The present studies were undertaken to investigate the effect of C-atrial natriuretic peptide (ANP)(4-23) and several peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of natriuretic peptide receptor (NPR)-C on cell proliferation in the absence or presence of angiotensin (ANG) II, endothelin (ET)-1, and arginine vasopressin (AVP) in A-10 vascular smooth muscle cells (VSMC). The peptide fragments used have either complete G(i) activator sequences K(461)-H(472) (peptide 1) and H(481)-H(492) (peptide 3) or partial G(i) activator sequences R(469)-K(480) (peptide 2) and I(465)-H(472) (peptide Y) with truncated COOH or NH(2) terminus, respectively. The other peptide used had no structural specificity (Q(473)-K(480), peptide X) or was the scrambled peptide control for peptide 1 (peptide Z). ANG II, ET-1 and AVP significantly stimulated DNA synthesis in these cells as determined by [(3)H]thymidine incorporation that was inhibited by peptides 1, 2, and 3 and not by peptides X, Y, and Z in a concentration-dependent manner, with an apparent K(i) between 1 and 10 nM. In addition, C-ANP(4-23), which interacts with NPR-C, also inhibited DNA synthesis stimulated by vasoactive peptides; however, the inhibition elicited by C-ANP(4-23) was not additive with the inhibition elicited by peptide 1. On the other hand, basal DNA synthesis in these cells was not inhibited by C-ANP(4-23) or the peptide fragments. Furthermore, vasoactive peptide-induced stimulation of DNA synthesis was inhibited by PD-98059 and wortmannin, and this inhibition was potentiated by peptide 1. In addition, peptide 1 also inhibited vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced expression of G(i)alpha proteins. These data suggest that C-ANP(4-23) and small peptide fragments containing 12 amino acids irrespective of the region of the cytoplasmic domain of NPR-C inhibit proliferative responses of vasoactive peptides through G(i)alpha protein and MAP kinase/phosphatidylinositol 3-kinase/AKT pathways.