Endocrine Abstracts

CNP, the third member of the mammalian natriuretic peptide family, exerts the majority of its biological effects via the guanylyl cyclase B (GC-B) receptor, resulting in accumulation of intracellular cGMP. Previous studies reveal numerous inhibitory effects of natriuretic peptides on the MAPK family in bone, neuronal and cardiovascular tissues. However, we recently reported that CNP stimulates ERK, p38MAPK and JNK phosphorylation in pituitary GH3 somatotrophs. As CNP is known to antagonise GnRH signalling in alphaT3-1 gonadotrophs, we investigated the effects of CNP signalling in LbetaT2 gonadotrophs. Western blotting for phosphoERK, phospho-p38MAPK and phosphoJNK in total protein extracts from serum-starved LbetaT2 cells showed the expected enhancement in phosphorylation following treatment with 100 nM GnRH. However, in the presence of a 30 min CNP pre-treatment (100 nM), basal phosphorylation was dramatically enhanced, suggesting direct effects of CNP on phosphoMAPK’s, without altering the effects of GnRH. Time-course studies (0–90 min) in LbetaT2 cells with 100 nM CNP showed a time-dependent, potent phosphorylation of ERK, p38MAPK and JNK (within 5 min), effects that were maintained for 60 min. In comparison, similar experiments with atrial natriuretic peptide (ANP, 100 nM) showed a modest but transient (5 min) enhancement of phosphoERK, and no effect on phosphoJNK. The levels of the MAPK phosphatase, MKP-1, showed a concomitant increase following CNP, but not ANP treatment. To confirm the presence of pharmacologically active GC-A or GC-B receptors, cGMP enzymeimmunoassays were performed. ANP potently and dose-dependently increased cGMP accumulation but, surprisingly, CNP failed to stimulate cGMP accumulation. These data suggest that CNP, but not ANP, can potently enhance ERK, p38MAPK and JNK phosphorylation, in the apparent absence of a conventional GC-B/cGMP signalling pathway, as indicated by the lack of cGMP response. The mechanism for this intriguing response remains to be elucidated. Funded by a BBSRC Project grant (BBD0015601).