No abstract

The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.

The supporting role of glial cells in maintaining neurons and in ion homeostasis has been studied in situ by perfusing the gliotoxin fluorocitrate (FC) through a microdialysis fiber in the CA1 area of urethane-anesthetized rats. Extracellular direct current potential, extracellular potassium concentration ([K+]o) and amino acid levels, extracellular pH (pHo), and evoked field activity were studied. Histology verified the swelling of glial cells after 4 hr of FC treatment. Massive neuron damage was evident after 8 hr. FC dialysis caused the rapid decrease of glutamine, pHo became progressively more acid, and [K+]o moderately elevated. Orthodromic transmission was variably blocked within 30 min to 4 hr. After 4 hr, spreading depression (SD) waves that originated from the neocortex invaded hippocampal CA1, [K+]o increased to higher levels, pHo became very acid, and there were steep increases in taurine, glutamate, and GABA levels. Simultaneously, the antidromic population spike (a-PS) became depressed and eventually disappeared. When a shorter dialysis probe that spared cortex was used to sample CA1, no SD was seen, a-PS was not abolished, and ion homeostasis was altered less markedly. Repeated SD provoked in hippocampus in the absence of FC caused only mild depression of a-PS. Dialysis of high-K+ solution in healthy neocortex or hippocampus caused only slight elevation of [K+]o at distances of 200-400 microns from the dialysis membrane. After treatment with FC, similar high-K+ dialysis raised [K+]o much more. We conclude the following: (1) recurrent SD waves injure neurons if and only if glial function has failed; (2) neurons can regulate [K+]o, albeit imperfectly; (3) glia is required for the normal fine tuning of [K+]o and particularly for the recovery of pathologically elevated [K+]o; and (4) glia are required for the regulation of pHo. The similarities between glial poisoning by FC and the reported changes in the penumbra of ischemic infarcts suggest that the extension of neuron loss into the penumbral region might depend on failure of glial protection.

An acidic, heat-stable protein of molecular weight about 12,000 is one of the components of the enzyme system that catalyzes the reductive deamination of glycine (1, 2). The biological activity of this protein depends on the presence of 1 g-atom of covalently bound selenium per mol of protein (2, 3). Selenium also is known to be essential for the biological activity of mammalian (4) and avian (5) glutathione peroxidase and formate dehydrogenase of Escherichia coli (6-8) and various anaerobic bacteria (3, 9, 10). The selenium-containing moieties of these two enzymes have not been identified. In this communication evidence is presented that the organoselenium moiety of the clostridial glycine reductase selenoprotein is a selenocysteine residue. We have thus identified an essential selenium-containing residue in a protein. MATERIALS AND METHODSThe selenoprotein of the glycine reductase system was isolated from sonic extracts of Clostridium sticklandii (1, 2). Chromatography on Affi-Gel 501, a mercuribenzoate-agarose preparation supplied by Bio-Rad Laboratories, was used as a final isolation step for some preparations that were otherwise difficult to free of the last traces of impurities. * Cells grown in the presence of 1 ,uM Na275SeO3 and 2 mM Na2S were the source of the 75Se-labeled protein (2). The yeast extract and tryptone in the culture medium supplied additional sulfur, and the resultant high ratio of sulfur to selenium suppressed nonspecific substitution of the latter for sulfur in the proteins and other constituents of the cell.Other reagents were purchased as follows: carboxypeptidase Selenocystine was reduced to selenocysteine with KBH4, and the Se-carboxamidomethyl, Se-carboxymethyl, Se-carboxyethyl, and Se-aminoethyl derivatives of the selenol were prepared by procedures similar to those used for synthesis of the corresponding S-alkyl derivatives of cysteine (11). On cellulose thin-layer sheets these derivatives migrated with the following RF values: in 2-propanol/HCOOH/H20 (60:3:15) Se-carboxamidomethylselenocysteine, 0.18 and Se-carboxymethylselenocysteine, 0.35; in tertiary butyl alcohol/methylethylketone/88% HCOOH/H20 (40:30:15:15) Se-carboxymethylselenocysteine, 0.47, Se-carboxyethylselenocysteine, 0.50, and selenocystine, 0.1 in 1-propanol/5% NH40H (70:30) Se-carboxymethylselenocysteine, 0.44; in CHC13/CH30H/15% NH40H (40:40:10) Se-aminoethylselenocysteine, 0.46; and in 2-propanol/28% NH4OH/H20 (60:1.5:30) Se-aminoethylselenocysteine, 0.79, and selenocystine, 0.72. In each instance the RF of the corresponding sulfur compound was similar. RESULTSThe unusual absorption spectrum of the selenoprotein of glycine reductase (Fig. 1) is due to its high content of phenylalanine relative to tyrosine and to the absence of tryptophan. Almost identical spectra are exhibited by the parvalbumin of dogfish (12) and the calcium-binding protein component of troponin from rabbit muscle (13). The spectrum of the latter, like that of the selenoprotein, exhibits a shoulder in the 295-to 320-nm region. In neither insta...

To detect what initiates spreading depression (SD), the early prodromal events were investigated in hippocampal CA1 of urethane-anesthetized rats. SD was provoked by microdialysis or focal microinjection of high- K+ solution. Extracellular DC potentials and extracellular potassium concentration ([K+]o) were recorded, and spontaneous and evoked potentials analyzed for current source-density (CSD). In the front of an approaching SD wave, several seconds before the onset of the typical sustained negative potential shift (delta Vo) and the increased [K+]o, fast electrical activity was detected. This consisted initially of small rhythmic (60–70 Hz) sawtooth wavelets, which then gave way to a shower of population spikes (PSs) of identical frequency. Prodromal wavelets and PSs were synchronized over considerable distances in the tissue. Sawtooth wavelets were identified as pacemakers of the prodromal PS burst. Simultaneous recording at three depths revealed that the spontaneous prodromal PSs occurred exactly in phase in dendrites and somata whereas synaptically transmitted PSs arose first in the proximal dendrites and were conducted from there into the soma membrane. During a spike burst, stratum (st.) pyramidale served as current sink, while in the proximal sublayer of st. radiatum spike- sinks gave way to spike sources that grew larger as the sinks in st. pyramidale began to subside. Blocking synaptic transmission did not abolish the prodromal spike burst, yet repetitive orthodromic activation inhibited it without altering the subsequent SD waveform. Complex changes in cell excitability were detected even before fast spontaneous activities. We concluded that, in the initial evolution of SD, changes in neuron function precede the regenerating depolarization by several seconds. We propose that the opening of normally closed electric junctions among neurons can best explain the long-distance synchronization and the flow current that occurs in the leading edge of a propagating wave of SD.