C-type natriuretic peptide (CNP) acts via its specific membrane-bound guanylyl cyclase receptor, GC-B, to stimulate cGMP production. Stimulation of cGMP impinges upon PKG and PKA signalling, leading to cell proliferation and cell cycle events. In endothelial cells, CNP is thought to perform a predominantly anti-mitogenic role, but the role of CNP on cell proliferation, cell cycle and DNA synthesis in pituitary cell lines has yet to be established. Using the mouse gonadotroph-derived αT3-1 and rat somatotroph-derived GH3 cells, we investigated the biological role of CNP by cGMP-enzymeimmunoassay, MTT cell proliferation, tritiated thymidine incorporation and flow cytometry. CNP concentration-dependently increased cGMP accumulation in both αT3-1 and GH3 cells, suggesting the presence of pharmacologically active GC-B receptors. Proliferation studies involved culture of 3×105 cells/well, plated in 24-well plates and treated for 48 h in the absence or presence of either CNP (10 nM, 100 nM), GnRH (100 nM), PACAP (10 pM100 nM; αT3-1 cells), TRH (100 nM) or EGF (10 nM; GH3 cells) in 2% FCS-supplemented media. Cell proliferation was determined by addition of either MTT reagent or tritiated thymidine for the last 4 h of culture. Only a 10% FCS positive control treatment stimulated cell proliferation, using the MTT assay (P<0.01) and CNP failed to alter this affect. However, PACAP-treated αT3-1 cells showed significantly enhanced thymidine incorporation (1.7-fold, P<0.01), but again CNP failed to attenuate this affect. Finally, flow cytometric analysis of cell cycle distribution in CNP treated αT3-1 and GH3 cells failed to show any significant alteration in the distribution of cells in either G0/G1, M or S-phase. These data suggest that although CNP potently stimulated cGMP accumulation in both αT3-1 and GH3 cells, this does not influence the proliferative capacity of the cell lines in the presence of CNP alone or in combination with other peptide and growth factor regulators of cell proliferation.
06 - 07 Nov 2006
Society for Endocrinology