Reduction of the serotonin 5-HT1B and 5-HT2A receptor-mediated contraction of human pulmonary artery by the combined 5-HT1B receptor antagonist and serotonin transporter inhibitor LY393558.

BACKGROUND: LY393558 is a combined antagonist of serotonin (5-HT) 5-HT1B receptors and inhibitor of serotonin transporter (SERT). LY393558 reduces 5-HT-induced vasoconstriction and remodelling of rat and/or mouse pulmonary arteries. The aim of our study was to examine the effect of LY393558 on the 5-HT-stimulated vasoconstriction of human pulmonary arteries (hPAs) and to determine the underlying mechanism(s). METHODS: Vascular effects of 5-HT receptor agonists, antagonists and a SERT inhibitor were examined in organ bath studies on intralobar hPAs obtained from patients during resection of lung carcinoma. RESULTS: Serotonin and agonists of the 5-HT1B receptor (5-carboxamidotryptamine, 5-CT) and 5-HT2A receptor (α-methyl-5-HT) contracted endothelium-intact hPAs in a concentration-dependent fashion. The 5-HT1B antagonists SB224289 and GR55562 reduced responses induced by 5-HT and 5-CT and the 5-HT2A antagonist ketanserin inhibited the effects of 5-HT and α-methyl-5-HT. Administration of the SERT inhibitor citalopram (at a concentration that failed to modify the 5-HT-induced vasoconstriction) in combination with SB224289 or GR55562 was more effective in inhibiting the response to 5-HT than the 5-HT1B antagonists alone. LY393558 showed the greatest antagonistic effect against the vasoconstriction elicited by 5-HT, 5-CT and α-methyl-5-HT. CONCLUSIONS: LY393558 reduces the 5-HT-induced contraction antagonizing 5-HT1B and 5-HT2A receptors probably due to synergic interaction between SERT inhibition and 5-HT1B receptor antagonism. Thus, it might represent a valuable future option in the pulmonary arterial hypertension therapy.

Serotonin discovery and stepwise disclosure of 5-HT receptor complexity over four decades. Part II. Some contributions of Manfred Göthert.

About 40% of the papers within the scientific oeuvre of Manfred Göthert (1939-2019) were dedicated to serotonin (5-hydroxytryptamine, 5-HT). He was not only the witness of the gradual definition of the fourteen 5-HT receptor subtypes but also was involved directly by identifying 5-HT1B, 5-HT1D and 5-HT3 receptors. Moreover, he identified presynaptic 5-HT receptors on central and/or peripheral serotoninergic, noradrenergic and/or cholinergic neurones. Two inhibitory (5-HT1B, 5-HT1D) and two facilitatory (5-HT3, 5-HT4) receptors were found, the 5-HT1B receptor representing a possible target for antidepressant drugs. Ten years earlier than electrophysiologists, he identified ligand-gated receptors like the 5-HT3 and the nicotinic acetylcholine (nACh) receptor as targets of halothane. Simultaneously with, but independent of, other authors he found that ethanol allosterically inhibits N-methyl-D-aspartate (NMDA) receptors, which are affected at an even lower concentration than 5-HT3 and nACh receptors. The latter two receptors were shown to be subject to allosteric inhibition also by cannabinoids via a mechanism unrelated to cannabinoid CB1 or CB2 receptors; cannabinoid inhibition of 5-HT3 receptors may represent a new target for the treatment of neuropathic pain.

Serotonin discovery and stepwise disclosure of 5-HT receptor complexity over four decades. Part I. General background and discovery of serotonin as a basis for 5-HT receptor identification.

This review contains background information on the serotonin system, furthermore the suggestion to introduce the term Contemporary Witness Report (CWR) for a novel type of review and, as the main part, an overview over the history of serotonin discovery as a basis for the identification of its receptor heterogeneity and the increase in complexity by genetic and allosteric variation. The present article conforms to CWRs in historical and autobiographical elements, in more emphasis on the author's work than in conventional reviews and in aspects neglected in previous reviews, but not in the main feature namely the work of a scientist with comprehensive expertise in a field in which, over long time, he/she continuously performed research and published. A scientist complying with these requirements is a contemporary witness in that field. His report on the scientific achievements in that period, a CWR, comprises confirmation and putative re-interpretation of data from a superior viewpoint. Identification of serotonin's vascular properties (publication year: 1912) as an "adrenaline mimicking substance" (without attempt to isolate it) by O'Connor preceded the discovery of serotonin in the gastrointestinal tract by Erspamer [1937] and in blood by Rapport [1948, 1949], who identified its structure as 5-hydroxytryptamine [1949]. Detection as a neurotransmitter in invertebrates suggested its occurrence in vertebrate CNS as well. This was verified by finding it in dog, rat and rabbit brain [1953]. The Falck-Hillarp technique [1962] visualized serotonin neurones as fluorescent structures. The neurotoxin 5,7-dihydroxytryptamine [1972] indirectly proved the involvement of 5-HT in multiple CNS functions.

Differences between human wild-type and C23S variant 5-HT2C receptors in inverse agonist-induced resensitization.

The aim of this study was to analyze functional properties of the naturally occurring C23S variant of the human 5-HT2C receptor. In HEK293 cells transiently expressing the unedited forms of the variant receptor (VR) or the wild-type receptor (WTR), surface expression was determined by [3H]mesulergine binding to membrane fragments. Function was examined by an aequorin luminescence-based Ca2+ assay. Surface expression of the VR was 116% of that of the WTR. The 5-HT-induced increase in cytosolic Ca2+ ([Ca2+]i), and its inhibition by the inverse agonist SB 206553 did not differ between VR- or WTR-expressing cells. Preexposure of VR- or WTR-expressing cells to 0.5 µM 5-HT (3 min-4.5 h) led to a practically identical time course and extent in the reduction of the 5-HT-induced increase in [Ca2+]i. In contrast, prolonged preexposure to the inverse agonist SB 206553 (1 µM) elevated the 5-HT-induced increase in [Ca2+]i for both isoreceptors. A preexposure time of 4.5 h was necessary to significantly elevate the Ca2+ response of the WTR, but the VR produced this elevation within 1 h with virtually no further effect after 4.5 h of preexposure. In conclusion, prolonged preexposure to 5-HT caused equally rapid and strong desensitization of both isoreceptors. The different time course of SB 206553-induced resensitization of the two isoreceptors might be therapeutically relevant for drugs exhibiting inverse agonist properties at 5-HT2C receptors, such as atypical antipsychotics and certain antidepressants.

Characterization of the naturally occurring Arg344His variant of the human 5-HT 3A receptor.

The present study aimed at examining the function and pharmacological properties of the naturally occurring Arg344His variant of the human 5-HT(3A) receptor, identified in a schizophrenic patient. In intact human embryonic kidney (HEK) 293 cells expressing the wild-type (WT) or the variant receptor, the function was analyzed by indirect measurement of agonist-induced Ca(2+) current through the 5-HT(3A) receptor channel by an aequorin luminescence-based Ca(2+) assay. In cell membrane patches cation currents were determined electrophysiologically including technically demanding single channel analyses. The pharmacological properties were analyzed by [(3)H]GR65630 binding to cell membrane fragments. The density of [(3)H]GR65630 binding sites in cells expressing the variant receptor was reduced to 55% of that in cells expressing the WT receptor, which, however, was not accompanied by an analogous decrease in 5-HT-induced Ca(2+) influx through the receptor channel. However, the single channel analysis suggests an increase in single receptor channel mean open time (which is known to be subject of many variables) but not in unitary current amplitude. Radioligand competition experiments revealed that the affinity of five 5-HT(3) receptor agonists and four antagonists for the variant receptor did not differ from that for the WT receptor. In conclusion, the variant receptor resembles the WT receptor in that it forms functional homopentameric 5-HT(3A) receptors with identical pharmacological properties. In view of the lack of reduction in Ca(2+) flux through the variant receptor channels in spite of the decrease in its density on the cell membrane, the increase in single receptor channel mean open time appears to compensate for the reduction in variant receptor density.

Association of major depression with rare functional variants in norepinephrine transporter and serotonin1A receptor genes.

Dysregulations of central noradrenergic and serotonergic neurotransmission have been suggested to contribute to the pathogenesis of neuropsychiatric disorders such as depression. The norepinephrine transporter (NET; SLC6A2) and the serotonin (5-HT)(1A) receptor (5-HT(1A) receptor; HTR1A) play an important role in central nervous monoaminergic homeostasis. As shown previously, variations in the human NET and 5-HT(1A) receptor genes can alter noradrenergic and serotonergic signaling in the brain: a single nucleotide polymorphism (SNP) in the coding region of the NET gene resulting in a F528C substitution increased plasma membrane expression of this NET variant, and a SNP in the human 5-HT(1A) receptor gene leading to the R219L receptor variant almost abolished cellular signal transduction subsequent to receptor activation. The present study aimed at investigating whether these NET and 5-HT(1A) receptor variants are associated with major depression (MD). The sample comprised 426 patients suffering from unipolar MD as well as 643 healthy control subjects for the variants of the 5-HT(1A) receptor and the NET. Both SNPs were shown to be associated with MD. In conclusion, our results favor the hypothesis that monoaminergic neurotransmission in general and the F528C NET and R219L 5-HT(1A) receptor variants in particular are involved in the pathogenesis of depression.

Regulatory mechanisms underlying agmatine homeostasis in humans.

Regulation of agmatine homeostasis has so far only been poorly defined. In the present study, three mechanisms regulating human agmatine homeostasis were investigated. 1) Enzymatic regulation: expression of arginine decarboxylase, diamine oxidase, and ornithine decarboxylase in human colon neoplastic tissue was, at the mRNA level, about 75% and 50% lower and 150% higher, respectively, than in the adjacent normal tissue; expression of agmatinase was unchanged. 2) Bacteria-derived agmatine: ten representative bacteria strains of the human intestinal microbiota considerably differed in agmatine production and its efflux into their surrounding fluid, suggesting that the composition of the intestinal microbiota influences the agmatine availability in the gut lumen for absorption. 3) Regulation of blood plasma agmatine concentration by the human liver: at low concentrations in portal venous blood plasma, agmatine either slightly increased or further decreased in blood plasma through liver passage. Above a threshold of 14 ng/ml agmatine in the portal venous blood plasma, substantial hepatic agmatine removal from blood occurred. Taken together, a perturbation of agmatine homeostasis has been proven to be involved in the regulation of malignant cell proliferation. The amount of agmatine available for absorption, which is an important physiological source of agmatine in the human organism, should differ considerably depending on the composition of the bacterial flora in the chyme since the various species of intestinal bacteria largely differ in their ability to form agmatine. Finally, evidence has been presented that the liver plays a crucial physiological role in the maintenance of agmatine homeostasis in the human organism.

Anandamide effects on 5-HT(3) receptors in vivo.

Studies in knockout mouse strains have shown that some cannabimimetic effects persist in animals lacking cannabinoid CB(1) and CB(2) receptors. These residual effects are thought to result, in part, from a cannabinoid-modulation of ion channels. This study investigates the role of 5-HT(3) receptors as a potential in vivo target for cannabinoids. Mice deficient in CB(1) and CB(2) receptors were treated with Delta(9)-tetrahydrocannabinol and anandamide, in the presence of the 5-HT(3) antagonist ondansetron. We show that the cannabinoid receptor-independent anandamide analgesia, but not catalepsy, is completely blocked by ondansetron. Thus, 5-HT(3) receptors seem to be involved in cannabinoid analgesia.

Methanandamide allosterically inhibits in vivo the function of peripheral nicotinic acetylcholine receptors containing the alpha 7-subunit.

Methanandamide (MAEA), the stable analog of the endocannabinoid anandamide, has been proven in Xenopus oocytes to allosterically inhibit the function of the alpha7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 micromol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 micromol/kg) inhibited the electrically induced tachycardia (maximally by 15-20%; abolished by the CB(1) receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 micromol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB(1) receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 micromol/kg) and the selective alpha7-subunit antagonist methyllycaconitine (MLA; 3 and 10 micromol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the alpha7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 micromol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely alpha7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.

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.

Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization.

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/B) receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/B) receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT(3) receptors that is well suited for high-throughput screening.

S1P-receptors in PC12 and transfected HEK293 cells: molecular targets of hypotensive imidazoline I(1) receptor ligands.

The present study aimed at elucidating the molecular identity of the proposed "I(1)-imidazoline receptors", i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [(3)H]clonidine and [(3)H]lysophosphatidic acid on intact, alpha(2)-adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P(1)-, S1P(2)- or S1P(3)-receptor abolished specific [(3)H]lysophosphatidic acid binding. [(3)H]Clonidine binding was markedly decreased by siRNA targeting S1P(1)- and S1P(3)-receptors but not by siRNA against S1P(2)-receptors. Finally, in HEK293 cells transiently expressing human S1P(3)-receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the "I(1)-imidazoline receptors". The present results indicate that the "I(1)-imidazoline receptors" mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I(1) imidazoline receptors represent a mixture of S1P(1)- and S1P(3)-receptors and/or hetero-dimers of both.

Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E.

Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT(3)) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT(3A) and 5-HT(3B) subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT(3) receptors, which cannot solely be explained on the basis of the 5-HT(3A) and 5-HT(3B) subunits. After our discovery of further putative 5-HT(3) serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT(3Ea) are able to form functional 5-HT(3) receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT(3A). To investigate whether the novel subunits modulate 5-HT(3) receptor function, we performed radioligand-binding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT(3C),5-HT(3D), 5-HT(3E), and 5-HT(3Ea) subunits alone cannot form functional receptors. Coexpression with 5-HT(3A), however, results in the formation of functional heteromeric complexes with different serotonin efficacies. Potencies of two agonists and antagonists were nearly identical with respect to homomeric 5-HT(3A) and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT(3A/D) and 5-HT(3A/E) receptors, which is consistent with the increased surface expression compared with 5-HT(3A) receptors. In contrast, 5-HT(3A/C) and 5-HT(3A/Ea) receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT(3) subunits are able to form heteromeric 5-HT(3) receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT(3A) receptors.

Characterization of an antiproliferative effect of imidazoline receptor ligands on PC12 cells.

The present study aimed at investigating the influence of imidazoline receptor ligands on the proliferation of PC12 cells and the involvement of the sphingosine-1-phosphate (S1P) signaling system in this effect. In cultured PC12 cells, S1P (0.3-100 nM) and the I(1) imidazoline receptor ligands moxonidine (0.3 and 1 mM), agmatine (1 mM), idazoxan (10-100 microM) and efaroxan (1-100 microM) concentration-dependently reduced protein contents which were used as estimates for cell number. The antiproliferative effects elicited by the compounds were abolished after knock-down of S1P(1), S1P(2) and S1P(3) receptors by RNA interference indicating an involvement of S1P receptors. In conclusion, the present data add further evidence to the recent finding that effects of imidazoline receptor ligands in PC12 cells are mediated by homo- and heterodimers of members of the S1P receptor family.

5-Hydroxytryptamine-induced contraction of human temporal arteries coexpressing 5-HT2A receptors and wild-type or variant (Phe124Cys) 5-HT1B receptors: increased contribution of 5-HT1B receptors to the total contractile amplitude in arteries from Phe124Cys heterozygous individuals.

OBJECTIVES: Expression studies of the rare Phe124Cys sequence variant of the human 5-HT1B receptor in HEK293 cells demonstrated that 5-hydroxytryptamine (5-HT) and sumatriptan had both three times higher binding affinity and agonist potency at the variant receptor than wild-type receptor. We examined whether in-vivo expression of the variant compared to the wild-type Phe/Phe genotype at codon 124 of the 5-HT1B receptor in human temporal arteries modifies their agonist-induced contraction. METHODS: Rings of arteries, coexpressing 5-HT1B and 5-HT2A receptors, from 98 patients undergoing neurosurgery were set up to measure contraction. Blood sample genotyping was performed by PCR using a mutagenic primer which induces a NheI restriction site in the Cys but not in the Phe allele. RESULTS: Three patients exhibited the Cys/Phe genotype, probably yielding coexpression of both the 124Phe and the 124Cys 5-HT1B receptors. In 95 Phe/Phe patients, exclusively the 124Phe receptor was expressed. The contractile potencies of 5-HT and sumatriptan were not significantly different in arteries from Cys/Phe or Phe/Phe individuals. The 5-HT1B receptor-selective antagonist SB224289 was five-fold more potent in blocking the effects of 5-HT in arteries from three Cys/Phe than from 30 Phe/Phe individuals (P < 0.03). The fraction of 5-HT effects via 5-HT1B receptors, related to the total contractile amplitude via 5-HT1B and 5-HT2A receptors, was enhanced from 0.42 +/- 0.03 in 88 Phe/Phe individuals to 0.75 +/- 0.10 in three Cys/Phe individuals (P < 0.05). CONCLUSIONS: Although the potency of 5-HT1B receptor agonists does not differ between arteries from Phe/Phe and Cys/Phe individuals, the contribution of 5-HT1B receptors to the mediation of the effects of 5-HT is increased in Cys/Phe individuals.

Functional and molecular identification of 5-hydroxytryptamine receptors in rabbit pulmonary artery: involvement in complex regulation of noradrenaline release.

The rabbit pulmonary artery was used to examine whether presynaptic serotonin (5-HT) receptors modulate noradrenaline (NA) release also in this blood vessel and to confirm their presence with molecular biology techniques. Arteries preincubated with [3H]NA were superfused in the presence of the alpha2-adrenoceptor blocker rauwolscine and the effects of 5-HT receptor ligands on the electrically evoked 3H overflow were determined. The 5-HT4 receptor agonist cisapride inhibited 3H overflow in a manner sensitive to blockade by atropine. The 5-HT1B/1D receptor agonist 5-carboxamidotryptamine inhibited 3H overflow only in the presence of atropine. The 5-HT4 and 5-HT1B/1D receptor agonists 5-HT and 5-methoxytryptamine reduced 3H overflow in the absence and presence of atropine, and this effect was blocked by methiothepin, a non-selective 5-HT receptor antagonist, in the presence of atropine. PCR with cDNAs derived from reverse transcribed blood vessel mRNA suggested the expression of the 5-HT1B, 5-HT1D and 5-HT4 receptors, the latter being highly homologous to the human one. In conclusion, the cholinergic nerves are endowed with excitatory 5-HT4 receptors mediating release of acetylcholine which, in turn, activates muscarine receptors on the sympathetic nerves leading to inhibition of NA release. Blockade of the presynaptic muscarinic receptors involved is necessary to disclose an inhibition of NA release via 5-HT1B/1D receptors. Taking results reported in the literature into account, the 5-HT1D and 5-HT4 receptors identified by molecular biology techniques probably are located predominantly on the noradrenergic and cholinergic neurons, respectively.

WIN 55,212-2-induced reduction of cocaine hyperlocomotion: possible inhibition of 5-HT(3) receptor function.

We examined the effect of WIN 55,212-2 (an agonist of cannabinoid receptors) and its enantiomer WIN 55,212-3, as well as of ondansetron (an antagonist of serotonin (5-HT)(3) receptors) on the cocaine-induced locomotor hyperactivity in rats. WIN 55,212-2, but not WIN 55,212-3, in doses of 3 and 6 mg/kg which did not affect the basal locomotor activity, dose-dependently reduced the hyperactivation evoked by cocaine. The inhibitory effect of WIN 55,212-2 was not affected by rimonabant (an antagonist of cannabinoid receptors). Like in the case of WIN 55,212-2, the cocaine-induced hyperlocomotion was reduced in a dose-dependent manner by ondansetron (0.03-0.3 mg/kg). The obtained results indicate that the inhibitory effect of WIN 55,212-2 on cocaine hyperactivation is stereoselective and is not mediated by cannabinoid receptors. Moreover, together with the literature data they may suggest that this effect of WIN 55,212-2 involves inhibition of the 5-HT(3) receptor function.

Decreased agonist, but not antagonist, binding to the naturally occurring Thr92Lys variant of the h5-HT7(a) receptor.

In the present study on transfected human embryonic kidney (HEK)293 cells, we aimed at establishing whether expression of the naturally occurring Thr92Lys variation of the Gs-coupled h5-HT7(a) receptor leads to changes of ligand binding properties, of agonist-evoked cAMP formation and/or of antagonist-mediated blockade of the latter. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulated [3H]cAMP accumulation in whole cells were determined. Saturation binding experiments in membranes of transiently transfected cells expressing either the wild-type or the variant receptor revealed a single binding site in both cases and no difference in Bmax between both receptor isoforms. In competition binding experiments in membranes of stably transfected cells, the Thr92Lys variant exhibited a 2.8-11 times lower binding affinity of the ligands 5-hydroxytryptamine (5-HT), 5-CT, 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4yl)-1H-indole (RU24969), (+/-)-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and sumatriptan compared to the wild-type receptor. However, the variant did not differ from the wild-type with respect to the binding properties of the antagonists (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolodine-1-sulfonyl)phenol hydrochloride (SB-269970), risperidone, mesulergine and clozapine. In agreement with the decreased binding affinity of 5-HT, 5-CT, RU24969 and 8-OH-DPAT for the variant receptor, these agonists were less potent in stimulating [3H]cAMP accumulation in cells stably expressing the Thr92Lys h5-HT7(a) receptor. Sumatriptan did not stimulate cAMP accumulation in spite of its affinity for both receptor isoforms pointing to a putative weak antagonistic property of this drug at the h5-HT7 receptor. SB-269970 and clozapine were equipotent at both the variant and the wild-type receptor in producing a rightward shift of the 5-HT concentration-response curve for its stimulant effect on [3H]cAMP accumulation. In view of, e.g., the purported involvement of the 5-HT7 receptor in the regulation of circadian rhythm, it may be concluded that the decrease in affinity of 5-HT and other 5-HT receptor agonists at the (Thr92Lys) h5-HT7 receptor may be associated with changes of sleep physiology and of actions of new 5-HT7 receptor agonists designed to treat circadian dysregulation.

The naturally occurring Arg219Leu variant of the human 5-HT1A receptor: impairment of signal transduction.

The present study in transfected HEK293 cells aimed to investigate whether the pharmacological and/or transductional properties of the naturally occurring Arg219Leu variant (VAR) in the third intracellular loop of the h5-HT1A receptor differ from those of the wild-type receptor. Binding of [3H]8-hydroxy-2-(di-n-propylamino)tetraline ([H]8-OH-DPAT) and of [35S]GTPgammaS to membranes, as well as inhibition of forskolin-stimulated [3H]cAMP formation by 5-HT receptor agonists in whole cells, were estimated. The VAR and wild-type h5-HT1A receptors were found to be expressed at virtually identical densities. The VAR and wild-type receptors did also not differ with respect to the potencies of 5-HT receptor agonists and antagonists in inhibiting [3H]8-OH-DPAT binding. The ability of 5-HT to stimulate [35S]GTPgammaS binding (a measure of G protein coupling) to the VAR receptor and of the agonists 5-HT, buspirone and urapidil to inhibit forskolin-stimulated cAMP accumulation in HEK293 cells expressing the VAR receptor was decreased by 60-90%. In conclusion, the Arg219Leu variation of the human 5-HT1A receptor does not change the binding properties, but is associated with a drastic impairment of signal transduction. In patients carrying this variation, disturbances of 5-HT1A receptor-mediated functions and diminished responses to drugs acting via this receptor may occur.

Central and peripheral components of the pressor effect of anandamide in urethane-anaesthetized rats.

1. We wanted to search for the mechanism(s) responsible for the brief pressor response induced by anandamide in urethane-anaesthetized rats. 2. The anandamide-induced pressor effect was not modified by the antagonists of cannabinoid CB(1) and vanilloid TRPV(1) receptors, SR 141716A (3 micromol kg(-1)) and capsazepine (1 micromol kg(-1)), respectively, by bilateral vagotomy and by pithing. Replacement of urethane by pentobarbitone virtually abolished the pressor effect of anandamide, both in pithed and vagotomized and in 'intact' rats (i.e. not treated in this manner). 3. The pressor effect of anandamide was reduced by the nonselective TRPV family inhibitor ruthenium red (3 micromol kg(-1)) and by the blocker of L-type calcium channels nifedipine (1 micromol kg(-1)), both in pithed urethane-anaesthetized rats and in 'intact' urethane-anaesthetized rats. The nonselective beta-adrenoceptor antagonist propranolol (0.1 or 0.3 micromol kg(-1)) and the nonselective NMDA receptor antagonist MK-801 (1 micromol kg(-1)) diminished the anandamide-induced vasopressor response in 'intact' but not in pithed rats. The inhibitory effect of propranolol in 'intact' rats was mimicked by the beta(2)-adrenoceptor antagonist ICI 118551 (1 micromol kg(-1)), but not by the beta(1)-adrenoceptor antagonist CGP 20712 (1 micromol kg(-1)). 4. The present study revealed that two mechanisms may be responsible for the anandamide-induced pressor response in urethane-anaesthetized rats. The first involves the central nervous system (probably the medulla oblongata) and is sensitive to propranolol and MK-801. The second, which is located peripherally (most probably in blood vessels), is sensitive to nifedipine, ruthenium red and pentobarbitone and, hence, probably represents a Ca(2+)-dependent mode of action.