Irreversible blockade of central 5-HT1A receptor binding sites by the photoaffinity probe 8-methoxy-3'-NAP-amino-PAT

A new photoaffinity ligand derived from the potent 5-HT agonist, 8-OH-DPAT, has been synthesized. In the dark, this compound, 8-methoxy-2-(N-n-propyl,N-3-(2-nitro-4-azidophenyl)aminopropyl) aminotetralin or 8-methoxy-3'-NAP-amino-PAT, displaced [3H]8-OH-DPAT and [3H]5-HT bound to 5-HT1A and 5-HT1 sites in hippocampal membranes with IC50 values of 6.6 and 18.1 nM respectively. The apparent affinity of 8-methoxy-3'-NAP-amino-PAT for the 5-HT1A binding sites was at least 20 times higher than for the other 5-HT receptor sites (5-HT2 and 5-HT3) or the dopamine-related [3H]spiperone and [3H]7-OH-DPAT binding sites. Under UV irradiation (lambda = 366 nm), 8-methoxy-3'-NAP-amino-PAT produced an irreversible blockade of 5-HT1A sites which could be prevented by prior site occupancy by a saturating concentration (10 microM) of reversible 5-HT ligands such as 5-HT itself, 8-OH-DPAT or LSD. The blockade of 5-HT1A binding sites was concentration-dependent, and two successive irradiations of rat brain membranes in the presence of 30 nM 8-methoxy-3'-NAP-amino-PAT were found to be more efficient that a single exposure to 100 nM of the photosensitive ligand. Thus, a 55-60% irreversible blockade of 5-HT1A binding sites was achieved following 2 cumulative irradiations of hippocampal membranes with 30 nM 8-methoxy-3'-NAP-amino-PAT. Under such conditions, cortical 5-HT2 receptor binding sites as well as striatal 5-HT3 and dopamine-related binding sites remained unaltered.     

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (K_Aapp 8.6 μM) and inhibited the specific binding of [³H]5-HT to 5-HT₁ sites. The PAT-induced inhibition of [³H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC₅₀ 2 nM) with the 5-HT_1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [³H]5-HT bound to hippocampal membranes was markedly increased by Mn²⁺ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (K_i 1.4 μM) [³H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K⁺-evoked release of [³H]5-HT previously taken up or newly synthesized from [³H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K⁺-induced depolarization on the synthesis of [³H]5-HT from [³H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.     

2-(Alkylamino)tetralin derivatives: interaction with 5-HT1A serotonin binding sites

8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) is a selective 5-HT1A serotonin agonist. Derivatives of 8-OH-DPAT with amine substituents larger or more bulky than n-propyl appear to be inactive in a presynaptic biochemical assay measuring agonist-induced feedback inhibition of 5-HT synthesis but have never been examined in brain binding assays. A series of N-phenylalkyl derivatives of 8-methoxy-2-aminotetralin was evaluated at [3H]-8-OH-DPAT-labeled 5-HT1A sites in rat brain hippocampal membranes. All of the phenylalkyl derivatives displayed significant affinity for these sites and, of the agents examined, the 3-phenylpropyl 8-hydroxy analogue appears to be optimal and had an affinity (Ki = 1.9 nM) comparable to that of 8-OH-DPAT (Ki = 1.2 nM). In addition, the presence of an oxygen-containing substituent at the 8-position of the tetralin ring is not necessary for good affinity, and secondary amines and tertiary amines displayed equal affinity at central 5-HT1A binding sites. 5-HT1A sites are found both pre- and postsynaptically; thus, differences observed in the biochemical assay as compared to the results of the present binding study could be due to different structural requirements of these two receptors. This seems unlikely, however, because there was little difference in the affinities of several selected analogues for striatal versus hippocampal binding sites. Because we have now demonstrated that amine substituents larger than propyl, and an unsubstituted 8-position, are well tolerated by central 5-HT1A sites, future studies aimed at the development of new serotonergic tetralin analogues need not be limited to N-propyl or 8-hydroxy derivatives of 2-aminotetralin.     

The selective labelling of central 5-HT1A receptor binding sites by [3H]5-methoxy-3-(di-n-propylamino)chroman

Investigations on the pharmacological properties of a series of chroman derivatives indicated that 5-methoxy-3-(di-n-propylamino)chroman (5-MeO-DPAC) acts in the nM range on 5-HT1A sites but recognizes very poorly other 5-HT sites and D2 sites in rat brain membranes. As expected from these observations, the tritiated derivative [3H]5-MeO-DPAC bound to a single class of specific sites which exhibited the same pharmacological properties as 5-HT1A sites labelled by [3H]8-OH-DPAT in hippocampal and cortical membranes. In contrast to [3H]8-OH-DPAT, [3H]5-MeO-DPAC did not bind to presynaptic striatal sites (possibly associated with 5-HT reuptake in serotoninergic terminals), which indicated that this new chroman derivative was even more selective than the [3H]tetralin ligand for the in vitro labelling of 5-HT1A sites. Comparison of the chemical structures of 5-MeO-DPAC and other 5-HT1A ligands suggests that electronic enrichment due to isosteric O-substitution in the chroman derivative may play an important role in the highly selective recognition of the 5-HT1A receptor by this drug.     

Chloroamphetamine did not prevent the effects of chronic antidepressants on 5-hydroxytryptamine inhibition of forskolin-stimulated adenylate cyclase in rat hippocampus.

Administration of p-chloroamphetamine (PCA, 10 mg/kg i.p. on two occasions) to rats resulted in a severe depletion of [3H]paroxetine binding sites, a measure of presynaptic serotonergic terminals, in both cortex and hippocampus, but did not affect [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT) binding or 5-hydroxytryptamine (5-HT)-induced inhibition of forskolin-stimulated adenylate cyclase in hippocampal membranes. Administration of either imipramine (15 mg/kg i.p. for 2 weeks) or lithium (0.2% for 2 weeks) to PCA-treated rats did not affect [3H]8-OH-DPAT binding but reduced the degree of inhibition of forskolin-stimulated adenylate cyclase by 5-HT in hippocampal membranes. It is concluded that the effects of imipramine and Li+ on 5-HT1A receptor-mediated responses in the hippocampus are exerted postsynaptically, possibly at a level distal to the receptor.     

Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists

We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) tetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC50 values of CAT, PAT and 5-HT and the KB of spiperone suggest that this receptor may correspond to the 5-HT1A binding site.     

3H]8-OH-DPAT labels the 5-hydroxytryptamine uptake recognition site and the 5-HT1A binding site in the rat striatum

The binding of [3H]8-hydroxy-2-(di-N-propylamino)-tetralin ([ 3H]8-OH-DPAT) to rat hippocampal and striatal membranes has been compared. In the hippocampus, low concentrations of [3H]8-OH-DPAT bound to a single, high affinity site which was sensitive to inhibition by spiperone, buspirone and ergotamine but not by mianserin, quipazine or (-)-propranolol. This is consistent with a selective labeling of the 5-HT1A receptor. In the striatum, [3H]8-OH-DPAT bound to two sites with high and low affinity (KD's 1.18 and 109 nM). The high affinity component was blocked by low concentrations of buspirone, spiperone and ergotamine. The low affinity component was blocked only by high concentrations of buspirone and spiperone, and was not displaced by ergotamine at concentrations up to 1 microM. The ergotamine-resistant component of striatal [3H]8-OH-DPAT binding was blocked by low concentrations of the 5-HT uptake inhibitors fluvoxamine and paroxetine, and by relatively low concentrations of 5-HT itself. Thus [3H]8-OH-DPAT labels the 5-HT transporter in the rat striatum. Unlike [3H]imipramine binding, the binding of [3H]8-OH-DPAT to the 5-HT transporter was independent of external sodium ions. It is therefore suggested that 8-OH-DPAT acts as substrate for the 5-HT transporter and labels the 5-HT recognition site of the transporter complex.     

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.     

Physical evidence of the coupling of solubilized 5-HT1A binding sites with G regulatory proteins

Previous investigations (El Mestikawy et al., J Neurochem 51: 1031-1040, 1988) have shown that 5-HT1A binding sites (R[5-HT1A]) solubilized by CHAPS from rat hippocampal membranes can be modulated by guanine nucleotides, as expected from their solubilization together with associated G regulatory proteins (G). Studies of the hydrodynamic properties of solubilized R[5-HT1A] have been presently carried out in order to assess in a more direct way the presence of R[5-HT1A]-G complexes in the soluble extract. Under control conditions, the sedimentation of a CHAPS extract from hippocampal membranes through a 5-30% sucrose gradient (200,000 g, 17 hr, 4 degrees) gave two maxima of [3H]8-OH-DPAT binding activity corresponding to sedimentation coefficients of 8.0 S and 10.0 S, respectively. Running the gradient in the presence of 1 microM GTP revealed a significant reduction of the 10.0 S peak, as expected from the loss of material (probably a G protein) normally associated with R[5-HT1A]. Conversely, attempts to prevent the dissociation of R[5-HT1A]-G by treatment of CHAPS soluble hippocampal extracts with the cross-linking reagent disuccinimidyl suberate (0.1 mM) resulted in a significant increase (+70%) in [3H]8-OH-DPAT binding activity associated with the appearance of a new sedimenting material with a higher coefficient (16.5 S). Furthermore, [3H]8-OH-DPAT binding became almost completely insensitive to guanine nucleotides as expected from the irreversible coupling by disuccinimidyl suberate of R[5-HT1A] with G protein(s). WGA-agarose chromatography of CHAPS soluble hippocampal extract supplemented with GTP allowed the physical separation of R[5-HT1A] from the bulk of G proteins, and a concomitant decrease of [3H]8-OH-DPAT high affinity binding capacity. Partial recovery of the latter could be achieved by reconstituting R[5-HT1A]-G complexes upon the addition of a mixture of pure bovine Gi + Go to G-deprived soluble extracts. Finally in vivo treatment with Pertussis toxin (5 micrograms intracerebroventricularly, 48 hr before killing) resulted in a significant reduction of the specific binding of [3H]8-OH-DPAT (-36%) to hippocampal membranes and corresponding CHAPS soluble extracts, and a marked decrease in the inhibitory effect of GppNHp. Accordingly the G protein associated with R[5-HT1A] belongs probably to the Gi or Go families.     

5-HT1A-receptors mediate stimulation of adenylate cyclase in rat hippocampus

Serotonin (5-HT) stimulated adenylate cyclase activity in homogenates of rat hippocampus. This effect was pharmacologically characterised with a series of agonists and antagonists of various structural classes. These compounds where also tested in radioligand binding studies using selective ligands for the various subtypes of 5-HT1 and 5-HT2 receptors. 5-HT1A, 5-HT1B and 5-HT1C recognition sites were labelled with [3H]8-OH-DPAT([3H]8-hydroxy-2-(di-n-propylamino)-tetralin) in pig cortex membranes, [125I]CYP([125I]iodocyanopindolol) in rat cortex and [3H]mesulergine in pig choroid plexus membranes, respectively. The rank order of potency of 13 agonists stimulating adenylate cyclase activity in homogenates of rat hippocampus was in good agreement with the rank order of affinity of these agonists for the 5-HT1A binding site: N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT) greater than 5-carboxamidotryptamine (5-CT) greater than 8-OH-DPAT greater than 5-HT greater than 5-methoxytryptamine (5-OCH3T) greater than d-LSD greater than 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) greater than alpha-methylserotonin (alpha-CH3-5-HT) greater than dopamine greater than 2-methylserotonin (2-CH3-5-HT). The correlation between the respective potencies and affinities of these agonists was r = 0.934, P less than 0.001. There was no correlation between stimulation of adenylate cyclase activity by these agonists and their affinity for 5-HT1B, 5-HT1C or 5-HT2 binding sites. r = 0.381-0.108, P less than 0.20-0.73.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-HT receptor regulation of neurotransmitter release

Serotoninergic neurons in the central nervous system impinge on many other neurons and modulate their neurotransmitter release. This review focuses on 1) the function of presynaptic 5-hydroxytryptamine (5-HT) heteroreceptors on axon terminals of central cholinergic, dopaminergic, noradrenergic, or GABAergic neurons and 2) the role of GABAergic interneurons expressing 5-HT heteroreceptors in the regulation of acetylcholine, dopamine, or noradrenaline release. In vitro studies on slices or synaptosomes and in vivo microdialysis experiments have shown that 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(2C), 5-HT(3), and/or 5-HT(4) heteroreceptors mediate this modulation. 5-HT(1B) receptors on neocortical cholinergic, striatal dopaminergic, or hippocampal GABAergic axon terminals are examples for release-inhibiting 5-HT heteroreceptors; 5-HT(3) receptors on hippocampal GABAergic or 5-HT(4) receptors on hippocampal cholinergic axon terminals are examples for release-facilitating 5-HT heteroreceptors. GABA released from GABAergic interneurons upon activation of facilitatory 5-HT receptors, e.g., 5-HT(2A) or 5-HT(3) receptors, mediates inhibition of the release of other neurotransmitters such as prefrontal neocortical dopamine or neocortical acetylcholine release, respectively. Conversely, attenuated GABA release in response to activation of inhibitory 5-HT heteroreceptors, e.g., 5-HT(1A) or 5-HT(1B) receptors on GABAergic interneurons is involved in paradoxical facilitation of hippocampal acetylcholine and striatal dopamine release, respectively. Such 5-HT heteroreceptors are considered potential targets for appropriate 5-HT receptor ligands which, by enhancing the release of a relevant neurotransmitter, can compensate for its hypothesized deficiency in distinct brain areas. Examples for such deficiencies are the impaired release of hippocampal or neocortical acetylcholine, striatal dopamine, and hippocampal or neocortical noradrenaline in disorders such as Alzheimer's disease, Parkinson's disease, and major depression, respectively.     

Inactivation of 5-HT(1A) receptors in hippocampal and cortical homogenates

5-HT(1A) receptor function can be assessed in rat hippocampal and cortical membrane preparations as agonist-stimulated [35S]GTPgammaS binding. Membranes were preincubated in vitro with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT]-stimulated [35S]GTPgammaS binding and [3H]8-OH-DPAT binding assays were used to assess 5-HT(1A) receptor function and density, respectively. EEDQ decreased both R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding in hippocampal and cortical membranes. The E(max) but not the EC(50) of R(+)-8-OH-DPAT to stimulate [35S]GTPgammaS binding was decreased by EEDQ in both preparations. Additionally, the IC(50) for EEDQ to reduce R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding was the same for both brain regions in both assays. In contrast to EEDQ alone, agonist-stimulated [35S]GTPgammaS binding was not reduced in hippocampal membranes preincubated with EEDQ and the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl- cyclohexanecarboxamide maleate (WAY 100,635), suggesting that EEDQ acts directly on the receptor. Due to parallel reductions in receptor density and maximal functional response, it is concluded that there is little or no reserve for 5-HT(1A) receptor coupling to G(alpha) in these preparations. In addition, the sensitivity of hippocampal and cortical 5-HT(1A) receptors to inactivation by EEDQ in vitro is the same.     

Chronic effects of imipramine and lithium on postsynaptic 5-HT1A and 5-HT1B sites and on presynaptic 5-HT3 sites in rat brain

The effects of chronic treatment with imipramine, a tricyclic antidepressant, or lithium, an antimanic-depressive illness drug, on postsynaptic serotonin-1A (5-HT1A) and 5-HT1B sites and on presynaptic 5-HT3 sites in the frontal cortex and hippocampus from rat brains were studied. Chronic i.p. administration (21 days) of imipramine reduced the maximum number of binding sites (Bmax) for postsynaptic 5-HT1A as monitored by the radioligands 3H-5-HT or 3H-8-hydroxy-2-(di-n-propylamino)tetralin (3H-8-OH-DPAT), but did not change the Bmax for postsynaptic 5-HT1B and presynaptic 5-HT3 in either the frontal cortex or the hippocampus. Chronic i.p. administration (21 days) of lithium reduced the Bmax for postsynaptic 5-HT1A sites in the hippocampus, but not in the frontal cortex. There was a specific difference between imipramine and lithium regarding the inhibitory effect on postsynaptic 5-HT1A sites in the frontal cortex. In addition, lithium decreased the affinity of presynaptic 5-HT3 sites in the hippocampus. These findings may be also consistent with the inhibitory effect of lithium on presynaptic autoreceptors, which results in an increase of 5-HT release. It is concluded that enhanced 5-HT neurotransmission which develops during chronic treatment with imipramine or lithium seems tob e related to the down-regulation of postsynaptic 5-HT1A receptors in addition to postsynaptic 5-HT2 receptors, which may also have an important role in the antidepressant effects of these drugs.     

Fentanyl and sufentanil inhibit agonist binding to 5-HT1A receptors in membranes from the rat brain.

The influence of several opioid narcotics and related drugs, on the binding of [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin. ([3H]8-OH-DPAT), a serotonergic agonist, to 5-HT1A receptors was determined in membranes from the brain of the rat. Sufentanil and fentanyl inhibited binding of [3H]8-OH-DPAT to hippocampal membranes, with IC50 values of 5.5 and 3.4 microM, respectively. In contrast, IC50 values for meperidine, alfentanil and naloxone exceeded 100 microM. The inhibition of binding by sufentanil appeared to be competitive insofar as 10 microM sufentanil increased the apparent KD from 1.0 +/- 0.1 to 3.9 +/- 0.3 nM, without affecting the number of binding sites and the inhibition was easily reversed. The binding of [3H]8-OH-DPAT to hippocampal membranes was inhibited by 5'-guanylylimidodiphosphate, a stable analogue of GTP, in a concentration-dependent manner. None of the opioid drugs examined altered the sensitivity of binding of [3H]8-OH-DPAT to guanine nucleotides. These results suggest that certain opioid narcotics, disrupt serotonergic neurotransmission as a result of direct interactions with 5-HT1A receptors. No effects of opioid narcotics on 5-HT1A receptor-G protein coupling were noted.     

Stimulation and inhibition of adenylyl cyclase by distinct 5-hydroxytryptamine receptors

5-Hydroxytryptamine (serotonin, 5-HT) stimulates basal adenylyl cyclase activity in membranes from guinea pig or rat hippocampi, but 5-HT inhibits forskolin-stimulated adenylyl cyclase activity in these same membranes. The opposing effects of 5-HT on adenylyl cyclase activity indicate that distinct 5-HT receptors, positively and negatively coupled to adenylyl cyclase, are present in these membranes. Stimulation of adenylyl cyclase activity is mediated by two distinct 5-HT receptors. The receptor with lower affinity for 5-HT, designated as RL, is apparently homologous with a 5-HT receptor present in rat collicular membranes, but it is not homologous with the stimulatory receptor characterized in neuroblastoma hybrid cell (NCB-20) membranes. The receptor with higher affinity for 5-HT is homologous with the 5-HT1A binding site. The magnitude of stimulation by 5-HT1A receptors is variable with respect to stimulation by RL and is sometimes completely absent. Inhibition of forskolin-stimulated adenylyl cyclase activity, in membranes from either rat or guinea pig hippocampus or rat cortex, is a functional correlate of the 5-HT1A binding site. This inhibitory response was used to determine the pharmacological characteristics of drugs that reportedly have high affinity for 5-HT1A binding sites, such as 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) and (-)pindolol. PAPP inhibited adenylyl cyclase activity in guinea pig hippocampal membranes with an EC50 value of 27 +/- 3 nM. (-)Pindolol was a partial agonist in inhibiting adenylyl cyclase activity in guinea pig and rat hippocampal membranes. Because of the low intrinsic activity of (-)pindolol, it was tested as an antagonist of the inhibition produced by 5-HT1A receptor agonists in rat hippocampal membranes. The Kb of (-)pindolol was 40 nM as measured by a Schild plot. (-)Propranolol was a simple competitive antagonist at the rat hippocampal receptor with a Kb value of 550 nM. In summary, guinea pig and rat hippocampal membranes possess two distinct populations of 5-HT receptors, a 5-HT receptor that mediates inhibition of adenylyl cyclase activity and is pharmacologically homologous with the 5-HT1A binding site, and a stimulatory receptor that appears to be homologous with the 5-HT receptor first characterized in infant rat collicular membranes.     

Differential sensitivity of 3H-agonist binding to pre- and postsynaptic 5-HT1A receptors in bovine brain.

1. The full and weak partial 5-HT1A agonist ligands [3H]-8-OH-DPAT and [3H]-BMY-7378 were used to characterize the binding parameters of pre- and postsynaptic 5-HT1A binding sites in bovine dorsal raphe and hippocampal membranes, respectively. The Kd and Bmax values for the individual radioligands were indistinguisable across the regions tested, as were the Ki values generated by a series of agents acting at 5-hydroxytryptamine (5-HT) receptors. 2. The concentration-dependent allosteric attenuation of [3H]-8-OH-DPAT and [3H]-BMY-7378 binding produced by the nonhydrolyzable guanyl nucleotide, Gpp(NH)p, generated similar IC50 values within a particular region; however, these were significantly different between regions. While the maximal attenuation of [3H]-8-OH-DPAT and [3H]-BMY-7378 binding was similar in dorsal raphe, Gpp(NH)p produced a significantly greater attenuation of [3H]-8-OH-DPAT binding in hippocampal membranes when compared to [3H]-BMY-7378. The maximal attenuation of [3H]-8-OH-DPAT binding by Gpp(NHp) in hippocampus was also significantly greater than that seen with either radioligand in dorsal raphe. 3. Although exposure to Gpp(NH)p had no effect on the affinity constants of either radioligand in either region, it produced a concentration-dependent reduction in the maximal number of binding sites for both radioligands in both regions. While the percentage reduction in Bmax values were similar for both radioligands in the dorsal raphe, Gpp(NH)p reduced the Bmax of [3H]-8-OH-DPAT in hippocampus significantly more than that of [3H]-BMY-7378. 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low doses of 8-OH-DPAT prevent the impairment of spatial learning caused by intrahippocampal scopolamine through 5-HT(1A) receptors in the dorsal raphe

1. We studied the effects of low doses of 8-OH-DPAT, a 5-HT(1A) receptor agonist, on the impairment of spatial learning caused by scopolamine injected into the CA1 region of the dorsal hippocampus of rats performing a two-platform spatial discrimination task. 2. Bilateral injections of 4 microg (in 1 microl) of scopolamine into the CA1 region of the dorsal hippocampus 10 min before each training session impaired choice accuracy with no effect on choice latency and errors of omission. 3. Administered subcutaneously 20 min before each training session, 8-OH-DPAT 10 and 30 (but not 3) microg kg(-1) did not modify choice accuracy but prevented the impairment by intrahippocampal scopolamine. 4. Injection of 1.0 microg (in 0.5 microl) of WAY 100635, a 5-HT(1A) receptor antagonist, into the dorsal raphe 5 min before scopolamine had no effect on choice accuracy and latency or errors of omission and did not modify the effect of scopolamine, but completely antagonized the effect of 10 and 30 microg kg(-1) 8-OH-DPAT on scopolamine-induced impairment of choice accuracy. 5. The results confirm previous findings that stimulation of presynaptic 5-HT(1A) receptors in the dorsal raphe attenuates the deficit of spatial learning caused by blockade of cholinergic excitatory input on hippocampal pyramidal cells. 6. Drugs that stimulate presynaptic 5-HT(1A) receptors such as 5-HT(1A) receptor partial agonists may be useful in the symptomatic treatment of human memory disturbances associated with loss of cholinergic innervation to the hippocampus.     

In situ molecular sizes of the various types of 5-HT binding sites in the rat brain

The radiation inactivation technique has been used to estimate the molecular weights of 5-HT binding sites in various regions of the rat brain. Using 3H-5-HT or 3H-8-OH-DPAT as the ligand, the same molecular weight of 55,000-60,000 daltons was calculated for the postsynaptic 5-HT1A and 5-HT1B sites in the hippocampus and cerebral cortex. Studies with 3H-ketanserin as the selective ligand indicated a molecular weight in the same range for the post-synaptic 5-HT2 binding site in the cerebral cortex. In contrast, a higher value (67,000 daltons) was found for the presynaptic 5-HT3 site selectively labelled by 3H-8-OH-DPAT in the striatum and cerebral cortex. The curvilinear pattern of the radiation-induced inactivation of 5-HT1A and 5-HT1B binding sites suggested that both sites belong to complex polymeric structures. In contrast, the 5-HT2 and 5-HT3 sites may correspond to less cooperative structures since simple monoexponential inactivation curves were observed upon irradiation.     

5-HT1A receptor agonists inhibit carbachol-induced stimulation of phosphoinositide turnover in the rat hippocampus

The selective 5-HT1A agonists, 8-hydroxy-2-(di-n-dipropylamino)tetralin (8-OH-DPAT) and ipsapirone, and the 5-HT1A/5-HT1B agonist, 1-(m-trifluoromethylphenyl)piperazine, partially inhibited the carbachol-stimulated [3H]inositol phosphate formation in rat hippocampal slices. The effect of 8-OH-DPAT was antagonized by cyanopindolol. Selective 5-HT1B, 5-HT2 and 5-HT3 agonists were inactive. 8-OH-DPAT failed to affect the phosphoinositide turnover stimulated by KCl, quisqualate or noradrenaline in hippocampal slices and by carbachol in striatal or cortical slices. These results suggest that 5-HT1A receptors are negatively coupled to phosphoinositide phosphodiesterase in the hippocampus.     

Effect of (+/-)-methyl 3-ethyl-2,3,3a,4-tetrahydro-1H-indolo-[3,2,1-de] [1,5] naphthyridine-6-carboxylate hydrochloride (OM-853), a new vincamine analogue, on the metabolism and function of cerebral serotonergic neurons

Effect of (+/-)-methyl 3-ethyl-2,3,3a,4-tetrahydro-1H-indolo[3,2,1-de] [1,5] naphthyridine-6-carboxylate hydrochloride (OM-853), a new vincamine analogue, on the metabolism and function of cerebral 5-hydroxytryptamine (5-HT) neurons was investigated using male Wistar rats. The single administration of OM-853 (200 mg/kg, p.o.) induced the facilitation of metabolic turnover of 5-HT in various brain areas except the cerebral cortex, pons-medulla and cerebellum. In vitro addition of OM-853 inhibited the uptake of [14C]5-HT in striatal slices only at a high concentration (10(-4) M). On the other hand, a low concentration of OM-853 (10(-8)-10(-6) M) induced the increase of the spontaneous and high K+ (30 mM)-evoked releases of [14C]5-HT from striatal slices. OM-853 had more potent inhibitory effect on the binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-hydroxy DPAT) to 5-HT1A receptors and/or 5-HT autoreceptors than that of [3H]-ketanserin to 5-HT2 receptors. The stimulatory effect of OM-853 (10(-7) M) on [14C]5-HT release was antagonized by 10(-7) M 8-hydroxy DPAT, which is known to act at presynaptic 5-HT autoreceptors as an agonist. These results suggest that OM-853 may induce facilitation of 5-HT turnover by enhancing 5-HT release, probably via the inhibition of presynaptic 5-HT autoreceptor.