Endocrine Abstracts

Gonadotrophs in rats, mice and humans, express an intact natriuretic peptide system, in which C-type natriuretic peptide (CNP) is the predominant member. Despite showing an interaction between CNP and GnRH at the level of cGMP and Ca²⁺ signalling, the role of CNP in gonadotroph biology is poorly understood. In this study, we utilise a novel multiplex qRT-PCR assay, examining simultaneous expression of natriuretic peptide genes along with genes for gonadotroph transcription factors and signalling proteins, in a single PCR (Nppa, Nppb, Nppc, Npr1, Npr2, Npr3, Corin, Furin, Nr5a1, Nr0b1, cFos, Egr1, cJun, Mkp1, Mkp2, Actb, Gapdh, Rpl19). Initial expression screening in αT3-1, LβT2 and mouse pituitaries confirmed CNP (Nppc) and GC-B (Npr2) to be the predominant peptide and receptors present. We then challenged αT3-1 and LβT2 cells for 4 h with 100 nM GnRH, either chronically, or as 5 min pulse/hour, before examining gene expression. Chronic GnRH significantly inhibited Corin expression in αT3-1 cells (*P<0.05), but enhanced Npr3 expression in LβT2 cells (P<0.01). In contrast, pulsatile GnRH significantly increased Nppc, Furin, Npr2 and Npr3 expression in LβT2 cells (*P<0.05, ***P<0.001). As expected, both chronic and pulsatile GnRH caused significant changes in transcription factor and signalling protein gene expression in both cell lines. To establish the effects of CNP signalling on gonadotroph gene expression, αT3-1 and LβT2 cells were treated with 100 nM CNP for 0, 4, 8 and 24 h prior to multiplex qRT-PCR analyses. In αT3-1 cells, CNP stimulated cFos, cJun and Mkp2 expression, but inhibited Nr5a1. In contrast, CNP stimulated cJun, Egr1, Nr5a1, Nr0b1 and Mkp1 expression in LβT2 cells. Collectively, these data provide the first evidence that the gonadotroph natriuretic peptide system is sensitive to pulsatile GnRH, and has identified putative CNP-target genes.