Serotonergic regulation of blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor characterization

The present study investigated the site of action of 5-hydroxytryptamine (5-HT) and pharmacologically characterized the receptors involved in regulating blood glucose levels in the crayfish, Procambarus clarkii. Injection of 5-HT into intact animals increased glucose levels in a dose-dependent manner. In contrast, 5-HT failed to elicit a hyperglycemic response in eyestalk-ablated animals. Effects of several 5-HT receptor agonists and antagonists were examined. 5-CT, oxymetazoline (both 5-HT(1) receptor agonists) and alpha-methyl-5-HT (a 5-HT(2) receptor agonist), but not 1-phenylbiguanide, m-CPBG (both 5-HT(3) receptor agonists), or RS 67333 (a 5-HT(4) receptor agonist), induced hyperglycemic responses in a dose-dependent manner. In addition, 8-OH-DPAT (a 5-HT(1A) receptor agonist), L-694,247 (a 5-HT(1B/1D) receptor agonist), and DOI (a 5-HT(2A) receptor agonist) were effective in significantly increasing the glucose levels, whereas both BW 723C86 (a 5-HT(2B) receptor agonist) and m-CPP (a 5-HT(2C) receptor agonist) were ineffective. Finally, ketanserin (a 5-HT(2A) receptor antagonist), but not p-MPPF (a 5-HT(1A) receptor antagonist), GR 55562 (a 5-HT(1B/1D) receptor antagonist), SB 206553 (a 5-HT(2B/2C) receptor antagonist), or tropisetron (a 5-HT(3) receptor antagonist), was able to block 5-HT-induced hyperglycemia. The combined results support the hypothesis that 5-HT exerts its hyperglycemic effect by enhancing the release of hyperglycemic factor(s) from the eyestalks, and suggest that 5 HT-induced hyperglycemia is mediated by 5-HT(1)- and 5-HT(2)-like receptors.     

In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice

In the dorsal raphe nucleus (DR), extracellular serotonin (5-HT) regulates serotonergic transmission through 5-HT1A autoreceptors. In this work we used in vivo microdialysis to examine the effects of stressful and pharmacological challenges on DR 5-HT efflux in 5-HT1A receptor knockout (5-HT1A-/-) mice and their wild-type counterparts (5-HT1A+/+). Baseline 5-HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5-HT1A autoreceptors on DR 5-HT release. (R)-(+)-8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT, 0.5 mg/kg) reduced 5-HT levels to 30% of basal values in 5-HT1A+/+ mice, but not in 5-HT1A-/- mice. The selective 5-HT1B receptor agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129, 300 micro m) reduced dialysate 5-HT to the same extent (30-40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5-HT1B receptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5-HT in mutants, but not in 5-HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5-HT in a dose-dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5-HT1A-/- mice. The increased responsiveness of the extracellular 5-HT in the DR of 5-HT1A receptor knockout mice reflects a lack of the autoinhibitory control exerted by 5-HT1A autoreceptors.     

Regional changes in density of serotonin transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the 5-HT1B knockout

5-HT1A knockout (KO) mice display an anxious-like phenotype, whereas 5-HT1B KOs are over-aggressive. To identify serotoninergic correlates of these altered behaviors, autoradiographic measurements of 5-HT1A and 5-HT1B serotonin (5-HT) receptors and transporter (5-HTT) were obtained using the radioligands [3H]8-OH-DPAT, [125I]cyanopindolol and [3H]citalopram, respectively. By comparison to wild-type, density of 5-HT1B receptors was unchanged throughout brain in 5-HT1A KOs, and that of 5-HT1A receptors in 5-HT1B KOs. In contrast, decreases in density of 5-HTT binding were measured in several brain regions of both genotypes. Moreover, 5-HTT binding density was significantly increased in the amygdalo-hippocampal nucleus and ventral hippocampus of the 5-HT1B KOs. Measurements of 5-HT axon length and number of axon varicosities by quantitative 5-HT immunocytochemistry revealed proportional increases in the density of 5-HT innervation in these two regions of 5-HT1B KOs, whereas none of the decreases in 5-HTT binding sites were associated with any such changes. Several conclusions could be drawn from these results: (i) 5-HT1B receptors do not adapt in 5-HT1A KOs, nor do 5-HT1A receptors in 5-HT1B KOs. (ii) 5-HTT is down-regulated in several brain regions of 5-HT1A and 5-HT1B KO mice. (iii) This down-regulation could contribute to the anxious-like phenotype of the 5-HT1A KOs, by reducing 5-HT clearance in several territories of 5-HT innervation. (iv) The 5-HT hyperinnervation in the amygdalo-hippocampal nucleus and ventral hippocampus of 5-HT1B KOs could play a role in their increased aggressiveness, and might also explain their better performance in some cognitive tests. (v) These increases in density of 5-HT innervation provide the first evidence for a negative control of 5-HT neuron growth mediated by 5-HT1B receptors.     

Serotonin decreases generation of dopaminergic neurons from mesencephalic precursors via serotonin type 7 and type 4 receptors

Inductive signals mediating the differentiation of neural precursors into serotonergic (5-HT) or dopaminergic neurons have not been clarified. We have recently shown that in cell aggregates obtained from rat mesencephalic precursors, reduction of serotonin levels induces a marked increase in generation of dopaminergic neurons. In the present study we treated rat neurospheres with antagonists of the main subtypes of 5-HT receptors, 5-HT transport inhibitors, or 5-HT receptor agonists, and studied the effects on generation of dopaminergic neurons. Cultures treated with Methiothepin (5-HT(1,2,5,6,7) receptor antagonist), the 5-HT(4) receptor antagonist GR113808;67:00-.or the 5-HT(7) receptor antagonist SB 269970 showed a significant increase in generation of dopaminergic cells. Treatment with the 5-HT(1B/1D) antagonist GR 127935, the 5-HT(2) antagonist Ritanserin, the 5-HT transporter inhibitor Fluoxetine, the dopamine and norepinephrine transport inhibitor GBR 12935, or with both inhibitors together, or 5-HT(4) or 5-HT(7) receptor agonists induced significant decreases in generation of dopaminergic cells. Cultures treated with WAY100635 (5-HT(1A) receptor antagonist), the 5-HT(3) receptor antagonist Ondasetron, or the 5-HT(6) receptor antagonist SB 258585 did not show any significant changes. Therefore, 5-HT(4) and 5-HT(7) receptors are involved in the observed serotonin-induced decrease in generation of dopaminergic neurons from proliferating neurospheres of mesencephalic precursors. 5-HT(4) and 5-HT(7) receptors were found in astrocytes and serotonergic cells using double immunolabeling and laser confocal microscopy, and the glial receptors appeared to play a major role.     

The influence of substitution at aromatic part of 1,2,3,4-tetrahydroisoquinoline on in vitro and in vivo 5-HT(1A)/5-HT(2A) receptor activities of its 1-adamantoyloaminoalkyl derivatives.

Further structure-activity relationship (SAR) studies with the 1,2,3,4-tetrahydroisoquinoline (THIQ) class of 5-HT(1A) ligands led to the synthesis of new 1-adamantoyloaminoalkyl derivatives. The impact of substituent variations in the aromatic part of THIQ moiety on 5-HT(1A) and 5-HT(2A) receptor affinities, as well as in vivo functional properties of the investigated compounds were discussed. It was found that those modifications reduced the binding affinity for 5-HT(1A) receptors (in comparison with unsubstituted THIQ derivatives); however, the majority of new compounds still remained potent 5-HT(1A) ligands (K(i)=4.9-46 nM) and most of them showed features of partial agonists of postsynaptic 5-HT(1A) receptors. At the same time, their 5-HT(2A) receptor affinity was slightly increased (K(i)=40-1475 nM), which resulted in a loss of 5-HT(2A)/5-HT(1A) selectivity. 5-Br,8-OCH3 derivative-the most potent, mixed 5-HT(1A)/5-HT(2A) ligand-produced activation of presynaptic 5-HT(1A) receptors and showed properties of a 5-HT(2A) receptor antagonist.     

5-hydroxytryptamine induces mast cell adhesion and migration

The neurotransmitter serotonin (5-hydroxytryptamine (5-HT)) is implicated in enhancing inflammatory reactions of skin, lung, and gastrointestinal tract. To determine whether 5-HT acts, in part, through mast cells (MC), we first established that mouse bone marrow-derived MC (mBMMC) and human CD34(+)-derived MC (huMC) expressed mRNA for multiple 5-HT receptors. We next determined the effect of 5-HT on mouse and human MC degranulation, adhesion, and chemotaxis. We found no evidence that 5-HT degranulates MC or modulates IgE-dependent activation. 5-HT did induce mBMMC and huMC adherence to fibronectin; and immature and mature mBMMC and huMC migration. Chemotaxis was accompanied by actin polymerization. Using receptor antagonists and pertussis toxin, we identified 5-HT(1A) as the principal receptor mediating the effects of 5-HT on MC. mBMMC from the 5-HT(1A) receptor knockout mouse (5-HT(1A)R(-/-)) did not respond to 5-HT. 5-HT did induce accumulation of MC in the dermis of 5-HT(1A)R(+/+) mice, but not in 5-HT(1A)R(-/-) mice. These studies are the first to demonstrate an effect of 5-HT on MC. Furthermore, both mouse and human MC respond to 5-HT through the 5-HT(1A) receptor. Our data are consistent with the conclusion that 5-HT promotes inflammation by increasing MC at the site of tissue injury.     

Effects of 5-hydroxytryptamine on cat spinal motoneurons

The effects of 5-hydroxytryptamine (5-HT) on spinal motoneurons were examined in pentobarbital-anaesthetized cats and in nonanaesthetized decerebrate cats by intracellular recording and extracellular iontophoresis of 5-HT. 5-HT first induced a depolarization and then a long-lasting hyperpolarization (up to 60 min) with unchanged input resistance. The slow hyperpolarization was prevented by the 5-HT antagonists ketanserin (5-HT2), methysergide, and spiperone (5-HT1,2) and mimicked by the agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (5-HT2). The post-spike after hyperpolarization was enhanced after application of 5-HT. A depolarization was induce by the 5-HT agonists (+/-)-8-hydroxy-(2)-(di-n-propylamino)tetralin (5-HT1A) and 1-(2-methoxyphenyl)piperazine (5-HT1). Possible mechanisms for the 5-HT-induced hyperpolarization and its intracellular medication are discussed. The present data suggest multiple effects of 5-HT on cat spinal motoneurons.     

Effect of vasoactive intestinal peptide on serotonin metabolism in the suprachiasmatic area of the rat: mechanism of action

Vasoactive intestinal peptide (VIP) inhibits serotonin (5-HT) uptake in the suprachiasmatic area (SCA) of the rat. The present study investigates the possibility of a functional relationship between 5-HT uptake mechanisms and 5-HT autoreceptor activity in this effect of VIP in the SCA. The hypothesis of a linkage between these two mechanisms of 5-HT regulation has been recently proposed. We investigated the possibility of the presence of 5-HT autoreceptors in the SCA. Using superfusion system, exogenous 5-HT (500 and 50 nM) increased the release of newly synthesized 3H-5-HT. In contrast, 5 nM of exogenous 5-HT inhibited this release. This latter effect was antagonized by methiothepin (10(-7) M). In contrast, the concentration of methiothepin required to inhibit the VIP effect was 10(-6) or 10(-5) M, the same molarity found to decrease the 5-HT uptake. On the other hand, the increase of the 3H-5-HT in the synaptic cleft, induced by VIP, did not modify the inhibition of 3H-5-HT release induced by 5 nM of exogenous 5-HT. We conclude that the effect of VIP on 5-HT metabolism in the SCA is linked to the 5-HT uptake mechanism but not to the activity of 5-HT presynaptic autoreceptors. In our experimental conditions, the activity of 5-HT autoreceptors is independent of the 5-HT uptake processes.     

Cell surface expression of 5-hydroxytryptamine type 3 receptors is controlled by an endoplasmic reticulum retention signal

Two subunits of the 5-hydroxytryptamine type 3 (5-HT3) have been identified (5-HT3A and 5-HT3B) that assemble into homomeric (5-HT3A) and heteromeric (5-HT3A+5-HT3B) complexes. Unassembled 5-HT3B subunits are efficiently retained within the cell. In this study, we address the mechanism controlling the release of 5-HT3B from the endoplasmic reticulum (ER). An analysis of chimeric 5-HT3A receptor(R).5-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression. To investigate this possibility, truncated 5-HT3B subunits were constructed and assessed for their ability to access the cell surface in COS-7 and ts201 cells. Using this approach, we have identified the presence of an ER retention signal located within the first cytoplasmic loop between transmembrane domains I and II of 5-HT3B. Transplantation of this signal (CRAR) into the homologous region of 5-HT3A results in the ER retention of this subunit until rescued by co-assembly with wild-type 5-HT3A. The mutation of this ER retention signal in 5-HT3B (5-HT3BSGER) does not lead to cell surface expression, suggesting the presence of other signals or mechanisms to control the surface expression of 5-HT3BRs. The generation of truncated 5-HT3BSGER constructs confirmed that the CRAR signal does play an important role in the ER retention of 5-HT3B.     

Arene- and quinoline-sulfonamides as novel 5-HT7 receptor ligands

Novel arene- and quinolinesulfonamides were synthesized using different solutions and a solid-support methodology, and were evaluated for their affinity for 5-HT(1A), 5-HT(2A), 5-HT(6), and 5-HT(7) receptors. Compound 54 (N-Ethyl-N-[4-(1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinolin-2-yl)butyl]-8-quinolinesulfonamide) was identified as potent 5-HT(7) antagonist (K(i)=13 nM, K(B)=140 nM) with good selectivity over 5-HT(1A), 5-HT(2A), 5-HT(6) receptors. In the FST in mice, it reduced immobility in a manner similar to the selective 5-HT(7) antagonist SB-269970.     

5-HT(2A) receptors: location and functional analysis in intestines of wild-type and 5-HT(2A) knockout mice

The distribution and function of the 5-hydroxytryptamine (5-HT(2A)) receptor were investigated in the intestines of wild-type (5-HT(2A) +/+) and knockout (5-HT(2A) -/-) mice. In 5-HT(2A) +/+ mice, rats, and guinea pigs, 5-HT(2A) receptor immunoreactivity was found on circular and longitudinal smooth muscle cells, neurons, enterocytes, and Paneth cells. Muscular 5-HT(2A) receptors were concentrated in caveolae; neuronal 5-HT(2A) receptors were found intracellularly and on the plasma membranes of nerve cell bodies and axons. Neuronal 5-HT(2A) immunoreactivity was detected as early as E14 in ganglia, intravillus nerves, and the deep muscle plexus. The 5-HT(2A) -/- colon did not express 5-HT(2A) receptors and did not contract in response to exogenous 5-HT. 5-HT(2A) -/- enterocytes were smaller, Paneth cells fewer, and muscle layers thinner (and showed degeneration) compared with those of 5-HT(2A) +/+ littermates. The 5-HT(2A) receptor may thus be required for the maintenance and/or development of enteric neuroeffectors and other enteric functions, although gastrointestinal and colonic transit times in 5-HT(2A) -/- and +/+ mice did not differ significantly.     

Functional characterization of alternatively spliced 5-HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode,Ascaris suum

Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.     

Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.     

Characterization of a Novel Serotonin Receptor Subtype (5-HTls) in Rat CNS: Interaction with a GTP Binding Protein

Three pharmacologically distinct high-affinity [3H]serotonin ([3H]5-HT) binding sites were identified in spinal cord synaptosomes. [3H]5-HT competition studies using selective 5-HT1A receptor ligands indicated that approximately 25% of high-affinity synaptosomal [3H]5-HT binding was inhibited by 5-HT1A-selective compounds, an estimate consistent with [3H](+-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) saturation experiments in which 5-HT1A receptors were directly labeled. [3H]5-HT competition studies using high-affinity 5-HT1B compounds performed in the presence of 100 nM 8-OH-DPAT (to block 5-HT1A receptors) indicated that approximately 26% of all specific, high-affinity [3H]5-HT binding to spinal cord synaptosomes was to 5-HT1B receptors. [3H]5-HT competition studies performed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (to block 5-HT1A and 5-HT1B receptors, respectively) indicated that the remaining 49% of [3H]5-HT binding did not possess the pharmacologic profile previous reported for 5-HT1C, 5-HT1D, 5-HT1E, 5-HT2, or 5-HT3 receptors. This residual 49% of [3H]5-HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (subsequently referred to as "5-HT1S") displayed high affinity and saturability (KD = 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 microM GTP or the nonhydrolyzable form of GTP, 5'-guanylylimidodiphosphate, inhibited [3H]5-HT binding to 5-HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice

In previous studies, we have found that 5-hydroxytryptamine (5-HT) is a potent stimulant of duodenal mucosal bicarbonate secretion (DMBS) in mice. The aim of the present study was to determine the intracellular signaling pathways and 5-HT receptor subtypes involved in 5-HT-induced DMBS. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. 5-HT receptor involvement in DMBS was inferred from pharmacological studies by using selective 5-HT receptor antagonists and agonists. The expression of 5-HT(4) receptor mRNA in duodenal mucosa and epithelial cells was analyzed by RT-PCR. cAMP-dependent signaling pathway inhibitors MDL-12330A, Rp-cAMP, and H-89 and Ca(2+)-dependent signaling pathway inhibitors verapamil and W-13 markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and short-circuit current (I(sc)), whereas cGMP-dependent signaling pathway inhibitors NS-2028 and KT-5823 failed to alter these responses. Both SB-204070 and high-dose ICS-205930 (selective 5-HT(4) receptor antagonists) markedly inhibited 5-HT-stimulated bicarbonate secretion and I(sc), whereas methiothepine (5-HT(1) receptor antagonist), ketanserin (5-HT(2) receptor antagonist), and a low concentration of ICS-205930 (5-HT(3) receptor antagonist) had no effect. RS-67506 (partial 5-HT(4) receptor agonist) concentration-dependently increased bicarbonate secretion and I(sc), whereas 5-carboxamidotryptamine (5-HT(1) receptor agonist), alpha-methyl-5-HT (5-HT(2) receptor agonist), and phenylbiguanide (5-HT(3) receptor agonist) did not significantly increase bicarbonate secretion or I(sc). RT-PCR analysis confirmed the expression of 5-HT(4) receptor mRNA in murine duodenal mucosa and epithelial cells. These results demonstrate that 5-HT regulates DMBS via both cAMP- and Ca(2+)-dependent signaling pathways and 5-HT(4) receptors located in the duodenal mucosa and/or epithelial cells.     

Endogenous 5-HT2B receptor activation regulates neonatal respiratory activity in vitro

An implication of 5-HT(2B) receptors in central nervous system has not yet been clearly elucidated. We studied the role of different 5-HT(2) receptor subtypes in the medullary breathing center, the pre-B?tzinger complex, and on hypoglossal motoneurons in rhythmically active transversal slice preparations of neonatal rats and mice. Local microinjection of 5-HT(2) receptor agonists revealed tonic excitation of hypoglossal motoneurons. Excitatory effects of the 5-HT(2B) receptor agonist BW723C86 could be blocked by bath application of LY272015, a highly selective 5-HT(2B) receptor antagonist. Excitatory effects of the 5-HT(2A/B/C) receptor agonist alpha-methyl 5-HT could be blocked by the preferential 5-HT(2A) receptor antagonist ketanserin. Therefore, 5-HT-induced excitation of hypoglossal motoneurons is mediated by convergent activation of 5-HT(2A) and 5-HT(2B) receptors. Local microinjection of BW723C86 in the pre-B?tzinger complex increased respiratory frequency. Bath application of LY272015 blocked respiratory activity, whereas ketanserin had no effect. Therefore, endogenous 5-HT appears to support tonic action on respiratory rhythm generation via 5-HT(2B) receptors. In preparations of 5-HT(2B) receptor-deficient mice, respiratory activity appeared unaltered. Whereas BW723C86 and LY272015 had no effects, bath application of ketanserin disturbed and blocked rhythmic activity. This demonstrates a stimulatory role of endogenous 5-HT(2B) receptor activation at the pre-B?tzinger complex and hypoglossal motoneurons that can be taken up by 5-HT(2A) receptors in the absence of 5-HT(2B) receptors. The presence of functional 5-HT(2B) receptors in the neonatal medullary breathing center indicates a potential convergent regulatory role of 5-HT(2B) and -(2A) receptors on the central respiratory network.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

Serotonin 5-HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Radioligand binding studies were performed to characterize serotonin 5-HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5-HT binding, in the presence of pindolol (to block 5-HT1A and 5-HT1B receptors) and mesulergine (to block 5-HT1C receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5-HT-labeled 5-HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8-hydroxydipropylaminotetralin, trifluoromethylphenylpiperazine, mesulergine, 4-bromo-2,5-dimethoxyphenylisopropylamine, and ICS 205-930 had low affinity for [3H]5-HT-labeled 5-HT1D sites, indicating that the pharmacology of the 5-HT1D site is distinct from that of previously identified 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 sites. 5-HT1D sites in human brain have a similar pharmacology to the 5-HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imido)-triphosphate (Gpp(NH)p), modulated the binding of [3H]5-HT to 5-HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5-HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.