Increased ventilation in response to hypoxia has been appreciated for over a century, but the biochemistry underlying this response remains poorly understood. Here we define a pathway in which increased minute ventilation (&Vdot;E ) is signalled by deoxyhaemoglobin-derived S-nitrosothiols (SNOs). Specifically, we demonstrate that S-nitrosocysteinyl glycine (CGSNO) and S-nitroso-l-cysteine (l-CSNO)-but not S-nitroso-d-cysteine (d-CSNO)-reproduce the ventilatory effects of hypoxia at the level of the nucleus tractus solitarius (NTS). We show that plasma from deoxygenated, but not from oxygenated, blood produces the ventilatory effect of both SNOs and hypoxia. Further, this activity is mediated by S-nitrosoglutathione (GSNO), and GSNO activation by gamma-glutamyl transpeptidase (gamma-GT) is required. The normal response to hypoxia is impaired in a knockout mouse lacking gamma-GT. These observations suggest that S-nitrosothiol biochemistry is of central importance to the regulation of breathing.
ABSTRACTy-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTml/GGTml) mice. GGTdeficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTml/GGTml mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas.Plasma cyst(e)ine levels in GGTm'/GGTml mice are reduced to '20% of wild-type mice. Oral administration of Nacetylcysteine to GGTml/GGTml mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the y-glutamyl cycle in cysteine and GSH homeostasis.
Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.
Worldwide, millions of people are exposed to arsenic in drinking water that exceeds the World Health Organization standard of 10 g/liter by as much as 50 -300-fold, yet little is known about the molecular basis for arsenic excretion. Here we show that transport of arsenic into bile depends on the MRP2/cMOAT transporter and that glutathione is obligatory for such transport. Using reversed phase liquid chromatography/mass spectrometry, we demonstrate that two arsenic-glutathione complexes not previously identified in vivo, arsenic triglutathione and methylarsenic diglutathione, account for most of the arsenic in the bile. The structure of the compounds was also confirmed by nuclear magnetic resonance spectroscopy. Our findings may help explain the increased susceptibility of malnourished human populations to arsenic.
Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of ␥-glutamylcysteine synthetase (␥GCS-HS tm1 ), an essential enzyme in GSH synthesis. Embryos homozygous for ␥GCS-HS tm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.
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