Normal serotonin uptake by blood platelets and brain synaptosomes but selective impairment of platelet serotonin storage in mice with Chediack-Higashi syndrome

The Chediack-Higashi syndrome (CHS) is an autosomal recessive disorder reported in man and in several animal species including the "beige mice" (bg/bg). Among several manifestations of this genetic trait, deficiency of secretable substances - including serotonin - normally stored in platelet dense granules is a characteristic feature. The animal model of Chediak-Higashi syndrome used in the present study provides a unique opportunity to compare the kinetics of serotonin (5-hydroxytryptamine, 5-HT) uptake in platelets and brain synaptosomes in conditions of selective reduction of 5HT concentration in the platelets. The kinetics of 5HT uptake, as measured in the present study, was normal in synaptosomes and platelets from the same animals. The lower intraplatelet 5HT levels in bg/bg animals as compared to normal synaptosomes levels in the presence of normal uptake offer an indirect proof that the 5HT defect described in the CHS is due to an impaired 5HT storage mechanism. This is supported by the observation that spontaneous release of 5HT was markedly increased in platelets from CH5 mice but was normal in synaptosomes from the same animals. Thus platelets are a reliable model to study 5HT uptake, but not 5HT storage and release in brain synaptosomes.     

Effects of cadmium treatment in vitro on the uptake and release of [3H]serotonin by human blood platelets

Effects of cadmium treatment on human platelets were studied with respect to uptake and release of 5-[3H]hydroxytryptamine (5-HT). The uptake of 5-[3H]HT in the presence of varying concentrations of CdCl2 (0.001-10 mM) was inhibited significantly with respect to control platelets and the inhibition was maximum at 1 mM CdCl2 concentration. From studies on the kinetics of 5-[3H]HT uptake a higher Km and significantly lower Vmax for CdCl2-treated platelets were observed. CdCl2 stimulated spontaneous release but inhibited thrombin-induced release of 5-[3H]HT. Spontaneous release of 5-[3H]HT induced by CdCl2 was not significantly altered in the presence of externally available CaCl2 (1 mM).     

Plasma serotonin levels and the platelet serotonin transporter

Serotonin (5HT) is a platelet-stored vasoconstrictor. Altered concentrations of circulating 5HT are implicated in several pathologic conditions, including hypertension. The actions of 5HT are mediated by different types of receptors and terminated by a single 5HT transporter (SERT). Therefore, SERT is a major mechanism that regulates plasma 5HT levels to prevent vasoconstriction and thereby secure a stable blood flow. In this study, the response of platelet SERT to the plasma 5HT levels was examined within two models: (i) in subjects with chronic hypertension or normotension; (ii) on platelets isolated from normotensive subjects and pretreated with 5HT at various concentrations. The platelet 5HT uptake rates were lower during hypertension due to a decrease in Vmax with a similar Km; also, the decrease in Vmax was primarily due to a decrease in the density of SERT on the platelet membrane, with no change in whole cell expression. Additionally, while the platelet 5HT content decreased 33%, the plasma 5HT content increased 33%. Furthermore, exogenous 5HT altered the 5HT uptake rates by changing the density of SERT molecules on the plasma membrane in a biphasic manner. Therefore, we hypothesize that in a hypertensive state, the elevated plasma 5HT levels induces a loss in 5HT uptake function in platelets via a decrease in the density of SERT molecules on the plasma membrane. Through the feedback effect of this proposed mechanism, plasma 5HT controls its own concentration levels by modulating the uptake properties of platelet SERT.     

Shape change and uptake of 5-hydroxytryptamine in human blood platelets: action of neuropsychotropic drugs.

Psychotropic drugs acted as agonists, antagonists or mixed agonists-antagonists of the shape change mediated by the 5HT receptor in blood platelets of man incubated in a protein-poor medium. Regarding inhibition of 5HT uptake some of the drugs were equally potent in both reserpinized and normal platelets, whereas others showed lower or higher potencies in the former than in the latter. There was no correlation between the potencies of the drugs as shape change inhibitors and as uptake inhibitors. It is concluded that 1) human platelets may serve as models for the neuronal 5HT receptors of some areas of the central nervous system, 2) platelets can be used to determine the subcellular site of action of 5HT uptake inhibitors and 3) the receptors responsible of the shape change, and the site of the 5HT transport at the plasma membrane are different entities.     

Phorbol ester treatment inhibits thrombin but not stable GTP analogue-induced platelet granule secretion despite inhibition of phosphatidate formation with both agonists

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.     

Removal of 5-hydroxytryptamine by rat lungs.

Isolated rat lungs perfused with Krebs solution removed free radioactive 5-Hydroxytryptamine (5 HT or serotonin) from the fluid perfusing them. However, when platelets labelled with 111In-oxine to tag them individually, and with 14C-labelled 5 HT were also perfused via the lungs, there was no significant removal of the labelled 5 HT from the platelets. Paper chromatography showed that 46.7% of the platelet bound 5 HT was metabolished to an unidentified material. Rat lungs remove free 5 HT from plasma but not when it is platelet bound.     

Inhibition of serotonin-amplified human platelet aggregation by ketanserin, ritanserin, and the ergoline 5HT2 receptor antagonists-LY53857, sergolexole, and LY237733

Human platelets are known to possess 5HT2 receptors which, when activated, amplify the aggregation response produced by other aggregating agents. Several 5HT2 receptor antagonists, including ketanserin and ritanserin, are known to antagonize serotonin-mediated aggregation of human platelets. In the present study, we document the ability of three ergoline 5HT2 receptor antagonists, LY53857, sergolexole, and LY237733, to antagonize the serotonergic component of the human platelet aggregation response. Potencies of the ergoline esters (LY53857 and sergolexole) and the ergoline amide (LY237733) to inhibit serotonin-amplified platelet aggregation responses were similar to the potencies of ketanserin and ritanserin under the conditions of our study. Furthermore, all five 5HT2 receptor antagonists were capable of fully inhibiting the serotonergic component of the platelet aggregation response. In contrast to these potent ergoline esters and amides, 1-isopropyl dihydrolysergic acid (up to 10(-5)M), a putative metabolite of the ergoline esters, was ineffective under these in vitro conditions. These data are consistent with the high potency of these ergolines as antagonists of 5HT2 receptors and further support the involvement of 5HT2 receptors on human platelets in the amplifying response to serotonin.     

Hyperserotonemia in autism: the potential role of 5HT-related gene variants

Increased platelet serotonin level (PSL) has been consistently found in a portion of autistic patients. Suggested mechanisms for hyperserotonemia in autism have been increased synthesis of serotonin (5HT) by tryptophan hydroxylase (TPH), increased uptake into platelets through 5HT transporter (5HTt), diminished release from platelets through 5HT2A receptor (5HT2Ar) and decreased metabolism by monoamine oxydase (MAOA). The allelic influence of genes, encoding the mentioned 5HT elements, on PSL was investigated in 63 autistic subjects. Our study shows that 5HTt-LPR and -1438AG 5HT(2Ar) genotypes did not significantly affect PSL. However, significantly higher PSLs were observed in subjects with "cc" genotype of a218c TPH and subjects with "4" genotype of uVNTR MAOA. In addition, when TPH-cc and MAOA-4 were combined as "high 5HT" genotypes, a correlative increase in PSL was observed with the increase in the number of "high 5HT" genotypes. These results suggest a possible synergistic effect of genes regulating 5HT synthesis/degradation in dysregulation of the peripheral 5HT homeostasis of autistic patients.     

Effects of reserpine and emipramine on vesicular serotonin uptake and storage in intact human platelets

The effects of reserpine and imipramine on intact human platelets have been investigated, utilizing brief thrombin treatment to evaluate serotonin (5HT) uptake into and loss from the vesicular (thrombin-releasable) compartment. Less than five seconds after its addition, reserpine (10^?6M) almost completely inhibited the uptake of 5HT into storage vesicles; but induced an outward flux of stored 5HT from vesicles only after more than two minutes following its addition. Imipramine (10^?6M), acting over a 30-minute period, caused no loss of vesicular 5HT, but acted within five minutes to inhibit markedly the movement of cytoplasmic 5HT into storage vesicles. It thus seems likely that in human platelets, inhibition of vesicular 5HT uptake does not necessarily lead to the loss of vesicular 5HT.     

Maximal packet size for serotonin in storage vesicles of intact human platelets

A procedure has been developed to measure maximal packet size for serotonin in vesicles of intact, washed human platelets. Vesicles were trace-labelled with H³-5HT and subsequently permitted to accumulate larger amounts of C¹⁴-5HT from the extracellular medium. Parallel platelet aliquots were incubated with unlabelled 5HT, and total and thrombin-releasable 5HT were measured by an enzymatic assay. Prior to incubation, 5HT packet size ranged from 0.05 to 0.53 × 10^?17 moles/platelet. No net addition of 5HT to vesicles occurred when this compartment contained more than 2–3 × 10^?17 moles/platelet of 5HT, suggesting that maximal packet size was 2.8 × 10^?17 moles/platelet. Under these conditions, a typical vesicle would contain 4 × 10^?18 molesof 5 HT, at an estimated concentration of 0.6 M. Nevertheless, when vesicles were full, they continued to exchange C¹⁴-5HT from the extracellular medium with H³-5HT sequestered in vesicles.     

Whole blood serotonin and tryptophan levels in Tourette's disorder: effects of acute and chronic clonidine treatment

Whole blood serotonin (WB5HT) and tryptophan (WBTRP) levels were studied in 20 patients (aged 8 to 45 years) with Tourette's disorder under medication-free baseline conditions and following acute and chronic clonidine treatment. Compared to 87 normal controls, Tourette's disorder patients had lower mean baseline WBTRP levels (mean +/- SEM: Tourette's, 5993 +/- 304 ng/ml vs. 6822 +/- 169 ng/ml; p less than .03). No significant differences in mean baseline WB5HT levels were found. Three hours after an acute dose of clonidine (2.5 - 5.1 micrograms/kg, p.o. at 9:00 A.M.), no mean differences were observed (baseline vs. post 3 hours) in WB5HT or WBTRP levels. However, following chronic treatment (greater than 3 weeks) with clonidine (3-8 micrograms/kg/day, p.o.), WB5HT levels were increased in 9 of 14 Tourette's disorder patients. The mean increases in WB5HT levels following chronic clonidine treatment were significant when WB5HT levels were expressed per 10(9) platelets. (mean +/- SEM: baseline, 471 +/- 45 ng/10(9) platelets vs. chronic, 697 +/- 82 ng/10(9) platelets, p = .02). No mean differences in WBTRP levels were observed after chronic clonidine treatment. These findings are discussed in light of a proposed intermediary role of 5HT systems in the mode of action of clonidine in the treatment of Tourette's disorder.     

Platelet serotonin transport is altered in streptozotocin-induced diabetic rats

The present work was conducted to examine whether experimental diabetes (streptozotocin-induced) promotes changes in mean platelet volume, and platelet serotonin (5HT) uptake and content. These variables were measured in from four experimental groups: control, diabetic, diabetic+insulin, and non-diabetic+insulin. Animals treated fifteen days before with streptozotocin had platelets with higher 5HT uptake affinity, 5HT content, and volume. The insulin therapy reestablished the control values of all of these three variables. Non-diabetic animals treated one week with insulin did not show any variations. The effects of in vitro application of insulin, hyperglycaemic incubation medium, and streptozotocin on platelet amine uptake and release were also examined. Only those platelets incubated with streptozotocin showed an altered platelet 5HT uptake. No changes were observed for spontaneous 5HT release. The results are consistent with: a) an increase of platelet uptake capacity, as a consequence of an increase in platelet turnover, for explaining alterations of intraplatelet 5HT contents in experimental diabetes; b) a non-direct effect of insulin and glucose levels on platelet 5HT uptake -for explaining its dysfunctions in experimental diabetes-; c) the contribution of alterations in platelet 5HT transport for explaining the higher incidence of vascular complications in diabetic patients; d) the suitability of platelet as a model for investigating neuronal 5HT reuptake.     

Norepinephrine-mediated Regulation of 5HT<Subscript>1</Subscript> Receptor Functioning in Human Platelets

Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.     

Quantitative evaluation of the uptake capacity of intact thrombocytes using fluorescent dyes

A new approach to quantitative determination of fluorescent dye uptake by intact cells is suggested. Fluorescent amine acridine orange selectively accumulating in 5HT granules of platelets has been used. Fluorescence signal analysis allows the estimation of a relative granule volume and the ratios of acridine orange transfer over cytoplasmic and granule membranes. The following results were obtained in human and rabbit platelets: a relative granule size was 14 +/- 1 % and 29 +/- 2 % of the total cell volume, intra-granule to extra-granule dye concentration ratios were 2260 +/- 382 and 30000 +/- 5550, while intra-cytoplasm to extra-cytoplasm concentration ratios were 375 +/- 60 and 225 +/- 60, respectively.     

Effects of antimycin a plus 2-deoxyglucose on uptake and vesicular storage of serotonin in human platelets

The effects of the metabolic poisons antimycin A (4.1 μg/ml) and 2-deoxyglucose (32.2 mM) on the uptake and vesicular storage of serotonin in washed human platelets have been examined. Within 15 seconds after the addition of the metabolic poisons, H³-5HT begins to move from vesicles into the cytoplasm; by 30 minutes after poison addition, essentially all the platelet 5HT appears to be cytoplasmic. The metabolic poisons also act rapidly to decrease plasma-membrane uptake of H³-5HT from the extracellular medium by approximately 20% within 1 minute after their addition. This may represent a direct effect rather than one resulting from altered cytoplasmic or vesicular 5HT, since platelets with <10% of the normal number of vesicular storage sites exhibit a similar reduction after addition of the metabolic poisons.     

Specific uptake of serotonin by murine macrophages

In view of the reported immunomodulatory properties of the monoaminergic neurotransmitter, serotonin (5HT), this study aimed to assess whether 5HT specifically associates with immunocompetent cells. Although murine splenocytes appear to lack high-affinity membrane binding sites for 5HT, they do possess a specific, active, 5HT uptake system similar in affinity (Km = 40 nM) to that described in platelets. This uptake system appears to be confined to macrophages. Further, macrophages rapidly metabolize 5HT to its 5-hydroxyindole acetic acid metabolite. Specific uptake of serotonin by macrophages may thus constitute an important mechanism whereby this amine is able to regulate immune function.     

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.     

Ultrastructural observation of 5-hydroxytryptamine-storing granules in the domestic fowl thrombocytes

Summary Ultrastructural observations of domestic fowl thrombocytes were made with special reference to their intracellular storage site of 5HT. Using a modified method for sampling blood and isolating thrombocytes, a characteristic structure was confirmed in the cytoplasm; the structure was highly osmiophilic, rough-surfaced and quite larger than 5HT granules observed in mammalian platelets. Disappearance of the structure, associated with a marked reduction of 5HT content, was caused by in vivo treatment with reserpine; this indicated its function to store the amine. From various morphological patterns indicative of stepwise advancement of degranulation, it was concluded that the 5HT-storing organelle is the multigranular body, which contains smooth-surfaced granules aggregated with fine filamentous structures.Sponsored partly by the Ministry of Education of Japan.The authors wish to acknowledge the valuable reviews of Professors A. Inouye and H. Takagi.     

The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.