Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation

Effect of mercury on selenium utilization and selenoperoxidase activity in LNCaP cells

Abstract Formation of stable complexes with protein thiols is the best-known mechanism of mercury toxicity. However, the solubility product of Hg(2+) with sulfides, although very low, is higher than that with selenides, suggesting that the fully reduced form of selenium might also be a relevant target for Hg(2+). In cells, selenide is the suggested intermediate for selenoprotein biosynthesis and selenoenzymes, in turn, contain reduced selenium as the catalytic moiety. Thus, inhibition of biological functions of selenium could be seen as a different mechanism of Hg(2+) toxicity. To address this issue, we investigated selenoperoxidase (SeGPx) activity in LNCaP cells exposed to HgCl(2). Cells growing in standard medium express a low GPx activity, which increases on addition of selenium donors such as selenite, selenomethionine, or methyl-Se-cysteine. HgCl(2) added to the medium has different effects depending on the type of Se donor. A progressive decrease of SeGPx activity is observed in cells grown in standard medium exposed to HgCl(2), while coadministration of suprastoichiometric amounts of HgCl(2) prevents the increase of SeGPx activity only when selenite, but not selenomethionine or methyl-Se-cysteine, is the selenium source. From this evidence we conclude that HgCl(2): (a) does not inhibit directly SeGPxs, as confirmed on isolated enzymes; (b) does not interfere with the intermediates of the metabolic pathway of selenoprotein synthesis; and (c) decreases the bioavailability of selenium only when ionic complexes can be formed