Serotonin Uptake In Human Platelets Research Articles

Serotonin level and serotonin uptake in human platelets: A variable interrelation under marked physiological influences

Background Although it is known that platelet serotonin level (PSL) depends directly on platelet serotonin uptake (PSU) through the plasma membrane, reports on their interrelation are inconsistent. The aim of this study was to systematically explore the relationship between these two platelet serotonin parameters in large human population. Methods PSL and full-kinetics of PSU were determined on 318 blood donors (276 males, 42 females; 20–67 years). Results The overall correlation coefficient between PSL and maximal velocity of PSU was highly significant but unexpectedly low ( r = 0.269). Further analyses revealed lack of correlation among females, and variable association among males, depending on the subject age and season of measurements. Highly significant correlations were observed in spring–winter, while association was absent during summer–autumn. Lowering of PSL–PSU correlation with increased age was also demonstrated, showing modest interrelation among younger men and no interrelation in older population. By multiple regression analyses season was identified as the only independent predictor of PSL–PSU relationship. Conclusions The results show prominent influence of biological (sex, age) and, especially, environmental (seasons) physiology on the intraindividual relationship between PSL and PSU. Although serotonin transporter activity plays an important role in determining PSL, the observed correlations indicate that other factors may predominate.

Hyperforin activates nonselective cation channels (NSCCs)

A large body of evidence supports the preclinical antidepressant profile of hyperforin including inhibition of the synaptosomal uptake of several neurotransmitters by hyperforin and studies in behavioural models. In contrast to other antidepressants, hyperforin does not directly inhibit neurotransmitter transporters, but instead uptake inhibition seems to be the consequence of an elevated intracellular sodium concentration ([Na+]i). The mechanism of hyperforin-induced elevation of [Na+]i was investigated using two different cell types: human platelets and rat pheochromocytoma cells (PC12 cells). In both cell systems, hyperforin increased both [Na+]i and free intracellular Ca2+ concentration ([Ca2+]i). One pathway for Na+ and Ca2+ entry is mediated by nonselective cation channels (NSCCs), which can be blocked by SK&F 96365 and LOE 908. LOE 908 is a blocker of both NSCC1 and NSCC2 subclasses, while SK&F 96365 blocks NSCC2 only. Both SK&F 96365 and LOE 908 completely inhibited the hyperforin-induced influx of Na+ and Ca2+ into platelets and PC12 cells. This indicates that hyperforin is mainly active upon NSCC2. The effect of hyperforin is inhibited by La3+ and Gd3+, indicating that there is a potential homology with canonical transient receptor potential protein channels (TRPC channels). Moreover, La3+ and Gd3+ attenuate the effect of hyperforin on serotonin uptake in human platelets. Additionally, hyperforin induces barium influx in PC12 cells and this influx can be inhibited by SK&F 96365, LOE 908, Gd3+ and La3+. In summary, these findings suggest that hyperforin represents a new principle for preclinical antidepressant activity, modulating brain neurotransmission by inhibition of neurotransmitter uptake via activation of NSCCs.British Journal of Pharmacology (2005) 145, 75-83. doi:10.1038/sj.bjp.0706155.

The effect of the anorectic agent, d-fenfluramine, and its primary metabolite, d-norfenfluramine, on intact human platelet serotonin uptake and efflux

Dexfenfluramine, a drug formerly prescribed for treatment of obesity, caused heart valve damage and pulmonary hypertension in some people. The cause of the toxicity has not been defined, but 5-HT has been implicated. The objective of this study was to evaluate the effect of the anorectic agent, d-fenfluramine, and its major metabolite, d-norfenfluramine, on intact human platelet serotonin (5-HT) transport in vitro. The effects of d-fenfluramine and d-norfenfluramine on platelet uptake and efflux of 3H-5-HT were measured in buffer at pH 6.7, to optimize serotonin transporter (SERT) function, and at pH 7.4. Uptake of 3H-5-HT at pH 6.7 and 7.4 was inhibited by both agents at micro m concentrations (IC50, d-fenfluramine approximately 3 microM; d-norfenfluramine approximately 10 microM). However, no efflux of 3H-5-HT from labeled platelets at either pH 6.7 or 7.4 occurred at similar concentrations of d-fenfluramine or d-norfenfluramine. With inhibition of platelet dense granule 3H-5-HT uptake by reserpine, efflux of 3H-5-HT was observed at pH 6, but not at pH 7.4. Fluoxetine, a SERT inhibitor, was a more potent inhibitor of uptake (IC50 0.05 microM) than d-fenfluramine, but the anorectic agent, phentermine, had no effect. Therefore, d-fenfluramine and d-norfenfluramine inhibit human platelet uptake of 5-HT in vitro at tissue concentrations attainable in vivo, but they do not stimulate 5-HT efflux due to dense granule sequestration. Inhibition of platelet 5-HT uptake may play a role in the cardiopulmonary toxicity of d-fenfluramine, but other factors probably contribute, since similar toxicity has not been observed with fluoxetine.

Inhibition of platelet serotonin uptake by cytochrome P450 inhibitors miconazole and econazole

Serotonin uptake in human platelets was inhibited by cytochrome P450 inhibitors such as miconazole and econazole but not clotrimazole. There was a correlation between inhibition of serotonin uptake and inhibition of imipramine binding, suggesting that these P450 inhibitors may inhibit serotonin uptake via direct binding to the transporter. P450 inhibitor effects on serotonin uptake did not seem to be related to the effects of these compounds on intracellular calcium mobilization. Additionally, nitric oxide pathway stimulation does not appear to be involved.

Increase of human platelet serotonin uptake by atypical histamine receptors

Histamine and the guanosine 3',5'-cyclic monophosphate (cGMP)-inducing agent sodium nitroprusside both increased serotonin (5-HT) uptake and cGMP levels in isolated human platelets in vitro. Histaminergic stimulation was observed at concentrations ranging from 10 nM to 0.25 microM [mean effective concentration (EC50) = 0.1 microM histamine]. The inhibition produced by the H2-receptor antagonists tiotidine, metiamide, and cimetidine was 10-10(5) times more potent on histamine receptors regulating 5-HT uptake and cGMP generation in human platelets than on the histaminergic receptors H1, HIC, H2, and H3 in other tissues. The in vitro histamine-induced 5-HT uptake was prevented by preincubation of isolated human platelets in the presence of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine or the cGMP-lowering agent LY-83583. Histamine was ineffective in stimulating cAMP generation in human platelets and did not interact with effector sites known to downregulate 5-HT uptake, including imipramine, gamma-aminobutyric acid A, peripheral type benzodiazepine-binding sites, and V1a vasopressin receptors inducing human platelet shape change and aggregation. These atypical human platelet histaminergic receptors differ from the previously classified histamine receptors by their apparent high affinity to histamine H2-receptor antagonists and their apparent link with the soluble, nitric oxide-dependent guanylate cyclase. These findings suggest that human platelets express a new subtype H2h of histamine receptors.

Primary Structure of the Human Platelet Serotonin Uptake Site: Identity with the Brain Serotonin Transporter

A cDNA encoding the human platelet serotonin (5-HT) uptake site was isolated and sequenced using the PCR. The cDNA represents a approximately 3.1-kb mRNA transcript and contains an open reading frame encoding a hydrophobic polypeptide of 630 amino acids with 12 membrane-spanning segments, a calculated molecular mass of 70,320 Da, and an estimated isoelectrical point of 5.84. The human platelet 5-HT uptake site is identical with the human brain 5-HT transporter and approximately 92% homologous to the rat protein. Hydropathicity analysis indicates 12 membrane-spanning segments with two putative glycosylation sites within the second extracellular loop. The human platelet 5-HT uptake site contains two intraplasmatic consensus phosphorylation sites for cyclic AMP-dependent protein kinase recognition located in the cytoplasmatic N-terminal region and three potential protein kinase C phosphorylation sites. The identity of the human platelet 5-HT uptake site and the brain 5-HT transporter indicates that both proteins are encoded by the same single-copy gene, which has been assigned to the human chromosome 17. Our findings are likely to facilitate molecular pharmacologic and genetic investigations of the 5-HT transporter in psychiatric disorders.

Effect of nicotine on human blood platelet serotonin uptake and efflux

1. 1. Physostigmine administration has been previously shown to decrease the uptake of serotonin in human platelets. In order to test whether uptake could be inhibited as a nicotinic-cholinergic effect, the in vitro effects of nicotine on platelet 5HT uptake and efflux were examined. 2. 2. Nicotine stimulated release of serotonin from human blood platelets, and competitively inhibited human platelet serotonin uptake in a concentration-dependent fashion at in vitro concentrations as low as 20 μM for uptake. 3. 3. The kinetics of the nicotine effects on uptake were different from those of physostigmine. Unlike the effects of physostigmine, nicotine produced different kinetic changes, with an increase in Km and no consistent change in Vmax. 4. 4. The efflux and inhibition of uptake paralleled that previously reported in rat brain in vitro, and was likewise similar to concentrations found previously to augment extracellular amine in other tissue preparations. However, the effects of nicotine in human platelets were not reversible by nicotinic antagonism with hexamethonium. 5. 5. The results distinguish human platelet from rat brain with respect to nicotinic antagonism, and suggest that, at similar concentrations, nicotine may increase extracellular serotonin through differing mechanisms.

Biochemical and pharmacological characterization of histamine-mediated up-regulation of human platelet serotonin uptake. Evidence for a subclass of histamine H2 receptors (H2h) highly sensitive to H2 receptor antagonists.

The stimulatory effect of histamine: H (1.2 to 3-fold increase) on serotonin (5-HT) uptake by human platelets was observed after a 5 min incubation period in the presence of 2.5 X 10(-7) M histamine, followed by subsequent 5 min incubation of the platelets with 10(-7) M [3H] 5-HT. Methyl, ethyl and acetyl substituents in the side chain of H mimicked the stimulatory effect of H. In contrast, H analogs methylated at the position N-1 of the imidazole ring of H, as well as imidazole and histidine inhibited platelet 5-HT uptake. The cAMP-inducing agents forskolin and theophylline have no effect on 5-HT uptake when they are tested alone or in combinations with H. In contrast, the cGMP-inducing agent sodium nitroprusside (10(-7) M-10(-6) M) stimulated and potentiated H-mediated up-regulation of 5-HT uptake. Histamine H2 receptor agonists and antagonists are more potent than drugs acting on H1 receptors (H2 greater than H1). However, the inhibition constants Ki are not consistent with those determined for typical H1, H2, H3 receptors characterized in other tissues. This findings provide further evidence for the existence of multiple forms of H receptors and suggest the involvement of a subpopulation of H2 receptors, highly sensitive to H2 receptor antagonists (H2h), mediating 5-HT uptake in human platelets.

A modified assay method for determining serotonin uptake in human platelets

Effects of various experimental conditions on serotonin (5-HT) uptake in human platelets were examined. The experimental design allowed the evaluation of the effect of diffusion and other non-saturable processes on the affinity and maximum activity of the membrane pump for 5-HT uptake. Total 5-HT uptake was determined by incubating platelet-rich plasma (PRP) with increasing concentrations of serotonin at 37°C for 4 min. The passive uptake was measured by the addition of various 5-HT concentrations to PRP in buffer at 37°C, followed by immediate transfer to an ice-cold water bath. The difference between the total and passive uptake was linear for 6 min. The affinity ( K m) for active platelet serotonin uptake was 0.45 ± 0.09 γmol/ l and maximal rate of uptake ( V) was 10.7 ± 2.1 pmol/10 7 platelets/min. The described method provides a convenient and reliable measure of active 5-HT uptake suitable for clinical investigation. The effect of passive diffusion on kinetic parameters is discussed.

2-Nitroimipramine: A selective irreversible inhibitor of [ 3H] serotonin uptake and [ 3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [ 3H] imipramine binding and [ 3H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [ 3H] imipramine binding and [ 3H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.

Alterations in Human-Platelet Serotonin Uptake Following the Addition of Thrombin or A 23187

Alterations in Human-Platelet Serotonin Uptake Following the Addition of Thrombin or A 23187

Sex specific differences in chlorimipramine inhibition of serotonin uptake in human platelets

The uptake of 14C-serotonin in platelet-rich plasma was similar in clinically healthy men and women. Serotonin uptake inhibition by chlorimipramine was significantly less in women than in men, the extent of inhibition correlating with the stage of the menstrual cycle.