioimmunoassay and immunohistochemical procedures. The antiserum appears to be directed to the COOH-terminus of the peptide, as it fails to cross-react with met⁵-enkeph-alin, met³-enkephalin-arg⁶, met⁵-enkephalin-arg⁶-arg⁷, met⁶-enkephalin-lys⁶, and leu-enkephalin. However, it cross-reacts with phe-met-arg-phe by about 10% and with phe-met-arg-phe-NH₂ to an insignificant degree. The highest content of met⁵-enkephalin-arg⁶-phe⁷ was found in the striatum, which contains a dense network of immunoreactive varicose fibers and terminals, as well as immunoreac tive cell bodies. The met⁵-enkephalin-arg⁶-phe⁷ in striatum can be released in a Ca²⁺-dependent manner by a depolarizing concentration of KC1, raising the possibility of a neu-roregulatory role for met⁵-enkephalin-arg⁶-phe⁷. Characterization of the immunoreactive material by gel filtration and high pressure liquid chromatography revealed the presence of multiple forms of immunoreactive material in some brain regions.