A potential role for the secretogranin II-derived peptide EM66 in the hypothalamic regulation of feeding behaviour.

EM66 is a conserved 66-amino acid peptide derived from secretogranin II (SgII), a member of the granin protein family. EM66 is widely distributed in secretory granules of endocrine and neuroendocrine cells, as well as in hypothalamic neurones. Although EM66 is abundant in the hypothalamus, its physiological function remains to be determined. The present study aimed to investigate a possible involvement of EM66 in the hypothalamic regulation of feeding behaviour. We show that i.c.v. administration of EM66 induces a drastic dose-dependent inhibition of food intake in mice deprived of food for 18 hours, which is associated with an increase of hypothalamic pro-opiomelanocortin (POMC) and melanocortin-3 receptor mRNA levels and c-Fos immunoreactivity in the POMC neurones of the arcuate nucleus. By contrast, i.c.v. injection of EM66 does not alter the hypothalamic expression of neuropeptide Y (NPY), or that of its Y1 and Y5 receptors. A 3-month high-fat diet (HFD) leads to an important decrease of POMC and SgII mRNA levels in the hypothalamus, whereas NPY gene expression is not affected. Finally, we show that a 48 hours of fasting in HFD mice decreases the expression of POMC and SgII mRNA, which is not observed in mice fed a standard chow. Taken together, the present findings support the view that EM66 is a novel anorexigenic neuropeptide regulating hypothalamic feeding behaviour, at least in part, by activating the POMC neurones of the arcuate nucleus.

Comparisons between bupropion and dexamphetamine in a range of in vivo tests exploring dopaminergic transmission.

BACKGROUND AND PURPOSE: In the present study we investigated, in a range of in vivo tests whether the antidepressant bupropion, and its metabolites shared the dopamine releasing effect of the chemically related dexamphetamine. EXPERIMENTAL APPROACH: We compared bupropion and dexamphetamine in different neurochemical (microdialysis, DOPAC and HVA contents) and behavioural tests, assessing their effects in animals pretreated with a variety of agents (reserpine, sodium hydroxy-4-butyrate or haloperidol) known to modify dopaminergic transmission. KEY RESULTS: In mice, dexamphetamine, like bupropion, increased at low doses and reduced at high doses, locomotor activity. Dexamphetamine restored the locomotor activity in mice made akinetic by either sodium hydroxy-4-butyrate or reserpine, whereas bupropion did not. Moreover, bupropion prevented the dexamphetamine-induced reversal of akinetic effects of reserpine. Haloperidol abolished the locomotor-stimulant effects of dexamphetamine but did not suppress stimulation by bupropion. In microdialysis experiments, in chloral hydrate anesthetized rats, low doses of dexamphetamine (1 mg kg(-1)) markedly increased the extracellular dopamine concentration in striatum (340%), while bupropion (100 mg kg(-1)) produced only a moderate increase (150%). Finally, in rat striatum, as well as in the nucleus accumbens, bupropion increased the effect of haloperidol on DOPAC and HVA concentrations, whereas dexamphetamine reduced these haloperidol effects. CONCLUSIONS AND IMPLICATIONS: Considering only dopaminergic transmission, our results demonstrated that bupropion and metabolites displayed in vivo, as did bupropion in vitro, an inhibition of dopamine uptake and, contrast to dexamphetamine, were devoid of dopamine releasing effects.

Localization of neurotensin NTS2 receptors in rat brain, using.

The brain localization of the neurotensin receptor NTS2 was studied with [3H]levocabastine, using an autoradiographic procedure. This study suggests that NTS2 receptors are mainly intracellular. High densities of binding sites were observed in the cingulate, insular, temporal, occipital, enthorhinal cortex, amygdaloid complex, septohippocampal nuclei, medial thalamus, mammillary bodies and superior colliculi; a moderate labelling was observed in the anterior and medial hippocampus, olfactory tubercle, hypothalamus, periaqueductal gray matter, caudate putamen, nucleus accumbens, septum, lateral thalamus, dorsal raphe nucleus and cerebellum; finally, a low labelling was apparent in the ventral tegmentum area and substantia nigra. Thus it appears that NTS2 receptors are particularly abundant in the cerebral cortex, the limbic areas and some areas involved in pain perception.

Developmental changes of (3)H-labelled mu-opioid receptors in brainstems of intra-uterine growth-restricted rats.

The opioid mu-system is involved in brainstem-mediated respiratory control. Infants with intra-uterine growth restriction (IUGR) have more respiratory disorders in the early postnatal period. Using [(3)H]DAGO, a mu-selective ligand, and a computer-based image analysis of autoradiography, we compared the ontogeny and distribution of mu-opioid binding sites in the brainstem of IUGR and control rats in utero (E21), at birth (P0) and on postnatal days 1 (P1), P7, P10, P14 and P21. The ontogeny pattern was found to be similar in both groups. The density of the binding sites, which was low in E21, increased at P0, slightly declined at P1 and remained relatively constant thereafter. The distribution of DAGO-binding sites, also similar in both groups, was heterogeneous and was much denser in the dorsal areas of medulla and pons. In particular, binding sites were highly concentrated in nuclei involved in the cardio-respiratory function. However, DAGO-binding density was higher at all ages (except for P0 and P1) in IUGR than in control rats. Taken together, these results give at least a partial explanation for the effects of IUGR which lowers the Apgar score at birth and raises the incidence of respiratory disorders in infants.

The partial agonist properties of levocabastine in neurotensin-induced analgesia.

The antihistaminic drug levocabastine is a ligand for the low affinity neurotensin receptor (NTS2). Its intracerebroventricular administration to mice induced a significant analgesia in the writhing test but not in the hot plate test. In the writhing test, levocabastine decreased neurotensin-induced analgesia to a level not significantly different from the effects of levocabastine alone. In the hot plate test, levocabastine had no analgesic effect but completely reversed the neurotensin-induced analgesia. Mepyramine, another antihistaminic drug, did not share these levocabastine effects. Neither levocabastine nor mepyramine modified the colonic temperature or reversed the neurotensin-induced hypothermia. Thus, levocabastine behaves as a partial agonist at neurotensin NTS2 receptors, which are involved in visceral nociception, but not at yet unidentified neurotensin receptors involved in hypothermia.

Identification of the receptor subtype involved in the analgesic effect of neurotensin.

The neuropeptide neurotensin (NT) elicits hypothermic and naloxone-insensitive analgesic responses after brain injection. Recent pharmacological evidence obtained with NT agonists and antagonists suggests that these effects are mediated by a receptor distinct from the initially cloned high-affinity NT receptor (NTR1). The recent cloning of a second NT receptor (NTR2) prompted us to evaluate its role in NT-induced analgesia. Intracerebroventricular injections in mice of two different antisense oligodeoxynucleotides from the NTR2 markedly decreased NTR2 mRNA and protein and reduced NT-induced analgesia. This effect was specific, because NTR1 levels were unaffected, and sense or scramble oligodeoxynucleotides had no effect. Structure-activity studies revealed a close correlation between the analgesic potency of NT analogs and their affinity for the NTR2 and disclosed potent and selective agonists of this receptor. These data confirm that NTR1 is involved in the NT-elicited turning behavior and demonstrate that the NTR2 mediates NT-induced analgesia.

A C-terminal cyclic 8-13 neurotensin fragment analog appears less exposed to neprilysin when it crosses the blood-brain barrier than the cerebrospinal fluid-brain barrier in mice.

A C-terminal cyclic 8-13 neurotensin fragment analog. JMV 1193, a direct agonist of central neurotensin receptors, is able to cross both the cerebrospinal fluid-brain barrier and the blood-brain barrier. When administered intracerebroventricularly (i.c.v.), its hypothermic effect was potentiated by the enkephalinase inhibition induced either by thiorphan (simultaneous intracerebroventricular administration of 10 micrograms) or by the thiorphan prodrug. acetorphan (intravenous (i.v.) administration of 10 mg/kg). Such a potentiation was not observed when both JMV 1193 and acetorphan were administered intravenously. Therefore it appears that the sensitivity of JMV 1193 to enkephalinase depends on its route of administration. It is exposed to this peptidase after i.c.v. injection (when crossing the cerebrospinal fluid-brain barrier), while it is not after i.v. administration (when crossing the blood-brain barrier).

The nonpeptide neurotensin antagonist, SR 48692, used as a tool to reveal putative neurotensin receptor subtypes.

The nonpeptide neurotensin (NT) antagonist, SR 48692, was recently shown to inhibit NT binding to the cloned rat and human NT receptor and to antagonize NT effects in a variety of in vitro and in vivo assays. Here, we show that, in contrast to its antagonistic action on NT-induced hypomotility in the rat, SR 48692 failed to antagonize NT-induced hypothermia and analgesia in the mouse and rat. We suggest that these effects might be mediated through a subtype of SR 48692-insensitive NT receptor.

Tolerance to the hypothermic but not to the analgesic effect of [D-Trp11]neurotensin during the semichronic intracerebroventricular infusion of the peptide in rats.

The peptidase-resistant derivative of neurotensin, [D-Trp11]neurotensin, has been continuously infused intracerebroventricularly (75 ng/h) with an osmotic minipump for 10 days. On several days during this infusion the locomotor activity, the body temperature, the food intake, the body weight, and the nociceptive response in the plantar test were measured. A nonsignificant decrease of body temperature and a sustained analgesic effect were observed at each time considered. The response to a test dose of [D-Trp11]neurotensin (75 ng per rat) injected intracerebroventricularly at the 10th day of the chronic infusion revealed a complete tolerance to its hypothermic effect. Thus, it appears that the analgesic effect of [D-Trp11]neurotensin is independent of a hypothermic or an incapacitating effect of the peptide and does not give rise to tolerance after a 10-day continuous administration, in contrast to the hypothermic effect.

In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes.

The present study was designed to compare, with respect to structure-activity relationships, the receptors that subserve the hypothermic and analgesic effects of neurotensin (NT) to the receptor that mediates the effects of NT in mesencephalic dopamine (DA) neurons, and to compare these receptors to the cloned adult rat brain NT receptor and to newborn mouse and rat brain NT receptors. The results show that NT receptors in homogenates from newborn mouse and rat brain and from COS 7 cells transfected with the cloned high-affinity NT receptor from the adult rat brain displayed virtually identical structure-activity relationships toward a series of 12 peptide and pseudopeptide NT analogs, as assessed by the ability of the compounds to inhibit the binding of [125I]NT binding in these systems. Furthermore, when eight of these analogs were tested for their ability to inhibit [125I]NT binding and to potentiate K(+)-evoked DA release in primary cultures of rat mesencephalic neurons, it was found that they all behaved as agonists with binding and biological potencies quite similar to those observed in the other binding assays. Finally and strikingly, when seven of these analogs with checked metabolic stability were tested in vivo for their hypothermic and analgesic (tail-flick test) effects after i.c.v. injection in the mouse, they exhibited relative potencies that were completely different from those obtained in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and analgesic effects of N-[3-[(hydroxyamino) carbonyl]-1-oxo-2(R)-benzylpropyl]-L-isoleucyl-L-leucine, a new potent inhibitor of multiple neurotensin/neuromedin N degrading enzymes.

The synthesis of N-[3-[(hydroxyamino) carbonyl]-1-oxo-2(R)-benzylpropyl]-L-isoleucyl-L-leucine (JMV-390-1, 6a), a multipeptidase inhibitor based on the C-terminal sequence common to neurotensin (NT) and neuromedin N (NN), is described. This compound behaves as a full inhibitor of the major NT/NN degrading enzymes in vitro, e.g. endopeptidase 24.16, endopeptidase 24.15, endopeptidase 24.11, and leucine aminopeptidase (type IV-S), in the nanomolar range (IC50's from 30 to 60 nM). Compound 6a was found to increase endogenous recovery of NT and NN from slices of mice hypothalamus depolarized with potassium. In various assays commonly used to select analgesics, e.g. hot-plate test, tail-flick test, acetic acid-induced writhing test, in mice, compound 6a proved to be potent when intracerebroventricularly (icv) injected. The analgesic effects observed were totally (hot-plate test) or largely (tail-flick test) reversed by the opioid antagonist naltrexone. Furthermore, icv injection of compound 6a (10 micrograms/mouse) was found to significantly potentiate the hypothermic effects of NT or NN.

JMV 449: a pseudopeptide analogue of neurotensin-(8-13) with highly potent and long-lasting hypothermic and analgesic effects in the mouse.

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.

Effects of thiorphan, bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the recovery of neurotensin and neuromedin N released from mouse hypothalamus.

The effects of the endopeptidase 24.11 ('enkephalinase') inhibitor thiorphan, the aminopeptidase inhibitor bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the K(+)-evoked release of immunoreactive neurotensin and neuromedin N (iNT and iNN) from mouse hypothalamic slices were examined. (JMV 390-1 inhibits several metallopeptidases including endopeptidases 24.11, 24.15 and 24.16, and aminopeptidase N equipotently with Ki values around 50 nM.) Thiorphan increased the recovery of released iNT nearly 2-fold and had no effect on iNN. Bestatin produced a 4-fold increase in iNN recovery and was inactive on iNT. Finally, iNT and iNN recoveries were increased up to 4- and 5-fold, respectively, by JMV 390-1. These results show that in the mouse hypothalamus endopeptidase 24.11 participates with other metalloendopeptidases to the degradation of endogenously released NT while endogenously released NN is principally degraded by aminopeptidase(s).

Centrally administered [D-Trp11]neurotensin, as well as neurotensin protected from inactivation by thiorphan, modifies locomotion in rats in a biphasic manner.

Neurotensin injected intracerebroventricularly at the dose of 30 ng per rat was without intrinsic effect on locomotion. When associated with the enkephalinase inhibitor thiorphan (50 micrograms, intracerebroventricular) it decreased locomotor activity. On the contrary, the 3 micrograms dose of NT, which had a tendency to decrease locomotion, stimulated locomotor activity when associated with thiorphan (50 micrograms, intracerebroventricular). This effect was independent of endogenous enkephalins since it was not suppressed by a high dose of naloxone (2 mg/kg). Similarly, increasing doses of the enkephalinase-resistant peptide [D-Trp11]neurotensin had a biphasic effect on locomotion since doses lower than 60 ng were hypokinetic whereas higher doses were hyperkinetic. This latter effect was not modified by thiorphan. It was antagonized by the dopamine antagonist haloperidol (50 micrograms/kg, IP).

Calcium-dependent release of neuromedin N and neurotensin from mouse hypothalamus.

Neuromedin N (NN), a hexapeptide, was isolated from porcine spinal cord. Its C-terminal tetrapeptide sequence is identical to that of neurotensin (NT) and it exhibits NT-like effects when injected in the central nervous system. Both peptides were recently shown to be encoded in the same precursor molecule. We have just developed a sensitive and specific radioimmunoassay (RIA) for NN and showed that the peptide central nervous system distribution paralleled that of NT, the highest concentrations being found in the hypothalamus. Using this assay and a specific RIA for NT, we show here that NN and NT were simultaneously released from slices of mouse hypothalamus by K(+)-induced depolarization in a Ca(++)-dependent manner. The ratio of released NN over NT was 0.3 and was identical to the ratio of endogenous NN over NT. For both NN and NT, the releasable peptide pool represented 2% of the endogenous peptide pool. HPLC characterization of the releasable and endogenous immunoreactive material reacting with the NN and NT antisera showed that it coeluted with synthetic NN and NT, respectively. The present data further support the hypothesis that NN acts as a neuromodulator in the central nervous system.

Hypothermic effect of neuromedin N in mice and its potentiation by peptidase inhibitors.

The intracerebroventricular administration of neuromedin N (from 50 ng to 5 micrograms) elicited a dose- and time-dependent hypothermia in mice. Two aminopeptidase inhibitors, bestatin (50 micrograms) and puromycin (50 micrograms), the endopeptidase 24.11 inhibitor, thiorphan (10 micrograms), and the angiotensin-converting enzyme inhibitor, captopril (50 micrograms), were tested for their ability to potentiate the neuromedin N-induced hypothermia. Only bestatin significantly increased the response to the peptide. In addition, thiorphan, though devoid of effect on the neuromedin N-induced hypothermia when given alone, further potentiated the response elicited by neuromedin N and bestatin. The combinations of puromycin/thiorphan and bestatin/captopril did not potentiate the neuromedin N-induced hypothermia.

Potentiation by thiorphan and bestatin of the naloxone-insensitive analgesic effects of neurotensin and neuromedin n.

Neurotensin induced significant antinociceptive activity as measured in a variety of nociceptive tests 10 and 30 min following intracerebroventricular (i.c.v.) injection in mice. The lowest effective peptide doses were 25 ng in the writhing test, 25-50 ng in the tail-flick test, 50-100 ng in the hot-plate test and 2000 ng in the tail electrical stimulation test. The neurotensin related hexapeptide neuromedin N also displayed antinociceptive properties but only in the writhing and tail-flick tests. Furthermore, as compared to neurotensin, the neuromedin effects required higher doses. ED(50)'s for neurotensin and neuromedin in the writhing test were 70 ng and 1070 ng, respectively. Separate or combined injections of the endopeptidase 24.11 (enkephalinase) inhibitor thiorphan (l0?g) and the aminopeptidase inhibitor bestatin (50?g) did not affect tail-flick latencies. In contrast, i.c.v. injection of thiorphan together with an ineffective dose of neurotensin (25 ng) resulted in a significant antinociceptive effect. Bestatin did not modify tail-flick latencies in neurotensin-treated mice whether in the absence or presence of thiorphan. On the contrary, each of these peptidase inhibitors promoted antinociceptive effects of subthreshold doses of neuromedin (l?g) in the tail-flick test. Maximal antinociception was obtained by combining both inhibitors, thus conferring antinociceptive effects to neuromedin doses that were as low as 10 ng. Naloxone (0.5-2 mg/kg, s.c.) did not significantly reduced the antinociceptive effects of combinations of neurotensin and thiorphan and of neuromedin, thiorphan and bestatin. The data show that both neurotensin and neuromedin elicit analgesia in mice through an opiate independent mechanism. Furthermore, like enkephalin, neuromedin is readily degraded by brain endopeptidase 24.11 and bestatin sensitive aminopeptidase(s), whereas the resistance of neurotensin to aminopeptidase attack confers to this peptide a broader spectrum and longer duration of action than its congener neuromedin.

Naloxone-insensitive potentiation of neurotensin hypothermic effect by the enkephalinase inhibitor thiorphan.

The hypothermic effect of neurotensin (0.1 microgram) injected i.c.v. in mice is potentiated by the enkephalinase inhibitor thiorphan (10 micrograms, i.c.v.). This potentiation is not reversed by systemic naloxone. The hypothermic effect of neurotensin is not modified by the amino-peptidase inhibitor bestatin (50 micrograms, i.c.v.) nor by the angiotensin-converting enzyme inhibitor captopril (50 micrograms, i.c.v.). These data indicate the involvement of enkephalinase in the inactivation of neurotensin, at least when it is injected i.c.v.

Pharmacological characteristics of dopamine receptors involved in the dual effect of dopamine agonists on yawning behaviour in rats.

Increasing doses of apomorphine (APO) induced the dose-dependent appearance of yawns in rats at doses up to 0.1 mg X kg-1 and their disappearance from 0.1 to 0.6 mg X kg-1. A similar biphasic effect on yawning was observed with increasing doses of n-propyl norapomorphine, piribedil, S 584, bromocriptine, lergotrile, lisuride, CQ 32084 and L-DOPA. APO, n-propyl norapomorphine, piribedil and CQ 32084 had similar ED50 on the induction of sniffing and on the disappearance of yawns. All the neuroleptics tested antagonized the yawns induced by 0.1 mg X kg-1 APO. Increasing doses of haloperidol, chlorpromazine, mezilamine, metoclopramide and thioridazine made the yawns reappear in rats injected with 0.6 mg X kg-1 APO. The ID50 were similar to those for the antagonism of sniffing. On the other hand, increasing doses of clozapine, (+/-)- or (-)-sulpiride, veralipride and DAN 2163 did not make the yawns reappear in rats injected with 0.6 mg X kg-1 APO although sniffing was antagonized. These results are discussed in terms of the ability of sulpiride, veralipride and DAN 2163 to distinguish between the dopamine (DA) receptors involved in the appearance of yawns at low doses of DA agonists and in their disappearance at higher doses. The decreased APO-induced yawning observed concomitantly with increased sniffing in rats with 6-hydroxydopamine-lesioned olfactory tubercles suggests that yawning and sniffing could be mutually exclusive.