Decreased endothelial nitric-oxide synthase (eNOS) activity resulting from abnormal interaction between eNOS and its regulatory proteins in hypoxia-induced pulmonary hypertension.
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.
Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Japan. murata@jvm2.a.u-tokyo.ac.jp
Takahisa
T
Murata
Koichi
K
Sato
Masatoshi
M
Hori
Hiroshi
H
Ozaki
Hideaki
H
Karaki
D000818:Animals; D015153:Blotting, Western; D001835:Body Weight; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000473:pathology; D006056:Golgi Apparatus / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006976:Hypertension, Pulmonary / Q000378:metabolism*; D000860:Hypoxia; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III; D009929:Organ Size; D010766:Phosphorylation; D011233:Precipitin Tests; D011651:Pulmonary Artery; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism; D013997:Time Factors
D018841:HSP90 Heat-Shock Proteins; D009569:Nitric Oxide; D012694:Serine; D002217:Carbachol; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D006152:Cyclic GMP; D002118:Calcium