Novel pituitary actions of phoenixin in grass carp

Abstract

Phoenixin (PNX), a novel peptide recently identified by bioinformatics, is widely expressed at the tissue level and confirmed to be the ligand for the orphan receptor GPR173. Its mature peptide PNX20 is highly conserved and has emerged as a novel regulator for reproductive, appetite control, memory retention, plain perception and cardiovascular protection. However, little is known for the comparative aspects of PNX, especially in lower vertebrates, and its functional role in the pituitary is largely unclear. Using grass carp as a model for bony fish, the cDNAs of grass carp PNX and its receptor GPR173 were cloned. Using Southern blot, both PNX and GPR173 was confirmed to be a single-copy gene in the carp genome. As revealed by RT-PCR, PNX and GPR173 transcripts were widely expressed in different tissues and brain areas. At the pituitary level, transcript signals of PNX and GPR173 could be co-localized in lactotrophs and gonadotrophs. Using immunostaining, PNX immunoreactivity was also detected in these two pituitary cell types. In the primary culture of grass carp pituitary cells, static incubation with the synthetic peptide of carp PNX20 was effective in increasing prolactin (PRL) release, PRL cell content, and total production of PRL with a parallel rise in PRL mRNA level. The opposite was true for PRL secretion and transcript expression in the parallel experiment with the removal of endogenous PNX by immunoneutralization with PNX antiserum. Meanwhile, luteinizing hormone (LH) release was not altered by PNX20, despite parallel elevations in LH cell content and LH total production were noted with a mild increase in LHβ transcript expression. Interestingly, PNX immunoneutralization could reduce LHβ mRNA level with a parallel increase in LH secretion. Using a pharmacological approach, PNX20-induced LHβ mRNA expression was confirmed to be mediated by the AC/cAMP/PKA, PLC/IP3/PKC and Ca2+/CaM/CaMK-II pathways. Similar post-receptor signaling mechanisms were involved in PNX20-induced PRL release and transcript expression, except that the PKC component was missing in PRL responses. Besides the effects on basal secretion and transcript expression, PNX20 was also found to exhibit differential interactions with other LH and PRL regulators at the pituitary cell level. In parallel studies of PNX20 co-treatment with GnRH, PACAP, NKB, kisspeptin, somatostatin, estrogen, or dopamine, differential responses for LH/PRL secretion and/ or transcript expression, including additive/potentiating effects, inhibitory action, or lack of interaction could be observed depending on the target of co-treatment. Our results, as a whole, suggest that (i) PNX can serve as a novel regulator for LH and PRL in grass carp via direct action at the pituitary level, (ii) PNX regulation of LH and PRL are mediated by autocrine/paracrine mechanisms, presumably through AC/cAMP/PKA, PLC/IP3 (with/without PKC) and Ca2+/CaM/CaMK-II cascades coupled with GPR173 expressed in lactotrophs and gonadotrophs, and (iii) By interacting with other neuroendocrine factors, PNX probably can contribute to the physiological regulation of LH/PRL secretion and gene expression in the carp pituitary.