Figure 1. Mechanical stretch induces Ca²⁺-dependent NO production in NRVMs. A) Intracellular NO concentration ([NO]_i) was examined by DAF-based confocal analysis. Images shown were taken from cells before stretch (Control), 5 min after stretch (Stretch), and 10 min after SNAP (100 µmol/l) treatment (SNAP). NO donor SNAP treatment was used as a positive control. B) Confocal images were analyzed with IDL software to give quantitative NO levels. Digital images were taken from random fields (>50 pictures per time point were taken) and pixel density was calculated, normalized with control data (from 10 min to 0 min before stretch) and changes of [NO]_i ([NO]_i) were expressed as changes of fluorescence (F/F0). Error bars stand for SE. *P < 0.05. C) Total nitrate level ([NO]_t) was determined by nitrate reductase-based colorimetric method. Conditioned medium was examined. [NO]_t was converted to [NO₃^–] using KNO₃ (100 µmol/l) as a standard. *P < 0.05. D) Cells were incubated with Ca²⁺ chelator EGTA-AM (2 µmol/l), NOS inhibitor L-NAME (100 µmol/l), or iNOS inhibitor AMT (100 µmol/l) for 30 min at 37°C before stretch. [NO]_i was determined by DAF-based confocal analysis. Data of 10 min stretched cells were compared. *P < 0.05. E) Protein levels of iNOS and eNOS in NRVMs before and after 4 h stretch were detected by Western blot analysis. ß-Actin was blotted to monitor equal loading.