Table 1.
Primer Sequences (5′ to 3′).
Table 2.
NOX siRNA sequences (5′ to 3′).
Figure 1.
bFGF increased NOX-derived O2.− production.
bFGF (25 ng/mL) treatment for 24 hours increased DHE fluorescence (red) in CSMCs compared to control. Addition of the NOX inhibitor Apo (1.0 mM) and superoxide scavenger Tempol (1.0 mM) blocked bFGF induced O2.− production. DAPI stained nuclei are shown in blue. Data presented are representative images of three different experiments.
Figure 2.
Inhibition of NOX prevented bFGF upregulation of KCNN4 mRNA expression.
KCNN4 mRNA expression increased approximately 2.5 fold in both RCA (n = 4–6) and CSMCs (n = 15–17) with bFGF (50 ng/mL and 25 ng/mL, respectively) treatment compared to control. Addition of Apo (1.0 mM) abolished bFGF-induced increases in KCNN4 expression, and alone also significantly decreased basal KCNN4 expression in CSMCs. *P<0.05 vs respective control. #P<0.05 vs respective bFGF.
Figure 3.
Inhibition of NOX prevented bFGF upregulation of KCNN4 protein expression.
Treatment of RCA sections with bFGF (50 ng/mL) caused a significant increase in medial KCNN4 protein expression, while addition of Apo (1.0 mM) inhibited the response to bFGF; (A) representative images and (C) average relative percent positive KCNN4 staining (n = 3). (B) Demonstration of selectivity of anti-KCNN4 staining by antigen pre-absorption and distribution of nitrotyrosine and SM-actin. Positive staining is brown in all images. *P<0.05 vs control.
Figure 4.
Inhibition of NOX prevented bFGF upregulation of KCNN4 activity.
24-hour treatment with bFGF (25 ng/mL) increased TRAM-34 sensitive K+ channel activity, which was inhibited by addition of Apo (1.0 mM; n = 10–17). Reduction of internal calcium prevented bFGF induced channel activity. (A) Representative ensemble step currents (−70 mV to 100 mV) before and after addition of the KCNN4 specific channel blocker TRAM-34 (100 nM), (B) TRAM-34 sensitive currents at 100 mV in CSMCs treated with bFGF or bFGF+Apo, and in internal calcium-free (-Ca2+i) conditions. *P<0.05 vs control.
Figure 5.
NOX regulation of KCNN4 involves the AP-1 transcription factor.
Treatment with bFGF (25 ng/mL) for 24 hours increased AP-1 promoter reporter activity in CSMCs nucleofected with AP-1 luciferase promoter-reporter plasmid. Addition of Apo (1.0 mM) abolished the bFGF increase in AP-1 promoter activity (n = 9–12). Luciferase activity was absent in CSMCs nucleofected with a negative control plasmid (NegCtl). *P<0.05 vs AP-1.
Figure 6.
Porcine coronary smooth muscle expresses NOX1, NOX2, NOX4, and NOX5 isoforms.
Basal mRNA expression of NOX isoforms 1, 2, 4, and 5 in RCA and CSMCs. NOX4 mRNA expression was significantly higher in both porcine RCA (n = 12–13) and CSMCs (n = 13–15). *P<0.05 vs respective NOX1.
Figure 7.
Knockdown of NOX5 prevented bFGF upregulation of KCNN4 mRNA expression.
Infection with AdNOX5shRNA significantly reduced NOX5 mRNA expression (B) and prevented KCNN4 mRNA upregulation by bFGF (25 ng/mL) in CSMCs, while the control adenovirus (AdβGal) had no effect (A, n = 4). *P<0.05 vs control.
Figure 8.
Knockdown of neither NOX2 nor NOX4 prevented bFGF upregulation of KCNN4 mRNA expression.
Nucleofection with siNOX2 significantly reduced NOX2 expression but did not prevent upregulation of KCNN4 mRNA expression by bFGF (25 ng/mL) in CSMCs (A, n = 5–9). Similarly, nucleofection with siNOX4 significantly reduced NOX4 expression but did not prevent upregulation of KCNN4 mRNA expression by bFGF (25 ng/mL) in CSMCs (B, n = 5–9). *P<0.05 vs control.
Figure 9.
Knockdown of NOX5 prevented coronary smooth muscle cell migration.
Transduction with AdNOX5shRNA significantly reduced chemotactic-induced migration in CSMCs compared to control adenovirus. Addition of TRAM-34 (100 nM), inhibited migration ∼50% in control adenovirus cells, but had no effect in AdNOX5shRNA cells. Cell number per 20X field, n = 6–8. *P<0.05 vs other experimental groups.