Manganese Alters Rat Brain Amino Acids Levels

Manganese (Mn) is an essential element and it acts as a cofactor for a number of enzymatic reactions, including those involved in amino acid, lipid, protein, and carbohydrate metabolism. Excessive exposure to Mn can lead to poisoning, characterized by psychiatric disturbances and an extrapyramidal disorder. Mn-induced neuronal degeneration is associated with alterations in amino acids metabolism. In the present study, we analyzed whole rat brain amino acid content subsequent to four or eight intraperitoneal injections, with 25?mg MnCl₂/kg/day, at 48-h intervals. We noted a significant increase in glycine brain levels after four or eight Mn injections (p?<?0.05 and p?<?0.01, respectively) and arginine also after four or eight injections (p?<?0.001). Significant increases were also noted in brain proline (p?<?0.01), cysteine (p?<?0.05), phenylalanine (p?<?0.01), and tyrosine (p?<?0.01) levels after eight Mn injections vs. the control group. These findings suggest that Mn-induced alterations in amino acid levels secondary to Mn affect the neurochemical milieu.     

Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Zinc,Copper, Iron,and Selenium Levels in Brain and Liver of Mice Exposed to Acrylonitrile

The mechanism of toxicity of acrylonitrile (AN) has not been fully defined. The research described herein was undertaken to investigate the possible effects of AN on the levels of metallic elements in liver and brain of mice. Thirty-two mice were randomly assigned to four separate groups and treated intraperitoneal (i.p.) once daily for 1 week. Mice in the control group received normal saline, and mice in the three exposure groups received 5, 10, or 20 mg AN/kg b.w. Samples of brain and liver were collected immediately after decapitation. Tissue levels of trace elements (zinc, copper, iron) were determined with flame atomic absorption spectrophotometer or double channel atomic fluorescence absorption spectrophotometer (selenium). Mean brain weights of AN-treated mice were increased as a function of dose compared to controls, but there was no significant change in the ratio of liver/body weight in the four groups. While mean brain zinc decreased as a function of AN dosage, mean liver zinc of the low-dose group significantly increased (p < 0.05); mean liver copper in the medium-dose AN group was significantly higher compared to controls (p < 0.05); however, mean brain copper was increased, but the difference did not attain statistical significance in the three AN groups when compared with the controls (p > 0.05). Mean brain iron levels were significantly decreased in the middle-dose AN group (p < 0.05), but there were no consistent changes in liver iron. Tissue levels of selenium in brain and liver were similar for the control and AN treatment groups. AN induces significant and differential changes in the levels of zinc, copper, and iron in brain and liver. These changes likely play a pivotal role in mediating AN toxicity, most likely via changes in cellular redox status.     

Alterations of oxidative stress biomarkers due to in utero and neonatal exposures of airborne manganese

Neonatal rats were exposed to airborne manganese sulfate (MnSO₄) (0, 0.05, 0.5, or 1.0 mg Mn/m³) during gestation (d 0–19) and postnatal days (PNDs) 1–18. On PND19, rats were killed, and we assessed biochemical end points indicative of oxidative stress in five brain regions: cerebellum, hippocampus, hypothalamus, olfactory bulb, and striatum. Glutamine synthetase (GS) and tyrosine hydroxylase (TH) protein levels, metallothionein (MT), TH and GS mRNA levels, and reduced and oxidized glutathione (GSH and GSSG, respectively) levels were determined for all five regions. Mn exposure (all three doses) significantly (p=0.0021) decreased GS protein levels in the cerebellum, and GS mRNA levels were significantly (p=0.0008) decreased in the striatum. Both the median and high dose of Mn significantly (p=0.0114) decreased MT mRNA in the striatum. Mn exposure had no effect on TH protein levels, but it significantly lowered TH mRNA levels in the olfactory bulb (p=0.0402) and in the striatum (p=0.0493). Mn eposure significantly lowered GSH levels at the median dose in the olfactory bulb (p=0.032) and at the median and high dose in the striatum (p=0.0346). Significantly elevated (p=0.0247) GSSG, which can be indicative of oxidative stress, was observed in the cerebellum of pups exposed to the high dose of Mn. These data reveal that alterations of oxidative stress biomarkers resulting from in utero and neonatal exposures of airborne Mn exist. Coupled with our previous study in which similarly exposed rats were allowed to recover from Mn exposure for 3 wk, it appears that many of these changes are reversible. It is important to note that the doses of Mn utilized represent levels that are a hundred- to a thousand-fold higher than the inhalation reference concentration set by the United States Environmental Protection Agency.     

Manganese Uptake and Efflux in Cultured Rat Astrocytes

Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific high-affinity transport system for Mn2+. Saturation kinetics are clearly indicated by both 1/v versus 1/s plots (Km = 0.30 +/- 0.03 microM; Vmax = 0.30 +/- 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2+, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 microM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn(2+)-free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 +/- 2.2% of 54Mn2+ transported extracellularly, and 37.2 +/- 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.     

The methylmercury-L-cysteine conjugate is a substrate for the L-type large neutral amino acid transporter

Methylmercury (MeHg) is a potent neurotoxin. The mechanism(s) that governs MeHg transport across the blood-brain barrier and other biological membranes remains unclear. This study addressed the role of the L-type large neutral amino acid transporter, LAT1, in MeHg transport. Studies were carried out in CHO-k1 cells. Over-expression of LAT1 in these cells was associated with enhanced uptake of [(14)C]-MeHg when treated with L-cysteine, but not with the D-cysteine conjugate. In the presence of excess L-methionine, a substrate for LAT1, L-cysteine-conjugated [(14)C]-MeHg uptake was significantly attenuated. Treatment of LAT-1 over-expressing CHO-k1 cells with L-cysteine-conjugated MeHg was also associated with increased leakage of lactate dehydrogenase into the media as well as reduced cell viability measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. In contrast, knock-down of LAT1 decreased the uptake of l-cysteine-conjugated MeHg and attenuated the effects of MeHg on lactate dehydrogenase leakage and CHO-k1 cell viability. These results indicate that the MeHg-L-cysteine conjugate is a substrate for the neutral amino acid transporter, LAT1, which actively transports MeHg across membranes.     

trans-10,cis-12-Conjugated Linoleic Acid Instigates Inflammation in Human Adipocytes Compared with Preadipocytes

We showed previously in cultures of primary human adipocytes and preadipocytes that lipopolysaccharide and trans-10,cis-12-conjugated linoleic acid (10,12-CLA) activate the inflammatory signaling that promotes insulin resistance. Because our published data demonstrated that preadipocytes are the primary instigators of inflammatory signaling in lipopolysaccharide-treated cultures, we hypothesized that they played the same role in 10,12-CLA-mediated inflammation. To test this hypothesis, we employed four distinct models. In model 1, a differentiation model, CLA activation of MAPK and induction of interleukin-8 (IL-8), IL-6, IL-1β, and cyclo-oxygenase-2 (COX-2) were greatest in differentiated compared with undifferentiated cultures. In model 2, a cell separation model, the mRNA levels of these inflammatory proteins were increased by 10,12-CLA compared with bovine serum albumin vehicle in the adipocyte fraction and the preadipocyte fraction. In model 3, a co-culture insert model, inserts containing ∼50% adipocytes (AD50) or ∼100% preadipocytes (AD0) were suspended over wells containing AD50 or AD0 cultures. 10,12-CLA-induced IL-8, IL-6, IL-1β, and COX-2 mRNA levels were highest in AD50 cultures when co-cultured with AD0 inserts. In model 4, a conditioned medium (CM) model, CM collected from CLA-treated AD50 but not AD0 cultures induced IL-8 and IL-6 mRNA levels and activated phosphorylation of MAPK in naive AD0 and AD50 cultures. Consistent with these data, 10,12-CLA-mediated secretions of IL-8 and IL-6 from AD50 cultures were higher than from AD0 cultures. Notably, blocking adipocytokine secretion prevented the inflammatory capacity of CM from 10,12-CLA-treated cultures. These data suggest that CLA instigates the release of inflammatory signals from adipocytes that subsequently activate adjacent preadipocytes.     

Determining the oxidation states of manganese in PC12 and nerve growth factor-induced PC12 cells

Excessive brain Mn can produce toxicity with symptoms resembling parkinsonism. This syndrome, called "manganism," correlates with loss of dopamine in the striatum and cell death in the striatum and globus pallidus. A common hypothesis is that cell damage in Mn toxicity is caused by oxidation of important cell components by Mn3+. Determination of the amount of Mn3+ present, under a range of conditions, in neuronal cells and brain mitochondria represents an important step in evaluating the "damage through oxidation by Mn3+ hypothesis." In an earlier paper we used X-ray absorption near-edge structure (XANES) spectroscopy to determine the amount of Mn2+ and Mn3+ in brain mitochondria under a range of conditions. Here we extend the study to investigate the evidence for formation of Mn3+ through oxidation of Mn2+ by ROS in PC12 cells and in PC12 cells induced with nerve growth factor (NGF) to display a phenotype more like that of neurons. Although the results suggest that very small amounts of Mn3+ might be present at low Mn levels, probably in Mn superoxide dismutase, Mn3+ is not stabilized by complex formation in these cells and therefore does not accumulate to detectable amounts.     

Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting and resistance vessels

Bradykinin (BK) is a potent dilator of the perinatal pulmonary circulation. We investigated segmental differences in BK-induced dilation in newborn pig large conducting pulmonary artery and vein rings and in pressurized pulmonary resistance arteries (PRA). In conducting pulmonary arteries and veins, BK-induced relaxation is abolished by endothelial disruption and by inhibition of nitric oxide (NO) synthase with nitro-L-arginine (L-NA). In PRA, two-thirds of the dilation response is L-NA insensitive. Charybdotoxin plus apamin and depolarization with KCl abolish the L-NA-insensitive dilations, findings that implicate the release of endothelium-derived hyperpolarizing factor (EDHF). However, endothelium-disrupted PRA retain the ability to dilate to BK but not to ACh or A-23187. In endothelium-disrupted PRA, dilation was inhibited by charybdotoxin. Thus in PRA, BK elicits dilation by multiple and duplicative signaling pathways. Release of NO and EDHF contributes to the response in endothelium-intact PRA; in endothelium-disrupted PRA, dilation occurs by direct activation of vascular smooth muscle calcium-dependent potassium channels. Redundant signaling pathways mediating pulmonary dilation to BK may be required to assure a smooth transition to extrauterine life.