tautomycin and cobaltous-chloride

We studied hypoxia-induced dynamic changes in the balance between PKA and PKA-counteracting phosphatases in the microfluidic environment in single cells using picosecond fluorescence spectroscopy and intramolecular fluorescence resonance energy transfer (FRET)-based sensors of PKA activity. First, we found that the apparent PKA activity in bone cells (MC3T3-E1 cells) and endothelial cells (bovine aortic endothelial cells) is rapidly and sensitively modulated by the level of O(2) in the media. When the O(2) concentration in the glucose-containing media was lowered due to O(2) consumption by the cells in the microfluidic chamber, the apparent PKA activity increases; the reoxygenation of cells under hypoxia leads to a rapid ( approximately 2 min) decrease of the apparent PKA activity. Second, lack of glucose in the media led to a lower apparent PKA activity and to a reversal of the response of the apparent PKA activity to hypoxia and reoxygenation. Third, the apparent PKA activity in cells under hypoxia was predominantly regulated via a cAMP-independent pathway since 1) changes in the cAMP level in the cells were not detected using a cAMP FRET sensor, 2) the decay of cAMP levels was too slow to account for the fast decrease in PKA activity levels in response to reoxygenation, and 3) the response of the apparent PKA activity due to hypoxia/reoxygenation was not affected by an adenylate cyclase inhibitor (MDL-12,330A) at 1 mM concentration. Fourth, the immediate onset of ROS accumulation in MC3T3-E1 cells subjected to hypoxia and the sensitivity of the apparent PKA activity to redox levels suggest that the apparent PKA activity change during hypoxia and reoxygenation in this study can be linked to a redox potential change in response to intermittent hypoxia through the regulation of activities of PKA-counteracting phosphatases such as protein phosphatase 1. Finally, our results suggest that the detection of PKA activity could be used to monitor responses of cells to hypoxia in real time.

Topics: 3T3 Cells; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Biosensing Techniques; Cattle; Cell Hypoxia; Cobalt; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelial Cells; Enzyme Inhibitors; Fluorescence Resonance Energy Transfer; Glucose; Imines; Mice; Microfluidics; Osteoblasts; Oxidation-Reduction; Oxygen; Phosphoprotein Phosphatases; Phosphorylation; Pyrans; Reactive Oxygen Species; Spectrometry, Fluorescence; Spiro Compounds; Stress, Mechanical; Time Factors; Transfection