Serotonin Transporter Gene Polymorphism and Personality Traits Measured by MMPI

Polymorphisms of the serotonin transporter gene are known to be associated with some personality traits measured by means of various psychological inventories. In the present work we attempted to find an association between genetic variants of serotonin transporter (loci VNTR-17 and 5-HTTLPR) and psychological traits scored by the MMPI inventory in 125 mentally healthy donors. No statistically significant differences in personality traits were found between carriers of differentVNTR-17 genotypes. At locus 5-HTTLPR, significant between-genotype differences were revealed on the Schizophrenia scale (F= 3.49; P = 0.034) and on the validity scale F (F = 3.24; P = 0.042). The ss genotype carriers had the lowest scores on these scales. The score on the Psychopathic Deviate scale was significantly lower in the carriers of the ssgenotype than in the combined group of the carriers of genotypes lland ls(t= 2.07; P = 0.041). The differences on the validity scale K between the carriers of the ll and ssgenotypes were also statistically significant (t= 2.49; P = 0.015). These results suggest that polymorphism of the serotonin transporter gene may be associated with the expression of schizoid traits (namely, social introversion, internal tension, bizarre thoughts and actions) in mentally healthy individuals. In the context of social adaptation, the personality profile configuration and data of statistical analysis indicate that the carriers of the ss genotype are more inclined to observe social norms than the carriers of the lland ls genotypes.     

Biallelic and triallelic approaches of 5-HTTLPR polymorphism are associated with food intake and nutritional status in childhood

BackgroundThe 5-HTT gene contains polymorphisms in its promoter region, the insertion/deletion (5-HTTLPR) that creates long (L) or short (S) alleles (biallelic approach) and SNP (rs25531) in L allele (triallelic approach).ObjectivesThe aim of this study is to investigate the association of the 5-HTTLPR and rs25531 polymorphisms, using bi- and triallelic approach, with dietary intake and anthropometric parameters in children followed until 8 years old.MethodsThe sample were 303 children who were recruited at birth and examined at 1, 3 to 4 and 7 to 8 years old. The polymorphisms were analyzed by polymerase-chain-reaction-based methods.ResultsIn the biallelic approach, children with the S/S genotype presented a higher body mass index Z-score in the three developmental stages and higher sum of skinfolds at 3 to 4 and 7 to 8 years old than carriers of the L allele. In the triallelic approach, S/S, Lg/S plus Lg/Lg genotypes were associated with higher energy intake daily at 1 year old and with waist circumference at 3 to 4 years old.ConclusionsIn the biallelic approach, the 5-HTTLPR polymorphism is associated with food intake, body mass index Z-score and sum of skinfolds in children, reinforcing the role of the serotonin transporter in childhood obesity. Our data indicate that the biallelic approach is more sensible than the triallelic approach for detected associations with food intake and nutritional status in childhood. Identifying susceptibility genes in early life could provide the foundations for interventions in lifestyle to prevent children to become obese adults.     

Association of several polymorphic loci of serotoninergic genes with unipolar depression

Serotoninergic system is one of the major brain neurotransmitter systems that is involved in the development of depressive spectrum disorders. Regulatory genes of this system are the principle candidate genes predisposing to unipolar depression. Using PCR-RFLP analysis, we have conducted a study of polymorphic loci of several genes of this system: C1019G of serotonin receptor 1A gene, (HTR1A); A-1438G of serotonin receptor 2A gene, (HTR2A); G861C of serotonin receptor 1B gene, (HTR1B); Stin2VNTR and 5-HTTLPR of serotonin transporter gene (SLC6A4) in patients with unipolar depression from Tatar and Russian population. The results of the study suggest that genotype 10/10 of the SLC6A4 gene as well as genotype G/G and allele G of the HTR2A gene can predispose to increased risk of unipolar depression development in ethnic Russians. In contrast, genotype 12/10 of the SLC6A4 gene is a marker of low risk of the disease in both groups.     

Regulation of the neuronal norepinephrine transporter by endothelin-1 and -3 in the rat anterior and posterior hypothalamus

We previously reported that endothelin-1 and endothelin-3 modulate norepinephrine neuronal release and tyrosine hydroxylase activity and expression in the hypothalamus. In the present study we sought to establish the role of endothelin-1 and -3 in the regulation of norepinephrine uptake in the anterior and posterior hypothalamus. Results showed that in the anterior hypothalamus endothelin-3 increased neuronal norepinephrine uptake whereas endothelin-1 decreased it. Conversely, in the posterior hypothalamic region both endothelins diminished the neuronal uptake of the amine. Endothelins response was concentration dependent and maintained at all studied times. Endothelins also modified the kinetic and internalization of the NE neuronal transporter. In the anterior hypothalamic region endothelin-3 increased the V_max and the B_max whereas endothelin-1 decreased them. However, in the posterior hypothalamic region both endothelins diminished the V_max as well as B_max. Neither endothelin-1 nor endothelin-3 modified neuronal norepinephrine transporter K_d in the studied hypothalamic regions. These findings support that in the posterior hypothalamic region both endothelins diminished neuronal norepinephrine transporter activity by reducing the amine transporter expression on the plasmatic membrane. Conversely, in the anterior hypothalamic region endothelin-3 enhanced neuronal norepinephrine transporter activity by increasing the expression of the transporter on the presynaptic membrane, whereas endothelin-1 induced the opposite effect. Present results permit us to conclude that both endothelins play an important role in the regulation of norepinephrine neurotransmission at the presynaptic nerve endings in the hypothalamus.     

Clinical Pharmacology of 3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”): The Influence of Gender and Genetics (CYP2D6, COMT, 5-HTT)

The synthetic psychostimulant MDMA (±3,4-methylenedioxymethamphetamine, ecstasy) acts as an indirect serotonin, dopamine, and norepinephrine agonist and as a mechanism-based inhibitor of the cytochrome P-450 2D6 (CYP2D6). It has been suggested that women are more sensitive to MDMA effects than men but no clinical experimental studies have satisfactorily evaluated the factors contributing to such observations. There are no studies evaluating the influence of genetic polymorphism on the pharmacokinetics (CYP2D6; catechol-O-methyltransferase, COMT) and pharmacological effects of MDMA (serotonin transporter, 5-HTT; COMT). This clinical study was designed to evaluate the pharmacokinetics and physiological and subjective effects of MDMA considering gender and the genetic polymorphisms of CYP2D6, COMT, and 5-HTT. A total of 27 (12 women) healthy, recreational users of ecstasy were included (all extensive metabolizers for CYP2D6). A single oral weight-adjusted dose of MDMA was administered (1.4 mg/kg, range 75–100 mg) which was similar to recreational doses. None of the women were taking oral contraceptives and the experimental session was performed during the early follicular phase of their menstrual cycle. Principal findings show that subjects reached similar MDMA plasma concentrations, and experienced similar positive effects, irrespective of gender or CYP2D6 (not taking into consideration poor or ultra-rapid metabolizers) or COMT genotypes. However, HMMA plasma concentrations were linked to CYP2D6 genotype (higher with two functional alleles). Female subjects displayed more intense physiological (heart rate, and oral temperature) and negative effects (dizziness, sedation, depression, and psychotic symptoms). Genotypes of COMT val158met or 5-HTTLPR with high functionality (val/val or l/*) determined greater cardiovascular effects, and with low functionality (met/* or s/s) negative subjective effects (dizziness, anxiety, sedation). In conclusion, the contribution of MDMA pharmacokinetics following 1.4 mg/kg MDMA to the gender differences observed in drug effects appears to be negligible or even null. In contrast, 5-HTTLPR and COMT val158met genotypes play a major role.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01447472","term_id":"NCT01447472"}}NCT01447472     

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.     

Maintenance of Chronic Fatigue Syndrome (CFS) in Young CFS Patients Is Associated with the 5-HTTLPR and SNP rs25531 A > G Genotype

Earlier studies have shown that genetic variability in the SLC6A4 gene encoding the serotonin transporter (5-HTT) may be important for the re-uptake of serotonin (5-HT) in the central nervous system. In the present study we investigated how the 5-HTT genotype i.e. the short (S) versus long (L) 5-HTTLPR allele and the SNP rs25531 A > G affect the physical and psychosocial functioning in patients with chronic fatigue syndrome (CFS). All 120 patients were recruited from The Department of Paediatrics at Oslo University Hospital, Norway, a national referral center for young CFS patients (12–18 years). Main outcomes were number of steps per day obtained by an accelerometer and disability scored by the Functional Disability Inventory (FDI). Patients with the 5-HTT SS or SL_G genotype had a significantly lower number of steps per day than patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. Patients with the 5-HTT SS or SL_G genotype also had a significantly higher FDI score than patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. Thus, CFS patients with the 5-HTT SS or SL_G genotype had worse 30 weeks outcome than CFS patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. The present study suggests that the 5-HTT genotype may be a factor that contributes to maintenance of CFS.     

5-HTTLPR Expression Outside the Skin: An Experimental Test of the Emotional Reactivity Hypothesis in Children

Background

There is increasing evidence that variation in the promoter region of the serotonin transporter gene SLC6A4 (i.e., the 5-HTTLPR polymorphism) moderates the impact of environmental stressors on child psychopathology. Emotional reactivity −the intensity of an individual’s response to other’s emotions− has been put forward as a possible mechanism underlying these gene-by-environment interactions (i.e., G×E). Compared to children homozygous for the L-allele (LL-genotypes), children carrying an S-allele (SS/SL-genotypes), specifically when they have been frequently exposed to negative emotions in the family environment, might be more emotionally reactive and therefore more susceptible to affective environmental stressors. However, the association between 5-HTTLPR and emotional reactivity in children has not yet been empirically tested. Therefore, the goal of this study was to test this association in a large-scale experiment.

Methods

Children (N = 521, 52.5% boys, Mage = 9.72 years) were genotyped and randomly assigned to happy, angry or neutral dynamic facial expressions and vocalizations. Motor and affective emotional reactivity were assessed through children’s self-reported negative and positive affect (n = 460) and facial electromyography activity (i.e., fEMG: the zygomaticus or “smile” muscle and the corrugator or “frown” muscle, n = 403). Parents reported on their negative and positive parenting behaviors.

Results

Children mimicked and experienced the emotion they were exposed to. However, neither motor reactivity nor affective reactivity to these emotions depended on children’s 5-HTTLPR genotype: SS/SL-genotypes did not manifest any stronger response to emotional stimuli than LL-genotypes. This finding remained the same when taking the broader family environment into account, controlling for kinship, age, gender and genetic ancestry, and when including a tri-allelic factor.

Conclusions

We found no evidence for an association between the 5-HTTLPR polymorphism and children’s emotional reactivity. This finding is important, in discounting one potential underlying endophenotype of G×E between the 5-HTTLPR and affective environmental stressors.     

5-HTTLPR and Early Childhood Adversities Moderate Cognitive and Emotional Processing in Adolescence

Background

Polymorphisms in the promoter region of the serotonin transporter gene (5-HTTLPR) and exposure to early childhood adversities (CA) are independently associated with individual differences in cognitive and emotional processing. Whether these two factors interact to influence cognitive and emotional processing is not known.

Methodology and Principal Findings

We used a sample of 238 adolescents from a community study characterised by the presence of the short allele of 5-HTTLPR (LL, LS, SS) and the presence or absence of exposure to CA before 6 years of age. We measured cognitive and emotional processing using a set of neuropsychological tasks selected predominantly from the CANTAB® battery. We found that adolescents homozygous for the short allele (SS) of 5-HTTLPR and exposed to CA were worse at classifying negative and neutral stimuli and made more errors in response to ambiguous negative feedback. In addition, cognitive and emotional processing deficits were associated with diagnoses of anxiety and/or depressions.

Conclusion and Significance

Cognitive and emotional processing deficits may act as a transdiagnostic intermediate marker for anxiety and depressive disorders in genetically susceptible individuals exposed to CA.     

Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

The Striatal Transporter for Dopamine in the Rat May Be Kinetically Up-Regulated Following 3 Weeks of Withdrawal from Cocaine Self-Administration

Abstract: Rotating disk electrode voltammetry was used to measure the inwardly directed V_max and K_m of dopamine with its transporter in striatal suspensions prepared from nonhandled control rats, rats that had been trained to self-administer cocaine for 20 days (at 26 mg/day per rat) via a jugular catheter and subsequently withdrawn for 3 weeks, and rats that had received saline (155 mM NaCl) via a jugular catheter on the same schedule as the rats that had received cocaine. Because a limited number of animals was available from the self-administration procedure, the velocity of dopamine transport as a function of [dopamine] was measured by incremental addition of dopamine to a given striatal preparation. In nonhandled controls the values of V_max, K_m, and turnover, observed in this experimental paradigm, were increased relative to results obtained in studies of the velocity-[dopamine] relationship where dopamine was added to suspensions, one concentration per suspension. The kinetics of the association of dopamine with the transporter were unchanged. The V_max to K_m ratios obtained in the two experiments were statistically indistinguishable, suggesting that the two types of experiments probe the same transporter. Also, the increased velocity observed in the experiment involving sequential additions to the same preparation is evidence of trans acceleration, suggesting that the movement of dopamine across the membrane is carrier mediated as opposed to being mediated via channels or pores and that the rate-limiting step in inwardly directed transport is the reorientation of the unloaded transporter from the inwardly to the outwardly facing forms. Saline-treated rats in the self-administration paradigm exhibited kinetic parameters of transport indistinguishable from those observed in nonhandled controls. In contrast, V_max and K_m of the transporter were increased in suspensions prepared from rats that self-administered cocaine and were withdrawn for 3 weeks, relative to saline-treated and nonhandled animals. Combined, these results suggest that the striatal uptake of dopamine is mediated by a transporter and that it is kinetically up-regulated following withdrawal from repeated cocaine administered in a self-administration paradigm.     

Variability of Reaction Kinetics for Ribulose-1,5-bisphosphate Carboxylase in a Barley Population

The photosynthetic enzyme ribulose bisphosphate carboxylase-oxygenase [EC 4.1.1.39] (RuBPCase) plays a key role in the carbon reduction system of plants. In this study, we determined the kinetic variability of RuBPCase among 46 varieties of Hordeum vulgare L. at two ages. The V_max CO₂ and K_m CO₂ of RuBPCase was determined for each cultivar. Varietal differences were found in K_m CO₂ and V_max CO₂ for one and four genotypes, respectively. One variety exhibited atypical behavior in both K_m and V_max. A comparison of varieties and age showed a significant interaction between these factors for K_m but not for V_max. These data indicate the presence of kinetic variability in RuBPCase within the H. vulgare population and perhaps between plant ages.     

Possible Existence of Quaternary Structure in the High-Affinity Serotonin Transport Complex

Deletion-mutants of the cloned mouse serotonin transporter (SERT) rendered dominant negative-mutant effects upon wild-type transporter activities in heterologous expression studies; such effects were transporter-selective and did not influence the activities of co-expressed neuronal GABA transporter. Heterologous expression of linear concatenates (up to four copies) of SERT further revealed discernable uptake activities for both transporter-dimer and -tetramer, but not for the trimer. Kinetic and pharmacological analyses revealed that the monomer, dimer, and tetramer manifested comparable transport K_mand potencies for known serotonin uptake inhibitors; the tetramer was distinct from the others only in manifesting notably reduced transport V_max. Surprisingly, equivalent cocaine congener-binding activities were observed for all concatenates, including the functionally inactive trimer. These findings collectively support the existence of quaternary structure in the active 5-HT transport complex; such structure is likely to be a critical determinant of ligand transport activities, but apparently not of transporter–inhibitor interactions.     

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [³H]-5-hydroxytryptamine uptake into whole cells, but the V_max for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.     

Action of sulphite on the substrate kinetics of chloroplastic NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The kinetics of NADP-GPD from spinach chloroplasts are biphasic vs NADPH and PGA. Thus, two maximum velocities exist with an intermediary plateau and two K_m values. Activation by NADPH + DTT increases V_max of both sections, but does not change the substrate affinities. Sulphite reduces the maximum activities of both sections vs NADPH, however, it causes normal substrate kinetics vs PGA; even V_max is reduced. Sulphite, present only during the activation process, suppresses the enzyme form with the higher V_max. The kinetics vs NADH are also biphasic; the activity is strongly reduced by preincubation of the chloroplasts with NADH + DTT or at NADH concentrations > 0.4mM. Using NADH as cofactor, inverted peaks in the kinetics vs PGA occur; sulphite is active in a similar way as when NADPH is used as cofactor. The biphasic kinetics are discussed with respect to additional potential for regulation of enzyme activity according to illumination and NADPH concentrations respectively.     

Exploring the Impact of BDNF Val66Met Genotype on Serotonin Transporter and Serotonin-1A Receptor Binding

Background

The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism (rs6265) may impact on the in-vivo binding of important serotonergic structures such as the serotonin transporter (5-HTT) and the serotonin-1A (5-HT_1A) receptor. Previous positron emission tomography (PET) studies on the association between Val66Met and 5-HTT and 5-HT_1A binding potential (BP_ND) have demonstrated equivocal results.

Methods

We conducted an imaging genetics study investigating the effect of Val66Met genotype on 5-HTT or 5-HT_1A BP_ND in 92 subjects. Forty-one subjects (25 healthy subjects and 16 depressive patients) underwent genotyping for Val66Met and PET imaging with the 5-HTT specific radioligand [¹¹C]DASB. Additionally, in 51 healthy subjects Val66Met genotypes and 5-HT_1A binding with the radioligand [carbonyl-¹¹C]WAY-100635 were ascertained. Voxel-wise and region of interest-based analyses of variance were used to examine the influence of Val66Met on 5-HTT and 5-HT_1A BP_ND.

Results

No significant differences of 5-HTT nor 5-HT_1A BP_ND between BDNF Val66Met genotype groups (val/val vs. met-carrier) were detected. There was no interaction between depression and Val66Met genotype status.

Conclusion

In line with previous data, our work confirms an absent effect of BDNF Val66Met on two major serotonergic structures. These results could suggest that altered protein expression associated with genetic variants, might be compensated in vivo by several levels of unknown feedback mechanisms. In conclusion, Val66Met genotype status is not associated with changes of in-vivo binding of 5-HTT and 5-HT_1A receptors in human subjects.     

Implications of the Alternating Access Model for Organic Anion Transporter Kinetics

Many transport proteins, including the clinically important organic anion transporters (OATs), appear to function via an “alternating access” mechanism. In analyzing the kinetics of these transporters, the terms K _m and V _max are often treated in the field as denoting, respectively, the affinity of the substrate for the transporter and the turnover (conformational switch) rate of the substrate–transporter complex. In fact, the expressions for both these parameters have very complex forms comprising multiple rate constants from conformational switch as well as association/dissociation steps in the cycling of the transporter and, therefore, do not have straightforward physical meanings. However, if the rapid equilibrium assumption is made (namely, that the association/dissociation steps occur far more rapidly than the conformational switch steps), these expressions become greatly simplified and their physical meaning clear, though still distinct from the conventional interpretations. V _max will be a function of not just the rate of substrate–transporter complex turnover but also the rate of the “return” conformational switch and will vary largely with the slower of these two steps (the rate-limiting step). K _m will be seen to be related to substrate affinity by a term that varies inversely with the substrate–transporter complex turnover rate, essentially because the greater this rate, the greater the extent to which transporters will be distributed in a conformation inaccessible to substrate. Here, an intuitive approach is presented to demonstrate these conclusions. The phenomena of trans-stimulation and trans-inhibition are discussed in the context of this analysis.     

Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide

The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10–200 μM) stimulation of 5-HT transport that was rapid and sustained over 0–10 minutes of incubation. When platelets were incubated with diamide (10–200 μM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 μM, while washed platelets incubated with diamide (10–200 μM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (V_max control = 28.4 ± 1.4 vs. V_max diamide = 60.9 ± 4.1 pM/10⁸ platelets/4 min) but did not significantly alter the K_m values. Ouabain, an inhibitor of platelet Na⁺-K⁺ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.