Search

Aims: Human retinopathy and models of experimental retinopathy of prematurity (ROP) in rodents are related to an hyperproduction of vascular endothelial growth factor (VEGF), a common mechanism also associated with [...]

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, with a major role in stimulation of feeding behavior in mammals. MCH signals in the brain occur via two seven-transmembrane G protein-coupled receptors, namely MCH1 (SLC-1, MCH1, MCH-R1, or MCH-1R) and MCH2 (SLT, MCH2, MCH-R2, or MCH-2R). In this study, we demonstrate that the pro-MCH131–165peptide neuropeptide-glutamic acid-isoleucine (NEI)-MCH is more potent than MCH in stimulating feeding in the rat. Using rat MCH1-expressed human embryonic kidney 293 cells, we show that NEI-MCH exhibits 5-fold less affinity in a binding assay and 2-fold less potency in a cAMP assay than MCH. A similar 7- to 8-fold shift in potency was observed in a Ca2+iassay using rat MCH1 or human MCH2-transfected Chinese hamster ovary cell models. This demonstrates that NEI-MCH is not a better agonist than MCH at either of the MCH receptors. Then, we compared the proteolysis resistance of MCH and NEI-MCH to rat brain membrane homogenates and purified proteases. Kinetics of peptide degradation using brain extracts indicated a t1/2of 34.8 min for MCH and 78.5 min for NEI-MCH with a specific pattern of cleavage of MCH but not NEI-MCH by exo- and endo-proteases. Furthermore, MCH was found highly susceptible to degradation by aminopeptidase M and endopeptidase 24.11, whereas NEI-MCH was fully resistant to proteolysis by these enzymes. Therefore, our results strongly suggest that reduced susceptibility to proteases of NEI-MCH compared with MCH account for its enhanced activity in feeding behavior. NEI-MCH represents therefore the first MCH natural functional “superagonist” so far described.

Several studies have shown that melanin-concentrating hormone (MCH) is an orexigenic peptide in rat. In the present study, a structure-activity relationship with MCH analogs was performed in rat, both in vitro and in vivo. On rat recombinant SLC-1 receptor, both cAMP inhibition and [125I]S36057 binding were measured. In vivo, these analogs were injected intracerebroventricularly in rats and their effects were evaluated upon food intake. First, data obtained with the rat recombinant receptor were highly correlated with those obtained from its human counterpart. Second, agonist potencies in the cAMP assay were also highly correlated with binding affinities. These peptides could be classified into several groups according to their potency at the SLC-1 receptor (from subnanomolar activity to complete inactivity). Indeed, there was a strong correlation between their effects upon food intake and the results obtained at the rat SLC-1 receptor. The present report describes for the first time the rat SLC-1 receptor pharmacology and clearly establishes the relevance of the SLC-1 receptor in feeding behavior.

The purpose of this study in the hamster cheek pouch was to determine whether or not vascular endothelial growth factor (VEGF) induced changes in plasma extravasation and if so, the mechanism(s) involved.The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and the number of vascular leakage sites, as shown by fluorescein isothiocyanate (FITC‐dextran, 150 kD) extravasation, was counted. Drugs and VEGF were applied topically. VEGF from 0.05 to 0.5 μg ml−1(1.2 to 12 nM) produced a dose‐dependent increase in the number of microvascular leakage sites from virtually none in basal conditions to up to 250 in some pouches. The effects of VEGF (0.1 μg ml−1or 2.4 nM) were blocked in a concentration‐dependent manner by the non‐specific heparin growth factor antagonist TBC‐1635 (0.1, 1 and 3μM). The placenta growth factor (PlGF‐1: 0.1 and 0.5 μg ml−1or 3.4 and 17 nM) did not increase plasma extravasation, per se, but abolished the effects of VEGF (2.4 nM).The increases in microvascular leakage produced by VEGF (2.4 nM) were partially but significantly (P<0.05) inhibited by genistein (5 and 10 μM, up to 33% inhibition), LY 294002 (30 μM, 41%), bisindolylmaleimide (1 μM, 65%) and virtually abolished by indomethacin (3 μM, 88%) and L‐nitro‐arginine (10 μM, 95%), these drugs being inhibitors of tyrosine kinase, phosphatidylinositol‐3‐kinase, protein kinase C, cyclo‐oxygenase and nitric oxide synthase respectively. None of these inhibitors, at the concentration tested, induced alone an increase in plasma extravasation.These results indicate that the VEGF‐induced plasma extravasation may involve the stimulation of VEGF‐R2 (Flk‐1/KDR) and the activation of phosphatidylinositol‐3‐kinase and protein kinase C. The production of both nitric oxide and prostaglandin is required to observe an increase in vascular leakage.