In the pulmonary artery isolated from 1-week
hypoxia-induced pulmonary hypertensive rats, endothelial NO production stimulated by
carbachol was decreased significantly in in situ visualization using diaminofluorescein-2 diacetate and also in cGMP content. This change was followed by the decrease in
carbachol-induced endothelium-dependent relaxation.
Protein expression of endothelial
NO synthase (eNOS) and its regulatory
proteins,
caveolin-1 and
heat shock protein 90, did not change in the hypoxic pulmonary artery, indicating that chronic
hypoxia impairs eNOS activity at posttranslational level. In the hypoxic pulmonary artery, the increase in intracellular Ca(2+) level stimulated by
carbachol but not by
ionomycin was reduced. We next focused on changes in Ca(2+) sensitivity of the eNOS activation system. A morphological study revealed
atrophy of endothelial cells and a peripheral condensation of eNOS in hypoxic endothelial cells preserving co-localization between eNOS and Golgi or plasma membranes. However, eNOS was tightly coupled with
caveolin-1, and was dissociated from
heat shock protein 90 or
calmodulin in the hypoxic pulmonary artery in either the presence or absence of
carbachol. Furthermore, eNOS Ser(1177) phosphorylation in both conditions significantly decreased without affecting Akt phosphorylation in the hypoxic artery. In conclusion, chronic
hypoxia impairs endothelial Ca(2+) metabolism and normal coupling between eNOS and
caveolin-1 resulted in eNOS inactivity.