FIGURE 3.

Inhibition of NO/cGMP signaling prevents shear-induced c-fos, fra-1, fra-2, and fosB/ΔfosB mRNA expression. A, serum-deprived hPOBs were placed in a flow chamber and were incubated for 1 h with either culture medium alone (lanes 1 and 2) or with medium containing 4 mm l-NAME (lane 3), 10 μm ODQ (lane 4), or 100 μm (R_p)-8-pCPT-PET-cGMPS ((Rp)-cGMPS, lane 5). Cells were then either kept under static conditions (lane 1) or were exposed to laminar flow for 20 min (lanes 2–5). Ten minutes after the cessation of flow, total RNA was extracted, and c-fos, fra-1, fra-2, fosB/ΔfosB, or gapd mRNA levels were determined by semi-quantitative RT-PCR as described in Fig. 1A. B, MC3T3 cells were treated as described for hPOBs in A, but c-fos, fra-1, and fra-2 mRNA levels were quantified after 60 min by real time RT-PCR as described in Fig. 1B. p < 0.05 for the comparison between control cells receiving no drug and cells treated with l-NAME (L-N), ODQ, or (R_p)-cGMPS (Rp). C, MC3T3 cells were kept under static conditions (gray bars) or were exposed to laminar flow for 20 min (black bars); 5 min after the cessation of flow, media were collected from the chamber, and nitrate plus nitrite (NO_x) concentrations were measured. D, MC3T3 cells expressing human VASP were incubated for 1 h with medium alone (lanes 1 and 2) or with medium containing 4 mm l-NAME (lane 3), 10 μm ODQ (lane 4), or 100 μm (R_p)-8-pCPT-PET-cGMPS (Rp-cGMPS, lane 5). Cells were kept under static conditions (lane 1) or were exposed to 20 min of fluid shear stress (lanes 2–5), and 10 min after the cessation of flow, cell lysates were analyzed by SDS-PAGE/Western blotting using antibodies specific for VASP phosphorylated on Ser²³⁹ (upper panel) or α-tubulin (lower panel).