Is the platelet serotonin transporter different in venous vs. arterial blood?

The binding of labelled paroxetine to the serotonin transporter (SERT) of platelet membranes has been studied in both venous and mixed venous/arterial blood of the rat. In addition, we studied the inhibition of paroxetine binding to SERT by quipazine and N-methyl-quipazine (NMQ). The results indicate differences in affinity for the two test drugs, quipazine and NMQ, in venous vs. mixed venous/arterial blood. This suggests different post-translational modifications of SERT in platelets of arterial vs. venous blood.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 4: 3-Alkyl-4-halo-6-nitroquipazines.

On the basis of the structure-activity relationship (SAR) of 4-chloro-6-nitroquipazine (Ki = 0.03 nM) and 3-fluoropropyl-6-nitroquipazine (Ki = 0.32 nM), 3-alkyl-4-halo-6-nitroquipazines were synthesized and tested for their potential abilities in vitro to displace [3H]citalopram binding to the rat cortical membranes. Binding affinities of 3b and 4d were Ki = 2.70+/-0.32 and 2.23+/-0.46 nM, respectively. The syntheses of 3-alkyl-4-halo-6-nitroquipazine, their in vitro binding affinities, and the SAR of C3, C4 position in 6-nitroquipazine are described.

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.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter: Part 3. A potential 5-HT transporter imaging agent, 3-(3-[18F]fluoropropyl)-6-nitroquipazine.

3-(3-[18F]Fluoropropyl)-6-nitroquipazine ([18F]FPNQ) as a 5-HT transporter imaging agents was designed, synthesized, and evaluated. FPNQ was selected due to its potent in vitro biological activity (K(i)=0.32 nM) in rat brain cortical membranes. The 18F-labeled FPNQ was prepared by reaction of the propyl mesylate as a precursor with tetra-n-butylammonium [18F]fluoride generated under NCA conditions. The precursor mesylate was synthesized from commercially available hydrocarbostyril in nine steps in 21% overall yield. The specific activity of the [18F]FPNQ determined by radioreceptor assay was 27.0 GBq/micromol. Tissue distribution studies in mice showed the highest uptake in the frontal cortex (5.79 %ID/g) at 60 min post-injection.

Biochemical evidence for the presence of serotonin transporters in the rat cochlea.

Cochlear serotonergic innervation is constituted by efferent fibers projecting both to the area below the inner and the outer hair cells. Previous detection of serotonin (5-HT) metabolites and 5-HT receptor mRNAs suggests the existence of serotonergic synaptic activity in the cochlea. The present study explores this possibility through the effect of 6-nitroquipazine (6-NQ), a 5-HT selective reuptake inhibitor, on the basal turnover of 5-HT. The concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) were quantified by high performance liquid chromatography with electrochemical detection in blood-free cochleae of rats treated with 6-NQ or saline and kept under silent conditions. Treatment with 6-NQ induced a significant increase of the cochlear concentration of 5-HT and a significant reduction of 5-HIAA concentration with respect to saline treatment. These findings could indicate that 6-NQ induced the blockade of the 5-HT selective reuptake to the cochlear serotonergic fibers. This suggests that plasma membrane 5-HT transporters are present in cochlear serotonergic fibers. Even though the role of serotonergic innervation on cochlear physiology remains unknown, the existence of cochlear serotonergic synaptic activity is strongly supported by present contributions.

Role of 5-HT3 receptors in the mechanisms of central pain syndrome.

Electrophysiological and behavioral studies showed that spinal 5-HT3 receptors are involved in the regulation of pain sensitivity in rats. Intrathecal administration of the 5-HT3 receptor antagonist tropine (200 microg) produced allodynia, reduced the threshold, decreased the latency, and increased the number of spikes in the late component of the nociceptive flexion reflex. Intrathecal administration of 5-HT3 receptor agonist quipazine (200 mg) abolished nociceptive flexion reflex and alleviated spinal pain syndrome produced by impairment of GABAergic inhibition in the lumbar spinal segments. Our results indicate that spinal 5-HT3 receptors are involved in the modulation of pain sensitivity: activation of these receptors inhibits nociceptive reactions, while blockade of 5-HT3 receptors potentiates the nociceptive response via modulation of excitability of GABAergic interneurons.

Synthesis, radiolabeling and preliminary biological evaluation of radiolabeled 5-methyl-6-nitroquipazine, a potential radioligand for the serotonin transporter.

5-Methyl-6-nitroquipazine, a novel analogue of the potent and selective serotonin transporter inhibitor 6-nitroquipazine was synthesized and radiolabeled with tritium and the positron emitter carbon-11. [3H]5-methyl-6-nitroquipazine was found to have a K(d)=51+/-7 pM. The high affinity and the facile labeling of [11C]5-methyl-6-nitroquipazine makes it a promising radioligand for visualization of the serotonin transporter with positron emission tomography.

The Interactions of the 5-HT(3) receptor with arylpiperazine, tropane, and quinuclidine ligands.

The serotonin 5-HT(3) receptor subtype is unique among the receptors for this neurotransmitter because it has been demonstrated to be a ligand-gated ion channel capable of mediating rapid intercellular communication. This review covers the authors work performed during more than a decade in the development of 5-HT(3) receptor ligands belonging to the classes of arylpiperazines, tropanes, and quinuclidine derivatives. The discussion is focused mainly on what the authors have learned about the interaction of these structurally different ligands with their receptor and shows the way their ideas evolved along with the progress of the project. Furthermore, a summary of the most significant structure-affinity relationships, derived from the original work, is reported to support the discussion.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 2: 4-substituted 6-nitroquipazines.

Eleven 4-substituted derivatives of 6-nitroquipazine were synthesized and evaluated for their abilities to displace [3H]citalopram binding to the rat cortical synaptic membranes. Among them, 4-chloro-6-nitroquipazine was shown to possess the highest binding affinity (K(i=)0.03 nM) which was approximately 6 times higher than that of 6-nitroquipazine (K(i)=0.17 nM) itself. In this paper, we describe the syntheses of 4-substituted 6-nitroquipazine derivatives, the results of corresponding biological evaluation and the SAR study.

In vitro and in vivo ligand binding to the 5HT(3) serotonin receptor characterised by time-resolved fluorescence spectroscopy.

The binding of the fluorescein-labelled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilised ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT(3)R), was characterised by frequency-domain time-resolved fluorescence spectroscopy (TRFS). Detailed understanding of how ligands interact with the homopentameric receptor was obtained. While a 1:1 stoichiometry was observed for the GR-flu-receptor complex, the agonist quipazine bound cooperatively to the receptor, suggesting multiple binding sites for this ligand. The GR-flu-binding site of the receptor was proven to provide an acidic environment as shown by determining the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation experiments indicated a hindered but still high mobility for the receptor-bound GR-flu. Hence, the binding site is expected to present a wide opening to the ligand. Finally, we succeeded in measuring the binding of GR-flu to 5HT(3) receptors in live cells. These results show that the purified and the native receptor behave identically and demonstrate that time-resolved fluorescence measurements are suited to selectively investigate biomolecular interactions in live cells.

Serotonin transporter inhibitors: synthesis and binding potency of 2'-methyl- and 3'-methyl-6-nitroquipazine.

Racemic 2'-methyl- and 3'-methyl-6-nitroquipazine ligands were selected as targets, synthesized and evaluated at the serotonin transporter employing an in vitro competitive inhibition assay with [3H]paroxetine and rat cortical membrane. The 2'-methyl-6-nitroquipazine was found to be 50 times more potent than the 3'-methyl-substituted counterpart and of comparable potency to the known high affinity agent 5-iodo-6-nitroquipazine.

Conserved helix 7 tyrosine functions as an activation relay in the serotonin 5HT(2C) receptor.

The function of the helix VII Tyr in the conserved Asn-Pro-X-X-Tyr segment of rhodopsin-like G protein coupled receptors has been investigated in many receptors. Various effects of site-directed mutation of this locus have been found, including altered coupling, sequestration and agonist affinity. We report the first constitutively active mutations of this Tyr. In the serotonin 5HT(2C) receptor, substituting Ala or Cys for Tyr resulted in a marked increase in the basal level of inositol phosphate accumulation in transfected COS-1 cells. This constitutive signaling was abolished by the inverse agonist SB206553. Introducing Phe at this locus eliminated both basal and agonist-stimulated signaling. All three mutant receptors showed an increase in binding affinity for the structurally dissimilar agonists 5-hydroxytryptamine (5HT), (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and quipazine, suggesting that both the activating and inactivating mutations stabilize a high affinity state. These results implicate the conserved Tyr in the conformational rearrangements that occur during agonist complexing and receptor activation.

Characteristics of agonist displacement of [3H]ketanserin binding to platelet 5-HT2A receptors: implications for psychiatric research.

Brain 5-HT(2A) receptors exist in two agonist affinity states as a function of their coupling to Gq protein. This has not yet been shown in platelets. We examined [3H]ketanserin's saturable binding to platelet 5-HT2A receptors and characteristics of agonist displacement curves of [3H]ketanserin binding in healthy control subjects. [3H]ketanserin saturation curves showed a trend for a two-site model, reflecting two independent binding sites. At low [3H]ketanserin concentrations, agonist displacement curves were flat and best fit a two-site model, indicating the existence of two agonist affinity states. Guanylyl 5'-imidotriphosphate [Gpp(NH)p] induced a significant rightward shift in agonist displacement curves to fit a one-site model. Platelet membrane 5-HT2A receptors exist in two agonist affinity states that are regulated by Gq protein. Platelet 5-HT2A receptors provide an accessible model for examining possible dysregulation in the agonist affinity or coupling efficiency to the phosphoinositide system in psychiatric disorders and their modulation by psychotropic medications.

Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 1. Mapping the central 5-HT3 receptor binding site by arylpiperazine derivatives.

Synthesis and pharmacological evaluation of a series of condensed quinoline and pyridine derivatives bearing a N-methylpiperazine moiety attached to the 2-position of the quinoline or pyridine nucleus are described. 5-HT receptor binding studies revealed subnanomolar affinity for the 5-HT3 receptor subtype in some of the compounds under study. The most active compound (5b) displayed a Ki value about 1 order of magnitude higher than that of quipazine along with a higher selectivity. The potential 5-HT3 agonist/antagonist activity of four selected compounds was assessed in vitro on 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Compound 5j acted as a 5-HT3 agonist in this assay with an EC50 value close to that reported for quipazine, while 5b was a partial agonist with an EC50 value of about 0.25 nM, and compound 5c possessed antagonist properties with an IC50 value (approximately 8 nM) in the same range as those of previously characterized 5-HT3 receptor antagonists. Qualitative and quantitative structure-affinity relationship studies carried out by making use of theoretical molecular descriptors allowed to elucidate the role of the main pharmacophoric components and to develop a model for the interaction of the 5-HT3 ligands related to quipazine with their receptor.

Evaluation of three transporter ligands as quantitative markers of serotonin innervation density in rat brain.

Direct counting of axon terminals (varicosities) labeled by uptake/storage of a tritiated monoamine provides a means to test radioligands of the corresponding membrane transporter as quantitative markers of regional monoamine innervation density in brain tissue. In autoradiographs from alternate rat brain slices, counts of [3H]5-HT-labeled axon terminals were matched with densitometric measurements of the specific binding of tritiated cyanoimipramine (CYI), citalopram (CITAL), and 6-nitroquipazine (6-NTQ), under conditions of hypo-, normo-, or hyper-5-HT innervation of the neostriatum. A total of 267 pairs of data were subjected to a multilevel analysis (iterative generalized least square procedure). With all three ligands, there was a linear relationship between the density of 5-HT innervation and the density of specific binding and no change in the slope of the regression lines as a function of 5-HT innervation density. Thus, none of these ligands gave any sign of down- or up-regulation of the 5-HT transporter consequent to 5-HT hypo- or hyper-innervation. The regression lines for CYI and CITAL were not significantly different from one another and crossed the ordinate near zero, whereas the regression line for 6-NTQ was less steep and had a higher intercept with the ordinate. In addition, the dispersion of values around the regression line (residuals) was lower with CYI and CITAL than 6-NTQ. It was concluded that both CYI and CITAL may serve as quantitative markers of 5-HT innervation density, at least in vitro, whereas 6-NTQ demonstrates a certain lack of specificity and sensitivity. Further work will be needed to assess the potential of CYI and CITAL for positron emission tomographic studies of living brain. Such empirical testing should also be applicable for screening radioligands of the dopamine or the noradrenaline transporters.

The interaction of RS 25259-197, a potent and selective antagonist, with 5-HT3 receptors, in vitro.

1. A series of isoquinolines have been identified as 5-HT3 receptor antagonists. One of these, RS 25259-197 [(3aS)-2-[(S)-1-azabicyclo[2.2.2]oct-3-yl]-2,3,3a,4,5,6-hexahydro- 1- oxo-1H-benzo[de]isoquinoline-hydrochloride], has two chiral centres. The remaining three enantiomers are denoted as RS 25259-198 (R,R), RS 25233-197 (S,R) and RS 25233-198 (R,S). 2. At 5-HT3 receptors mediating contraction of guinea-pig isolated ileum, RS 25259-197 antagonized contractile responses to 5-HT in an unsurmountable fashion and the apparent affinity (pKB), estimated at 10 nM, was 8.8 +/- 0.2. In this tissue, the -log KB values for the other three enantiomers were 6.7 +/- 0.3 (R,R), 6.7 +/- 0.1 (S,R) and 7.4 +/- 0.1 (R,S), respectively. The apparent affinities of RS 25259-197 and RS 25259-198, RS 25233-197 and RS 25233-198 at 5-HT3 receptors in membranes from NG-108-15 cells were evaluated by a [3H]-quipazine binding assay. The -log Ki values were 10.5 +/- 0.2, 8.4 +/- 0.1, 8.6 +/- 0.1 and 9.5 +/- 0.1, respectively, with Hill coefficients not significantly different from unity. Thus, at these 5-HT3 receptors, the rank order of apparent affinities was (S,S) > (R,S) > (S,R) = (R,R). 3. RS 25259-197 displaced the binding of the selective 5-HT3 receptor ligand, [3H]-RS 42358-197, in membranes from NG-108-15 cells, rat cerebral cortex, rabbit ileal myenteric plexus and guinea-pig ileal myenteric plexus, with affinity (pKi) values of 10.1 +/- 0.1, 10.2 +/- 0.1, 10.1 +/- 0.1 and 8.3 +/- 0.2, respectively. In contrast, it exhibited low affinity (pKi <6.0) at 28 other receptors in binding assays, including adrenoceptors (alpha1A, alpha 1B, alpha2A, alpha 2B ,beta1, beta2), muscarinic (M1-M4), dopamine (D1, D2), opioid and other 5-HT(5-HTlA, 5-HTlD, 5-HT2C, 5-HT4) receptors.4. RS 25259-197 was tritium labelled (specific activity: 70 Ci mmol-1) and evaluated in pharmacological studies. Saturation studies with [3H]-RS 25259-197 in membranes from NG-108-15 and cloned homomeric a subunits of the 5-HT3 receptor from N1E-1 15 cells expressed in human kidney 293E1 cells,revealed an equilibrium dissociation constant (Kd) of 0.05 +/- 0.02 and 0.07 +/- 0.01 nM, and Bmax of610 +/- 60 and 1068 +/- 88 fmol mg-1, respectively. Competition studies in NG-108-15 cells indicated a pharmacological specificity entirely consistent with labelling a 5-HT3 receptor, i.e. RS 25259-197> granisetron> (S)-zacopride> tropisetron> (R)-zacopride> ondansetron> MDL 72222.5. In contrast to the majority of radioligands available to label 5-HT3 receptors, [3H]-RS 25259-197 labelled a high affinity site in hippocampus from human post-mortem tissue with an equilibrium dissociation constant (Kd) of 0.15 +/- 0.07 nM and density (BmaX) of 6.8 +/- 2.4 fmol mg-1 protein. Competition studies in this tissue indicated a pharmacological specificity consistent with labelling of a 5-HT3receptor.6. Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5-HT3receptor sites by [3H]-RS 25259-197. High densities of sites were seen in nuclear tractus solitaris and area postrema, a medium density in spinal trigeminal tract, ventral dentate gyrus and basal medial amygdala,and a low density of sites in hippocampal CAl, parietal cortex, medium raphe and cerebellum.7 In conclusion, the functional, binding and distribution studies undertaken with the radiolabelled and non-radiolabelled RS 25259-197 (S,S enantiomer) established the profile of a highly potent and selective5-HT3 receptor antagonist.

Binding potency of 6-nitroquipazine analogues for the 5-hydroxytryptamine reuptake complex.

The in-vitro inhibition constants (Ki) of nine structural analogues of the potent 5-hydroxytryptamine (5-HT)-uptake inhibitor, 6-nitroquipazine, were determined to assess the structure-affinity relationship of these derivatives. The goal of these studies was to determine those positions on 6-nitroquipazine that could be derivatized without significantly decreasing the affinity of the drug for the binding site, so that radiolabels such as 123I, 76Br or 18F might be appended for in-vivo imaging studies of the 5-HT reuptake system. Using bromine as a steric probe, the rank order of potency of bromine-substituted 6-nitroquipazine analogues for inhibiting the binding of [3H]paroxetine to the 5-HT reuptake binding site was: 8- < 3- < 7- < 4- < 5-bromo. The in-vitro equipotent molar ratio (EPMR, Ki (analogue)/Ki(6-nitroquipazine)) of the 5-bromo analogue was 0.57, indicating that this analogue had greater affinity for the 5-HT reuptake complex than 6-nitroquipazine. Derivatization at the 5-position with fluorine and iodine also resulted in potent compounds with EPMR values of 1.1 and 0.83, respectively. Substitution of quipazine with bromo, cyano, and formyl groups at the 6-position produced less potent compounds than the 6-nitro group. Based upon the high affinities of the 5-bromo-, 5-fluoro- and 5-iodo-6-nitroquipazines for the 5-HT reuptake complex, these compounds are candidates for radiolabeling for in-vivo studies of the 5-HT reuptake site.

In vivo imaging of the 5-hydroxytryptamine reuptake site in primate brain using single photon emission computed tomography and [123I]5-iodo-6-nitroquipazine.

Previous experiments have demonstrated that 5-iodo-6-nitro-2-piperazinylquinoline (5-I-6-NQP) is a potent and selective ligand for studying brain 5-hydroxytryptamine (5-HT) reuptake sites. We performed in vivo imaging in non-human primates using single photon emission computed tomography (SPECT) and the 123I-labeled compound [123I]5-I-6-NQP. These studies showed rapid brain uptake, with slow egress of the tracer from the brainstem, a region rich in 5-HT reuptake sites. Loss of the tracer from regions with a lower density of these sites, such as cerebellum, was relatively more rapid. Pretreatment of animals with paroxetine increased the washout of tracer from the brainstem to rates similar to that seen in cerebellum. Brainstem to cerebellar ratios of tracer accumulation were > 2 by 8 h after injection, and in paroxetine pretreated animals remained close to 1. These results indicate that the radiotracer has characteristics suitable for use as a SPECT imaging agent of serotonin reuptake sites.

[125I]5-iodo-6-nitroquipazine: a potent and selective ligand for the 5-hydroxytryptamine uptake complex. I. In vitro studies.

In search of a potent and selective radioiodinated ligand for the 5-hydroxytryptamine (serotonin or 5-HT) uptake complex, we synthesized and evaluated the in vitro properties of [125I]5-iodo-6-nitroquipazine. The binding properties and pharmacological profile of this radioligand were studied in rat brain homogenates, and it was found to display high affinity and selectivity for the serotonin uptake complex. Scatchard analysis of the binding data indicated a single population of sites with a Kd of 23 +/- 6 pM and a Bmax of 430 +/- 50 fmol/mg protein (mean +/- S.E.M., n = 7). Inhibitors of serotonin uptake were the most efficient competitors for [125I]5-iodo-6-nitroquipazine binding with Ki values similar in rank order and magnitude to those obtained in studies of other established serotonin uptake blockers. Inhibitors of dopamine and norepinephrine uptake as well as a wide variety of postsynaptic receptor agents were relatively ineffective in inhibiting [125I]5-iodo-6-nitroquipazine binding to rat brain membranes. Serotonin was the only monoaminergic neurotransmitter capable of effectively competing for [125I]5-iodo-6-nitroquipazine binding sites and gave a Ki value of 2.8 +/- 0.6 microM. Lesions of the serotonergic system with p-chloroamphetamine resulted in a dramatic loss (> 90%) of [125I]5-iodo-6-nitroquipazine binding to rat cortical membranes. Non-radiolabeled 5-iodo-6-nitroquipazine potently inhibited the binding of [3H]paroxetine to serotonin reuptake sites in rat cortical membranes with a Ki of 0.17 +/- 0.06 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

[125I]5-iodo-6-nitroquipazine: a potent and selective ligand for the 5-hydroxytryptamine uptake complex. II. In vivo studies in rats.

The in vivo regional distribution and pharmacological profile of [125I]5-iodo-6-nitroquipazine in the rat brain were studied to evaluate this compound as a potential in vivo imaging agent of the 5-hydroxytryptamine (serotonin or 5-HT) uptake complex. This radioligand penetrated the blood-brain barrier quickly and efficiently, with 1.9% of injected dose found in the whole brain at 5 min post i.v. injection. The regional brain distribution of radioactivity at time points later than 2 h was highly correlated with the known distribution of serotonin uptake sites and terminals. Coadministration of 2 mg/kg paroxetine inhibited > 90% of the total in vivo binding of [125I]5-iodo-6-nitroquipazine. Other serotonin uptake inhibitors, such as fluoxetine and sertraline, were also effective inhibitors of [125I]5-iodo-6-nitroquipazine brain binding in vivo. Non-serotonergic uptake blockers (desipramine, nomifensine, and GBR-12909) and the postsynaptic serotonin receptor agent LSD had no effect on [125I]5-iodo-6-nitroquipazine binding in vivo even at high doses. Lesioning of the serotonergic system by p-chloramphetamine produced approximately 90% decrease in specific in vivo binding. Extraction and analysis of brain radioactivity indicated that approximately 95% of the extractable radioactivity was unmetabolized [125I]5-iodo-6-nitroquipazine. These results indicate that [125I]5-iodo-6-nitroquipazine is a specific, useful radioligand for studying serotonergic uptake sites and terminals in animals, and an 123I-radiolabeled form of the drug would be an excellent candidate for non-invasive single photon emission computed tomography (SPECT) imaging of these sites in the living human brain.