Lung serotonin uptake kinetics from indicator-dilution and constant-infusion methods

The kinetics of the pulmonary endothelial uptake of serotonin (5-HT) were evaluated in isolated dog lung lobes using three methods. In method A serotonin was infused at various constant rates to provide a range of capillary concentrations that included Km. The arterial and venous concentrations measured by high-performance liquid chromatography were then used to determine the effect of concentration on the rate of 5-HT uptake. In method B trace doses of 5-[3H]HT and a reference indicator (indocyanine green dye) were injected during each constant infusion of unlabeled 5-HT to provide a measure of unidirectional 5-HT uptake at each background concentration. In method C boluses containing different amounts of unlabeled 5-HT, along with the 5-[3H]HT and the dye, were injected such that each bolus resulted in a range of concentrations and provided a measure of the unidirectional uptake at each concentration. Each method provided the data needed to calculate the maximum uptake rate (Vmax) and the concentration at Vmax/2 (Km), assuming that the uptake kinetics can be represented by the Michaelis-Menten equation. However, the mathematical model underlying each method involved different assumptions about the returning flux of the 5-HT which entered the endothelial cell and the heterogeneity of vascular transit times. The results obtained, considered in light of the different assumptions involved, indicate that all three methods can provide reasonable estimates of the mass transfer kinetic constants if the constant infusions of 5-HT are of short duration and/or the boluses are adequately dispersed prior to reaching the capillary bed.     

Serotonin-activated signal transduction via serotonin receptors on Jurkat cells

Serotonin (5-hydroxytryptamine) (5HT) a neurotransmitter and vasoactive amine, is a major storage product of platelets that are released at sites of inflammation. Several different subtypes of serotonin receptors have been defined. 5HT receptors have been divided into three major families based on molecular, biochemical, and pharmacologic properties. Binding of serotonin to the 5HT1 family results in inhibition of adenylate cyclase whereas binding to the 5HT2 family results in stimulation of phosphatidylinositol turnover and mobilization of intracellular Ca2+. 5HT has been shown to have effects on lymphoid cells. The question of whether human T lymphocytes express receptors for 5HT and transduce signals through 5HT receptors has not been adequately addressed. As a model system, Jurkat cells (a transformed human T lymphocyte line) were examined to determine if they expressed 5HT receptors and whether 5HT stimulated an increase in inositol phosphates or affected adenylate cyclase activity. The results show that Jurkat cells bind 5HT with an average dissociation constant of 90 nM and that 5HT stimulates an increase in inositol phosphate and intracellular Ca2+ levels. These results link the 5HT receptor on Jurkat cells to the 5HT2 family; however, studies with 5HT receptor agonists and antagonists failed to clearly classify the 5HT receptor on Jurkat cells as a known member of the 5HT2 family.     

Expression of serotonin receptors in bone

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.     

Structure-affinity relationship studies on arylpiperazine derivatives related to quipazine as serotonin transporter ligands. Molecular basis of the selectivity SERT/5HT3 receptor

A series of quipazine derivatives, previously synthesized to probe the 5-HT(3) receptor, was evaluated for its potential interaction with serotonin transporter (SERT). Some of them show nanomolar affinity for the rodent SERT comparable to or slightly higher than quipazine or N-methylquipazine. Subsequently a candidate was selected on the basis of its SERT affinity and submitted to a molecular manipulation of the basic moiety. The structure-affinity relationships obtained provided information on the role of the fused benzene ring of quipazine in the interaction with the SERT binding site and on the stereoelectronic requirements for the interaction of both the heteroaromatic component and the basic moiety. Moreover, the comparison of the structure-affinity relationships obtained in the present work with those concerning the interaction of these heteroarylpiperazine derivatives with 5-HT3 receptor suggested some molecular determinants of the selectivity SERT/5HT3 receptor.     

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.     

Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C

The serotonin 5-HT(1A) receptor couples to heterotrimeric G proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor-interacting proteins in 5-HT(1A) receptor signaling. We have found that the ubiquitous Ca(2+)-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT(1A) receptor in Chinese hamster ovary fibroblasts. The human 5-HT(1A) receptor contains two putative CaM binding motifs, located in the N- and C-terminal juxtamembrane regions of the third intracellular loop of the receptor. Peptides encompassing both the N-terminal (i3N) and C-terminal (i3C) CaM-binding domains were tested for CaM binding. Using in vitro binding assays in combination with gel shift analysis, we demonstrated Ca(2+)-dependent formation of complexes between CaM and both peptides. We determined kinetic data using a combination of BIAcore surface plasmon resonance (SPR) and dansyl-CaM fluorescence. SPR analysis gave an apparent K(D) of approximately 110 nm for the i3N peptide and approximately 700 nm for the i3C peptide. Both peptides also caused characteristic shifts in the fluorescence emission spectrum of dansyl-CaM, with apparent affinities of 87 +/- 23 nm and 1.70 +/- 0.16 microm. We used bioluminescence resonance energy transfer to show that CaM interacts with the 5-HT(1A) receptor in living cells, representing the first in vivo evidence of a G protein-coupled receptor interacting with CaM. Finally, we showed that CaM binding and phosphorylation of the 5-HT(1A) receptor i3 loop peptides by protein kinase C are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT(1A) receptor phosphorylation and desensitization. These data suggest that the 5-HT(1A) receptor contains high and moderate affinity CaM binding regions that may play important roles in receptor signaling and function.     

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: evidence that the same G protein-coupled receptor is responsible for maintaining meiosis arrest in both species

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.     

Hyperglycemic properties of serotonin receptor antagonists

Several serotonin (5-HT) receptor antagonists with varying specificities for the 5-HT receptor types, were studied with regard to their effects on blood glucose levels in mice. The non-selective antagonists, metergoline and methysergide, proved to be hyperglycemic at doses commonly used to antagonize 5-HT receptors. In contrast, ritanserin (a 5-HT2 and 5-HT1c antagonist) and MDL 72222 (a 5-HT3 antagonist) were effective only at doses which surpassed the dose range considered to be selective for their respective receptors. The results suggest that 5-HT systems play a role in maintaining glucose homeostasis and that 5-HT1 receptors may be particularly important in this function. Furthermore, the inherent hyperglycemic properties of non-selective serotonin antagonists described here, are pertinent to studies using these agents to investigate glucose metabolism.     

Effect of serotonin on intracellular free calcium of rat cerebrovascular smooth muscle cells in culture

Smooth muscle cells were dissociated from conducting cerebral arteries of adult rats and maintained in culture for 2-4 days. The calcium-sensitive fluorescent probe, fura-2, was used to study the effect of the vasoconstrictor serotonin (5-HT) on the level of free intracellular Ca2+ in these cells. The baseline level of free intracellular calcium was 39 +/- 3.6 nM. In 74 out of 110 cells, 5-HT application transiently increased the free Ca2+ content. This effect was dose-dependent and was suppressed by nanomolar concentrations of the 5-HT2 receptor antagonist, ketanserin. The 5-HT induced rise in free intracellular calcium was not prevented by the presence of Co2+, La3+, or nifedipine, blockers of voltage-sensitive calcium channels. These results indicate that 5-HT mobilizes intracellular Ca2+ in cultured smooth muscle cells derived from the rat cerebrovasculature. The mobilization of intracellular Ca2+ appears to be triggered by a 5-HT2 type receptor, although further pharmacological experiments are required to verify this hypothesis.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.     

Further evidence that the serotonin receptor in the rat stomach fundus is not 5HT1A or 5HT1B

The nature of the receptor mediating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT1 or 5HT2 receptors. We have explored the possibility that such receptors in the rat fundus may better correlate with 5HT1A or 5HT1B receptor subtypes as defined by radiolabeled ligand binding studies with brain cortical membranes. Meta chlorophenylpiperazine (CPP) and meta trifluoromethylphenylpiperazine (TFMPP), selective ligands for the 5HT1B receptor and LY165163, a selective ligand for the 5HT1A receptor, have been evaluated for their agonist and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY165163 showed no agonist activity in this smooth muscle in concentrations up to 10(-4)M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors in the rat fundus was similar for all three phenylpiperazines in spite of the reported selectivity of MCPP and TFMPP for 5HT1B and of LY165163 for 5HT1A receptors. Furthermore, the affinity of these agents for serotonin receptors in the rat stomach fundus did not agree with their reported affinity for either 5HT1A or 5HT1B binding sites in rat cortical membranes. Thus, the similarity in affinities of these phenylpiperazine derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HT1A and 5HT1B binding sites argues against the possibility that the serotonin receptor in the rat fundus is of the 5HT1A or 5HT1B subtype of serotonin receptor.     

Synthesis and Pharmacology of the enantiomers of the potential atypical antipsychotic agents 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT.

The optically pure enantiomers of the potential atypical antipsychotic agents 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 5) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}t etralin [5-OMe-(2,6-di-OMe)-BPAT, 6] were synthesized and evaluated for their in vitro binding affinities at alpha1-, alpha2-, and beta-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2 receptors. In addition, their intrinsic efficacies at serotonin 5-HT1A receptors were established in vitro. (S)- and (R)-5 had high affinities for dopamine D2A, D3, and serotonin 5-HT1A receptors, moderate affinities for alpha1-adrenergic and serotonin 5-HT2 receptors, and no affinity (Ki > 1000 nM) for the other receptor subtypes. (S)- and (R)-6 had lower affinities for the dopamine D2A and the serotonin 5-HT1A receptor, compared to (S)- and (R)-5, and hence showed some selectivity for the dopamine D3 receptor. The interactions with the receptors were stereospecific, since the serotonin 5-HT1A receptor preferred the (S)-enantiomers, while the dopamine D2A and D3 receptors preferred the (R)-enantiomers of 5 and 6. The intrinsic efficacies at the serotonin 5-HT1A receptor were established by measuring their ability to inhibit VIP-induced cAMP production in GH4ZD10 cells expressing serotonin 5-HT1A receptors. Both enantiomers of 5 behaved as full serotonin 5-HT1A receptor agonists in this assay, while both enantiomers of 6 behaved as weak partial agonists. The potential antipsychotic properties of (S)- and (R)-5 were evaluated by establishing their ability to inhibit d-amphetamine-induced locomotor activity in rats, while their propensity to induce extrapyramidal side-effects (EPS) in man was evaluated by determining their ability to induce catalepsy in rats. Whereas (R)-5 was capable of blocking d-amphetamine-induced locomotor activity, indicative of dopamine D2 receptor antagonism, (S)-5 even enhanced the effect of d-amphetamine, suggesting that this compound has dopamine D2 receptor-stimulating properties. Since both enantiomers also were devoid of cataleptogenic activity, they are interesting candidates for further exploring the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.     

Activation of peripheral serotonin2 receptors induces hypothermia in mice

The effects of peripherally administered serotonin (5-HT) on the rectal temperature were investigated. 5-HT i.p. induced a dose-dependent hypothermia in mice. The hypothermic effects of 5-HT were strongly antagonized by the 5-HT1 and 5-HT2 receptor antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. However, the 5-HT1 receptor antagonist pindolol and the 5-HT3 receptor antagonist ICS 205-930 were without effect. In addition, the peripheral 5-HT2 receptor antagonist xylamidine strongly reduced 5-HT-induced hypothermia. These results indicate that the activation of the peripheral 5-HT2 receptors induces hypothermia, although the central 5-HT2 receptors have been suggested to relate to hyperthermia.     

The effects of glycogen synthase kinase-3beta in serotonin neurons

Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase in brain. Increasing evidence has shown that GSK3 acts as a modulator in the serotonin neurotransmission system, including direct interaction with serotonin 1B (5-HT1B) receptors in a highly selective manner and prominent modulating effect on 5-HT1B receptor activity. In this study, we utilized the serotonin neuron-selective GSK3β knockout (snGSK3β-KO) mice to test if GSK3β in serotonin neurons selectively modulates 5-HT1B autoreceptor activity and function. The snGSK3β-KO mice were generated by crossbreeding GSK3β-floxed mice and ePet1-Cre mice. These mice had normal growth and physiological characteristics, similar numbers of tryptophan hydroxylase-2 (TpH2)-expressing serotonin neurons, and the same brain serotonin content as in littermate wild type mice. However, the expression of GSK3β in snGSK3β-KO mice was diminished in TpH2-expressing serotonin neurons. Compared to littermate wild type mice, snGSK3β-KO mice had a reduced response to the 5-HT1B receptor agonist anpirtoline in the regulation of serotonergic neuron firing, cAMP production, and serotonin release, whereas these animals displayed a normal response to the 5-HT1A receptor agonist 8-OH-DPAT. The effect of anpirtoline on the horizontal, center, and vertical activities in the open field test was differentially affected by GSK3β depletion in serotonin neurons, wherein vertical activity, but not horizontal activity, was significantly altered in snGSK3β-KO mice. In addition, there was an enhanced anti-immobility response to anpirtoline in the tail suspension test in snGSK3β-KO mice. Therefore, results of this study demonstrated a serotonin neuron-targeting function of GSK3β by regulating 5-HT1B autoreceptors, which impacts serotonergic neuron firing, serotonin release, and serotonin-regulated behaviors.     

Evidence of fast serotonin transmission in frog slowly adapting type 1 responses

The Merkel cell-neurite (MCN) complex generates slowly adapting type 1 (SA1) response when mechanically stimulated. Both serotonin (5-HT) and glutamate have been implicated in the generation of normal SA1 responses, but previous studies have been inconclusive as to what their roles are or how synaptic transmission occurs. In this study, excised dorsal skin patches from common water frogs (Rana ridibunda) were stimulated by von Frey hairs during perfusion in a tissue bath, and single-unit spike activity was recorded from SA1 fibres. Serotonin had no significant effect on the SA1 response at low (10?μM) concentration, significantly increased activity in a force-independent manner at 100?μM, but decreased activity with reduced responsiveness to force at 1?mM. Glutamate showed no effect on the responsiveness to force at 100?μM. MDL 72222 (100?μM), an ionotropic 5-HT3 receptor antagonist, completely abolished the responsiveness to force, suggesting that serotonin is released from Merkel cells as a result of mechanical stimulation, and activated 5-HT3 receptors on the neurite. The metabotropic 5-HT2 receptor antagonist, ketanserin, greatly reduced the SA1 fibre's responsiveness to force, as did the non-specific glutamate receptor antagonist, kynurenic acid. This supports a role for serotonin and glutamate as neuromodulators in the MCN complex, possibly by activation and/or inhibition of signalling cascades in the Merkel cell associated with vesicle release. Additionally, it was observed that SA1 responses contained a force-independent component, similar to a dynamic response observed during mechanical vibrations.