To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Cocaine- and amphetamine-regulated transcript (CART) was originally isolated from rat brain, but CART is also synthesized and stored in the anterior pituitary. The localization of pituitary CART and factors regulating its synthesis are largely unknown. The regulation of pituitary CART synthesis and release in response to CRH and glucocorticoids was examined in vitro and in vivo. CART immunoreactivity (CART-IR) was released from anterior pituitary segments. This release was increased 15-fold in response to corticotropin-releasing hormone (CRH). Intraperitoneal administration of CRH to rats significantly increased plasma CART-IR. Furthermore, CART-IR content and plasma CART-IR were significantly increased in adrenalectomized rats, and anterior pituitary CART mRNA expression, CART-IR content, and plasma CART-IR were significantly decreased in corticosterone-treated rats. Plasma CART-IR showed a pattern of diurnal variation similar to that of ACTH and corticosterone, and plasma CART-IR was positively correlated with corticosterone. CART-IR was detectable in the medium of the corticotroph cell line AtT-20. Dual in situ hybridization for prepro-CART (ppCART) mRNA expression and immunocytochemistry for ACTH showed localization of ppCART mRNA to a subpopulation of ACTH-immunoreactive cells. These findings demonstrate that pituitary CART expression and release are regulated by CRH and the glucocorticoid environment and that pituitary CART is partly localized to corticotrophs.

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.

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The present series of studies aimed to further our understanding of the role of melanin-concentrating hormone (MCH) neurones in the central regulation of luteinising hormone (LH) release in the female rat. LH release was stimulated when MCH was injected bilaterally into the rostral preoptic area (rPOA) or medial preoptic area (mPOA), but not when injected into the zona incerta (ZI), of oestrogen-primed ovariectomised rats. In rats that were steroid-primed to generate a surge-like release of LH, MCH administration into the ZI blocked this rise in LH release: no such effect occurred when MCH was injected into the rPOA or mPOA. In vitro, MCH stimulated gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants. Double-label immunohistochemistry showed GnRH-immunoreactive neurones in the vicinity of and intermingled with immunoreactive MCH processes. MCH is the endogenous ligand of the MCH type 1 receptor (MCH1-R). Previously, we have shown a role for melanocortin-5 receptors (MC5-R) in the stimulatory action of MCH, so we next investigated the involvement of both MCH1-R and/or MC5-R in mediating the actions of MCH on GnRH and hence LH release. The stimulatory action of MCH in the rPOA was inhibited by administration of antagonists for either MCH1-R or MC5-R. However, in the mPOA, the action of MCH was blocked only by the MC5-R antagonist. LH release was stimulated by an agonist for MC5-R injected into the rPOA or mPOA; this was blocked by the MC5-R antagonist but not the MCH1-R antagonist. These results indicate that both MCH1-R and MC5-R are involved in the central control of LH release by MCH.