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Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of L-glutamate to α-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native β-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to α-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion. hypoglycemia; hyperinsulinism/hyperammonemia syndrome; islet of Langerhans. [ABSTRACT FROM AUTHOR]

Islets of Langerhans contain gamma-aminobutyrate (GABA) and may use it as an intercellular transmitter. In beta-cells, GABA is stored in synaptic-like microvesicles and secreted through Ca(2+)-dependent exocytosis. Vesicular inhibitory amino acid transporter (VIAAT), which is responsible for the storage of GABA and glycine in neuronal synaptic vesicles, is believed to be responsible for the storage and secretion of GABA in beta-cells. However, a recent study by Chessler et al. indicated that VIAAT is expressed in the mantle region of islets. In the present study, we investigated the precise localization of VIAAT in rat islets of Langerhans and clonal islet cells and found that it is present in alpha-cells, a minor population of F-cells and alphaTC6 cells, and clonal alpha-cells but not in beta-cells, delta-cells, or MIN6 m9-cells (clonal beta-cells). Combined biochemical, immunohistochemical, and electronmicroscopical evidence indicated that VIAAT is specifically localized with glucagon-containing secretory granules in alpha-cells. ATP-dependent uptake of radiolabeled GABA, which is energetically coupled with a vacuolar proton pump, was detected in digitonin-permeabilized alphaTC6 cells as well as in MIN6 m9 cells. These results demonstrate that functional neuronal VIAAT is present in glucagon-containing secretory granules in alpha-cells and suggest that the ATP-dependent GABA transporter in beta-cells is at least immunologically distinct from VIAAT. Because glucagon-containing secretory granules also contain vesicular glutamate transporter and store L-glutamate, as demonstrated by Hayashi et al., the present results suggest more complex features of the GABAergic phenotype of islets than previously supposed. [ABSTRACT FROM AUTHOR]

1-Bromopropane is used as a cleaning agent or adhesive solvent in the workplace. The present study investigated the long-term effects of exposure to 1-bromopropane on biochemical components in the central nervous system (CNS) of rats. Four groups, each of nine male Wistar rats, were exposed to 200, 400, or 800 ppm 1-bromopropane or fresh air only, 8 h per day, 7 days a week for 12 weeks. We measured the levels of neuron-specific γ-enolase, glia-specific β-S100 protein, creatine kinase (CK) subunits B and M, heat shock protein Hsp27 (by enzyme immunoassay), enzymatic activity of CK and levels of glutathione (GSH), oxidized glutathione (GSSG) and sulfhydrul (SH) base in the cerebrum, cerebellum, brainstem and spinal cord. γ-Enolase decreased dose-dependently in the cerebrum, which showed a decrease in wet weight, at 400 ppm or over, but no change was noted in β-S100 protein in any brain region or spinal cord. Hsp27 decreased in the cerebellum, brainstem and spinal cord. Protein-bound SH base, non-protein SH base and total glutathione decreased in every brain region. CK activity decreased dose-dependently at 200 ppm or over, and the ratio of CK activity to CK-B concentration tended to decrease in all regions. The decrease in γ-enolase in the cerebrum suggests the involvement of biochemical changes in neurons with decrease in the wet weight of the cerebrum. Glutathione depletion and changes in proteins containing SH base as a critical site might be the underlying neurotoxic mechanism of 1-bromopropane. The biochemical changes in the cerebrum indicate that long-term exposure to 1-bromopropane has effects on the CNS. [Copyright &y& Elsevier]

Investigates secretory granule-mediated co-secretion of L-glutamate and glucagon which triggers glutamatergic signal transmission in islets of Langerhans. Stimulation of glutamate receptors in the islets; Neuronal storage of L-glutamate; Regulatory molecule for the endocrine function.

5-Hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from L-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT[sub 2] receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca[sup 2+] in rat pinealocytes in culture. Omission of Ca[sup 2+] from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca[sup 2+]-dependent release of 5-HT, which was blocked by phentolamine, an α antagonist, but not by propranolol, a β antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H[sup +]-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca[sup 2+]-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated L-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca[sup 2+]-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles. [ABSTRACT FROM AUTHOR]

Investigates whether L-glutamate is secreted through exocytosis by clonal mouse pancreatic alpha-cells. Expression of receptors and transporters by pancreatic islet cells for L-glutamate and use it as an intercellular signaling molecule; Release of L-glutamate from cultured cells after the addition of potassium chloride in the presence of calcium and glucose in the medium.

The sperm acrosome exhibits a low pH. However, the mechanism of acidification in the acrosome remains unclear. Vacuolar-type proton ATPase (V-ATPase) has been shown to play a principle role in generating and maintaining the acidity of organelles such as lysosomes and endosomes. In this study, we examined whether V-ATPase is localized in the acrosome membranes using immunohistochemical techniques. Sections of rat testis were immunostained using antibodies against V-ATPase. Under light microscopic observation, the perinuclear region in spermatids at an early stage of development was heavily immunostained. At the electron microscopic level, gold particles showing the presence of V-ATPase were localized to the acrosome membranes in the developing spermatids. V-ATPase was also localized to the membrane of vesicles locating between the trans-Golgi area and the acrosome. These observations suggest that V-ATPase may play a role in acrosome acidification. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian pineal gland uses L-glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, δ, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT-PCR analyses with specific probes indicated the expression of mRNA of δ2 but not that of δ1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl-terminal region of the δ2 receptor recognized a single 110-kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The δ2 antibodies recognized a 110-kDa polypeptide of pineal membranes and specifically immunostained huge glial-like cells with the occasional presence of several long, branching processes in a pineal cell culture. δ2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin-positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The δ2-positive cells constitute a very minor population among total pineal cells (∼0.03%). Double immunolabeling with δ2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes δ2-positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein- or vimentin-positive glial-like cells. These results indicated that the δ2 glutamate receptor is expressed in a novel subpopulation of pineal glial-like cells in culture and suggest the presence of a glutamate-mediated intercellular... [ABSTRACT FROM AUTHOR]

Abstract: Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis. Besides melatonin, we showed previously that pinealocytes accumulate L-glutamate in microvesicles and secrete it through an exocytic mechanism. The secreted glutamate binds to the class II metabotropic glutamate receptor and inhibits norepinephrine-stimulated melatonin synthesis in neighboring pinealocytes through an inhibitory cyclic AMP cascade. In this study, it was found that, in addition to metabotropic receptors, pinealocytes express functional ionotropic receptors. RT-PCR and northern analyses indicated the expression of mRNA for GluR1, KA2, and NR2C in pineal gland. The presence of GluR1 protein was confirmed by immunological techniques, but neither KA2 nor NR2C was detected. Consistent with this observation, the presence of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, non-N-methyl-D-aspartate receptor agonists, transiently stimulated increased the intracellular Ca2+ concentration of cultured pinealocytes, whereas N-methyl-D-aspartate did not. These responses were prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, a selective antagonist for non-N-methyl-D-aspartate receptors, by L-type Ca2+ channel blockers such as nifedipine, or by omitting Ca2+ or Na+ in the medium. In the presence of Ca2+ and Na+, (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate evoked glutamate secretion from the cultured cells, which was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, L-type Ca2+ channel blockers, type E or B botulinum neurotoxin, or incubation at <20°C. These results strongly suggest that GluR1 is functionally expressed in pinealocytes and triggers microvesicle-mediated exocytosis of L-glutamate via activation of L-type Ca2+ channels. It is possible that GluR1 participates in a signaling cascade that enhances and expands the L-glutamate signal throughout the pineal gland. [ABSTRACT FROM AUTHOR]

A congenital diaphragmatic hernia (CDH) is an anomaly caused by defects in the diaphragm; the resulting limited thorax cavity in turn restricts lung growth (pulmonary hypoplasia). This condition is related to pulmonary hypertension. Despite advances in neonatal CDH therapy, the mortality for severe pulmonary hypoplasia remains high. Therefore, it is essential to establish prenatal therapeutic interventions. Vitamin D was reported to have beneficial effects on adult pulmonary hypertension. This study aims to evaluate the efficacy of prenatal vitamin D administration for CDH. First, serum 25-hydroxyvitamin D [25(OH)D] levels in umbilical cord blood were evaluated among CDH newborns. Second, Sprague Dawley rat CDH models were exposed to nitrofen on embryo day 9 (E9). Randomly selected rats in the nitrofen-treated group were infused with calcitriol from E9 to E21. Samples from CDH pups diagnosed after birth were used for lung weight measurements, blood gas analysis, and immunohistochemical analysis. Third, microarray analysis was performed to examine the effect of vitamin D on gene expression profiles in CDH pulmonary arterial tissues. Serum 25(OH)D levels in the umbilical cord blood of newborns who did not survive were significantly lower than those who were successfully discharged. Prenatal vitamin D showed no significant effect on CDH incidence or lung weight but attenuated alveolarization and pulmonary artery remodeling accompanied the improved blood gas parameters. Vitamin D inhibited several gene expression pathways in the pulmonary arteries of CDH rats. Our results suggest that prenatal vitamin D administration attenuates pulmonary vascular remodeling by influencing several gene pathways in CDH. • Vitamin D concentration in human CDH newborns was significantly lower than in healthy newborns. • CDH-related mortality was correlated with low vitamin D concentrations. • Prenatal Vitamin D attenuated the alveolarization and pulmonary artery remodeling in the CDH rats. • Isolated pulmonary arteries from CDH rats demonstrated the specific gene expression pattern. [ABSTRACT FROM AUTHOR]

Non-alcoholic steatohepatitis (NASH) is becoming a growing public health problem along with the increase of metabolic syndrome worldwide. Extracellular nucleotides are known to serve as a danger signal by initiating purinergic signaling in many inflammatory disorders, although the role of purinergic signaling in the progression of NASH remains to be clarified. Vesicular nucleotide transporter (VNUT) is a key molecule responsible for vesicular ATP release to initiate purinergic signaling. Here, we studied the role of VNUT in the progression of nonalcoholic steatohepatitis. VNUT was expressed in mouse hepatocytes and associated, at least in part, with apolipoprotein B (apoB)-containing vesicles. High glucose stimulation evoked release of appreciable amount of ATP from hepatocytes, which disappeared in hepatocytes of Vnut knockout (Vnut −/−) mice. Glucose treatment also stimulated triglyceride secretion from hepatocytes, which was inhibited by PPADS and MRS211, antagonists of P2Y receptors, and clodronate, a VNUT inhibitor, and was significantly reduced in Vnut −/− mice. In vivo, postprandial secretion of triglyceride from hepatocytes was observed, while the serum triglyceride level was significantly reduced in Vnut −/− mice. On a high-fat diet, the liver of wild type mice exhibited severe inflammation, fibrosis, and macrophage infiltration, which is similar to NASH in humans, while this NASH pathology was not observed in Vnut −/− mice. These results suggest that VNUT-mediated vesicular ATP release regulates triglyceride secretion and involves in chronic inflammation in hepatocytes. Since blockade of vesicular ATP release protects against progression of steatohepatitis, VNUT may be a pharmacological target for NASH. Unlabelled Image • VNUT is expressed in hepatocytes and responsible for vesicular ATP storage and release upon high glucose stimulation. • VNUT-mediated vesicular ATP release regulates secretion of triglyceride in the liver. • Vnut gene knockout protects against diet-induced steatohepatitis and fibrosis. • VNUT-mediated vesicular ATP release involves in the progression of non-alcoholic steatohepatitis. • VNUT may be a pharmacological target for NASH for which no effective treatment is currently available. [ABSTRACT FROM AUTHOR]

Abstract: Vesicular monoamine transporters (VMATs) are involved in chemical transduction in monoaminergic neurons and various endocrine cells through the storage of monoamines in secretory vesicles. Mammalian pinealocytes contain more 5-hydroxytryptamine (5-HT) than any other cells and are expected on contain VMAT, although no information is available so far. Upon the addition of ATP, radiolabeled 5-HT was taken up by a particulate fraction prepared from cultured rat pinealocytes. The 5-HT uptake was inhibited significantly by bafilomycin A1 (an inhibitor of vacuolar H+-ATPase), 3,5-di-tert-butyl-4-hydroxybenzyli-denemalononitrile (a proton conductor), or reserpine (an inhibitor of VMAT). RT-PCR analysis suggested that VMAT type 1 (VMAT1), but not type 2, is expressed. Antibodies against VMAT1 recognized a single polypeptide with an apparent molecular mass of ~55 kDa, and specifically immunostained pinealocytes. VMAT1 immunoreactivity was high in the vesicular structures in the varicosities of long branching processes and was associated with 5-HT, but not with synaptophysin, a marker protein for microvesicles. The 5-HT immunoreactivity in the long branching processes disappeared upon incubation with reserpine. These results indicate that 5-HT, at least in part, is stored in vesicles other than microvesicles in pinealocytes through a mechanism similar to that of various secretory vesicles. [ABSTRACT FROM AUTHOR]

• We demonstrated the levels of 40 cytokines in fetal pleural fluids. • Several pro-inflammatory cytokines and CCL chemokines were highly correlated with each other. • The levels of IL-1β had the strongest causal link to the fetal adverse outcome among the cytokines. Few studies have investigated the distribution of multiple cytokines in fetal pleural effusion, and its clinical implications are uncertain. This study aimed to determine cytokine levels in fetal pleural effusion and their clinical role in affected fetuses. We obtained fetal pleural fluid samples from 18 infants and investigated the profiles of 40 cytokines using multiplex immunoassay. Relationships among cytokines were estimated by Spearman correlation analysis. Possible associations of cytokine levels with fetal adverse outcomes, including perinatal demise and neurodevelopmental impairment, were studied using univariate logistic regression analysis. Several pro-inflammatory cytokines and CCL chemokines were highly correlated with each other. In contrast, CXCL chemokines had relatively weak correlations with other cytokines. The levels of IL-1β, IL-2, and CCL20 were significantly associated with the occurrence of fetal adverse outcomes. Based on our findings, IL-1β had the strongest causal link to adverse outcomes among the cytokines [odds ratio (OR): 19.74; 95% confidence interval (CI): 1.14–341.9; p = 0.040]. Cytokine levels in fetal pleural effusion varied considerably among cases with or without adverse outcomes. These results provide important information for further clarifying the pathophysiology of fetal pleural effusion and a novel clinical implication that could predict the occurrence of adverse outcomes. [ABSTRACT FROM AUTHOR]

Background: Endometriosis is a common disease occurring in 1-2% of all women of reproductive age. Although there is increasing evidence on the association between endometriosis and adverse perinatal outcomes, little is known about the effect of pre-pregnancy treatments for endometriosis on subsequent perinatal outcomes. Thus, this study aimed to evaluate maternal and neonatal outcomes in pregnant women with endometriosis and to investigate whether pre-pregnancy surgical treatment would affect these outcomes.Methods: This case-control study included 2769 patients who gave birth at Nagoya University Hospital located in Japan between 2010 and 2017. Maternal and neonatal outcomes were compared between the endometriosis group (n = 80) and the control group (n = 2689). The endometriosis group was further divided into two groups: patients with a history of surgical treatment such as cystectomy for ovarian endometriosis, ablation or excision of endometriotic implants, or adhesiolysis (surgical treatment group, n = 49) and those treated with only medications or without any treatment (non-surgical treatment group, n = 31).Results: In the univariate analysis, placenta previa and postpartum hemorrhage were significantly increased in the endometriosis group compared to the control group (12.5% vs. 4.1%, p <  0.01 and 27.5% vs. 18.2%, p = 0.04, respectively). In the multivariate analysis, endometriosis significantly increased the odds ratio (OR) for placenta previa (adjusted OR, 3.19; 95% confidence interval [CI], 1.56-6.50, p <  0.01) but not for postpartum hemorrhage (adjusted OR, 1.14; 95% CI, 0.66-1.98, p = 0.64). Other maternal and neonatal outcomes were similar between the two groups. In patients with endometriosis, patients in the surgical treatment group were significantly associated with an increased risk of placenta previa (OR. 4.62; 95% CI, 2.11-10.10, p <  0.01); however, patients in the non-surgical treatment group were not associated with a high risk (OR, 1.63; 95% CI, 0.19-6.59, p = 0.36). Additionally, other maternal and neonatal outcomes were similar between the two groups.Conclusion: Women who have had surgical treatment for their endometriosis appear to have a higher risk for placenta previa. This may be due to the more severe stage of endometriosis often found in these patients. However, clinicians should be alert to this potential increased risk and manage these patients accordingly. [ABSTRACT FROM AUTHOR]