Intracellular peptides of natriuretic peptide receptor-C inhibit vascular hypertrophy via Gqalpha/MAP kinase signaling pathways

OBJECTIVE: The present studies were undertaken to investigate if the activation of natriuretic peptide receptor-C (NPR-C) that has been shown to inhibit cell proliferation could also modulate the hypertrophic responses of vasoactive peptides in A10 vascular smooth muscle cells (VSMC). METHODS: For these studies A10 VSMC were incubated in the presence of angiotensin II (AngII), endothelin (ET-1) or arginine vasopressin (AVP) alone or in combination with C-ANP4-23 or NPR-C peptides for 24 h and were used for Western blotting and [3H]leucine incorporation. The different peptide fragments used were K461KYRITIERRNH472 (peptide-1) and H481RELREDSIRSH492 (peptide-3) with complete Gi activator sequences and R469RNHQEESNIGK480 (peptide-2) and I465TIERRNH472 (peptideY) with partial Gi activator sequences. The other peptide used had no structural specificity, (Q473EESNIGK480; peptide X) or was the scrambled peptide control for peptide-1 (ITIYKKRRNHRE; peptide Z). RESULTS: AngII, ET-1 and AVP significantly stimulated protein synthesis in these cells as determined by 3H-leucine incorporation, which was inhibited by peptides 1, 2 and 3 and not by peptides X, Y or Z in a concentration-dependent manner with an apparent Ki between 1-10 nM. In addition, C-ANP4-23 also inhibited protein synthesis stimulated by AngII, ET-1 and AVP; whereas basal protein synthesis in these cells was not inhibited by C-ANP4-23 or by the peptide fragments. Furthermore, AngII-, AVP- and ET-1-induced stimulation of protein synthesis was inhibited by PD98059 (MEK inhibitor) and wortmannin (P13K inhibitor) and this inhibition was potentiated by peptide-1. In addition, peptide-1 was also able to inhibit vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced the expression of Gqalpha protein in these cells. CONCLUSIONS: These data suggest that C-ANP4-23 and small peptide fragments containing 12 amino acids from different regions of cytoplasmic domain of NPR-C could modulate vasoactive peptide-stimulated protein synthesis through Gqalpha/MAP kinase/P13K and AKT pathways.