S-Palmitoylation of the serotonin transporter promotes its cell surface expression and serotonin uptake.

The neurotransmitter serotonin (5-HT) is transported back into serotonergic neurons by the serotonin transporter (SERT). SERT is a main target of antidepressants, and much effort has therefore focused on finding relationships between SERT and depression. However, it is not fully understood how SERT is regulated at the cellular level. Here, we report post-translational regulation of SERT by S-palmitoylation, in which palmitate is covalently attached to cysteine residues of proteins. Using AD293 cells (a human embryonic kidney 293-derived cell line with improved cell adherence) transiently transfected with FLAG-tagged human SERT, we observed S-palmitoylation of immature SERT containing high-mannose type N-glycans or no N-glycan, which is presumed to be localized in the early secretory pathway, such as the endoplasmic reticulum. Mutational analysis by alanine substitutions shows that S-palmitoylation of immature SERT occurs at least at Cys-147 and Cys-155, juxtamembrane cysteine residues within the first intracellular loop. Furthermore, mutation of Cys-147 reduced cellular uptake of a fluorescent SERT substrate that mimics 5-HT without decreasing SERT on the cell surface. On the other hand, combined mutation of Cys-147 and Cys-155 inhibited SERT surface expression and reduced the uptake of the 5-HT mimic. Thus, S-palmitoylation of Cys-147 and Cys-155 is important for both the cell surface expression and 5-HT uptake capacity of SERT. Given the importance of S-palmitoylation in brain homeostasis, further investigation of SERT S-palmitoylation could provide new insights into the treatment of depression.

P2Y2 receptor mediates dying cell removal via inflammatory activated microglia.

Microglial removal of dying cells plays a beneficial role in maintaining homeostasis in the CNS, whereas under some pathological conditions, inflammatory microglia can cause excessive clearance, leading to neuronal death. However, the mechanisms underlying dying cell removal by inflammatory microglia remain poorly understood. In this study, we performed live imaging to examine the purinergic regulation of dying cell removal by inflammatory activated microglia. Lipopolysaccharide (LPS) stimulation induces rapid death of primary rat microglia, and the surviving microglia actively remove dying cells. The nonselective P2 receptor antagonist, suramin, inhibited dying cell removal to the same degree as that of the selective P2Y2 antagonist, AR-C118925. This inhibition was more potent in LPS-stimulated microglia than in non-stimulated ones. LPS stimulation elicited distribution of the P2Y2 receptor on the leading edge of the plasma membrane and then induced drastic upregulation of P2Y2 receptor mRNA expression in microglia. LPS stimulation caused upregulation of the dying cell-sensing inflammatory Axl phagocytic receptor, which was suppressed by blocking the P2Y2 receptor and its downstream signaling effector, proline-rich tyrosine kinase (Pyk2). Together, these results indicate that inflammatory stimuli may activate the P2Y2 receptor, thereby mediating dying cell removal, at least partially, through upregulating phagocytic Axl in microglia.

GPR3 accelerates neurite outgrowth and neuronal polarity formation via PI3 kinase-mediating signaling pathway in cultured primary neurons.

During neuronal development, immature neurons extend neurites and subsequently polarize to form an axon and dendrites. We have previously reported that G protein-coupled receptor 3 (GPR3) levels increase during neuronal development, and that GPR3 has functions in neurite outgrowth and neuronal differentiation in cerebellar granular neurons. Moreover, GPR3 is transported and concentrated at the tips of neurite, thereby contributing to the local activation of protein kinase A (PKA). However, the signaling pathways for GPR3-mediated neurite outgrowth and its subsequent effects on neuronal polarization have not yet been elucidated. We therefore analyzed the signaling pathways related to GPR3-mediated neurite outgrowth, and also focused on the possible roles of GPR3 in axon polarization. We demonstrated that, in cerebellar granular neurons, GPR3-mediated neurite outgrowth was mediated by multiple signaling pathways, including those of PKA, extracellular signal-regulated kinases (ERKs), and most strongly phosphatidylinositol 3-kinase (PI3K). In addition, the GPR3-mediated activation of neurite outgrowth was associated with G protein-coupled receptor kinase 2 (GRK2)-mediated signaling and phosphorylation of the C-terminus serine/threonine residues of GPR3, which affected downstream protein kinase B (Akt) signaling. We further demonstrated that GPR3 was transiently increased early in the development of rodent hippocampal neurons. It was subsequently concentrated at the tip of the longest neurite, and was thus associated with accelerated polarity formation in a PI3K-dependent manner in rat hippocampal neurons. In addition, GPR3 knockout in mouse hippocampal neurons led to delayed neuronal polarity formation, thereby affecting the dephosphorylation of collapsing response mediator protein 2 (CRMP2), which is downstream of the PI3K signaling pathway. Taken together, these findings suggest that the intrinsic expression of GPR3 in differentiated neurons constitutively activates PI3K-mediated signaling pathway predominantly, thus accelerating neurite outgrowth and further augmenting polarity formation in primary cultured neurons.

GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice.

Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice.

Potential role of inducible GPR3 expression under stimulated T cell conditions.

G protein-coupled receptor 3 (GPR3) constitutively activates Gαs proteins without any ligands and is predominantly expressed in neurons. Since the expression and physiological role of GPR3 in immune cells is still unknown, we examined the possible role of GPR3 in T lymphocytes. The expression of GPR3 was upregulated 2 h after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation and was sustained in Jurkat cells, a human T lymphocyte cell line. In addition, the expression of nuclear receptor 4 group A member 2 (NR4A2) was highly modulated by GPR3 expression. Additionally, GPR3 expression was linked with the transcriptional promoter activity of NR4A in Jurkat cells. In mouse CD4+ T cells, transient GPR3 expression was induced immediately after the antigen receptor stimulation. However, the expression of NR4A2 was not modulated in CD4+ T cells from GPR3-knockout mice after stimulation, and the population of Treg cells in thymocytes and splenocytes was not affected by GPR3 knockout. By contrast, spontaneous effector activation in both CD4+ T cells and CD8+ T cells was observed in GPR3-knockout mice. In summary, GPR3 is immediately induced by T cell stimulation and play an important role in the suppression of effector T cell activation.

Effects of flurbiprofen on the functional regulation of serotonin transporter and its misfolded mutant.

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

Role of the E3 ubiquitin ligase HRD1 in the regulation of serotonin transporter function.

To elucidate the regulation of serotonin transporter (SERT) function via its membrane trafficking, we investigated the involvement of the ubiquitin E3 ligase HRD1 (HMG-CoA reductase degradation protein), which participates in endoplasmic reticulum (ER)-associated degradation (ERAD), in the functional regulation of SERT. Cells transiently expressing wild-type SERT or a SERT C-terminal deletion mutant (SERTΔCT), a SERT protein predicted to be misfolded, were used for experiments. Studies using HRD1-overexpressing or HRD1-knockdown cells demonstrated that HRD1 is involved in SERT proteolysis. Overexpression of HRD1 promoted SERT ubiquitination, the effect of which was augmented by treatment with the proteasome inhibitor MG132. Immunoprecipitation studies revealed that HRD1 interacts with SERT in the presence of MG132. In addition, HRD1 was intracellularly colocalized with SERT, especially with aggregates of SERTΔCT in the ER. HRD1 also affected SERT uptake activity in accordance with the expression levels of the SERT protein. These results suggest that HRD1 contributes to the membrane trafficking and functional regulation of SERT through its involvement in ERAD-mediated SERT degradation.

Syntaxin 3 interacts with serotonin transporter and regulates its function.

Herein, we investigated the functional association of the serotonin transporter (SERT) with syntaxin-3 (STX3). We first overexpressed SERT and STX3 in various cells and examined their interaction, localization, and functional association. Immunoprecipitation studies revealed that STX3 interacted with SERT when expressed in COS-7 cells. Immunocytochemical studies revealed that SERT and STX3 were colocalized in the endoplasmic reticulum (ER) and Golgi apparatus. STX3 overexpression significantly reduced the uptake activity of SERT by attenuating its plasma membrane expression, suggesting that overexpressed STX3 anchors SERT in the ER and Golgi apparatus. STX3 knockdown did not affect the uptake activity of SERT but altered its glycosylation state. To elucidate the association of STX3 with SERT under physiological conditions, rather than overexpressing cells, we investigated this interaction in polarized Caco-2 cells, which endogenously express both proteins. Immunocytochemical studies revealed that SERT and STX3 were localized in microvilli-like structures at the apical plasma membrane. STX3 knockdown marginally but significantly decreased the serotonin uptake activity of Caco-2 cells, suggesting that STX3 positively regulates SERT function in Caco-2 cells, as opposed to SERT regulation by STX3 in overexpressing cells. Collectively, STX3 may colocalize with SERT during SERT membrane trafficking and regulate SERT function in an STX3-expressing lesion-dependent manner.

Improvement of a twice collection method of mouse oocytes by surgical operation.

Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.

Repulsive guidance molecule A suppresses angiogenesis.

The repulsive guidance molecule-a (RGMa) is a membrane-associated glycoprotein that has diverse functions in the developing and adult central nervous system. Here, we show that RGMa suppresses new blood vessel formation. Treatment of human umbilical artery endothelial cells (HUAEC) on Matrigel with recombinant RGMa inhibits vascular endothelial growth factor (VEGF)-induced and VEGF-independent tubular formation and migration. RGMa enhances adhesion presumably through dephosphorylation of focal adhesion kinase (FAK) at tyrosine-397. Neogenin, an RGMa receptor, in HUAEC is required for the effect of RGMa. In vivo Matrigel plug assay reveals that treatment with recombinant RGMa suppresses angiogenesis. Thus, we conclude that RGMa inhibits angiogenesis in vitro and in vivo suggesting that its manipulation would be an efficient therapeutic strategy for pro-angiogenic conditions.

Restrictions on monetary payments for human biological substances in Japan: The mu-shou principle and its ethical implications for stem cell research.

Introduction: Restrictions on financial gains from the sale of human body parts is a leading policy issue surrounding the use of human tissues and cells. However, discrepancies exist between regulations and reality. In stem cell research, in which diverse sources of tissues and cells can be used, unclear regulations can impede research. Thus, using the Japanese system as a case study, we examined the challenges in the implementation of the "no payment" or the mu-shou principle in stem-cell research over the years. Methods: We reviewed 28 Japanese laws and governmental guidelines and summarized the scope of restrictions on payments for the donation and supply of human biological samples (HBS). Results: As part of restrictions on financial rewards, the mu-shou principle emerged in Japanese laws and administrative documents in the 1990s. Although the Japanese mu-shou generally means "free" or "gratis" in English, its interpretation in research and development settings remains ambiguous. Traditionally, this principle was used to deny remuneration to donors. However, it is also inconsistently applied while processing and transferring human tissue after donation, which creates confusion among the various stakeholders. Recent policies have interpreted the principle in multiple ways: (1) treating the use of HBS for cell-processing as a non-profit activity; (2) a flexible interpretation of the principle to broaden the scope of user payments; and (3) removal of the principle itself to allow for commercial use. Conclusions: The inconsistencies in the monetary payment requirements for HBS could hinder research and development. After scrutinizing the principle's background, an effective approach is needed that considers the concerns of the providers, users, and society alike.

Identification of protein kinase C domains involved in its translocation induced by propofol.

Propofol is widely used for general anesthesia and sedation; however, the mechanisms of its anesthetic and adverse effects are not fully understood. We have previously shown that propofol activates protein kinase C (PKC) and induces its translocation in a subtype-specific manner. The purpose of this study was to identify the PKC domains involved in propofol-induced PKC translocation. The regulatory domains of PKC consist of C1 and C2 domains, and the C1 domain is subdivided into the C1A and C1B subdomains. Mutant PKCα and PKCδ with each domain deleted were fused with green fluorescent protein (GFP) and expressed in HeLa cells. Propofol-induced PKC translocation was observed by time-lapse imaging using a fluorescence microscope. The results showed that persistent propofol-induced PKC translocation to the plasma membrane was abolished by the deletion of both C1 and C2 domains in PKCα and by the deletion of the C1B domain in PKCδ. Therefore, propofol-induced PKC translocation involves the C1 and C2 domains of PKCα and the C1B domain of PKCδ. We also found that treatment with calphostin C, a C1 domain inhibitor, abolished propofol-induced PKCδ translocation. In addition, calphostin C inhibited the propofol-induced phosphorylation of endothelial nitric oxide synthase (eNOS). These results suggest that it may be possible to modulate the exertion of propofol effects by regulating the PKC domains involved in propofol-induced PKC translocation.

Survey of Japanese researchers and the public regarding the culture of human embryos in vitro beyond 14 days.

The International Society for Stem Cell Research (ISSCR) has eliminated its prohibition on research involving the culturing of human embryos beyond 14 days within the updated 2021 guidelines. We conducted a survey of Japanese researchers working in stem cell- or embryo-related research (n = 535) and the public (n = 3,000) about their attitudes toward the 14-day rule. Among the researchers, 46.2% agreed that embryos could be cultured beyond 14 days, a result that was slightly lower among the public (37.9%). Among those that disagreed with embryo culturing beyond 14 days, 9.5% of researchers and 5.1% of the public agreed with culturing embryos within 14 days. Among the public, higher comprehension levels correlated with both agreement and disagreement with the culture of embryos beyond 14 days compared with "cannot judge." Further research and pubic discourse are necessary in order to better understand the factors informing participant decisions regarding the 14-day rule.

Features and mechanisms of propofol-induced protein kinase C (PKC) translocation and activation in living cells.

Background and purpose: In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation. Experimental approach: Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation. We also examined PKC translocation using isomers and derivatives of propofol to identify the crucial structural motifs involved in this process. Key results: Propofol persistently translocated PKCα conventional PKCs and PKCδ from novel PKCs (nPKCs) to the plasma membrane (PM). Propofol translocated PKCδ and PKCη of nPKCs to the Golgi apparatus and endoplasmic reticulum, respectively. Propofol also induced the nuclear translocation of PKCζ of atypical PKCs or proteins other than PKCs, such that the protein concentration inside and outside the nucleus became uniform. CKAR analysis revealed that propofol activated PKC in the PM and Golgi apparatus. Moreover, tests using isomers and derivatives of propofol predicted that the structural motifs important for the induction of PKC and nuclear translocation are different. Conclusion and implications: Propofol induced the subtype-specific intracellular translocation of PKCs and activated PKCs. Additionally, propofol induced the nuclear translocation of PKCs and other proteins, probably by altering the permeability of the nuclear envelope. Interestingly, propofol-induced PKC and nuclear translocation may occur via different mechanisms. Our findings provide insights into the action mechanisms of propofol.

Novel missense COL2A1 variant in a fetus with achondrogenesis type II.

Achondrogenesis type II (ACG2) is a lethal skeletal disorder caused by pathogenic variants in COL2A1. We present a fetus with cystic hygroma and severe shortening of the limbs at 14 weeks of gestation. We performed postnatal genetic analysis of the parents and fetus to diagnose the disease. A novel missense variant of COL2A1 [NM_001844.5: c.2987G>A, (p. Gly996Asp)] was identified, which led to the ACG2 diagnosis.

Extracellular ATP differentially modulates Toll-like receptor 4-mediated cell survival and death of microglia.

The survival and death rates of inflammatory cells directly control their number and are substantially associated with the degree of inflammation. Microglia, key players in neuroinflammation, often cause excessive reactions implicated in neurological diseases. However, the mechanisms that determine microglial fate under pathological conditions remain to be elucidated. Here, we report that activation by lipopolysaccharide (LPS, a Toll-like receptor 4 ligand), an inflammation inducer, primarily promotes survival of microglia, but as its concentration is increased it induces cell death, resulting in decreased cell number. Moreover, extracellular ATP, which is released upon tissue damage, further enhanced the survival induced by a low LPS concentration and the death induced by a high LPS concentration. The survival-promoting effect of ATP was mimicked by non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), and also by the P2X(7) receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and was suppressed by the P2X(7) antagonists, Brilliant Blue G and A 438079. On the contrary, the death of LPS-activated microglia was not affected by adenosine 5'-O-(3-thiotriphosphate), but enhanced by adenosine, ATP breakdown product. Thus, extracellular ATP modulates microglial survival and death in different ways involving P2X(7) receptor activation and ATP degradation to adenosine, respectively. Such Toll-like receptor 4/purinergic signaling may provide a fine regulatory system of neuroinflammation through modulating the microglial cell number.

[A case having chyliform pleural effusion caused by former tuberculous pleurisy].

A 49-year-old male who had been treated for pulmonary tuberculosis and tuberculous pleurisy in 2007 was referred to our hospital with the complaint of dyspnea on exertion in Nov. 2009. Chest X-ray showed increased pleural effusion compared with that remaining after the previous treatment of pleurisy in 2008. A chest CT revealed that fluid collection was surrounded by thickened pleura. Thoracocentesis was performed, and yellow milky liquid was obtained. The pleural effusion contained few cells. The triglyceride concentration was 83 mg/dl, and the cholesterol level was very high at 628 mg/dl. Based on these findings we diagnosed this case as chyliform pleural effusion. Both smear of acid-fast bacilli and PCR-TB test of the pleural effusion were positive, but culture was negative for mycobacterium, suggesting that this chyliform pleural effusion was produced by the former episode of tuberculous pleurisy, not by the recent reactivation of tuberculous pleurisy. The ADA concentration in the pleural effusion was high at 91.7 IU/l. No increase in the amount of pleural effusion was observed after thoracocentesis without any anti-tuberculosis therapy.

Detailed neuronal distribution of GPR3 and its co-expression with EF-hand calcium-binding proteins in the mouse central nervous system.

The G-protein coupled receptor 3 (GPR3), a member of the class A rhodopsin-type GPR family, constitutively activates Gαs proteins without any ligands. Although there have been several reports concerning the functions of GPR3 in neurons, the physiological roles of GPR3 have not been fully elucidated. To address this issue, we analyzed GPR3 distribution in detail using fluorescence-based X-gal staining in heterozygous GPR3 knockout/LacZ knock-in mice, and further investigated the types of GPR3-expressing neurons using fluorescent double labeling with various EF-hand Ca2+-binding proteins. In addition to the previously reported GPR3-expressing areas, we identified GPR3 expression in the basal ganglia and in many nuclei of the cranial nerves, in regions related to olfactory, auditory, emotional, and motor functions. In addition, GPR3 was not only observed in excitatory neurons in layer V of the cerebral cortex, the CA2 region of the hippocampus, and the lateral nucleus of the thalamus, but also in γ-aminobutyric acid (GABA)-ergic interneurons in the cortex, hippocampus, thalamus, striatum, and cerebellum. GPR3 was frequently co-expressed with neuronal Ca2+-binding protein 2 (NECAB2) in neurons in various regions of the central nervous system, especially in the hippocampal CA2, medial habenular nucleus, lateral thalamic nucleus, dorsolateral striatum, brainstem, and spinal cord anterior horn. Furthermore, GPR3 also co-localized with NECAB2 at the tips of neurites in differentiated PC12 cells. These results suggest that GPR3 and NECAB2 are highly co-expressed in specific neurons, and that GPR3 may modulate Ca2+ signaling by interacting with NECAB2 in specific areas of the central nervous system.

Submarine groundwater discharge: A previously undocumented source of contaminants of emerging concern to the coastal ocean (Sydney, Australia).

Submarine groundwater discharge (SGD) is rarely considered as a pathway for contaminants of emerging concern (CECs). Here, we investigated SGD as a source of CECs in Sydney Harbour, Australia. CEC detection frequencies based on presence/absence of a specific compound were >90% for caffeine, carbamazepine, and dioxins, and overall ranged from 25 to 100% in five studied embayments. SGD rates estimated from radium isotopes explained >80% of observed CEC inventories for one or more compounds (caffeine, carbamazepine, dioxins, sulfamethoxazole, fluoroquinolones and ibuprofen) in four out of the five embayments. Radium-derived residence times imply mixing is also an important process for driving coastal inventories of these persistent chemicals. Two compounds (ibuprofen and dioxins) were in concentrations deemed a high risk to the ecosystem. Overall, we demonstrate that SGD can act as a vector for CECs negatively impacting coastal water quality.

Propofol induces the elevation of intracellular calcium via morphological changes in intracellular organelles, including the endoplasmic reticulum and mitochondria.

Propofol, most frequently used as a general anesthetic due to its versatility and short-acting characteristics, is thought to exert its anesthetic actions via GABAA receptors; however, the precise mechanisms of its adverse action including angialgia remain unclear. We examined the propofol-induced elevation of intracellular calcium and morphological changes in intracellular organelles using SHSY-5Y neuroblastoma cells, COS-7 cells, HEK293 cells, and HUVECs loaded with fluorescent dyes for live imaging. Although propofol (>50 µM) increased intracellular calcium in a dose-dependent manner in these cells, it was not influenced by the elimination of extracellular calcium. The calcium elevation was abolished when intracellular or intraendoplasmic reticulum (ER) calcium was depleted by BAPTA-AM or thapsigargin, respectively, suggesting that calcium was mobilized from the ER. Studies using U-73122, xestospongin C, and dantrolene revealed that propofol-induced calcium elevation was not mediated by G-protein coupled receptors, IP3 receptors, or ryanodine receptors. We performed live imaging of the ER, mitochondria and Golgi apparatus during propofol stimulation using fluorescent dyes. Concomitant with the calcium elevation, the structure of the ER and mitochondria was fragmented and aggregated, and these changes were not reversed during the observation period, suggesting that propofol-induced calcium elevation occurs due to calcium leakage from these organelles. Although the concentration of propofol used in this experiment was greater than that used clinically (30 µM), it is possible that the concentration exceeds 30 µM at the site where propofol is injected, leading the idea that these phenomena might relate to the various propofol-induced adverse effects including angialgia.