FIGURE 9.

sGC activation by cytoglobin-mediated nitrite reduction in human smooth muscle cells. 1 × 10⁶ control human smooth muscle cells or cells with different gene knockdown were cultured either under normoxic or hypoxic conditions. Gene knockdown was performed using either shRNA or siRNA as described under “Experimental Procedures.” A, cells were incubated with 10 μm nitrite in 1 ml of PBS in the presence or absence of oxypurinol (0.2 mm) or amidone (20 μm) for 10 min at 37 °C under near-anaerobic conditions by gently purging with argon. * indicates significant difference compared with corresponding control cells (p < 0.05). B, cells were cultured under hypoxic conditions (0.6% O₂, pO₂ = 4.6 torr) for 48 h, harvested, and then incubated with 10 μm nitrite in 1 ml of PBS for 10 min at 37 °C under severe hypoxia with pO₂ 0.13 or 0.33 torr. * indicates significant difference of Cygb knockdown cell compared with corresponding cells without treatment of shRNA (p < 0.01). A and B, nitrite dependent-sGC activation was determined from the increased cGMP formation compared with cells incubated under similar conditions without the addition of nitrite. C shows expression level of Cygb (a) or Mb (b) in control or with gene knockdown of normoxic cultured cells; different concentrations of pure Cygb or Mb were run in parallel as standards. c shows expression level of Cygb in normoxic or hypoxic cultured cells with or without Cygb shRNA. Band intensities were quantified by a high resolution Pharos FX-plus Molecular Imager.