Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Serotonin 5-HT1A Receptor Activation Increases Cyclic AMP Formation in the Rat Hippocampus In Vivo

Abstract: In vivo microdialysis was used to examine the efflux of cyclic AMP (cAMP) into the extracellular fluid of the ventral hippocampus in the freely moving rat. The changes in extracellular cAMP concentration were monitored in response to forskolin and the serotonin 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The basal level of hippocampal extracellular cAMP was 2.3 ± 0.2 pmol/ml (n = 6), after a 3-h postsur- gery stabilisation period. Perfusion of forskolin (100 μM) through the probe for 30 min significantly increased the efflux of cAMP, which returned to baseline levels within 90 min. 8-OH-DPAT (0.3 mg/kg s.c.) also significantly increased cAMP efflux, whereas a similar volume of saline had no effect. Desensitisation of the 8-OH-DPAT-induced increase in cAMP efflux was observed following a second administration of 8-OH-DPAT after a 4-h interval. Administration of 8-OH-DPAT did not alter the efflux of cAMP when forskolin was perfused through the probe. Pretreatment with WAY 100135 [N-tert-butyl 3–4-(2-methoxyphenyl)piperazine-1 -yl-2-phenylpropanamide dihydrochloride] (5 mg/kg s.c.), a specific 5-HT_1A receptor antagonist, prevented the 8-OH-DPAT-induced increase in cAMP efflux. The data indicate that the 8-OH-DPAT-induced increase in cAMP efflux in vivo is mediated by a 5-HT_1A receptor.     

[3H]Spiroxatrine: A 5-HT1A Radioligand with Agonist Binding Properties

Spiroxatrine has been reported to be a 5-HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5-HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5-HT1A antagonist radioligand. [3H]8-Hydroxydipropylaminotetralin [( 3H]8-OH-DPAT), a well-characterized 5-HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8-OH-DPAT binding produced KD values of 0.9 nM and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r = 0.98; p less than 0.001) exists between Ki values obtained for a series of drugs in competing for [3H]-spiroxatrine and [3H]8-OH-DPAT binding. Of special interest was the observation that 5-HT1A agonists such as serotonin, 8-OH-DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8-OH-DPAT-labelled 5-HT1A receptors. [3H]Spiroxatrine and [3H]8-OH-DPAT binding to 5-HT1A receptors was inhibited by guanosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration-dependent manner whereas adenosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of ATP) had no effect. The similarities in the 5-HT1A receptor radiolabelling properties of [3H]spiroxatrine and [3H]8-OH-DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has "agonist-like" binding properties in its interaction with the 5-HT1A receptor.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

Primary Structure of the Human Platelet Serotonin 5-HT2A Receptor: Identity with Frontal Cortex Serotonin 5-HT2A Receptor

Abstract: Previous radioligand binding studies have demonstrated human platelet serotonin_2A (5-HT_2A) receptor binding sites. Pharmacological similarities between platelet and frontal cortex 5-HT_2A receptor binding parameters have been demonstrated. However, it is not clear whether the platelet 5-HT_2A receptor primary structure is identical to that of the brain receptor. Three overlapping cDNAs were obtained to span completely the coding region of the 5-HT_2A receptor. These clones were sequenced with external and internal primers. The nucleotide sequence of human platelet 5-HT_2A cDNA was identical to that reported for the human frontal cortex 5-HT_2A receptor, except for nucleotide 102 (T → C), which has been reported to represent a normal DNA polymorphism that does not alter the amino acid sequence. This finding may have implications in the study of neuropsychiatric disorders for which altered platelet 5-HT_2A receptor binding has been demonstrated.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Characterization of Pharmacologically Active Anti-Peptide Antibodies Directed Against the First and Second Extracellular Loops of the Serotonin 5-HT1A Receptor

Abstract: The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5-HT_1A receptor were studied with three populations of anti-peptide antibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; residues 96–111), Ab-2 (loop II; sequence G-W-R-T-P-E-D-R-S-D-P-D-A-C-T-I-S-K-D-H-G; residues 173–193), and Ab-12 (produced against loop I but cross-reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N-terminal domain of loop II for Ab-2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab-1 and Ab-12 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5-HT_1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5-HT_1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT_1A receptor (K_DH = 0.54 ± 0.09 nM and K_DL = 13.74 ± 4.9 nM) for the agonist 8-hydroxy-2-(di-n-[³H]propylamino)tetralin ([³H]8-OH-DPAT) binding, but Ab-1 and Ab-12 revealed only one site (K_D of ≈2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodies decreased the B_max of the [³H]8-OH-DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Differential Regulation of Somatodendritic Serotonin 5-HT1A Receptors by 2-Week Treatments with the Selective Agonists Alnespirone (S-20499) and 8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract : Single treatment with the serotonin (5-hydroxytryptamine) 5-HT_1A receptor agonists 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT) and alnespirone (S-20499) reduces the extracellular 5-HT concentration (5-HT_ext) in the rat midbrain and forebrain. Given the therapeutic potential of selective 5-HT_1A agonists in the treatment of affective disorders, we have examined the changes in 5-HT_1A receptors induced by 2-week minipump administration of alnespirone (0.3 and 3 mg/kg/day) and 8-OH-DPAT (0.1 and 0.3 mg/kg/day). The treatment with alnespirone did not modify baseline 5-HT_ext but significantly attenuated the ability of 0.3 mg/kg s.c. alnespirone to reduce 5-HT_ext in the dorsal raphe nucleus (DRN) and frontal cortex. In contrast, the ability of 8-OH-DPAT (0.025 and 0.1 mg/kg s.c.) to reduce 5-HT_ext in both areas was unchanged by 8-OH-DPAT pretreatment. Autoradiographic analysis revealed a significant reduction of [³H]8-OH-DPAT and [³H]WAY-100635 {³H-labeled N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridyl)cyclohexanecarboxamide · 3HCl} binding to somatodendritic 5-HT_1A receptors (but not to postsynaptic 5-HT_1A receptors) of rats pretreated with alnespirone but not with 8-OH-DPAT. In situ hybridization analysis revealed no change of the density of the mRNA encoding the 5-HT_1A receptors in the DRN after either treatment. These data indicate that continuous treatment for 2 weeks with alnespirone, but not with 8-OH-DPAT, causes a functional desensitization of somatodendritic 5-HT_1A receptors controlling 5-HT release in the DRN and frontal cortex.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

A Naturally Occurring Amino Acid Substitution of the Human Serotonin 5-HT2A Receptor Influences Amplitude and Timing of Intracellular Calcium Mobilization

Abstract: Recently, two naturally occurring amino acid substitutions were identified in the C-terminal region of the serotonin 5-HT_2A receptor. One of these, His ⁴⁵²Tyr, has a rarer allele Tyr frequency of 9%. If ⁴⁵²Tyr alters 5-HT_2A function, it would thus be a candidate allele for human neurobehavioral variation. The present study was designed to evaluate the potential influence of the ⁴⁵²His and ⁴⁵²Tyr alleles on cellular 5-HT_2A functions. Platelet 5-HT_2A binding and 5-HT-induced Ca²⁺ response were compared in eight ⁴⁵²His/⁴⁵²His homozygous and eight ⁴⁵²His/⁴⁵²Tyr heterozygous individuals matched for sex, age, and diagnosis (all were patients with seasonal affective disorder). There was no difference in 5-HT_2A binding measured using ¹²⁵I-lysergic acid diethylamide. Nor were levels of G-protein subunits or PKC α, δ, ε, or ζ significantly altered. However, when Ca²⁺ response was stimulated by 2, 5, 10, or 25 µM 5-HT, significant differences were found. In ⁴⁵²His/⁴⁵²Tyr heterozygotes, ⁴⁵²Tyr was associated with both smaller peak amplitude in Ca²⁺ mobilization and a different time course of response, with slower peak latency and longer half-time in ⁴⁵²His/⁴⁵²Tyr heterozygotes compared with ⁴⁵²His/⁴⁵²His homozygotes. The overall difference in the response of the 5-HT_2A receptor in individuals with ⁴⁵²Tyr was a blunting of the shape of the Ca²⁺ mobilization peak. The data reported here suggest that the primary sequence of this intracellular domain is important in function of the receptor and that the ⁴⁵²His and ⁴⁵²Tyr 5-HT_2A alleles should be carefully evaluated for effects on human neurobehavioral variation.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.     

Serotonin 5-HT1D and 5-HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro

Abstract: The K⁺-evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5-hydroxytryptamine (5-HT); pD₂ = 8.95], 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT; pD₂ = 7.35), and sumatriptan (pD₂ = 8.43). These inhibitions were prevented by the selective 5-HT_1D receptor antagonist N -[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)(1,1-biphenyl)-4-carboxamide (GR-127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K⁺ depolarization; pD₂ values were 9.37 (5-HT), 9.00 (8-OH-DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5-HT (pD₂ values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8-OH-DPAT (respective pD₂ values of 9.15 and 9.00); sumatriptan (0.1 µ M ) was ineffective. The 5-HT_1A receptor antagonist ( S )-(+) N-tert -butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropionamide dihydrochloride [(+)-WAY 100135] did not prevent the inhibition of glutamate release by 5-HT but blocked the inhibition by 8-OH-DPAT of the NMDA/AMPA-evoked cGMP responses. It is suggested that presynaptic 5-HT_1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5-HT_1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.     

Characterisation of Recombinant Serotonin 5-HT1A Receptors Expressed in Chinese Hamster Ovary Cells: The Agonist [3H]Lisuride Labels Free Receptor and Receptor Coupled to G Protein

Abstract: Cholinesterases form a family of serine esterases that arise in animals from at least two distinct genes. Multiple forms of these enzymes can be precisely localized and regulated by alternative mRNA splicing and by co- or posttranslational modifications. The high catalytic efficiency of the cholinesterases is quelled by certain very selective reversible and irreversible inhibitors. Owing largely to the important role of acetylcholine hydrolysis in neurotransmission, cholinesterase and its inhibitors have been studied extensively in vivo. In parallel, there has emerged an equally impressive enzyme chemistry literature. Cholinesterase inhibitors are used widely as pesticides; in this regard the compounds are beneficial with concomitant health risk. Poisoning by such compounds can result in an acute but usually manageable medical crisis and may damage the CNS and the PNS, as well as cardiac and skeletal muscle tissue. Some inhibitors have been useful for the treatment of glaucoma and myasthenia gravis, and others are in clinical trials as therapy for Alzheimer's dementia. Concurrently, the most potent inhibitors have been developed as highly toxic chemical warfare agents. We review treatments and sequelae of exposure to selected anticholinesterases, especially organophosphorus compounds and carbamates, as they relate to recent progress in enzyme chemistry.     

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.     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Detection of a Novel Serotonin Receptor Subtype (5-HT1E) in Human Brain: Interaction with a GTP-Binding Protein

[3H]Serotonin (5-hydroxytryptamine, [3H]5-HT) was used as a radioligand probe of brain 5-HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 microM pindolol (to block 5-HT1A and 5-HT1B receptors), and 100 nM mesulergine (to block 5-HT1C and 5-HT2 receptors). One of these sites demonstrated high affinity for 5-carboxyamidotryptamine (5-CT) and ergotamine, consistent with the known pharmacology of the 5-HT1D receptor; the second site demonstrated low affinity for 5-CT and ergotamine. Computer-assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5-CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5-CT and ergotamine, respectively) for 45% of the sites. To investigate the non-5-HT1D component of the binding, 100 nM 5-CT (to block 5-HT1A, 5-HT1B, and 5-HT1D receptors) was coincubated with [3H]5-HT, membranes, and mesulergine. The remaining [3H]5-HT binding (hereafter referred to as "5-HT1E") displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5-CT and ergotamine displayed the highest selectivity for the 5-HT1D site versus the 5-HT1E site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.