Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.     

Expression of sarco/endoplasmic reticulum Ca ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg³³⁴, Arg³⁹⁶ and Arg⁶³⁸ were directly assigned to the ETF and ii) Arg¹⁹⁸ was assigned as the only tryptic site to the LTF. Arg⁶⁷¹, Lys⁷¹²/Lys⁷¹³ and Lys⁷²⁸ were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl₂, EGTA or AlF₄⁻ strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg¹⁰⁰² as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.