Subject(s)
Cell Hypoxia/physiology , Endothelial Growth Factors/genetics , Endothelium, Vascular/physiology , Lymphokines/genetics , Oxidative Stress , Pulmonary Artery/physiology , Animals , Base Sequence , Cells, Cultured , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Endothelial Growth Factors/metabolism , Endothelium, Vascular/cytology , Lymphokines/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Rats , Reactive Oxygen Species/metabolism , Response Elements , Time Factors , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Xanthine Oxidase/metabolismABSTRACT
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% O(2), 75% N(2), and 5% CO(2)) resulted in stable PO(2) values of approximately 150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O(2), 95% N(2), and 5% CO(2) ) was associated with a reduction in PO(2) values to stable levels of approximately 25 Torr. Hypoxic PASMCs became increasingly fluorescent at approximately 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.
Subject(s)
Cell Hypoxia/physiology , Free Radicals/metabolism , Muscle, Smooth, Vascular/metabolism , Pulmonary Artery/metabolism , Thiourea/analogs & derivatives , Animals , Catalase/pharmacology , Cell Hypoxia/drug effects , Diffusion Chambers, Culture/instrumentation , Evaluation Studies as Topic , Fluoresceins , Fluorescence , Hydrogen-Ion Concentration , Male , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Partial Pressure , Pulmonary Artery/cytology , Pulmonary Artery/drug effects , Rats , Rats, Sprague-Dawley , Thiourea/pharmacologySubject(s)
Endothelial Growth Factors/genetics , Hypoxia/physiopathology , Lymphokines/genetics , Muscle, Smooth, Vascular/cytology , Oxidative Stress/genetics , Pulmonary Artery/cytology , Animals , DNA/analysis , Gene Expression , RNA, Messenger/metabolism , Rats , Signal Transduction , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was shown to reverse the hypoxic pressor response (HPR) in the isolated, blood-perfused rat lung model. EHNA, an adenosine deaminase inhibitor, showed reversal of the HPR in a dose-dependent manner (EC50 = 129 +/- 30 microM). We found that the reversal of HPR by EHNA was not mediated by the adenosine receptors because the EHNA effect was not blocked by the adenosine receptor antagonist, 8-p-sulfophenyl-theophylline (67 microM; n = 6). Pretreatment with a cy-clic-3',5'-adenosine monophosphate (cAMP)-dependent protein kinase inhibitor, Rp-adenosine-3',5'-cyclic monophosphorothioate (0.5 mM; n = 4), blocked EHNA reversal of the HPR. As an alternative mechanism of action, EHNA inhibition of cyclic nucleotide phosphodiesterase(s) isozymes was studied in endothelium intact and denuded pulmonary arteries. Using anion-exchange chromatography the cyclic nucleotide phosphodiesterase (PDE) separated into predominantly PDE families 2 and a mixture of 3 and 4. DEAE fractions showing cAMP hydrolysis activated by 5 microM cyclic-3',5'-guanosine monophosphate (cGMP) had a Km for cAMP of 6.3 microM and an apparent Kact for cGMP of 1.4 microM. EHNA was shown to inhibit PDE2 competitively. In intact vessels, the IC50 for EHNA was 3.3 microM using 0.03 microM [3H]-cAMP substrate assayed in the presence of 2 microM cGMP and in denuded vessels 3.7 microM at 0.03 microM [3H]-cAMP substrate in the presence of 5 microM cGMP. Fractions in which cAMP hydrolysis was inhibited or not affected by 5 microM cGMP (PDE3 and 4, respectively) showed an IC50 of > 200 microM for EHNA. We conclude that reversal of the hypoxic pressor response by EHNA in the isolated, perfused rat lung model occurs with a mechanism involving in part inhibition of smooth muscle PDE2.
