ABSTRACT
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.
Subject(s)
Cysteine/metabolism , Glutathione/metabolism , Nitroso Compounds/metabolism , Respiratory Physiological Phenomena , S-Nitrosothiols , Solitary Nucleus/metabolism , Sulfhydryl Compounds/metabolism , gamma-Glutamyltransferase/metabolism , Animals , Cell Hypoxia , Cysteine/analogs & derivatives , Glutathione/analogs & derivatives , Isoxazoles/pharmacology , Mice , Oxygen/blood , Rats , S-Nitrosoglutathione , gamma-Glutamyltransferase/antagonists & inhibitorsABSTRACT
Front-surface detection of emission from fluorophores in the presence and absence of light-scattering particles was contrasted to right-angle and wave-guide detection. We found that front-surface detection was the least prone to the reabsorption, inner-filtering, and scattering effects that can plague fluorescent measurements. Front-surface detection was thus used to assess the use of protein and ANS fluorescence as a means of monitoring events in bacterial fermentations. Protein fluorescence appeared to track well changes in optical density during balanced growth. However, during the lag associated with diauxic growth and after exposure to ampicillin, protein fluorescence became decoupled from cellular growth in a manner consistent with prior observations and the known effect of ampicillin on cells. ANS proved to be nontoxic and capable of reporting the occurrence of protein release from cells. The spectral shifts of tryptophan indicated that the incorporation of tryptophan into cellular protein can be monitored.
