JMV 449

Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT.

We recently reported that H-Lys psi (CH2NH)Lys-Pro-Tyr-Ile-Leu-OH (JMV 449), a pseudopeptide analogue of neurotensin-(8-13) with a reduced CH2NH bond in position 8-9, was about 3 times more potent than neurotensin in binding to mouse brain membranes and in contracting the guinea-pig ileum, and was markedly more resistant to degradation than neurotensin when exposed to rat brain membranes. In the present study, we compared the time courses and dose-response relationships for the ability of i.c.v. injected neurotensin and JMV 449 to elicit hypothermia and analgesia (tail-flick test) in the mouse. The results show that the pseudopeptide analogue behaved as a highly potent and long-lasting neurotensin agonist in these two in vivo bioassays. The analogue should prove very useful for studying the effects of chronic neurotensin receptor stimulation in vitro and in vivo.

Biochemical, histochemical and coimmunoprecipitation experiments have indicated the existence of antagonistic dopamine D2 (D2R) and neurotensin 1 (NTS1R) receptor-receptor interactions in the dorsal and ventral striatum indicating a potential role of these receptor-receptor interactions in Parkinson's disease and schizophrenia. By means of Bioluminiscence Resonance energy transfer (BRET(2)) evidence has for the first time been obtained in the current study for the existence of both D2LR/NTS1R and D2SR/NTS1R heteromers in living HEK293T cells. Through confocal laser microscopy the NTS1R(GFP2) and D2R(YFP) were also shown to be colocated in the plasma membrane of these cells. A bioinformatic analysis suggests the existence of a basic set of three homology protriplets (TVM, DLL and/or LRA) in the two participating receptors which may contribute to the formation of the D2R/NTS1R heteromers by participating in guide-clasp interactions in the receptor interface. The CREB reporter gene assay indicated that the neurotensin receptor agonist JMV 449 markedly reduced the potency of the D2R like agonist quinpirole to inhibit the forskolin induced increase of the CREB signal. In contrast, the neurotensin agonist was found to markedly increase the quinpirole potency to activate the MAPK pathway as also studied with luciferase reporter gene assay measuring the degree of SRE activity as well as with ERK1/2 phosphorylation assays.