Natriuretic peptide receptor‐C‐mediated attenuation of vascular smooth muscle cell hypertrophy involves Gqα/PLCβ1 proteins and ROS‐associated signaling

Figures

• 

  Figure 1. Effect of C‐ ANP4‐23 treatment on enhanced protein synthesis and cell volume in VSMC from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours, Protein synthesis (A) and cell volume (B) were determined as described in “Materials and Methods.” Results are expressed as a % of WKY CTL, taken as 100%. Values are means ± SEM of five separate experiments using different cell populations from different animals. **P < .01, ***P < .001 vs WKY CTL, ### P < .001 vs SHR CTL

• 

  Figure 2. Effect of C‐ANP4‐23 treatment on enhanced levels of Gqα and PLCβ1 in VSMC from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against Gqα (A) and PLCβ1 (B) as described in “Materials and Methods.” Dynein was used as a loading control. The protein bands were quantified by densitometric scanning. The results are expressed as a % of WKY, which is taken as 100%. Values are mean ± SEM of five separate experiments using different cell populations from different animals. *P < .05, ***P < .001 vs WKY CTL, ### P < .001vs SHR CTL

• 

  Figure 3. Effect of C‐ANP4‐23 and/or GqI treatment on proteins synthesis in VSMCs from SHR and age‐matched WKY rats. VSMC from 16‐week‐old SHR and age‐matched WKY rats were preincubated in the absence (control) or presence of Gq inhibitor YM‐245890 (10 μmol·L−1) for 1 hour prior to the treatment with C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. Protein synthesis was determined by [3H] leucine incorporation as described in “Materials and Methods.” Results are expressed as percentage of control, taken as 100%. Values are means ± SEM of 5 separate experiments using different cell populations from different animals. ***P < .001 vs WKY CTL, ## P < .01, ### P < .001 vs SHR CTL

• 

  Figure 4. Effect of C‐ANP4‐23 treatment on enhanced expression of AT1 receptor in VSMCs from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against AT1 as described in “Materials and Methods.” Dynein was used as a loading control. The proteins were quantified by densitometric scanning as described in Materials and Methods. Results are expressed as a % of WKY CTL, taken as 100%. Values are means ± SEM of 5 separate experiments using different cell populations from different animals. *P < .05, ***P < .001 vs WKY CTL, ### P < .001 vs SHR CTL

• 

  Figure 5. Effect of C‐ANP4‐23 treatment on enhanced O2 − production and NADPH oxidase activity in VSMC from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence (control) and presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours and O2 − production (A) and NADPH oxidase activity (B) were determined as described in “Materials and Methods”. Results are expressed as % of WKY CTL, taken as 100%. Values are means ± SEM of 5 separate experiments using different cell populations from different animals. *P < .05, **P < .01, ***P < .001 vs WKY CTL, ### P < .001 vs SHR CTL

• 

  Figure 6. Effect of C‐ANP4‐23 treatment on the enhanced levels of NADPH oxidase subunits p47phox and Nox4 in VSMC from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against NOX4 (A) and P47phox (B). Dynein was used as the loading control. The proteins were quantified by densitometric scanning as described in “Materials and Methods.” Results are expressed as % of WKY CTL, taken as 100%. Values are means ± SEM of 5 separate experiments using different cell populations from different animals. **P < .01 ***P < .001 vs WKY CTL, ## P < .01, ### P < .001 vs SHR CTL

• 

  Figure 7. Effect of C‐ANP4‐23 treatment on enhanced c‐Src activation in VSMCs from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against (phospho)‐c‐Src (top) and c‐Src (bottom) as described in “Materials and Methods.” The proteins were quantified by densitometric scanning as described in “Materials and Methods.” Results are expressed as a % of WKY CTL, taken as 100%. Values are means ± SEM of 5 separate experiments using different cell populations from different animals. ***P < 0.001 vs WKY CTL, ### P < .001 vs SHR CTL

• 

  Figure 8. Effect of C‐ ANP4‐23 treatment on the hyperphosphorylation of epidermal growth factor receptor (EGFR), insulin‐like growth factor receptor (IGFR), and platelet‐derived growth factor receptor (PDGFR) in VSMC from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against p‐EGFR/EGFR (A), p‐IGF‐1R/IGF‐1R (B), and p‐PDGFR/PDGFR (C). The proteins were quantified by densitometric scanning as described in “Materials and Methods.” Results are expressed as % of WKY CTL, taken as 100%. Values are means ± SD of 5 separate experiments using different cell populations from different animals. ***P < .001 vs WKY CTL, ## P < .01, ### P < .001 vs SHR CTL

• 

  Figure 9. Effect of C‐ANP4‐23 treatment on the enhanced phosphorylation of extracellular signal‐regulated kinase ERK1/2 and AKT in VSMC from SHR. Confluent VSMC from 16‐week‐old SHR and age‐matched WKY rats were incubated in the absence or presence of C‐ANP4‐23 (0.1 μmol·L−1) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against pERK1/2/ERK1/2 (A) and pAKT/AKT (B) as described in “Materials and Methods.” Results are expressed as % of WKY CTL taken as 100%. Values are means ± SEM of 5 separate experiments using different cell populations from different animals. ***P < .001 vs WKY CTL, ## P < .01, ### P < .001 vs SHR CTL

• 

  Figure 10. Schematic diagram summarizing the possible signaling mechanisms by which C‐ANP4‐23 attenuates the hypertrophy of VSMC from SHR