ausblenden:
Schlagwörter:
Animals
Bacterial Proteins/biosynthesis/genetics
*Biosensing Techniques
Carotid Body/drug effects/*metabolism
Cell Hypoxia
Enzyme Inhibitors/pharmacology
Female
*Fluorescence Resonance Energy Transfer
Green Fluorescent Proteins/biosynthesis/genetics
Heat-Shock Proteins/genetics
Kinetics
Luminescent Proteins/biosynthesis/genetics
Membrane Potentials
Mice, Inbred C57BL
NADPH Oxidases/antagonists & inhibitors/metabolism
Phenotype
Phosphoproteins/antagonists & inhibitors/metabolism
Reactive Oxygen Species/*metabolism
Response Elements
Signal Transduction
Tissue Culture Techniques
Transfection
Fret-hsp33
NADPH oxidase
Ros
carotid body
hypoxia
membrane potential
tissue oxygen
Zusammenfassung:
Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.
