Free radical production in hypoxic pulmonary artery smooth muscle cells

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in pri mary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Pr oduction of reactive species was detected by fluorescence microscopy with t he probe 2',7'-dichlorodihydrofluorescein, which is converted to the fluore scent dichlorofluorescein (DCF) in the presence of various oxidants. Flushi ng the airspace above the PASMC cultures with normoxic gas (20% O-2, 75% N- 2, and 5% CO2) resulted in stable PO2 values of similar to 150 Torr, wherea s perfusion of the airspace with hypoxic gas (0% O-2, 95% N-2, and 5% CO2) was associated with a reduction in PO2 values to stable levels of similar t o 25 Torr. Hypoxic PASMCs became increasingly fluorescent at similar to 500 % above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescenc e was attenuated by the addition of the antioxidants dimethylthiourea and c atalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactiv e oxygen or nitrogen species production.