Prolactin-Releasing Peptide (1-31) (rat)

A recently identified neuropeptide with PRL-releasing capabilities binds to and activates a previously known orphan G protein-coupled receptor, GPR10. We initiated a study to define the pharmacology of the peptide/receptor interaction and to identify the distribution of the peptide and its receptor in the central nervous system to elucidate sites of action of the peptide. The PRL-releasing peptide (PrRP) is a C-terminally amidated, 31-amino acid peptide derived from a 98-amino acid precursor. Radioiodinated PrRP-(1-31) binds to its receptor with high affinity (1 nM) and stimulates calcium mobilization in CHOK1 cells stably transfected with the receptor. A series of N-terminal deletions reveals that the PrRP-(12-31) amino acid is equipotent to PrRP-(1-31). Further N-terminal deletions reduce the affinity of the ligand considerably, although PrRP-(25-31) is still able to compete for binding and behaves as an agonist. The arginine residues at position 26 and 30 are critical for binding, as substitution with either lysine or citrulline reduces the affinity substantially. In situ hybridization reveals a distinct tissue distribution for both the peptide and receptor messenger RNAs. The receptor is expressed abundantly in the reticular thalamic nucleus, periventricular hypothalamus, dorsomedial hypothalamus, nucleus of the solitary tract, area postrema, anterior pituitary, and adrenal medulla. The peptide messenger RNA is expressed in the dorsomedial hypothalamus, nucleus of the solitary tract, ventrolateral reticular nucleus, and intestine. This tissue distribution suggests an alternative function of PrRP than its purported hypophysiotropic function, such as a potential role for PrRP in the central feedback control of neuroendocrine and autonomic homeostasis. Further work using selective agonists and antagonists should help define additional physiological roles of this novel mammalian neuropeptide.

Prolactin releasing peptide (PrRP) was originally isolated as an endogenous hypothalamic ligand for the hGR3 orphan receptor. It has been shown to release prolactin from dispersed pituitaries harvested from lactating female rats and only at very high doses in cycling females. PrRP is reported to have no effect on prolactin production from dispersed pituitary cells harvested from males. The CNS distribution of this peptide suggested a role for PrRP in the control of the hypothalamo-pituitary axis. The aim of this study was to examine the actions of PrRP (1-31) on circulating pituitary hormones following intracerebroventricular (ICV) injection in male rats and to investigate the mechanism of PrRP's effect by measurement of hypothalamic releasing factors in vitro. In our experiments, PrRP (1-31) did not release LH, FSH, TSH, growth hormone or prolactin directly from dispersed male pituitary cells in vitro. We have shown for the first time that following ICV injection of PrRP (1-31) 5 nmol there was a highly significant simulation of plasma LH that began at 10 minutes and was maintained over the course of the experiment (at 60 minutes PrRP 5 nmol 2.2 +/- 0.2 vs. saline 0.5 +/- 0.1 ng/ml, p<0.001). Plasma FSH increased at 20 minutes following ICV injection (PrRP 5nmol 10.8 +/- 2.0 ng/ml vs. saline 5.1 +/- 0.5, p<0.01). Total plasma testosterone increased at 60 minutes post injection (PrRP 5nmol 9.2 +/- 1.6 vs. saline 3.5 +/- 0.6 nmol/l, p<0.01). There was no significant alteration in plasma prolactin levels. PrRP significantly increased the release of LHRH from hypothalamic explants in vitro (PrRP 100nmol/l 180.5 +/- 34.5% of the basal secretion, p<0.05). PrRP (100nmol/l) also increased the following hypothalamic peptides involved in the control of pituitary hormone release, vasoactive intestinal peptide (VIP) 188.1 +/- 24.6% and galanin 153.8 +/- 13.0% (both p<0.001 vs. basal secretion) but had no effect on orexin A secretion. These results suggest a role for PrRP in the control of gonadotrophin secretion acting via a hypothalamic mechanism involving the release of LHRH.