Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells

Purpose. Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a ce ll culture model system. Studies have also shown that atrial natriuretic pe ptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent , inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated pro liferation of RCEC involved in wound repair. Methods. The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or oth er agents. At prescribed time intervals, the cells were stained by Giemsa a nd the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured b y radioimmunoassay. Results. Addition of EGF (1-100 ng/ml) to RCEC stimulated cell proliferatio n which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 muM) or SNP (10 muM to 1 mM), in the presence of EGF, d ose-dependently inhibited the growth factor-stimulated wound closure in RCE C. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 muM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibite d the wound closure with a corresponding increase in cGMP contents. Treatme nt of the cells with ODQ (10 nM to 10 muM), a soluble guanylate cyclase inh ibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, an d reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Ad dition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited t he EGF-stimulated wound closure in a dose-dependent manner. Treatment of RC EC with mitomycin C (5 muM) exerted a marked inhibitory effect on wound clo sure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated ce lls. Conclusions. The results demonstrate that ANP and SNP inhibit the EGF-stimu lated wound repair in RCEC. The effect of these agents is mediated via acti vation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exer t its inhibitory effect at the level of cell proliferation and not cell mig ration. The data suggest an important role for cGMP-dependent protein kinas e in proliferation of RCEC stimulated by EGF.