[Effects of obstructive sleep apnea style intermittent hypoxia on endothelin-1, nitric oxide, and nitric oxide synthase in endothelium: experiment with human umbilical vein endothelial cells].
OBJECTIVE
To investigate the effects of intermittent hypoxia (IH) on the expression of endothelin-1 (ET-1), nitric oxide (NO), and nitric oxide synthase (NOS) in endothelium, and to evaluate the role of functional disorder of endothelium in the mechanism of obstructive sleep apnea syndrome (OSAS) induced hypertension.


METHODS
Human umbilical vein endothelial cells (HUVECs) of the line ECV304 were cultured and divided into 4 groups: IH group (exposed to 1.5% O(2) for 15 s and 21% O(2) for 1 min 15 s, 3 min 45 s, 5 min 15 s, or 8 min 15 s respectively alternatively with 60 episodes), intermittent normal oxygen group (exposed to 21% O(2) for 15 s and 225 s alternatively with 60 episodes), continuous hypoxia group (exposed to 1.5% for 15 min), and blank control group. Then the culture fluid was collected. The levels of activity of ET-1 (EIA) NO, and NOS were detected by enzyme immune assay, nitric acid reductase method, and chemical colorimetric analysis respectively. RT-PCR was used to detect the mRNA expression of endothelial NOS (eNOS).


RESULTS
Compared with those of the intermittent normal oxygen group and blank control groups the ET-1 levels of the 4 IH sub groups were significantly higher (F = 28.453, P = 0.000), the NO levels ere significantly lower F = 65.252, P = 0.000), the NOS activity levels were significantly lower (F = 5.969, P = 0.008), and eNOS mRNA was significantly down-regulated (F = 16.630, P = 0.000). Compared with continuous hypoxia group with the exposure time equivalent to the accumulated hypoxia time (15 s with 60 episodes) of the IH group, the ET-1 level was significantly higher (t = 2.742, P = 0.024), the NO level was significantly lower (t = 3.347, P = 0.004), the NOS activity level was significantly lower (t = 3.989, P = 0.004), and the eNOS mRNA expression was down-regulated (t = 5.045, P = 0.000). In the different IH group along with the prolongation of the re-oxygenation time from 105 s to 495 s, while maintaining the duration of hypoxic episodes at 15 s, the ET-1 level increased (F = 213.254, P = 0.000), the NO level decreased (F = 15.747, P = 0.000), NOS activity level decreased (F = 2.933, P = 0.048), and eNOS mRNA expression was down-regulated (F = 5.954, P = 0.002). However, there was a tendency for ET-1 to decrease and the NO system indicators when the re-oxygenation time was 8 min 15 s.


CONCLUSIONS
Hypoxia and re-oxygenation both cause endothelium dysfunction and unbalance of NO/ET-1, which is important to OSAS with hypertension.
Zhao
Hai-yan
HY
Department of Respiratory Medicine, Tianjin Medical University General Hospital, Tianjin, China.
Chen
Bao-yuan
BY
Cao
Jie
J
Feng
Jing
J
Guo
Mei-nan
MN
D000704:Analysis of Variance; D015687:Cell Hypoxia; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020181:Sleep Apnea, Obstructive / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014471:Umbilical Veins / Q000166:cytology
D019332:Endothelin-1; D012333:RNA, Messenger; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen