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Endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) binds with high affinity and selectivity to the mu-opioid receptor. In the present study, [125I]endomorphin-2 has been used to characterize mu-opioid-binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin-2 (1 nM) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K(d) = 18.79 +/- 1.13 nM, B(max) = 635 +/- 24 fmol/mg protein) and the other shows low affinity and higher capacity (K(d) = 7.67 +/- 0.81 microM, B(max) = 157 +/- 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin-2 binding site was [d-1-Nal3]morphiceptin > endomorphin-2 >> [d-Phe3]morphiceptin > morphiceptin > [d-1-Nal3]endomorphin-2, indicating binding of these peptides to mu-opioid receptors. The uptake of 131I-labeled peptides administered intraperitoneally to tumor-bearing mice was also investigated. The highest accumulation in the tumor was observed for [d-1-Nal3)morphiceptin, which reached the value of 8.19 +/- 1.14% dose/g tissue.

Two lines of mice, previously selected for their sensitivity (BS) or their resistance (BR) to an anxiogenic benzodiazepine (BZ) receptor inverse agonist, methyl β-carboline-3-carboxylate (β-CCM), have recently been shown to present several differences in anxiety. In the present study, attempt was made to extend their behavioral profile in two situations classically used for testing antidepressant drugs. Reassessment of locomotor performance of these new populations confirmed that the motor activity of BR mice was lower than that of BS mice. In both the forced-swimming and the tail suspension tests, the immobility time of BS mice was significantly higher than that of BR mice. In the tail suspension test, two administrations of imipramine (30 mg/kg ip, 5 h and 30 min before testing) significantly reduced the immobility time of BS mice but not of BR mice. From these data, it appears that BS mice are more “depressed” than BR mice. Thus, these selectively bred lines may represent potentially useful animal models to investigate behavioral, neurochemical and neuroendocrine correlates of antidepressant action.

Although tissue plasminogen activator (tPA) is known to promote neuronal remodeling in the CNS, no mechanism of how this plastic function takes place has been reported so far. We provide here in vitro and in vivo demonstrations that this serine protease neutralizes inhibitory chondroitin sulfate proteoglycans (CSPGs) by promoting their degradation via the direct activation of endogenous type 4 disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4). Accordingly, in a model of compression-induced spinal cord injury (SCI) in rats, we found that administration of either tPA or its downstream effector ADAMTS-4 restores the tPA-dependent activity lost after the SCI and thereby, reduces content of CSPGs in the spinal cord, a cascade of events leading to an improved axonal regeneration/sprouting and eventually long term functional recovery. This is the first study to reveal a tPA-ADAMTS-4 axis and its function in the CNS. It also raises the prospect of exploiting such cooperation as a therapeutic tool for enhancing recovery after acute CNS injuries.

Objective— Cardiovascular dysfunction is a major cause of mortality in patients with sepsis. Recently, we showed that gene deletion or pharmacological inhibition of protein tyrosine phosphatase 1B (PTP1B) improves endothelial dysfunction and reduces the severity of experimental heart failure. However, the cardiovascular effect of PTP1B invalidation in sepsis is unknown. Thus, we explored the beneficial therapeutic effect of PTP1B gene deletion on lipopolysaccharide (LPS)-induced cardiovascular dysfunction, inflammation, and mortality. Approach and Results— PTP1B −/− or wild-type mice received LPS (15 mg/kg) or vehicle followed by subcutaneous fluid resuscitation (saline, 30 mL/kg). α-1–dependent constriction and endothelium-dependent dilatation, assessed on isolated perfused mesenteric arteries, were impaired 8 hours after LPS and significantly improved in PTP1B −/− mice. This was associated with reduced vascular expression of interleukin1-β, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, cyclooxygenase-2, and inducible nitric oxide synthase mRNA. PTP1B gene deletion also limited LPS-induced cardiac dysfunction assessed by echocardiography, left ventricular pressure–volume curves, and in isolated perfused hearts. PTP1B −/− mice also displayed reduced LPS-induced cardiac expression of tumor necrosis factor-α, interleukin1-β, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and Gp91 phox , as well as of several markers of cellular infiltration. PTP1B deficiency also reduced cardiac P38 and extracellular signal–regulated protein kinase 1 and 2 phosphorylation and increased phospholamban phosphorylation. Finally, PTP1B −/− mice displayed a markedly reduced LPS-induced mortality, an effect also observed using a pharmacological PTP1B inhibitor. PTP1B deletion also improved survival in a cecal ligation puncture model of sepsis. Conclusions— PTP1B gene deletion protects against septic shock-induced cardiovascular dysfunction and mortality, and this may be the result of the profound reduction of cardiovascular inflammation. PTP1B is an attractive target for the treatment of sepsis.

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the μ-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-β-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies.

In vitro, the ability of DEEP-NCS {1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-isothiocyanatophenyl)ethyl]-piperazine} to inhibit [3H]-dopamine uptake by rat striatal synaptosomes was concentration-dependent and inversely related to the protein concentration. This inhibition was irreversible and resulted from changes in Vmax and KM. DEEP-NCS was less potent on noradrenaline, serotonin and choline transport. One day after intrastriatal injections of DEEP-NCS (100 and 1000 pmol) in 20% dimethylsulphoxide, moderate decreases in the ex vivo dopamine uptake were observed in synaptosomes obtained from striatum injected with DEEP-NCS or solvent, and the contralateral uninjected striatum. In similar conditions, 300 pmol DEEP-NCS in 45% 2 hydroxypropyl-γ-cyclodextrin–0.5% dimethylsulphoxide solution sub-totally reduced ex vivo dopamine uptake and mazindol binding, and moderately decreased choline and serotonin transport. These reductions were specific to DEEP-NCS-injected striata. A clomipramine pretreatment (16 mg kg−1 i.p. 1 h before) was performed in following experiments, since it reduced the DEEP-NCS-elicited decrease in serotonin uptake without affecting other indices. One day after intrastriatal injection, DEEP-NCS elicited similar dose-dependent decreases in ex vivo dopamine uptake and mazindol binding (ID50=6.9-8 ng striatum−1). Changes in KM and Vmax for ex vivo dopamine transport produced by DEEP-NCS disappeared according to similar time-courses. The t½ for transporter recovery was 6.1 days. This value should correspond to its actual turnover rate in vivo, since no change in transporter mRNA level was observed in substantia nigra ipsilateral to 300 pmol DEEP-NCS-injected striatum. The results indicate that DEEP-NCS behaves as a potent, quite selective, irreversible inhibitor of the DAT, in vitro and in vivo. Its use in vivo suggests that the physiological half-life of the rat striatal DAT is close to 6 days. British Journal of Pharmacology (1999) 128, 51–60; doi:10.1038/sj.bjp.0702784

As part of our continuing studies on the structure–activity relationships of cyclic pentapeptides based on the structure of endomorphin-2, we report here the synthesis and biological activities of a new series of analogs incorporating 2′, 3′ or 4′-methylphenylalanine (MePhe) residues into positions 3 or 4 of the parent cyclopeptide, Dmt-c[ d -Lys-Phe-Phe-Asp]NH 2 (Dmt = 2′,6′-dimethyltyrosine). Analogs with MePhe in position 4 showed a row of magnitude increased μ-opioid receptor (MOP receptor) affinity as compared with a parent compound. The in vitro potencies of the new analogs were determined in calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. All analogs were strong μ/κ (MOP/KOP) receptor agonists and weak δ (DOP) receptor agonists. In the in vivo hot-plate test in mice, the MePhe 4 -modified peptides showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration which was most likely due to the concomitant activation of more than one opioid receptor type.

Obese individuals often have increased appetite despite normal plasma levels of the main orexigenic hormone ghrelin. Here we show that ghrelin degradation in the plasma is inhibited by ghrelin-reactive IgG immunoglobulins, which display increased binding affinity to ghrelin in obese patients and mice. Co-administration of ghrelin together with IgG from obese individuals, but not with IgG from anorectic or control patients, increases food intake in rats. Similarly, chronic injections of ghrelin together with IgG from ob/ob mice increase food intake, meal frequency and total lean body mass of mice. These data reveal that in both obese humans and mice, IgG with increased affinity for ghrelin enhances ghrelin’s orexigenic effect, which may contribute to increased appetite and overeating., Obesity is often associated with increased appetite and food intake, despite normal blood levels of the hunger hormone ghrelin. Here the authors show that ghrelin-reactive antibodies in the blood of obese mice and humans enhance the orexigenic effect of ghrelin by protecting it from degradation.

Spinal cord injury (SCI) induces a permanent disability in patients. To this day no curative treatment can be proposed to restore lost functions. Therefore, extensive experimental studies have been conducted to induce recovery after SCI. One of the most promising therapies is based on the use of olfactory ensheathing cells (OECs). OECs can be obtained from either the olfactory bulbs (OB-OECs) or from olfactory mucosa (OM-OECs), involving a less invasive approach for autotransplantation. However the vast majority of experimental transplantations have been focusing on OB-OECs although the OM represents a more accessible source of OECs. Importantly, the ability of OM-OECs in comparison to OB-OECs to induce spinal cord recovery in the same lesion paradigm has never been described. We here present data using a multiparametric approach, based on electrophysiological, behavioral, histological and magnetic resonance imaging experiments on the repair potential of OB-OECs and OM-OECs from either primary or purified cultures after a severe model of SCI. Our data demonstrate that transplantation of OECs obtained from OB or OM induces electrophysiological and functional recovery, reduces astrocyte reactivity and glial scar formation and improves axonal regrowth. We also show that the purification step is essential for OM-OECs while not required for OB-OECs. Altogether, our study strongly indicates that transplantation of OECs from OM represents the best benefit/risk ratio according to the safety of access of OM and the results induced by transplantations of OM-OECs. Indeed, purified OM-OECs in addition to induce recovery can integrate and survive up to 60 days into the spinal cord. Therefore, our results provide strong support for these cells as a viable therapy for SCI.

International audience

Our manuscript by Piekielna et al. entitled “Synthesis of mixed opioid affinity cyclic endomorphin-2 analogues with fluorinated phenylalanines” (DOI: 10.1021/acsmedchemlett.5b00056) was published in ACS Medicinal Chemistry Letters in April 2015.

Ethnopharmacological relevance Passiflora alata is a Southern American species that constitutes many traditional remedies as well as phytomedicines used for sedative and anxiolytic purposes in Brazil. However studies on repeated treatment effects are scarce. Aim of the study To evaluate behavioral, physiological and biochemical effects of the repeated treatment with an aqueous spray-dried extract of Passiflora alata leaves containing 2.5% (w/v) of flavonoids (PA) in mice. Material and methods Male adult CF1 mice were treated (p.o.) for 14 days with PA (2.5; 25 or 250 mg/kg). The feeding behavior was evaluated at the beginning (1 h after the first administration) and at the end of the treatment (15th day). The body weight gain and food consumption were monitored along the days. On day 15 mice were evaluated on plus maze, spontaneous locomotor activity, catalepsy and barbiturate sleeping time tests. Serum glucose, lipids, ALT and AST enzymes were determined. Liver, kidney, perirenal fat, epididymal and peritoneal fat were analyzed. Results The repeated treatment with the highest dose tested (250 mg/kg) did not alter the mice behavior on open field, elevated plus maze, catalepsy and barbiturate sleeping time tests. Repeated administration of PA 250 decreased mice feeding behavior and weight gain. PA 25 and PA 250 reduced mice relative liver weight and caused mild hepatic hydropic degeneration as well as a decrease in alanine aminotransferase (ALT) serum level. Conclusions These results indicate that Passiflora alata does not present central cumulative effects and point to the needs of further studies searching for its hepatotoxicity as well as potential anorexigenic.

Summary Depression and eating disorders are frequently associated, but the molecular pathways responsible for co-occurrence of altered mood, appetite and body weight are not yet fully understood. Neuropeptide Y (NPY) has potent antidepressant and orexigenic properties and low central NPY levels have been reported in major depression. In the present study, we hypothesized that in patients with major depression alteration of mood, appetite and body weight may be related to NPY-reactive autoantibodies (autoAbs). To test this hypothesis, we compared plasma levels and affinities of NPY-reactive autoAbs between patients with major depression and healthy controls. Then, to evaluate if changes of NPY autoAb properties can be causally related to altered mood and appetite, we developed central and peripheral passive transfer models of human autoAbs in mice and studied depressive-like behavior in forced-swim test and food intake. We found that plasma levels of NPY IgG autoAbs were lower in patients with moderate but not with mild depression correlating negatively with the Montgomery–Asberg Depression Rating Scale scores and with immobility time of the forced-swim test in mice after peripheral injection of autoAbs. No significant differences in NPY IgG autoAb affinities between patients with depression and controls were found, but higher affinity of IgG autoAbs for NPY was associated with lower body mass index and prevented NPY-induced orexigenic response in mice after their central injection. These data suggest that changes of plasma levels of anti-NPY autoAbs are relevant to altered mood, while changes of their affinity may participate in altered appetite and body weight in patients with depressive disorder.

In this study we have demonstrated that cyclohexane extract of Hypericum polyanthemum (POL) and its main phloroglucinol derivative uliginosin B (ULI) present antidepressant-like activity in rodent forced swimming test (FST). The involvement of monoaminergic neurotransmission on the antidepressant-like activity of ULI was evaluated in vivo and in vitro. POL 90 mg/kg (p.o.) and ULI 10 mg/kg (p.o.) reduced the immobility time in the mice FST without altering locomotion activity in the open-field test. The combination of sub-effective doses of POL (45 mg/kg, p.o.) and ULI (5 mg/kg, p.o.) with sub-effective doses of imipramine (10 mg/kg, p.o.), bupropion (3 mg/kg, p.o.) and fluoxetine (15 mg/kg, p.o.) induced a significant reduction on immobility time in FST. The pretreatment with SCH 23390 (15 μg/kg, s.c., dopamine D1 receptor antagonist), sulpiride (50 mg/kg, i.p., dopamine D2 receptor antagonist), prazosin (1 mg/kg, i.p., α1-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p., α2-adrenoceptor antagonist) and pCPA (100 mg/kg/day, i.p., p-chlorophenilalanine methyl ester, inhibitor of serotonin synthesis, for four consecutive days) before ULI administration (10 mg/kg, p.o.) significantly prevented the anti-immobility effect in FST. ULI was able to inhibit synaptosomal uptake of dopamine (IC50 = 90 ± 38 nM), serotonin (IC50 = 252 ± 13 nM) and noradrenaline (280 ± 48 nM), but it did not bind to any of the monoamine transporters. These data firstly demonstrated the antidepressant-like effect of POL and ULI, which depends on the activation of the monoaminergic neurotransmission in a different manner from the most antidepressants.

The biological effects of endomorphins (EMs) are short-lasting due to their rapid degradation by endogenous enzymes. Competing enzymatic degradation is an approach to prolong EM bioavailability. In the present study, a series of tetra- and tripeptides of similar to EMs structure was synthesized and tested in vitro and in vivo for their ability to inhibit degradation of EMs. The obtained results indicated that, among the series of analogs, the tetrapeptide Tyr-Pro-d-ClPhe-Phe-NH(2) and the tripeptide Tyr-Pro-Ala-NH(2), which did not bind to the μ-opioid receptors, were potent inhibitors of EM catabolism in rat brain homogenate. In vivo, these two peptides significantly prolonged the analgesic and antidepressant-like effects, induced by exogenous EMs, by blocking EM degrading enzymes. These new potent inhibitors may therefore increase the level and the half life of endogenous EMs and could be used in a new therapeutic strategy against pain and mood disorders, based on increasing of EM bioavailability.

The endogenous opioid system is involved in the control of gastrointestinal (GI) motility. The potential use of endogenous MOR ligands, endomorphins (EMs), as therapeutics is limited because of their rapid enzymatic degradation and short duration of action. Targeting enzymatic degradation is an approach to prolong EM activity. In the present study, we characterized the effects of novel blockers of EM degradation in GI tissue preparation in vitro. The effects of actinonin, diprotin A (DIP) and the novel peptide EM degradation blockers Tyr-Pro-DClPhe-Phe-NH(2) (EMDB-1), Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3) on EM-2-mediated inhibition of electrically induced cholinergic twitch contractions were compared in rat ileum in vitro using an organ bath. EMDB-1 and EMDB-2 significantly prolonged the inhibitory effect of EM-2 on smooth muscle contractility in rat ileum. EMDB-2 extended the EM-2 action for up to 60 min compared to 10 min in controls and was more potent than the conventional peptidase inhibitor DIP. EMDB-1 and EMDB-2 are potent EM degradation blockers, which prolong the inhibitory effects of EM-2 on smooth muscle contractility in rat ileum. These novel compounds may be of future use when targeting the endogenous opioid system in the treatment of GI motility disorders such as diarrhea.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)), a tetrapeptide present in the enzymatic digest of bovine beta-casein, is a selective ligand of the mu-opioid receptor. In the present study, we describe the synthesis of a series of novel morphiceptin analogs modified in positions 1-3. Two of the obtained analogs, [Dmt(1), D-Ala(2), D-1-Nal(3)]morphiceptin and [Dmt(1), D-NMeAla(2), D-1-Nal(3)]morphiceptin (Dmt-2',6'-dimethyltyrosine and d-1-Nal-3-(1-naphthyl)-D-alanine)) displayed very high mu-receptor affinity, resistance to enzymatic degradation, and remarkable supraspinally mediated analgesia, as shown in the hot-plate test after intracerebroventricular but not intravenous administration, which indicated that they could not cross the blood-brain barrier. Therefore, these two analogs were further tested in vitro and in vivo towards their possible peripheral analgesic activity and inhibitory effect on gastrointestinal (GI) motility. We report that both peptides showed strong antinociceptive effect in the writhing test after intraperitoneal administration, inhibited smooth muscle contractility in vitro and GI motility in vivo. Taken together, these findings indicate that the novel morphiceptin analogs which induce peripheral, but not central antinociception, inhibit GI transit, and possess exceptional metabolic stability, may provide an interesting approach to the development of peripherally restricted agents for the treatment of GI motility disorders, such as diarrhea or diarrhea-predominant irritable bowel syndrome.

International audience; Peptide E (a 25-amino acid peptide derived from proenkephalin A) and beta-endorphin (a 31-amino acid peptide derived from proopiomelanocortin) bind with high affinity to opioid receptors and share structural similarities but induce analgesic effects of very different intensity. Indeed, whereas they possess the same N-terminus Met-enkephalin message sequence linked to a helix by a flexible spacer and a C-terminal part in random coil conformation, in contrast with peptide E, beta-endorphin produces a profound analgesia. To determine the key structural elements explaining this very divergent opioid activity, we have compared the structural and pharmacological characteristics of several chimeric peptides derived from peptide E and beta-endorphin. Structures were obtained under the same experimental conditions using circular dichroism, computational estimation of helical content and/or nuclear magnetic resonance spectroscopy (NMR) and NMR-restrained molecular modeling. The hot-plate and writhing tests were used in mice to evaluate the antinociceptive effects of the peptides. Our results indicate that neither the length nor the physicochemical profile of the spacer plays a fundamental role in analgesia. On the other hand, while the functional importance of the helix cannot be excluded, the last 5 residues in the C-terminal part seem to be crucial for the expression or absence of the analgesic activity of these peptides. These data raise the question of the true function of peptides E in opioidergic systems.

A series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues, containing non-cyclic amino acids (Ala, D-Ala, beta-Ala, NMeAla, D-NMeAla or Sar) instead of Pro in position 2 was synthesized, where NMeAla = N-methylalanine and Sar = N-methylglycine, sarcosine. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor (MOR and DOR)-representative binding assays and bioassays in vitro, as well as in the mouse hot-plate test in vivo. Finally, the degradation rates of all analogues in the presence of either rat brain homogenate or selected proteolytic enzymes were determined. Analogues of EM-2 were generally more potent than the respective analogues of EM-1. EM-2 analogues with D-Ala or D-NMeAla were about twofold more potent than the parent peptide and were least prone to degradation by brain homogenate, dipeptydyl peptidase IV and aminopeptidase M. In the in vivo test, [D-Ala(2)]EM-2 and [D-NMeAla(2)]EM-2 showed much higher analgesic potency than EM-2 which confirmed the usefulness of structural modifications in obtaining new leads for pain-relief therapeutics.

In our previous paper we reported synthesis and biological activity of two cyclic analogs of endomorphin-2 (EM-2): Tyr-c(Lys-Phe-Phe-Asp)-NH(2) and Tyr-c(Asp-Phe-Phe-Lys)-NH(2), achieved by making an amid bond between Lys and Asp side-chains. The first analog did not bind to the mu-opioid receptor, the affinity of the second one was very low. In the present study, we describe the synthesis of four novel cyclic analogs of similar structure, but with d-amino acids in position 2 (D-Lys or D-Asp). All new analogs displayed high affinity for the mu-opioid receptor, were much more stable than EM-2 in rat brain homogenate and showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration. Analgesic effect of the most potent cyclic analog, Tyr-c(D-Lys-Phe-Phe-Asp)NH(2) was much stronger and longer lasting than that of EM-2. This analog elicited analgesia also after peripheral administration and this effect was reversed by concomitant i.c.v. injection of the mu-opioid antagonist, beta-funaltrexamine, which indicated that antinociception was mediated by the mu-opioid receptor in the brain. Central action of the cyclic analog gives evidence that it was able to cross the blood-brain barrier, most likely due to the increased lipophilicity. Our results demonstrate that cyclization might be a promising strategy to enhance bioavailability of peptides and may serve a role in the development of novel endomorphin analogs with increased therapeutic potential.

International audience; Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), exert anorexigenic activities. While alpha-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuron-expressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a stress-related neuropeptide involved in the regulation of motivated behavior, appetite and emotion including stimulation of satiety and anxiety. Although autoantibodies (autoAbs) reactive with alpha-MSH have been identified in human subjects and in rats, it remained unknown if these autoAbs are involved in the regulation of feeding and anxiety and if their production is related to stress. Here we show that repeated exposure of rats to anxiolytic mild stress by handling increases the levels and affinity of alpha-MSH reactive IgG autoAbs and that these changes are associated with adaptive feeding and anxiety responses during exposure of rats to a strong stress by food restriction. Importantly, an increase in affinity of alpha-MSH reactive autoAbs was associated with changes of their functional roles from stimulation to inhibition of alpha-MSH-mediated behavioural responses, suggesting that these autoAbs can be a carrier or a neutralizing molecule of alpha-MSH peptide, respectively. Using a model of passive transfer into the brain, we show that alpha-MSH autoAbs affinity purified from blood of rats exposed to repeated mild stress, but not from control rats, are able to increase acutely food intake, suppress anxiety and modify gene expression of hypothalamic neuropeptides in naive rats. These data provide the first evidence that autoAbs reactive with alpha-MSH are involved in the physiological regulation of feeding and mood, supporting a further role of the immune system in the control of motivated behavior and adaptation to stress.

International audience; Since depressive patients present alterations in the hypothalamo-pituitary-adrenal (HPA) axis that are normalised by antidepressants, this HPA axis has been considered as a target of their actions. We have investigated the mechanism of action of a cyclohexane extract of Hypericum caprifoliatum (HCP), which displays antidepressant like activity, by studying, in mice, the influence of HCP and of two established antidepressant drugs, imipramine and bupropion, administered either acutely or semi-chronically (once a day, three consecutive days), on serum and brain cortex corticosterone levels, either in basal conditions or shortly after a forced-swimming session (FSS). Administered acutely, imipramine (20 mg/kg, per os (p.o.)), bupropion (30 mg/kg, p.o.) and HCP (360 mg/kg, p.o.) significantly reduced the immobility time and had no effects on FSS-induced increase of serum and cortical corticosterone levels. Conversely, 3 days repeated treatment with imipramine or bupropion resulted in a significant reduction of immobility time and FSS-induced increase of serum and cortical corticosterone levels. In a different way, repeated treatment with HCP significantly reduced the immobility time and only cortical corticosterone levels in stressed mice. These results indicate that short-term treatments with antidepressants are sufficient to induce modifications in the HPA axis reactivity to stress; and that apparently HCP has an influence on corticosterone levels by a mechanism diverse from the other tested antidepressants.

International audience; Urotensin-II (U-II) and urotensin-II-related peptide (URP) have been identified as the endogenous ligands of the orphan G-protein-coupled receptor GPR14 now renamed UT. The occurrence of U-II and URP in the central nervous system, and the widespread distribution of UT in the brain suggest that U-II and URP may play various behavioral activities. Studies conducted in rodents have shown that central administration of U-II stimulates locomotion, provokes anxiety- and depressive-like states, enhances feeding activity and increases the duration of paradoxical sleep episodes. These observations indicate that, besides the endocrine/paracrine activities of U-II and URP on cardiovascular and kidney functions, these peptides may act as neurotransmitters and/or neuromodulators to regulate various neurobiological activities.

The mu-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) exhibit an extremely high selectivity for the mu-opioid receptor and thus represent a potential framework for modification into mu-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal(4)]endomorphin-2 analogs, [Sar(2),d-2-Nal(4)]endomorphin-2 and [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 (Dmt=2'6'-dimethyltyrosine; Sar=N-methylglycine, sarcosine; d-2-Nal=3-(2-naphthyl)-d-alanine). [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 possessed very high affinity for the mu-opioid receptor (IC(50)=0.01+/-0.001 nM) and turned out to be a potent and extremely selective mu-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA(2)=9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier mu-opioid receptor antagonist, [Dmt(1),d-2-Nal(4)]endomorphin-2 (antanal-2). The exceptional mu-opioid receptor in vitro activity and selectivity of [Dmt(1), Sar(2),d-2-Nal(4)]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.

The morphiceptin-derived peptide [Dmt1, d-1-Nal3]morphiceptin, labeled mu-opioid receptor (MOP) with very high affinity and selectivity in the receptor binding assays. In the mouse hot plate test, [Dmt1, d-1-Nal3]morphiceptin given intracerebroventricularly (i.c.v.) produced profound supraspinal analgesia, being approximately 100-fold more potent than the endogenous MOP receptor ligand, endomorphin-2. The antinociceptive effect of this new analog lasted up to 120min. Thus, [Dmt1, d-1-Nal3]morphiceptin is an interesting and extraordinarily potent analgesic, raising the possibility of novel approaches in the design of clinically useful drugs for pain treatment.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are two endogenous opioid peptides with high affinity and remarkable selectivity for the mu-opioid receptor. The neuroanatomical distribution of endomorphins reflects their potential endogenous role in many major physiological processes, which include perception of pain, responses related to stress, and complex functions such as reward, arousal, and vigilance, as well as autonomic, cognitive, neuroendocrine, and limbic homeostasis. In this review we discuss the biological effects of endomorphin-1 and endomorphin-2 in relation to their distribution in the central and peripheral nervous systems. We describe the relationship between these two mu-opioid receptor-selective peptides and endogenous neurohormones and neurotransmitters. We also evaluate the role of endomorphins from the physiological point of view and report selectively on the most important findings in their pharmacology.

To synthesize potent antagonists of the mu-opioid receptor, we prepared a series of endomorphin-1 and endomorphin-2 analogues with 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal) in position 4. Some of these analogues displayed weak antagonist properties. We tried to strengthen these properties by introducing the structurally modified tyrosine residue 2,6-dimethyltyrosine (Dmt) in place of Tyr1. Among the synthesized compounds, [Dmt1, d-2-Nal4]endomorphin-1, designated antanal-1, and [Dmt1, d-2-Nal4]endomorphin-2, designated antanal-2, turned out to be highly potent and selective mu-opioid receptor antagonists, as judged on the basis of two functional assays, the receptor binding assay and the hot plate test of analgesia. Interestingly, another analogue of this series, [Dmt1, d-1-Nal4]endomorphin-1, turned out to be a moderately potent mixed mu-agonist/delta-antagonist.

This study investigated, in mice, the antidepressant like effect of hyperfoliatin, a prenylated phloroglucinol derivative isolated from the aerial parts of Hypericum perfoliatum , as well as its action on monoaminergic systems. In the forced-swimming test, hyperfoliatin dose-dependently reduced immobility time. Immobility was interpreted as an expression of “behavioural despair”, which could be a component of depression syndrome. The effect of hyperfoliatin did not result from the stimulation of animal motor activity. Hyperfoliatin inhibited, in a concentration-dependent manner, the [ 3 H]-dopamine, [ 3 H]-serotonin and [ 3 H]-noradrenaline synaptosomal uptakes, but did not prevent the binding of specific ligands to the monoamine transporters. These data suggest that the antidepressant-like effect of hyperfoliatin on the forced-swimming test is probably associated to monoamine uptake inhibition, due to a mechanism of action different from that of known antidepressants.

In this work, previously published and unpublished results on biological activity of Hypericum caprifoliatum, a native species to South Brazil, are presented. Lipophilic extracts obtained from this species showed an antidepressant-like activity in mice and rat forced swimming test. Results from in vivo experiments suggest an effect on the dopaminergic transmission. Besides that, in vitro experiments demonstrated that the extract and its main component (a phloroglucinol derivative) inhibit monoamine uptake in a concentration-dependent manner, more potently to dopamine, but this effect is not related to direct binding at the uptake sites. It was also observed that a 3-day treatment with lipophilic extract prevents stress-induced corticosterone rise in mice frontal cortex but not in plasma. The lipophilic and methanolic H. caprifoliatum extracts also demonstrated antinociceptive effect, which seems to be indirectly mediated by the opioid system. These results indicate that H. caprifoliatum presents a promising antidepressant-like effect in rodents which seems to be related to a mechanism different from that of other classes of antidepressants.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) are two recently isolated mu-opioid selective peptides with a potent antinociceptive activity, involved in a number of physiological processes, including food intake, vasomotricity, sexual behavior, as well as neuroendocrine and cardiorespiratory functions. The neuroanatomical distribution of endomorphins prompted us to study their antidepressant activity in two animal behavioral models of depression: forced-swimming and tail-suspension tests. In both tests, the intracerebroventricular (i.c.v.) injection of either endomorphin-1 or endomorphin-2 significantly decreased the duration of immobility, interpreted as an expression of 'behavioral despair', which could be related to the depression syndrome. These effects of endomorphins did not result from the stimulation of the animal motor activity. We have also demonstrated that the antidepressant-like effect of endomorphins was antagonized by the universal opioid antagonist, naloxone and the mu-opioid receptor selective antagonist, beta-funaltrexamine. In contrast, this effect was not antagonized by delta- and kappa-opioid receptor selective antagonists, naltrindole and nor-binaltorphimine, respectively. The results of the present study demonstrate that endomorphin-1 and endomorphin-2 produce potent antidepressant-like effects after i.c.v. injection in mice. We may suggest that endomorphins and the mu-opioid receptors might be involved in the physiopathology of depressive disorders, and that the endomorphinergic system could serve as a novel target for the development of antidepressant drugs.

The ability of several mu-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [(35)S]GTPgammaS binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [d-1-Nal(3)]Morphiceptin was more potent in G-protein activation (EC(50) value of 82.5+/-4.5 nM) than DAMGO (EC(50)=105+/-9 nM). [d-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [(35)S]GTPgammaS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists.

Endomorphins, endogenous mu-opioid receptor ligands, have been shown to exert antinociceptive, antidepressant, anxiolytic, and neuromodulatory effects, as well as to influence cardiovascular, respiratory, and gastrointestinal systems. In the present study, we designed and synthesized a series of tetrapeptides and tripeptides (amides and peptide acids) of similar to endomorphins structure, but with low mu-opioid receptor affinity, and tested them as possible inhibitors of endomorphin-degrading enzymes. The obtained results indicate that the tripeptides Tyr-Pro-Ala-NH2 and Tyr-Pro-Ala-OH, which do not bind to the mu-opioid receptors, are potent inhibitors of endomorphin-degrading enzymes in the rat brain. We suggest that the in vivo administration of these novel analogs may enhance physiological effects of endogenous endomorphins by decreasing the rate of their enzymatic cleavage.

Male Swiss albinos mice were submitted to two behavioural tests intended to determine their anxiety level: the elevated plus-maze test as well as the black and white compartments test. In addition they were submitted to the hole-board test. It was observed: (i) that the correlation between scores in the two first tests was weak, suggesting that they explore different components of anxiety; (ii) that the score on the latter test was better correlated with the response in the elevated plus-maze test than in the black and white compartments test. From these data three groups of animals were constituted, considered, respectively, as anxious, non anxious and intermediates. It was observed that both horizontal and vertical locomotion in an unfamiliar environment differed between groups, with higher activity in non anxious than in anxious. In the hole-board test, only animals classified as anxious displayed an obvious response to the anxiolytic drug diazepam (0.5 mg/kg). Finally in the forced-swimming test, the three groups demonstrated a similar immobility time, suggesting that the operated segregation was not depending on a helpless component. It is proposed that the selection of mice from a combination of either elevated plus-maze and black and white compartments tests or a combination of hole-board test and black and white compartments test, allows to distinguish high or low anxiety animals among a population of mice.

Cyclic analogs of the opioid peptides endomorphin-2 and morphiceptin of the type Tyr-X-Phe-Phe-Y-NH2 and Tyr-X-Phe-D-Pro-Y-NH2 (X = Lys or Asp and Y = Lys or Asp), respectively, were synthesized in order to test their structure-activity relationships. Antinociceptive activity of the new analogs was assessed in the hot-plate test after intracerebroventricular administration in mice. The strong analgesic effect was observed for the analogs with Asp in position 2, while the analogs with Lys in the second position were inactive. Antinociception caused by Asp2 analogs was dose-dependent and reversed by the concomitant administration of the universal opioid antagonist naloxone and by the selective kappa antagonist, nor-BNI. However, receptor binding studies revealed poor affinity of all cyclic analogs at the mu-opioid receptor and no affinity at delta- and kappa-opioid receptors. It is most likely that the new cyclic analogs produced their antinociception by the release of dynorphin A, which subsequently acted on the kappa-opioid receptor.

In the present study we investigated and compared the in vivo analgesia of centrally administered endomorphin-2 and morphiceptin, and their analogs modified in position 3. Two series of analogs were synthesized by introducing unnatural aromatic amino acids in the d configuration: 3-(1-naphthyl)- d -alanine ( d -1-Nal), 3-(2-naphthyl)- d -alanine ( d -2-Nal), 3-(4-chlorophenyl)- d -alanine ( d -ClPhe), 3-(3,4-dichlorophenyl)- d -alanine ( d -Cl 2 Phe). Antinociceptive activity of endomorphin-2, morphiceptin, and their analogs was compared in the mouse hot-plate test, performed after i.c.v. administration of the peptides at a dose of 10 μg/animal. The best results were obtained for two morphiceptin analogs, [ d -Phe 3 ]morphiceptin and [ d -1-Nal 3 ]morphiceptin, which showed greatly improved analgesic activity, as compared to morphiceptin. In the endomorphin-2 series none of the modifications produced analogs more potent than the parent compound, but [ d -1-Nal 3 ]endomorphin-2 was the best analog. Antinociception induced by endomorphin-2 was reversed by concomitant i.c.v. administration of [ d -Phe 3 ]endomorphin-2, [ d -2-Nal 3 ]endomorphin-2, and [ d -2-Nal 3 ]morphiceptin, indicating that these analogs were weak μ-opioid antagonists.

Analogs of morphiceptin (Tyr-Pro-Phe-Pro-NH 2 ), a μ-selective opioid receptor ligand, with position 3-modifications, including altered size, lipophilicity, and electronic character, while maintaining aromaticity were synthesized. The activity of the new analogs in in vitro assays and in in vivo hot-plate test of analgesia was compared and the results were consistent. [ d -1-Nal 3 ]Morphiceptin was the most potent analog of this series with a 26-fold increase in μ-opioid receptor affinity, a 15-fold potency increase in the GPI assay, and a significant potency increase in the hot-plate analgesic test, as compared with morphiceptin. [ d -Qal 3 ]Morphiceptin was found to be a weak antagonist in the GPI assay.

Addition of NaCl (90--290 mM) to a 10 mM Na(+) medium did not significantly modify B(max) and K(d) values for [3H]mazindol binding to the dopamine neuronal transporter (DAT) studied on rat striatal membranes at 20 degrees C. Addition of NaCl differentially affected the ability of other uptake inhibitors and substrates to block the [3H]mazindol binding. Ratios of 50% inhibiting concentrations calculated for 290 and 90 mM NaCl allowed to distinguish three groups of agents: substrates which were more potent in the presence of 290 mM NaCl (group 1; ratio1) and two groups of uptake inhibitors displaying ratio values either ranging around two (group 2: WIN 35,428, cocaine, methylphenidate, pyrovalerone) or close to unity (group 3: BTCP, mazindol, benztropine, nomifensine). However, agents from these three groups recognize mutually exclusive binding sites since in interaction studies the presence of WIN 35,428 (group 2) or mazindol (group 3) increased the 50% inhibiting concentrations of D-amphetamine (group 1) and WIN 35,428 on the [3H]mazindol binding to theoretical values expected for a competition of all of these compounds for the same binding domain on the DAT.

Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37°C produced an inhibition of the specific uptake of [ 3 H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC 50 = 1.85 ± 0.1 nM) instead of incubation alone (IC 50 = 25 ± 3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V o ) decreased for increasing concentrations of inhibitor (K i ≥ 20 nM). TI slowly isomerized to a more stable complex TI * (K i * ≤ 5 nM) with a value of t 1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (V s ) is set to zero were generally preferred, suggesting that formation of TI * could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V 0 , and V s tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in K M and a decrease in V max ; these effects were improved for 10 nM GBR 12783 and at 20°C. These results are discussed in relation to previous data concerning [ 3 H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.

Endomorphins (EMs), two endogenous μ-opioid receptor selective ligands, are attractive lead compounds for opioid-based pain management studies. However, these peptides are quickly degraded by peptidases, in particular by dipeptidylpeptidase IV (DPP IV) and aminopeptidase M (APM). Targeting enzymatic degradation is one approach to prolong endomorphin activity. In this study we characterized the action of two new inhibitors of similar to endomorphins structure, Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3), which were designed earlier in our laboratory. The presented data give evidence that EMDB-2 and EMDB-3 are potent inhibitors of enzymes responsible for endomorphin cleavage. These compounds are stable and easily synthesized. EMDB-2 and EMDB-3 are competitive inhibitors of both, DPP IV and APM, with K(i) values in micromolar range. They are less potent than diprotin A in protecting EMs against DPP IV but more potent than actinonin in protecting these peptides against APM.