Abstract 663: The Effects of Hypoxia and Metabolic Inhibitors on Cellular ATP and ATP/ADP ratio in Human Coronary Artery Smooth Muscle cells
Background: Although ATP is principally considered as the body’s energy currency, it is also an important signaling molecule. As availability of oxygen is an important factor in the production of ATP, it is simply assumed that the latter is tightly regulated by the former. However, how oxygen tension over the physiological range may regulate ATP production is still unclear. Here, we address the fundamental question of whether changes in environmental oxygen tension over the physiological range result in significant changes in cellular ATP in Human Coronary Artery Smooth Muscle cells (HCASMCs).
Methods: Cells were seeded 24-48 hours before cellular ATP was measured in the cell population using CellTiter-Glo® Luminescent Cell Viability Assay according to manufacturer’s instruction (n=4-6). Percent change of cellular ATP was determined and normalized to control (untreated). For the change of ATP/ADP ratio, single cells were transient transfected with Perceval or PercevalHR, fluorescent reporters of ATP/ADP ratio. The fluorescent signal was collected in a confocal microscope (LSM510) under different experimental conditions, including hypoxia.
Results: Application of metabolic inhibitors for 10 minutes decreased the ATP to 104.6%±2.0% (5 mM 2-Deoxy-D-glucose/2DG),103.1% ±2.9% (6μM Oligomycin/Oligo) and 84.4%±2.7% (2DG+ Oligo) in 10 mM Glucose physiological saline (PSS); and 55.3%±0.3% (5 mM 2DG), 92.4%±3.7% (6μM Oligo) and 36.7%±2.7% (2DG+ Oligo) in glucose free PSS. Hypoxia for 10 minutes also caused a reduction in ATP to 67.0%±1.6% (10%O2), 52.7%±0.6% (5%O2) and 36.2%±1.8% (1%O2) in 10 mM glucose PSS; 71.3%±2.4% (10%O2), 57.9%±1.1% (5%O2) and 40.4%±3.2% (1%O2) in glucose free PSS. Inhibiting metabolism with 2DG in single cells resulted in a decrease in Perceval signal by 50.2±14.4% (n=3). Transient hypoxia (1%O2) also induced a significant reduction in Perceval signal by 19.3%±5.3% (n=3). By inhibiting the electron transport chain for 10 mins, we also observed PercevalHR signal declined by 43.6%±3.6% (1μM Rotenone, n=3), 33.0%±11.4% (1μM Antimycin, n=4), 39.5%±9% (6μM Oligo, n=3) and 16.9%±4.4% (1μM CCCP, n=3).
Conclusions: Our studies suggest hypoxia causes a reduction in cellular ATP and a decrease in the ATP/ADP ratio in HCASMCs.
Author Disclosures: M. Yang: None. T. Kamishima: None. O.L. Roberts: None. D. Caroline: None. J.M. Quayle: None.
- © 2015 by American Heart Association, Inc.