SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), a novel 5-ht5A receptor-selective antagonist, enhances 5-HT neuronal function: Evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain

This study utilised the selective 5-ht(5A) receptor antagonist, SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), to investigate 5-ht5A receptor function in guinea pig brain. SB-699551-A competitively antagonised 5-HT-stimulated [35S]GTPgammaS binding to membranes from human embryonic kidney (HEK293) cells transiently expressing the guinea pig 5-ht5A receptor (pA2 8.1+/-0.1) and displayed 100-fold selectivity versus the serotonin transporter and those 5-HT receptor subtypes (5-HT(1A/B/D), 5-HT2A/C and 5-HT7) reported to modulate central 5-HT neurotransmission in the guinea pig. In guinea pig dorsal raphe slices, SB-699551-A (1 microM) did not alter neuronal firing per se but attenuated the 5-CT-induced depression in serotonergic neuronal firing in a subpopulation of cells insensitive to the 5-HT1A receptor-selective antagonist WAY-100635 (100 nM). In contrast, SB-699551-A (100 or 300 nM) failed to affect both electrically-evoked 5-HT release and 5-CT-induced inhibition of evoked release measured using fast cyclic voltammetry in vitro. SB-699551-A (0.3, 1 and 3 mg/kg s.c.) did not modulate extracellular levels of 5-HT in the guinea pig frontal cortex in vivo. However, when administered in combination with WAY-100635 (0.3 mg/kg s.c.), SB-699551-A (0.3, 1 or 3 mg/kg s.c.) produced a significant increase in extracellular 5-HT levels. These studies provide evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain.     

G-protein activation at 5-HT1A receptors by the 5-ht1F ligand LY334370 in guinea-pig brain sections and recombinant cell lines

1. G-protein activation by the 5-ht_1F receptor agonist 5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate ({"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370) was investigated by use of autoradiography of receptor-activated G-proteins in guinea-pig brain sections and [³⁵S]-GTPγS binding responses in cell lines stably expressing human 5-HT_1A (h 5-HT_1A) receptors.
2. {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 (10 μM) caused little or no stimulation of [³⁵S]-GTPγS binding in guinea-pig brain regions enriched in 5-ht_1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [³H]-sumatriptan plus 10 nM 5-carboxamidotryptamine (5-CT).
3. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 (10 μM) to guinea-pig brain sections resulted in an increase of [³⁵S]-GTPγS binding in hippocampus (123±17%), lateral septum (58±14%), dorsal raphe (57±10%), entorhinal (37±11%) and cingulate cortex (28±10%). This distribution fits with the G-protein activation mediated by 5-HT_1A receptors as found with lisuride (10 μM), and labelling of 5-HT_1A receptors by 140 pM [¹²⁵I]-4-(2′-methoxy-phenyl)-1-[2′-(n-2′′-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI).
4. The {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-mediated [³⁵S]-GTPγS response was antagonized by the selective, silent 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635, 1 μM) in each of the brain structures investigated. The distribution pattern of the [³⁵S]-GTPγS binding response and the antagonist profile suggest that the {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-induced response in guinea-pig brain is mediated by 5-HT_1A receptors.
5. The maximal {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-induced [³⁵S]-GTPγS binding response (83 to 94%) in membranes of recombinant C6-glial/h 5-HT_1A and HeLa/h 5-HT_1A cells was close to that of 5-HT, suggesting {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 to exert high intrinsic activity at h 5-HT_1A receptors.
6. In conclusion, in guinea-pig brain sections and recombinant cell lines the 5-ht_1F receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 causes G-protein activation that is mediated by 5-HT_1A receptors. Caution should be taken when employing this ligand as a putative selective 5-ht_1F agonist.

     

5-HT1D receptors in guinea-pig and pigeon brain

Summary The characteristics of high affinity [³H]5-HT (5-hydroxytryptamine) binding to non 5-HTIA non 5-HT_1A sites were examined in crude membranes prepared from different regions of guinea-pig and pigeon brains. The coupling of these sites to adenylate cyclase was examined, and its pharmacological profile investigated. In the presence of 100 nmol/1 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) and 100 nmol/l mesulergine, [³H]5-HT labelled with nanomolar affinity an apparently homogeneous population of recognition sites in guinea-pig and pigeon brain membranes. The rank order of affinities of agonists and antagonists (5-CT (5-carboxamidotryptamine) > 5-HT > RU 24969 pyridinyl)-1H indole succinate) > yohimbine rauwolscine > DP-5-CT (N,N dipropyl-5-carboxamidotryptamine) mianserin > 8-OH-DPAT > mesulergine > SDZ 21-009 ((±)-4(3-tert-butyl-amino-2-hydroxypropoxy)-in-dol-2 carbonic acid isopropyl ester) > (-)propranolol), as well as their individual pKD values, were very similar to those at porcine caudate 5-HT_1D sites and clearly different from those at rat cortex 5-HT_1B sites. In the substantia nigra of the guinea-pig the 5-HT receptor-mediated inhibition of forskolin-stimulated adenylate cyclase had a pharmacological profile fully comparable to that of 5-HT_1D binding sites (5-CT > 5-HT > yohimbine > RU 24969 > 8-OH-DPAT > SDZ 21-009 = isamoltane > (–)pindolol > (–)propranolol). The rank order of potency of agonists and antagonists in this system closely paralleled their corresponding rank order of potency in the calf substantia nigra (5-HT_1D), but was clearly different from that in rat substantia nigra (5-HT_1B) These results demonstrate the existence of 5-HT_1D recognition sites in the guinea-pig and pigeon brain and their similarity to 5-HT_1D sites of higher mammals, in terms of both drug affinity profile and second messenger coupling. No evidence of the presence of 5-HTIB sites was obtained. The present findings also suggest that 5-HT_1D sites may be present in the brain of the majority of vertebrate species located higher than the sauropside-mammalian divergence in the phylogenic tree, whereas 5-HT_1B sites are only found in some (e.g., mouse, rat, hamster) but not in other rodents (e.g. guinea-pig).     

Effects of triiodothyronine and imipramine on basal 5-HT levels and 5-HT(1) autoreceptor activity in rat cortex

Clinical studies have shown that triiodothyronine (T3) both augments and accelerates the therapeutic response to antidepressant drugs, particularly tricyclics. There is evidence that this effect is mediated by the serotonergic system. We show here that T3 administered daily for 7 days over the range 0.02-0.5 mg/kg increases basal serotonin (5-hydroxytryptamine, 5-HT) levels, as measured by in vivo microdialysis in rat cortex, in a dose-dependent fashion. All the doses of T3 examined reduced 5-HT(1A) autoreceptor activity, as measured by the effect of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.05 mg/kg s.c.) to decrease 5-HT levels in frontal cortex. T3 administered daily for 14 days at 0.02 mg/kg also reduced 5-HT(1B) autoreceptor activity, as measured by the effect of locally administered 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129, 10 microM) to decrease 5-HT levels. In animals administered imipramine (10 mg/kg/day by osmotic minipump) concurrently with T3 injections, no further changes in either 5-HT(1A) or 5-HT(1B) autoreceptor activity were seen. We suggest that the effect of T3 to accelerate the therapeutic actions of antidepressant drugs may be due to a combination of the actions of T3 at autoreceptors and the actions of the drugs at postsynaptic 5-HT(1A) receptors.     

The pharmacological properties of the presynaptic serotonin autoreceptor in the pig brain cortex conform to the 5-HT1D receptor subtype

Summary The effects of serotonin receptor agonists and antagonists on the electrically (3 Hz) evoked ³H overflow were determined on pig brain cortex slices preincubated with ³H-serotonin and superfused with physiological salt solution containing indalpine (an inhibitor of serotonin uptake) plus phentolamine. The potencies of the serotonin receptor agonists and antagonists were compared with their affinities for 5-HT_1A, 5-HT_1B, 5-HT_1c, and 5-HT_1D binding sites in pig or rat tissue membranes; in addition, the potencies of the agonists were compared to their potencies in inhibiting adenylate cyclase activity in membranes of calf substantia nigra. In the superfusion experiments on pig brain cortex slices the following rank orders of potencies were obtained: agonists, serotonin > 5-methoxytryptamine = 5-carboxamidotryptamine >R U 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) > SDZ 21009 (4(3-terbutylamino- 2-hydroxypropoxy)indol- 2-carbonic-acid-isopropylester) yohimbine cyanopindolol > 8-OHDPAT (8-hydroxy-2-(di-n-propylamino)tetralin) CGS 12066 B (7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrrolo[1,2-a]quinoxaline); ipsapirone and urapidil were ineffective; antagonists (antagonism determined against 5methoxytryptamine as an agonist), metitepine > metergoline > mianserin. Propranolol, spiperone or mesulergine did not produce a shift of the concentration-response curve for 5-methoxytryptamine. The potencies of the serotonin receptor agonists in pig brain cortex slices were significantly correlated with their affinities for 5-HT_1c and 5-HT_1D binding sites in membranes of the pig choroid plexus and caudate nucleus, respectively, but not with their affinities for 5-HT_1A and 5-HT_1B sites in membranes of the cerebral cortex of pig and rat, respectively. The agonist potencies in decreasing ³H overflow were also significantly correlated with their potencies in inhibiting adenylate cylase activity in calf substantia nigra (i.e., a 5-HT_1D receptor-mediated effect). In conclusion, the pig brain cortical 5-HT autoreceptor probably belongs to the 5-HT_1D subtype. The involvement of 5-HT_1c recognition sites was excluded by the low potency of mianserin as an antagonist and, in particular, by the ineffectiveness of the 5-HT_1c receptor antagonist mesulergine.E. S. and M. G. were supported by grants of the Deutsche ForschungsgemeinschaftSend offprint requests to M. Göthert at the above address     

5.HT1 receptors in the vertebrate brain

Summary The regional distribution of high affinity [³3H]5-HT recognition sites in the brain of several vertebrates (pigeon, rat, mouse, guinea-pig, cat, dog, monkey and human) was analyzed using in vitro autoradiography. The presence of subtypes of 5-HT₁ binding sites was investigated by selective displacements with 8-OH-DPAT, mesulergine and (±)SDZ 21-009 at appropriate concentrations to block 5-HT_1A, 5-HT_1c and 5-HT_1B sites respectively. In addition, 5-HT_1A, and 5-HT_1c sites were directly visualized with the more selective radioligands [³H]8-OH-DPAT and [³H]mesulergine, respectively. In the pigeon brain, total [³H]5-HT binding sites were enriched in all telencephalic areas. Densely labelled regions were also present in the optic tectum and the brainstem. No binding was observed in the cerebellum. 8-OH-DPAT and mesulergine only displaced a small proportion of [³H]5-HT binding in most of the areas where high concentrations of 5-HT₁ sites were found. (±)SDZ 21-009 did not affect [³H]5-HT binding in the regions examined. Taking into account our pharmacological studies, these results suggest that the majority of 5-HT₁ sites belong to the 5-HT_1D subtype in the pigeon brain. In the mammalian species investigated high levels of [³H]5-HT binding were found in the neo-cortex, hippocampal formation, basal ganglia and related structures (substantia nigra), raphe dorsalis, nucleus superior colliculus and choroid plexus. However, these brain areas were differentially enriched in subtypes of 5-HT₁ recognition sites. 5-HT_1A sites were observed in the neo-cortex, the hippocampal formation and the raphe nucleus, whereas 5-HT_1C sites accounted for all 5-HT₁ binding in the choroid plexus. In the mouse and rat brain, 5-HT_1B binding sites were enriched in the basal ganglia and associated regions (substantia nigra). These areas were enriched in 5-HT_1D sites in the brain of the other mammals studied. In these animals, no site with a 5-HT_1B pharmacological profile were detected.These results indicate that 5-HT_1A 5-HT_1c and 5-HT_1D sites are present already in the lower vertebrate species investigated and that 5-HT_1B appear to be exclusive of the myomorph rodents (mouse, rat). Furthermore, the different subtypes of the 5-HT₁, receptors present a conserved regional distribution with the 5-HT_1D sites being enriched in the basal ganglia and the 5-HT_1A sites predominating in the hippocampal formation.     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

Time course of the effects of adrenalectomy and corticosterone replacement on 5-HT1A receptors and 5-HT uptake sites in the hippocampus and dorsal raphe nucleus of the rat brain: an autoradiographic analysis

Previous studies have shown that adrenalectomy (ADX) increases the binding of³H-DPAT to 5-HT_1A receptors in the hippocampus (HIP) and this effect is partially overcome by corticosterone (CORT) replacement. The present study investigated the time course of the effects of ADX with or without CORT replacement on serotonin (5-HT) pre- and postsynaptic systems in the HIP and dorsal raphe nucleus (DR) by quantitative autoradiography. In the HIP, ADX for 7, 10 or 14 days caused a significant increase in³H-DPAT binding in the CA₁ region (pyramidal layer), CA_2,3 region (molecular and pyramidal layers) and in the dentate gyrus (molecular and granular layers) which returned to control levels when measurements were made 35 days post-ADX. A decrease in³H-DPAT binding was observed 14 days after ADX in the DR but not in the median raphe nucleus (MR). Although replacement with CORT did not lead to a reversal in³H-DPAT binding at early time points, binding was restored to control levels 7–28 days after CORT replacement in all regions of the HIP. In the DR, CORT did not cause a reversal in³H-DPAT binding at any of the time points examined. In contrast to the effects seen on the 5-HT_1A receptor subtype, no significant change was noted on the binding of³H-CN-IMI to uptake sites for 5-HT in the HIP or DR after ADX or CORT replacement. The results of this study indicate that long-term alterations in the HPA axis lead to changes in the 5-HT_1A receptor system that are both region-specific and time-dependent.     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

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.     

Chronic fluoxetine-induced desensitization of 5-HT1A and 5-HT1B autoreceptors: regional differences and effects of WAY-100635

Desensitization of 5-HT(1A) and 5-HT(1B) autoreceptors is thought to be the mechanism underlying the therapeutic effects of fluoxetine and other selective serotonin reuptake inhibitors when these are administered chronically. The blockade of 5-HT(1A) autoreceptors occurring on administration of a selective serotonin reuptake inhibitor together with a 5-HT(1A) autoreceptor antagonist is responsible for the acute increase in 5-hydroxytryptamine (serotonin, 5-HT) levels observed under these circumstances. The effects of repeated administration of selective serotonin reuptake inhibitors together with 5-HT(1A) receptor antagonists have not been widely studied. In this work, we found that the effects of fluoxetine (5 mg/kg, i.p., daily for 12 days) to desensitize 5-HT(1B) autoreceptors in the frontal cortex, as measured by the effect of the locally administered 5-HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129), and to desensitize 5-HT(1A) autoreceptors as measured by the action of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 50 microg/kg, s.c.) to reduce 5-HT levels in cortex, were prevented by concomitant administration of the 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635; 0.3 mg/kg, s.c.). 5-HT(1B) receptor activity in the hypothalamus, as measured by the effects of locally administered CP 93129, and 5-HT(1A) autoreceptor activity, as determined by the effects of subcutaneous 8-OH-DPAT to reduce 5-HT levels in hypothalamus, were not altered either by fluoxetine alone or by fluoxetine in the presence of WAY-100635. The data suggest that the regulation of extracellular levels of 5-HT in the cortex and hypothalamus is subject to different autoregulatory mechanisms.     

Functional behavioral homology between rat 5-HT1B and guinea pig 5-HT1D receptors in the modulation of prepulse inhibition of startle

The serotonin (5-HT) 1B receptor in rats and mice appears to be homologous to the 5-HT_1D receptor found in other mammals, such as guinea pigs and humans. The present series of experiments explored the functional similarity between the rat 5-HT_1B receptor and the guinea pig 5-HT_1D receptor on two behavioral measures known to be influenced by 5-HT_1B receptor manipulations in rats: prepulse inhibition of the startle response (PPI) and locomotor activity. Because the 5-HT_1B agonist RU 24969 disrupts PPI and stimulates locomotor behavior in rats, it was predicted that the 5-HT_1D agonist, SDZ 219–964, would demonstrate a similar behavioral profile in guinea pigs. In support of this hypothesis, SDZ 219–964 was found to disrupt PPI dose-dependently (1.0 and 2.0 mg/kg) without significantly affecting startle amplitude and to increase locomotor activity (0.5–2.0 mg/kg) in guinea pigs. In guinea pigs, RU 24969 failed to affect PPI, although it did increase locomotor activity, indicating that RU 24969 may have activity at the 5-HT_1D receptor. As expected, RU 24969 in rats disrupted PPI (2.5 and 5.0 mg/kg) and significantly increased locomotor activity (1.25–5.0 mg/kg). In rats, however, SDZ 219–964 had generalized, stimulatory effects on startle reactivity, without independent effects on PPI or locomotor activity. The spatial patterns of locomotion exhibited by guinea pigs treated with SDZ 219–964 versus those of rats treated with RU 24969 demonstrate important qualitative differences in structure, indicating that the neural substrates subserving these effects may be different. It is concluded that a functional similarity exists between 5-HT_1D and 5-HT_1B receptors with regard to the modulation of sensorimotor inhibition and, to a lesser extent, locomotor activity.     

A Search for New 5-HT1A/5-HT2A Receptor Ligands. In Vitro and in vivo Studies of 1-[ω-(4-Aryl-1-piperazinyl)alkyl]indolin-2(1H)-ones

A series of 1-[ω-(4-aryl-1-piperazinyl)alkyl]indolin-2(1H)-one derivatives 2–14 was synthesized in order to obtain ligands with a dual 5-HT_1A/5-HT_2A activity. The majority of those compounds ( 2–5, 7, 10–13 ) exhibited a high 5-HT_1A (K_i = 2 – 44 nM) and/or 5-HT_2A affinity (K_i = 51 and 39 for 5 and 7 , respectively). Induction of lower lip retraction (LLR) and behavioral syndrome and inhibition of these efects evoked by 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) were used for determination the agonistic and antagonistic activity, respectively, at 5-HT_1A receptors. The 5-HT_2A antagonistic activity was assessed by the blocking effect on the head twitches induced by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in mice. Two of the tested compounds, 1-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-6-fluoroindolin-2(1H)-one ( 5 ) and 1-{3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl}indolin-2(1H)-one ( 7 ), demonstrated a high 5-HT_1A/5-HT_2A affinity and an in vivo antagonistic activity towards both receptor subtypes.     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

Postsynaptic 5-HT(1A) receptors mediate an increase in locomotor activity in the monoamine-depleted rat

RATIONALE: In animal models of reduced dopamine transmission, such as haloperidol-induced catalepsy or monoamine-depleted animals, serotonin (5-hydroxytryptamine; 5-HT) 5-HT(1A) agonists appear to enhance motor activity. However, the exact mechanism remains unclear. OBJECTIVE: The objective of the present study was to demonstrate that 5-HT(1A) agonists can increase locomotor activity by activation of postsynaptic 5-HT(1A) heteroreceptors without the involvement of somatodendritic 5-HT(1A) autoreceptors which are known to regulate 5-HT neuronal activity. METHODS: The effects of the 5-HT(1A) full agonist R-(+)-8-hydroxy-2-(di- n-propylamino)tertralin ( R-(+)-8-OHDPAT) on locomotor activity in reserpinized (i.e., monoamine-depleted) rats were studied. RESULTS: The present data demonstrate that R-(+)-8-OHDPAT significantly increased locomotor activity in monoamine-depleted animals at a dose as low as 0.01 mg/kg. The partial 5-HT(1A) agonist/D(2) antagonist buspirone (3 mg/kg) also elevated locomotor activity. The effects of these 5-HT(1A) compounds were found to be similar to the locomotor-stimulating effect of the dopamine precursor 3,4-dihydroxyphenylalanine (150 mg/kg, 15 min after 50 mg/kg benserazide). The 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY 100635; 0.2 mg/kg) blocked the R-(+)-8-OHDPAT (0.03 mg/kg)-mediated increase in locomotion. Blockade of 5-HT synthesis with DL- p-chlorophenylalanine (pCPA), a tryptophan hydroxylase inhibitor, prior to reserpinization did not affect R-(+)-8-OHDPAT-induced locomotion. CONCLUSIONS: The present data indicate that R-(+)-8-OHDPAT can increase motor activity in monoamine-depleted rats through postsynaptic 5-HT(1A) receptors and not necessarily through 5-HT(1A) autoreceptor-mediated alterations in 5-HT synthesis and release. A potential mechanism of 5-HT(1A)-mediated modulation of non-monoaminergic motor circuits in the brain is discussed. Taken together, the results suggest that 5-HT(1A) agonists would provide a novel approach to the amelioration of antipsychotic-induced side effects and the symptomatic treatment of Parkinson's disease.     

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

Interaction of arylpiperazines with 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D receptors: do discriminatory 5-HT1B receptor ligands exist?

Summary The effects of several putative 5-HT₁ receptorsubtype selective ligands were investigated in biochemical models for 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors (inhibition of forskolin-stimulated adenylate cyclase activity in calf hippocampus, rat and calf substantia nigra, respectively) and 5-HT_1C receptors (stimulation of inositol phosphates production in pig choroid plexus). Following compounds were studied: 5-HT (5-hydroxytryptamine), TFMPP (1-(mtrifluoromethylphenyl)piperazine), mCPP (1-m-chlorophe-nyl)piperazine, 1 CGS 12066 (7-trifluoromethyl-4-(4-methyl1-piperazinyl)-pyrrolo[1,2-a]quinoxaline 1), isamoltane (CGP 361A, 1-(2-(1-pyrrolyl)-phenoxy)-3-isopropylamino-2-propranol), quipazine, 1-NP (1-(1-naphthyl)piperazine), and PAPP (LY165163, 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine). Among reported 5-HT_1B receptor selective drugs, TFMPP had similar potency at 5HT_1A, 5-HT_1B and 5-HT_1C receptors, mCPP did not separate between 5-HT_1B and 5-HT_1C receptors, CGS 12066 was equipotent at 5-HT_1B and 5-HT_1D receptors, and isamoltane was only slightly 5-HTIB versus 5-HT_1A selective. Quipazine showed equal potency at 5-HTIB and 5-HT_1C receptors and 1-NP did not discriminate between the four receptor subtypes. PAPP described as 5-HT_1A receptor selective, was equally potent at 5-HT_1A and 5-HT_1D receptors. The potencies determined in second messenger studies were in good agreement with the affinity values determined in radioligand binding studies. Thus 5-HT_1A, 5-HT_1B, 5-HT_1C and 5-HT_1D receptors have different pharmacological profiles as predicted from radioligand binding studies. Despite claims to the contrary, none of the tested compounds had actual selectivity for a given 5-HT₁ receptor subtype. Of interest were the properties of several of these drugs, which behaved as agonists at some receptors and as antagonists at others (e. g. quipazine, 1-NP, PAPP and isamoltane). Send offprint requests to D. Hoyer at the above address     

The effect of the selective 5-HT1A agonists alnespirone (S-20499) and 8-OH-DPAT on extracellular 5-hydroxytryptamine in different regions of rat brain

1. We have examined the effects of the systemic administration of the selective 5-HT_1A agonist alnespirone (S-20499) on in vivo 5-hydroxytryptamine (5-HT) release in the dorsal raphe nucleus, the median raphe nucleus and four forebrain areas innervated differentially by both (dorsal striatum, frontal cortex, ventral hippocampus and dorsal hippocampus).
2. Alnespirone (0.1–3 mg kg⁻¹, s.c.) dose-dependently reduced extracellular 5-HT in the six areas examined. In forebrain, the maximal reductions occurred in striatum and frontal cortex (maximal reduction to 23 and 29% of baseline, respectively). Those in dorsal and ventral hippocampus were more moderate (to ca 65% of baseline). In contrast, the decrease in 5-HT elicited in the median raphe nucleus was more marked than that in the dorsal raphe nucleus (to ca 30 and 60% of baseline, respectively). The selective 5-HT_1A antagonist WAY-100635 (0.5 mg kg⁻¹, s.c.) prevented the decrease in 5-HT induced by alnespirone (0.3 mg kg⁻¹, s.c.) in frontal cortex.
3. 8-OH-DPAT (0.025, 0.1 and 0.3 mg kg⁻¹, s.c.) also reduced extracellular 5-HT in a regionally-selective manner (e.g., to 32% of baseline in striatum and to 69% in dorsal hippocampus at 0.1 mg kg⁻¹, s.c.). In midbrain, 8-OH-DPAT reduced the dialysate 5-HT slightly more in the median than in the dorsal raphe nucleus at all doses examined.
4. Doses of both compounds close to their respective ED₅₀ values (0.3 mg kg⁻¹ alnespirone, 0.025 mg kg⁻¹ 8-OH-DPAT) reduced 5-HT to a comparable extent in all regions examined. However, the reductions attained at higher doses were more pronounced for 8-OH-DPAT.
5. These data show that the reduction of 5-HT release elicited by alnespirone and 8-OH-DPAT is more important in forebrain areas innervated by 5-hydroxytryptaminergic neurones of the dorsal raphe nucleus. This regional selectivity seems unlikely to be accounted for by differences in the sensitivity of 5-HT_1A autoreceptors controlling 5-HT release, given the dissimilar effects of these two 5-HT_1A agonists in regions rich in cell bodies and nerve terminals. This suggests the presence of complex mechanisms of control of 5-HT release by 5-HT_1A receptors.

     

Adenovirus-mediated expression of 5-HT1B receptors in cardiac ventricle myocytes; coupling to inwardly rectifying K channels

The 5-HT_1B receptor is expressed on nerve terminals where it inhibits neurotransmitter release. When expressed ectopically in fibroblasts, the 5-HT_1B receptor inhibits adenylyl cyclase. However, in the central nervous system, the effect of this receptor on neurotransmitter release appears to be cAMP-independent. We therefore investigated alternative effector systems that might be activated by the 5-HT_1B receptor. We constructed a recombinant adenovirus that allows expression of high levels of the 5-HT_1B receptor in a variety of cells. We chose cardiac ventricle myocytes because they express a muscarinic-gated, inwardly rectifying K⁺ channel (i_KACh). In infected ventricle cells, both 5-HT and the muscarinic receptor agonist, carbachol, elicited a similar inwardly rectifying K⁺ current. The currents elicited by these agonists were pertussis-toxin sensitive and were not additive. These results suggest a common signal transduction pathway for 5-HT_1B and muscarinic receptors in ventricle cells.     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.