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Departments of 1 Pharmacology and 3 Structural and Cellular Biology and 2 Biotechnical Services Laboratory, College of Medicine, University of South Alabama, Mobile, Alabama 36688
This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2',7'-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O2, 75% N2, and 5% CO2) resulted in stable PO2 values of ~150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O2, 95% N2, and 5% CO2 ) was associated with a reduction in PO2 values to stable levels of ~25 Torr. Hypoxic PASMCs became increasingly fluorescent at ~500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.
fluorescence microscopy; hypoxia; reactive oxygen species; reactive nitrogen species; vascular smooth muscle cells
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