C-type natriuretic peptide inhibits vascular smooth muscle proliferation via NPR-C-mediated ERK1/2 phosphorylation

1Catherine M. Panayiotou, 2Amrita Ahluwalia & 1Adrian J. Hobbs 1Wolfson Institute for Biomedical Research, UCL, Gower Street, London WC1E 6AE & 2Clinical Pharmacology, WHRI, Charterhouse Square, London EC1M 6BV.

C-Type natriuretic peptide (CNP) is a member of the natriuretic peptide family, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and urodilatin that coordinate regulation of cardiovascular homeostasis. CNP has recently been described as an endothelium-derived vasorelaxant mediator that possesses anti-atherogenic properties including inhibition of smooth muscle proliferation, leukocyte recruitment and platelet aggregation, and protects against ischaemia/reperfusion injury. Many of these cytoprotective properties are mediated via the G i-coupled natriuretic peptide receptor-C (NPR-C; (Ahluwalia & Hobbs, 2005) . Since Gi-protein coupled receptors are known to govern extracellular-regulated kinase (ERK 1/2) activation and cell growth, in this study we investigated if CNP/NPR-C signalling regulates ERK 1/2 phosphorylation and cell proliferation in primary rat aortic smooth muscle cells.

Aortic smooth muscle cells (RAoSMC) were isolated from male Sprague-Dawley rats (200 – 250 g) by enzymatic (collagenase) dissociation. RAoSMC, passage 2-15, were treated with CNP (1 μM), the selective NPR-C antagonist M372049 (10 μM; (Veale et al., 2000) ), the G i-protein inhibitor Pertussis Toxin (PTx; 100 ng/ml for 16 h), or the ERK 1/2 pathway inhibitor PD98059 (30 μM). Cells were then lysed and whole cell homogenates were analysed by immunoblot for the presence of ERK 1/2 phosphorylation. Cell proliferation was assessed by 5-bromo-2’-deoxyuridine (BrdU) incorporation. Data are expressed as mean values ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison test was used to assess differences between groups.

ERK 1/2 phosphorylation was transiently, but significantly increased in the presence of CNP with a peak effect observed 10 min after treatment (250.1 ± 71.79 % increase; n=8; P<0.05 versus control)). CNP-induced ERK 1/2 phosphorylation was blocked by PD98059 (96.6 ± 8.9 % inhibition; n=6), M372049 (64.6 ± 17.7 % inhibition; n=3) and PTx (180.7 ± 17.2 % inhibition; n=8; all P<0.05 versus CNP alone). CNP inhibited vascular smooth muscle growth by 38.8 ± 2.9 % (n ≥6; P<0.05 versus control), an effect that was reversed by M372049 (64.9 ± 3.9 % reversal; n=18) and PD98059 (61.3 ± 6.3 % reversal; n=6; both P<0.05 versus CNP). These observations suggest that the anti-proliferative properties of CNP are mediated, at least in part, via NPR-C and Gi-dependent ERK 1/2 phosphorylation. Such findings add to the anti-atherogenic profile of CNP/NPR-C signalling and may aid the development of novel therapeutic intervention in the treatment of inflammatory cardiovascular diseases such as atherosclerosis, sepsis and coronary heart disease.

Ahluwalia A & Hobbs AJ (2005). Trends Pharmacol Sci: 26, 162-167.
Veale C.A (2000). Bioorg. Med. Chem. Lett10: 1949-1952.

 This work was supported by The Wellcome Trust.