Serotonin (5-HT) transporter (SERT) regulates the level of 5-HT in placenta. Initially, we found that in gestational diabetes mellitus (GDM), whereas free plasma 5-HT levels were elevated, the 5-HT uptake rates of trophoblast were significantly down-regulated, due to impairment in the translocation of SERT molecules to the cell surface. We sought to determine the factors mediating the down-regulation of SERT in GDM trophoblast. We previously reported that an endoplasmic reticulum chaperone, ERp44, binds to Cys200 and Cys209 residues of SERT to build a disulfide bond. Following this posttranslational modification, before trafficking to the plasma membrane, SERT must be dissociated from ERp44; and this process is facilitated by insulin signaling and reversed by the insulin receptor blocker AGL2263. However, the GDM-associated defect in insulin signaling hampers the dissociation of ERp44 from SERT. Furthermore, whereas ERp44 constitutively occupies Cys200/ Cys209 residues, one of the SERT glycosylation sites, Asp208 located between the two Cys residues, cannot undergo proper glycosylation, which plays an important role in the uptake efficiency of SERT. Herein, we show that the decrease in 5-HT uptake rates of GDM trophoblast is the consequence of defective insulin signaling, which entraps SERT with ERp44 and impairs its glycosylation. In this regard, restoring the normal expression of SERT on the trophoblast surface may represent a novel approach to alleviating some GDM-associated complications.serotonin | ERp44 | insulin | serotonin transporter | gestational diabetes mellitus

Serotonin (5-HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT-knock out (KO), peripheral 5-HT (TPH1)-KO and wild-type (WT) mice, we explored the role of 5-HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT-KO placentas appeared only moderately in TPH1-KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT-KO and TPH1-KO showed 49- and 8-fold increase in TUNEL-positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1-KO mice was 16-fold lower than the rate in gestational age matched embryos of WT or SERT-KO mice. These findings highlight an important role of continuous 5-HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5-HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT-KO placentas is in caspase 3-independent pathway.

Hyperpermeability of the endothelial barrier and resulting microvascular leakage are a hallmark of sepsis. Our studies describe the mechanism by which serotonin (5-HT) regulates the microvascular permeability during sepsis. The plasma 5-HT levels are significantly elevated in mice made septic by cecal ligation and puncture (CLP). 5-HT-induced permeability of endothelial cells was associated with the phosphorylation of p21 activating kinase (PAK1), PAK1-dependent phosphorylation of vimentin (P-vimentin) filaments, and a strong association between P-vimentin and ve-cadherin. These findings were in good agreement with the findings with the endothelial cells incubated in serum from CLP mice. In vivo, reducing the 5-HT uptake rates with the 5-HT transporter (SERT) inhibitor, paroxetine blocked renal microvascular leakage and the decline in microvascular perfusion. Importantly, mice that lack SERT showed significantly less microvascular dysfunction after CLP. Based on these data, we propose that the increased endothelial 5-HT uptake together with 5-HT signaling disrupts the endothelial barrier function in sepsis. Therefore, regulating intracellular 5-HT levels in endothelial cells represents a novel approach in improving sepsis-associated microvascular dysfunction and leakage. These new findings advance our understanding of the mechanisms underlying cellular responses to intracellular/extracellular 5-HT ratio in sepsis and refine current views of these signaling processes during sepsis.

BackgroundCigarette smoking plays a major role in cardiovascular diseases. The acute effects of cigarette smoking produce central nervous system–mediated activation of the sympathetic nervous system. The overactive sympathetic nervous system stimulates the secretion of serotonin (5‐HT) and catecholamine into blood at supraphysiological levels. The correlation between these pathological conditions induced by smoking and the increased risk of thrombosis has not been thoroughly investigated. The goal of our study was to explore cigarette smoking–associated changes in platelet biology mediated by elevated 5‐HT and catecholamine levels in blood plasma.Methods and ResultsUsing blood samples collected from healthy nonsmokers and smokers (15 minutes after smoking), we determined that cigarette smoking increased the plasma 5‐HT/catecholamine concentration by several fold and the percent aggregation of platelets 2‐fold. Liquid chromatography–tandem mass spectrometry analysis of proteins eluted from platelet plasma membranes of smokers and nonsmokers demonstrated that GTPase‐activating proteins and proteins participating in the actin cytoskeletal network were differentially and significantly elevated in smokers' platelet membranes compared with those of nonsmokers. Interestingly, Matrix‐assisted laser desorption/ionization–mass spectrometry analyses of the glycans eluted from platelet plasma membranes of the smokers demonstrated that the level and structures of glycans are different from the nonsmokers' platelet surface glycans. Pharmacological blockade of 5‐HT or catecholamine receptors counteracted the 5‐HT/catecholamine‐mediated aggregation and altered the level and composition of glycan on platelet surfaces.ConclusionsBased on our findings, we propose that smoking‐associated 5‐HT/catecholamine signaling accelerates the trafficking dynamics of platelets, and this remodels the surface proteins and glycans and predisposes platelets to hyperactive levels. Smokers' platelets also had correspondingly higher resting concentrations of intracellular calcium and transglutaminase activity. These findings suggest a link among smoking, platelet 5‐HT, catecholamine signaling, and their downstream effectors—including phospholipase C and inositol‐1,4,5‐triphosphate pathways—resulting in an increased tonic level of platelet activation in smokers.

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine.

the images showing actin control were swapped. Upon request of the Editorial Office, the Authors were not able to provide matching images of the original membranes for Figures 2, 5, and 6. Since the Authors are unable to provide the original data used to generate these published figures, the Editors are unable to guarantee the accuracy of the results.

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