In vitro effect of phencyclidine and other psychomotor stimulants on serotonin uptake in human platelets

Phencylidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and 1-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (IC₅₀, 2.5 μM) was one-hundredth as potent as fluoxetine (IC₅₀, 22 νM) but ten times more potent than ketamine (IC₅₀, 25 μM) and d-amphetamine (IC₅₀, 24 μM) and three times more potent than 1-amphetamine (IC₅₀, 80 μM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed.     

Serotonergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in paired rats and three paradigms of aggressive behaviour were recorded, namely, latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). The effects of increasing or decreasing central serotonergic activity, by using a number of pharmacological agents with well defined effects on rat brain serotonin, were investigated on FIA and on FIA augmented by apomorphine, a dopamine receptor agonist. The results show that centrally administered serotonin, the serotonin precursor, 5-hydroxytryptophan administered with clorgyline, a selective MAO A inhibitor, quipazine, a serotonin receptor agonist, and fluoxetine, a selective inhibitor of neuronal re-uptake of serotonin, attenuated all paradigms of FIA and apomorphine induced potentiation of FIA. On the contrary, the other re-uptake inhibitor used, citalopram, appeared to have a dual effect and decreased LF and CAS, while increasing TPP. The serotonin synthesis inhibitor, p-chlorophenylalanine and the selective serotonin receptor (5-HT2) antagonist, ketanserin, augmented all paradigms of FIA per se and apomorphine induced augmentation of FIA. However, the other serotonin receptor antagonist used, metergoline, which blocks both 5-HT1 and 5-HT2 receptor subtypes, attenuated FIA per se but decreased only CAS in apomorphine induced increase in FIA. The data confirm the inhibitory effect of the central serotonergic system on aggressive behaviour and the inverse relationship existing between it and the central dopaminergic system in the modulation of FIA, as has also been confirmed in earlier biochemical investigations from this laboratory. The data has been discussed in the light of existing knowledge on serotonin receptor subtypes and the presence of modulatory serotonergic heteroreceptors on central dopaminergic neurones.     

Platelet serotonin content and uptake in spontaneously hypertensive rats

Platelet serotonin (5-HT) content and uptake were studied in male SHR and WKY at various ages. Blood was withdrawn from the carotid artery under anesthesia and 5-HT levels determined from platelet rich plasma (PRP) using a HPLC technique coupled with an electrochemical detection method. Platelet 5-HT uptake was studied by incubating PRP at 37 degrees C for 10 sec with increasing concentrations of 3H-5HT. Lineweaver- Burk plots of 3H-5HT uptake were linear suggesting simple Michaelis- Menten uptake kinetics. The SHR had more platelets than age-matched controls and consequently a higher blood circulating pool of 5-HT. Nevertheless, the 5-HT platelet levels were similar to those of their age-matched rats. The 5 week-old SHR and WKY had greater numbers of platelets and higher 5-HT platelet levels than the older rats of both strains. The affinity constants (Km) and the maximal velocities (Vmax) of platelet 5-HT uptake did not differ significantly between the 12 week- and the 6 month-old SHR and WKY. These data suggest that the SHR do not show the same impairment in platelet 5-HT metabolism as observed in essential hypertension in man.     

Assessment of blood platelets as a model for CNS response: comparative effects of caffeine on 5-HT uptake and release mechanisms in rat platelets and rat brain serotonin neurons

We have previously reported that caffeine significantly enhanced 5-HT uptake and reduced 5-HT release from crude synaptosomal fractions obtained from rat cerebral cortex and from midbrain raphe region. Blood platelets, as reported by many laboratories and also demonstrated in our own labs, have a very active mechanism for 5-HT uptake and storage. In this regard platelets bear a high degree of similarity to brain serotonin neurons. The present experiments were, therefore, carried out to investigate the effects of caffeine on 5-HT uptake and release from rat platelets in an attempt to assess the possibility of using platelets as a model for studying the CNS effects of caffeine. Platelet rich plasma was prepared from the trunk blood of decapitated rats. Effects of caffeine were investigated at 10(-7), 10(-6), 10(-5) and 10(-4)M, on both the high affinity 3H-5-HT uptake and the spontaneous 5-HT release from 3H-5-HT preloaded platelets. The results show that caffeine did not change 5-HT uptake into platelets. In brain synaptosomes the same concentration of caffeine, however, increased 5-HT uptake dose-dependently. The results also revealed that caffeine increased 5-HT release from rat platelets in a concentration-dependent manner. The concentrations 10(-6), 10(-5), and 10(-4)M increased release significantly compared to control. This finding is also in contrast to that observed in synaptosomes of brain serotonin neurons where caffeine decreased 5-HT release. It is concluded, therefore, that the rat blood platelet is not a suitable model for studying these CNS actions of caffeine. Furthermore, our observations imply that rat platelet serotonin uptake and release mechanisms are not identical to those mechanisms in brain serotonin neurons.     

Uptake, Release, and Subcellular Localization of l-Methyl-4-Phenylpyridinium in Blood Platelets

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.     

Reduced Vmax of 3H-serotonin uptake but unchanged 3H-imipramine binding in the platelets of untreated hypertensive subjects

The uptake of 3H-serotonin, endogenous serotonin content and 3H-imipramine binding were measured in platelets of subjects with essential hypertension and matched control volunteers. The uptake of 3H-serotonin and endogenous serotonin levels in platelets were significantly reduced while 3H-imipramine binding did not differ in the two experimental groups. These results provide further evidence that the uptake site for serotonin and the binding site for 3H-imipramine although associated, may be modified independently.     

Serotonin in pain and analgesia

The purpose of this article is to summarize recent findings on the role of serotonin in pain processing in the peripheral nervous system. Serotonin (5-hydroxtryptamine [5-HT]) is present in central and peripheral serotonergic neurons, it is released from platelets and mast cells after tissue injury, and it exerts algesic and analgesic effects depending on the site of action and the receptor subtype. After nerve injury, the 5-HT content in the lesioned nerve increases. 5-HT receptors of the 5-HT₃ and 5-HT_2A subtype are present on C-fibers. 5-HT, acting in combination with other inflammatory mediators, may ectopically excite and sensitize afferent nerve fibers, thus contributing to peripheral sensitization and hyperalgesia in inflammation and nerve injury.     

Influence of the 5-HT3A Receptor Gene Polymorphism and Childhood Sexual Trauma on Central Serotonin Activity

Background

Gene-environment interactions are important for understanding alterations in human brain function. The loudness dependence of auditory evoked potential (LDAEP) is known to reflect central serotonergic activity. Single nucleotide polymorphisms (SNPs) in the 5-HT3A serotonin receptor gene are associated with psychiatric disorders. This study aimed to investigate the effect between 5-HT3A receptor gene polymorphisms and childhood sexual trauma on the LDAEP as an electrophysiological marker in healthy subjects.

Methods

A total of 206 healthy subjects were recruited and evaluated using the childhood trauma questionnaire (CTQ) and hospital anxiety and depression scale (HADS). Peak-to-peak N1/P2 was measured at five stimulus intensities, and the LDAEP was calculated as the linear-regression slope. In addition, the rs1062613 SNPs of 5-HT3A (CC, CT, and TT) were analyzed in healthy subjects.

Results

There was a significant interaction between scores on the CTQ-sexual abuse subscale and 5-HT3A genotype on the LDAEP. Subjects with the CC polymorphism had a significantly higher LDEAP than T carriers in the sexually abused group. In addition, CC genotype subjects in the sexually abused group showed a significantly higher LDAEP compared with CC genotype subjects in the non-sexually abused group.

Conclusions

Our findings suggest that people with the CC polymorphism of the 5-HT3A gene have a greater risk of developing mental health problems if they have experienced childhood sexual abuse, possibly due to low central serotonin activity. Conversely, the T polymorphism may be protective against any central serotonergic changes following childhood sexual trauma.     

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.     

Serotonin and molecular neuroimaging in humans using PET

The serotonergic system is one of the most important modulatory neurotransmitter systems in the human brain. It plays a central role in major physiological processes and is implicated in a number of psychiatric disorders. Along with the dopaminergic system, it is also one of the phylogenetically oldest human neurotransmitter systems and one of the most diverse, with 14 different receptors identified up to this day, many of whose function remains to be understood. The system's functioning is even more diverse than the number of its receptors, since each is implicated in a number of different processes. This review aims at illustrating the distribution and summarizing the main functions of the serotonin (5-hydroxytryptamin, 5-HT) receptors as well as the serotonin transporter (SERT, 5-HTT), the vesicular monoamine transporter 2, monoamine oxidase type A and 5-HT synthesis in the human brain. Recent advances in in vivo quantification of these different receptors and enzymes that are part of the serotonergic system using positron emission tomography are described.     

Serotonin depletion produces long lasting increase in striatal glutamatergic transmission

The ability of serotonin (5-HT) to influence striatal glutamatergic transmission was examined by determining changes over time in glutamate extracellular levels, transporter expression and synaptosomal uptake in rats with lesion of serotonergic neurones. By 8 days after intraraphe injections of 5,7-dihydroxytryptamine, producing 80% decreases in striatal tissue 5-HT levels, no changes were observed in the glutamatergic transmission. When 5-HT depletion was almost complete (21 days post-lesion), high affinity glutamate uptake in striatal synaptosomal preparations was significantly increased (156% of control), although no changes in striatal GLT1, GLAST and EAAC1 mRNAs, and GLT1 protein were detected by in situ hybridization and immunohistochemistry. Meanwhile, the serotonin lesion produced large increases in basal extracellular levels of glutamate and glutamine (364% and 259%, respectively) determined in awake rats by in vivo microdialysis, whereas no change was observed in dopamine levels as compared with control rats. High potassium depolarization as well as L-trans-pyrrolidine-2,4-dicarboxylate, also induced larger increases in extracellular levels of glutamate in lesioned rats than in controls. Finally, similar changes in glutamate transmission were observed by 3 months post-lesion. These results suggest that 5-HT has a long lasting and tonic inhibitory influence on the striatal glutamatergic input, without affecting the basal dopaminergic transmission.     

Decreased platelet 3H-paroxetine binding in untreated panic disorder patients

Different findings support the involvement of the serotonin (5-HT) system in panic disorder. The presence of the 5-HT transporter in blood platelets similar to that in presynaptic serotonergic neurons, permits the investigation of this structure in periphery. We therefore evaluated the binding of 3H-paroxetine, a selective 5-HT reuptake inhibitor which is considered the ligand of choice for labelling the 5-HT transporter, in platelets of 20 drug-free patients with panic disorder. The same measurement was repeated after one year's treatment with different drugs. The results showed patients to have a lower number of 3H-paroxetine sites than a group of age- and sex-matched controls, thus suggesting the involvement of the 5-HT transporter in panic disorder. This abnormality reverted after one year of treatment with specific drugs that provoked the symptom remission in all cases, which would suggest a link with the clinical improvement.     

Altered platelet serotonin uptake kinetics in schizophrenia and depression

Platelet uptake of serotonin (5-HT) was studied in drug-free groups of normal controls, depressives and schizophrenics. The Michaelis constant was significantly higher in both the schizophrenics and the depressives than in the controls. The V_max did not differ significantly between the three groups. Uptake velocity at low substrate concentrations was significantly lower in the schizophrenic group and showed a similar trend in the depressives. There were no significant differences in K_m or V_max when depressives were subclassified as unipolar and bipolar. There were no significant correlations between 5-HT uptake kinetics and severity of illness. These findings raise the possibility of a structural defect in the 5-HT reuptake system in the major psychoses that reduces the affinity of the carrier for the amine. This alteration, if present in central serotonergic neurons, may play a role in the etiology and pathogenesis of depression and schizophrenia.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

Role of Ca(2+) and protein kinase C in the serotonin (5-HT) transport in human platelets

Accumulation of serotonin (5-HT) into human platelets was not affected by the presence of the extra-cellular calcium chelator EGTA, while decreased by platelet incubation with the membrane permeant chelator BAPTA-AM. Serotonin uptake also diminished upon platelet exposure to EGTA/thapsigargin or EGTA/ionomycin which increased the cytosolic [Ca(2+)] to levels lower than those inducing secretion of dense granules. The latter inhibition depended in part on changes of intra-granular pH, since the accumulation of acridine orange, which is driven into the dense granules by the intra-granular acid pH gradient, was slightly decreased in the presence of EGTA/thapsigargin. These compounds also inhibited the 5-HT uptake in platelets pre-incubated with reserpine and bafilomycin that prevent 5-HT from entering into the dense granules. Inhibitors of protease, protein phosphatase, Na(+)/H(+) exchanger or ciclo-oxygenase activities did not modify the serotonin accumulation. Addition of EGTA/thapsigargin to reserpine-treated, [(14)C]5-HT-loaded, platelets caused an imipramine-insensitive release of labelled serotonin. This release was reduced by both BAPTA-AM or protein kinase C inhibitor bisindoylmaleimide (GF). The latter compound, either alone or together with EGTA/thapsigargin, inhibited the 5-HT accumulation in reserpine-treated platelets. It is concluded that both cytosolic [Ca(2+)] and protein kinase C are involved in the regulation of the plasma membrane 5-HT transport.     

Propranolol modifies platelet serotonergic mechanisms in rats.

Though the mechanisms for the vascular actions of vasodilatory beta-blockers are mostly determined, some of their interactions with monoaminergic systems are not elucidated. Because there are evidences supporting a possible involvement of serotonin (5-HT) in the actions of beta-blockers, we studied the effect of propranolol on peripheral serotonergic mechanisms in normotensive and Goldblatt two-kidney - one clip (2K1C) hypertensive rats. In both groups of animals propranolol decreased systolic blood pressure, significantly increased whole blood serotonin concentration and at the same time it decreased platelet serotonin level. The uptake of the amine by platelets from hypertensive animals was lower than that of normotensive animals and it was decreased by propranolol only in the latter. In both groups propranolol inhibited potentiation of ADP-induced platelet aggregation by serotonin. In conclusion, this study provides evidence that propranolol modifies platelet serotonergic mechanisms in normotensive and renal hypertensive rats.     

Activators of protein kinase C decrease serotonin transport in human platelets.

Treatment of human platelets with activators of protein kinase C (PKC) for 5-20 min resulted in substantial reductions in the rate of platelet serotonin (5-HT) transport. The mean Vmax observed after 5 min treatment with 1 microM 4-beta-12-tetradecanoylphorbol 13-acetate (beta-TPA) was 66% (n = 16, P = 0.0001) of the control value. 5 min of treatment with 1 microM mezerein reduced uptake to 78% (n = 3, P = 0.01) of control. Both beta-TPA and mezerein had little effect on the Km of transport and had EC50 values of approx. 100 mM when a 20-min treatment period was used. The maximum effects of both were reached at approx. 20 min and could be blocked with staurospine. The beta-TPA effect was stereospecific, as alpha-TPA did not alter platelet 5-HT uptake. Although the PKC activators may have altered transmembrane ion-gradients for Na+ and Cl-, which are co-transported with 5-HT, minimizing ion-gradient changes had little effect on the observed reductions in transport. The PKC activators also had little or no effect on platelet 5-HT release or on the number (Bmax) of 5-HT transporters expressed at the platelet surface. The data indicate that PKC activation may down-regulate the activity of the 5-HT transporter in platelets. Apparently, most of this effect is mediated through mechanisms other than changes in ion-gradients, reductions in the number of available transporters, or increased 5-HT release. The apparent regulation of 5-HT transport by PKC may have important implications in platelet and neuronal functioning.     

Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.