M. Lebret

Initial characterization of human platelet mepacrine-labelled granules isolated using a short metrizamide gradient

Summary. A method for the purification of human platelet mepacrine‐labelled granules is described. Characterization of these isolated granules allowed them to be identified as the serotonin storage organelles or dense bodies. Each step of the purification procedure has been controlled in order to obtain a minimum of leakage of the granule content during initial isolation of the platelets from the blood, the platelet washing procedures, and platelet lysis and the subcellular separation. A key step in the procedure was the centrifugation of the labelled granules across a short, discontinuous metrizamide gradient. The pellet of isolated mepacrine‐fluorescent granules consisted almost entirely of granules with the typical appearance of dense bodies, as shown by electron microscopy, and was relatively free from membranes and other granule populations as evaluated by the presence of the different markers (tritiated lectin, beta‐glucuronidase, monoamine oxidase, platelet factor 4). The method is simple, reproducible and allows the highest enrichment in dense bodies obtained hitherto with human platelets: x 177 in calcium and x 115 in [14C]serotonin after fractionation of [14C]serotonin‐labelled whole platelets. Functional studies performed with the isolated granules showed that they rapidly accumulated [14C]serotonin.

Comparison between electronic cigarette refill liquid and nicotine on metabolic parameters in rats

AIMS Nicotine is known to promote body weight loss and to disturb glucose homeostasis and lipoprotein metabolism. Electronic cigarettes, as a substitute to nicotine, are becoming increasingly popular, although there is no evidence regarding their safety. Considering the dearth of information about e-cigarette toxicity, the present study was designed to compare nicotine alone to e-liquid with or without nicotine on metabolic parameters in Wistar rats. MAIN METHODS For this purpose, e-liquid with or without nicotine and nicotine alone (0.5mg/kg of body weight) were administered intra-peritoneally during 28 days. KEY FINDINGS Our results show a significant decrease in food and energy intake after nicotine or e-liquid with nicotine exposure, when compared to control or e-liquid without nicotine. Analysis of lipid status identified a significant decrease in cholesterol and LDL levels in e-cigarette groups, suggesting an improvement in lipid profile. Interestingly, e-liquid without nicotine induced hyperglycemia which is negatively correlated to hepatic glycogen level, acting like nicotine alone. Furthermore, an increase in liver biomarkers was observed in all treated groups. qRT-PCR analysis showed GSK3β up-regulation in e-liquid with nicotine as well as, surprisingly, in e-liquid without nicotine exposure. In contrast, PEPCK genes were only up-regulated in e-liquid with nicotine. SIGNIFICANCE While some features observed in rats may not be observed in human smokers, most of our data are consistent with, e-liquid per se i.e. without nicotine, not being neutral from a metabolic stand point since disrupting glucose homeostasis in rats.

Abnormal phosphoinositide metabolism and protein phosphorylation in platelets from a patient with the grey platelet syndrome.

Washed platelets isolated from one patient suffering from the inherited grey platelet syndrome were studied during thrombin‐induced activation. The agonist‐induced changes in (i) morphology, (ii) typical functional cell responses, (iii) membrane phospholipid metabolism and protein phosphorylation were studied and compared with the changes obtained with normal platelets. The morphology of the platelets as visualized by electron microscopy confirmed the almost total absence of intracellular alpha‐granules and marked vacuolization. During thrombin stimulation the morphological changes were clearly delayed as compared to normal platelets, the granule centralization and aggregation occurring only 15s after thrombin addition instead of 5 s in normal platelets. After 15 s, however, even though no alpha‐granules were observed, a ring‐like structure occurred centrally, indicating that they are not a prerequisite for this reaction. The whole release reaction, i.e. liberation of [14C]ser‐otonin from dense granules and β‐n‐acetylglucosaminidase activity from lysosomes, and the thromboxane synthesis were delayed and remained lower than in normal platelets. No thrombin‐induced phosphatidyl 4,5‐bisphosphate breakdown was measurable on 32P‐prelabelled platelets although [32P]phosphatidate formation occurred normally. Phosphorylation time courses of myosin light chain (P20) and of protein P43 (mol wt 43 000) markedly differed from those of controls, being less than half of the normal during the first 15 s and remaining subnormal even after complete aggregation. These results suggest that in platelets devoid of alpha‐granules a deficient transmembrane signalling system is likely responsible for the impaired physiological responses.

Abnormal calcium transport into microsomes of grey platelet syndrome

Summary. Calcium uptake into isolated membrane vesicles from two patients with a grey platelet syndrome has been investigated. An increase in calcium transport appears in both patients when compared to controls. Determination of the kinetic parameters of the calcium transport system gave similar apparent affinity for calcium and an increase in the calcium uptake velocity. This increase in calcium transport is correlated with the increase of the associated Ca2+ activated ATPase activity. The results would suggest a new relationship between the ultrastructural and functional abnormalities of the grey platelet syndrome.

Impact of electronic-cigarette refill liquid on rat testis.

Abstract Electronic cigarettes (e-cigarettes) are becoming the fashionable alternative to decrease tobacco smoking, although their impact on health has not been fully assessed yet. The present study was designed to compare the impact of e-cigarette refill liquid (e-liquid) without nicotine to e-liquid with nicotine on rat testis. For this purpose, e-liquid with nicotine and e-liquid without nicotine (0.5 mg/kg of body weight) were administered to adult male Wistar rats via the intraperitoneally route during four weeks. Results showed that e-liquid with or without nicotine leads to diminished sperm density and viability, such as a decrease in testicular lactate dehydrogenase activity and testosterone level. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis identified a reduction in cytochrome P450 side-chain cleavage (P450 scc) and 17 beta-hydroxysteroid dehydrogenase (17βHSD) mRNA level, two key enzymes of steroidogenesis. Following e-liquid exposure, histopathological examination showed alterations in testis tissue marked by germ cells desquamation, disorganization of the tubular contents of testis and cell deposits in seminiferous tubules. Finally, analysis of oxidative stress status pointed an outbreak of antioxidant enzyme activities such as superoxide dismutase, catalase and gluthatione-S-transferase, as well as an important increase in sulfhydril group content. Taken together, these results indicate that e-liquid per se induces toxicity in Wistar rat testis, similar to e-liquid with nicotine, by disrupting oxidative balance and steroidogenesis.

Isolation of Dense Bodies from Human Blood Platelets Using Discontinuous Metrizamide Gradients

ABSTRACT Functionally active dense bodies (5-HT storage organelles) have been isolated from human platelets following centrifugation of platelet lysates on a two step discontinuous metrizamide gradient. Their purification was followed using platelets loaded with either (i) [14C]-5HT or (ii) mepacrine, a fluorescent dye selectively accumulated by the dense bodies. The isolated granules gave the typical appearance of dense bodies when examined by electron microscopy and were able to rapidly incorporate 5-HT; this incorporation was unaffected by imipramine but was strongly inhibited by reserpine.