Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells.
There is indirect, contradictory evidence both for increased and reduced synthesis of the endothelium-derived vasodilator, nitric oxide, in the pulmonary circulation during acute hypoxia. Therefore, we decided to directly measure the effect of acute hypoxia on nitric oxide production by cultured pulmonary endothelium. Because increases in the intracellular free calcium concentration are known to initiate nitric oxide synthesis, we also studied cytosolic calcium levels. We measured the accumulation of the stable nitric oxide metabolite, nitrite, in the fluid used to superfuse the cultured bovine pulmonary artery endothelial cells at an oxygen tension (PO2) of either 20.3 (normoxia) or 4.9 kPa (hypoxia) (152 or 37 mmHg). Intracellular calcium levels were measured with dual-excitation microfluorimetry after loading the cells with the fluorescent calcium indicator, fura 2. Basal NO synthesis, measured as nitrite accumulation over 10 min, was significantly higher under hypoxic than normoxic conditions (8.3 +/- 2.2 versus 4.6 +/- 0.8 nM). Hypoxia transiently increased cytosolic calcium concentration (from 113 +/- 10 to 231 +/- 45 nM). Ryanodine and thapsigargin (which deplete intracellular calcium stores), but not the removal of extracellular calcium, inhibited the hypoxic increase in cytosolic calcium, indicating that it resulted primarily from release of intracellular calcium. Bradykinin-elicited NO synthesis was potentiated by hypoxia. Bradykinin-induced increase in cytosolic calcium was not inhibited by hypoxia. We conclude that hypoxia acutely increases cytosolic calcium levels and basal and bradykinin-stimulated nitric oxide synthesis in pulmonary artery endothelium.
Hampl
V
V
Dept of Medicine, VA Medical Center, Minneapolis, MN 55417, USA.
Cornfield
D N
DN
Cowan
N J
NJ
Archer
S L
SL
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D009573:Nitrites / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*
D009573:Nitrites; D009569:Nitric Oxide; D002118:Calcium