Apparent PKA activity responds to intermittent hypoxia in bone cells: a redox pathway?
| Autor: | Yan-Liang Zhang, Hesam Tavakoli, Mirianas Chachisvilis |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Time Factors
Physiology Microfluidics Biosensing Techniques Transfection Redox Fluorescence spectroscopy 3T3 cells Mice Physiology (medical) Bone cell Cyclic AMP Fluorescence Resonance Energy Transfer Phosphoprotein Phosphatases medicine Animals Spiro Compounds Enzyme Inhibitors Phosphorylation Protein kinase A Pyrans Osteoblasts Chemistry Endothelial Cells Intermittent hypoxia Articles 3T3 Cells Cobalt Cyclic AMP-Dependent Protein Kinases Cell Hypoxia Oxygen Glucose Spectrometry Fluorescence medicine.anatomical_structure Biochemistry Adenylyl Cyclase Inhibitors Biophysics Cattle Imines Stress Mechanical Reactive Oxygen Species Cardiology and Cardiovascular Medicine Oxidation-Reduction Adenylyl Cyclases |
| Zdroj: | American Journal of Physiology-Heart and Circulatory Physiology. 299:H225-H235 |
| ISSN: | 1522-1539 0363-6135 |
| DOI: | 10.1152/ajpheart.01073.2009 |
| Popis: | 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 O2 in the media. When the O2 concentration in the glucose-containing media was lowered due to O2 consumption by the cells in the microfluidic chamber, the apparent PKA activity increases; the reoxygenation of cells under hypoxia leads to a rapid (∼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 inhibitior (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 acitivity 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. |
| Databáze: | OpenAIRE |
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