Search for small molecules with antipsychotic activity based on neurotensin

A series of systemically active N-acylprolyltyrosines was constructed based on the structure of endogenous neuroleptic-like tridecapeptide neurotensin that exhibits activity on intracerebral administration. Among these dipeptides, N-caproyl-L-prolyl-L-tyrosine methyl ester (Dilept) was chosen for further development. The paper briefly describes the main concepts of Dilept design and presents new experimental data of the Dilept effects in a ‘translational’ model of schizophrenia, i.e., the deficit of prepulse inhibition caused by dopamine-mimetic apomorphine or ketamine, a noncompetitive blocker of N-methyl-D-aspartic acid. Dilept demonstrated effectiveness in this model in both intraperitoneal and oral administration, which makes it possible to predict its application as an antipsychotic agent.     

Antinociceptive properties of nasal delivery of neurotoxin-loaded nanoparticles coated with polysorbate-80

Neurotoxin-1 (NT) is an analgesic peptide which is endowed an exceptional specificity of action that blocks transmission of the nerve impulse. The aim of this study was to evaluate the potential application of nanoparticles technology as drug carrier system for the nasal delivery of NT. Mice were administered intranasally (i.n.) with NT (NT-P-NP), free NT solution (F-NT) and intravenously (i.v.) with NT (IV-NT) respectively. The NT levels in animal brain and antinociceptive activity of NT were analyzed. The result on brain transport showed that nanoparticles could exert enhanced delivery of NT into the brain significantly after i.n. administration. The results of antinociceptive activity showed that NT-P-NP increased immobility in the open-field test, both phases of formalin test were significantly inhibited by the NT-P-NP and NT-P-NP significantly inhibited the reaction time to thermal stimuli at 60 and 90 min. Both NT-P-NP and IV-NT were able to inhibit constrictions in acetic acid-induced writhing reaction. These data suggest that NT-loaded nanoparticles coated with polysorbate-80 could generate a significant improvement of drug levels in the brain. Intranasal administration of Neurotoxin-1 entrapped in nanoparticles coated with polysorbate-80 is an attractive alternative to intravenous administration.     

中脑导水管周围灰质内注射抗阿片肽血清对神经降压素增强电针镇痛的 …

本工作以钾离子透入引起大鼠甩尾反应作为痛反应指标,观察纳洛酮（ＮＸ）和抗阿片肽血清对大鼠中脑导水管周围灰质（ＰＡＧ）内微量注射神经降压素（ＮＴ）增强电针镇痛作用的影响。     

Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.     

Tonic suppression of baroreceptor reflex by endogenous neurotensin in the rat

We evaluated the modulatory role of endogenous neurotensin (NT) in baroreceptor reflex (BRR) response in Sprague-Dawley rats anesthetized with pentobarbital sodium. Intracerebroventricular (i.c.v.) administration of NT (15 or 30 nmol) significantly reduced the sensitivity of the BRR response. Blocking the endogenous activity of the tridecapeptide with its specific antagonist, (D-Trp11)-NT (4 or 8 nmol) or antiserum against NT (1:20); or inhibiting the aminopeptidases with bestatin (200 nmol), on the other hand, promoted a potentiation of BRR response. When administered together with bestatin (200 nmol), the suppressive effect of NT (15 nmol) on the BRR response was further enhanced, as was the augmentative action of (D-Trp11)-NT (4 nmol). Upon microinjection into the bilateral nucleus tractus solitarius (NTS), NT (600 pmol) and (D-Trp11)-NT (150 pmol) respectively elicited a reduction and enhancement of the BRR response. These results suggest that neurons that contain NT may participate in central cardiovascular regulation by tonically suppressing the BRR, possibly via an action on the NTS where baroreceptor afferents terminate.     

中脑导水管周围灰质内注射抗阿片肽血清对神经降压素增强电针镇痛的影响

本工作以钾离子透入引起大鼠甩尾反应作为痛反应指标,观察纳洛酮（ＮＸ）和抗阿片肽血清对大鼠中脑导水管周围灰质（ＰＡＧ）内微量注射神经降压素（ＮＴ）增强电针镇痛作用的影响。结果显示：（１）ＰＡＧ内微量注射ＮＴ后,大鼠电针镇痛效应明显提高;（２）ＰＡＧ内注射大剂量ＮＸ,可以显著减弱ＮＴ增强电针镇痛效应;（３）ＰＡＧ内微量注射抗甲脑啡肽血清和抗β内啡肽血清后,ＮＴ增强电针镇痛的作用明显降低;而ＰＡＧ内微量注射抗强啡肽Ａ１１３血清,对于ＮＴ增强电针镇痛的作用并无明显影响。提示：ＰＡＧ内ＮＴ在针刺镇痛过程中发挥重要作用,ＮＴ增强电针镇痛的作用与甲脑啡肽和β内啡肽有较密切的关系。     

Involvement of dopamine in the central effect of neurotensin on intestinal motility in rats

The effects of intracerebroventricular (ICV) administration of neurotensin (NT) before a meal on intestinal postprandial motility were examined in conscious rats chronically fitted with intraparietal Nichrome electrodes in the duodeno-jejunum. The effects were compared with those of two analogues, [D-Tyr¹¹]NT and [D-Trp¹¹]NT, resistant to degradation by brain peptidases. NT (10 μg ICV) delayed the occurrence of postprandial disruption of duodenal motility and blocked it on the jejunum. [D-Tyr¹¹]NT and [D-Trp¹¹]NT (1 μg ICV) elicited the same effects but at a ten-fold lower dose. NT administered peripherally just before a meal significantly lengthened the duration of the postprandial motor pattern. The central effect of NT on the fed pattern involved dopaminergic neurons as it was mimicked by dopamine, blocked by haloperidol and partly antagonized by either sulpiride or (+) SCH 23390. It is concluded that: 1) both D₁ and D₂ receptors are involved in the blocking effect of the postprandial disruption induced by central NT; 2) that [D-Tyr¹¹]NT and [D-Trp¹¹]NT are potent agonists at NT receptors in the brain.     

Botulinum type A neurotoxin digested with pepsin yields 132, 97, 72, 45, 42, and 18 kD fragments

Botulinum neurotoxin (NT) serotype A is a dichain protein made of a light and a heavy chain linked by at least one interchain disulfide; based on SDS-polyacrylamide gel electrophoresis their molecular masses appear as 147, 52, and 93 kD, respectively. Digestion of the NT with pepsin under controlledpH (4.3 and 6.0), time (1 and 24 hr), and temperature (25 and 30°C) produced 132, 97, 42, and 18 kD fragments. The three larger fragments were isolated by ionexchange chromatography. The 132 and 97 kD fragments are composed of 52 kD light chain and 72 and 45 kD fragments of the heavy chain, respectively. The sequences of amino terminal residues of these fragments were determined to identify the pepsin cleavage sites in the NT, which based on nucleotide sequence has 1295 amino acid residues (Binzet al., J. Biol. Chem. 265, 9153, 1990). The 42 kD fragment, beginning with residue 866, is the C-terminal half of the heavy chain. The 18 kD fragment, of which the first 72 residues were identified beginning with residue 1147, represents the C-terminal segment of the heavy chain. The 132 kD fragment (residue 1 to 1146) is thus a truncated version of the NT without its 18 kD C-terminal segment. The 97 kD fragment (residue 1 to 865) is also a truncated NT with its 42 kD C-terminal segment excised. These peptic fragments contain one or two of the three functional domains of the NT (binds receptors, forms channels, and intracellularly inhibits exocytosis of the neurotransmitter) that can be used for structure-function studies of the NT. This report also demonstrates for the first time that of the six Cys residues 453, 790, 966, 1059, 1234, and 1279 located in the heavy chain the later four do not form interchain disulfide links with the light chain; however, Cys 1234 and 1279 contained within the 18 kD fragment form intrachain disulfide. The electrophoretic behaviors of type A NT and its fragments in native gels and their comparison with botulinum NT serotypes B and E as well as tetanus NT suggest that each NT forms dimers or other aggregates and the aggregation does not occur when the 42 kD C-terminal half of the heavy chain is excised. Thus, the C-terminal half of the heavy chain appears important in the self-association to form dimers.     

Neurotensin enhances plasma adrenocorticotropin concentration by stimulating corticotropin-releasing hormone secretion.

The systemic administration of neurotensin (NT) dose-dependently increased plasma adrenocorticotropin (ACTH) concentration in rats, and this effect was annulled by (alpha-helical)-CRH9-41, an antagonist of corticotropin-releasing hormone (CRH). The systemic administration of [D-Trp11]-neurotensin (NT-A), a specific NT antagonist, dose-dependently reduced the basal level of circulating ACTH, and this effect was blunted by NT injection. The ACTH inhibitory action of NT-A was completely overcome by the administration of CRH. Taken together, our findings suggest that NT plays a physiologic role in rats, as ACTH secretagogue, and that the mechanism underlying this action of NT involves the stimulation of CRH release.     

The protective effect of melatonin in lungs of newborn rats exposed to maternal nicotine

We investigated possible healing effects of melatonin (MEL) on biochemical and histological changes in the lungs of rat offspring caused by exposure to nicotine (NT) in utero. Pregnant rats were divided randomly into five groups. The SP group was treated with physiological saline. The EA group was treated with ethyl alcohol. The MEL group was treated with MEL. The NT group was treated with NT. The NT + MEL group was treated with NT and MEL. At the end of the study, the biochemistry and histopathology of lung tissue of the offspring were examined. Reduced alveolar development and increased numbers of alveolar macrophages and mast cells were observed in the NT group compared to the SP, EA and MEL groups. We also found increased malondialdehyde (MDA) levels and decreased total glutathione (GSH) levels in the NT group. Application of MEL ameliorated the histological and biochemical damage caused by NT. The number of alveoli was greater in the NT + MEL group than in the NT group. Also, the increased numbers of alveolar macrophages and mast cells resulting from exposure to NT were decreased following MEL treatment. We found that MEL caused a significant decrease in the level of MDA. Maternal exposure to NT caused significant structural and biochemical changes in the lungs of the offspring and administration of MEL ameliorated the changes.     

Incidence and Risk Factors for Neonatal Tetanus in Admissions to Kilifi County Hospital,Kenya

Background

Neonatal Tetanus (NT) is a preventable cause of mortality and neurological sequelae that occurs at higher incidence in resource-poor countries, presumably because of low maternal immunisation rates and unhygienic cord care practices. We aimed to determine changes in the incidence of NT, characterize and investigate the associated risk factors and mortality in a prospective cohort study including all admissions over a 15-year period at a County hospital on the Kenyan coast, a region with relatively high historical NT rates within Kenya.

Methods

We assessed all neonatal admissions to Kilifi County Hospital in Kenya (1999–2013) and identified cases of NT (standard clinical case definition) admitted during this time. Poisson regression was used to examine change in incidence of NT using accurate denominator data from an area of active demographic surveillance. Logistic regression was used to investigate the risk factors for NT and factors associated with mortality in NT amongst neonatal admissions. A subset of sera from mothers (n = 61) and neonates (n = 47) were tested for anti-tetanus antibodies.

Results

There were 191 NT admissions, of whom 187 (98%) were home deliveries. Incidence of NT declined significantly (Incidence Rate Ratio: 0.85 (95% Confidence interval 0.81–0.89), P<0.001) but the case fatality (62%) did not change over the study period (P = 0.536). Younger infant age at admission (P = 0.001) was the only independent predictor of mortality. Compared to neonatal hospital admittee controls, the proportion of home births was higher among the cases. Sera tested for antitetanus antibodies showed most mothers (50/61, 82%) had undetectable levels of antitetanus antibodies, and most (8/9, 89%) mothers with detectable antibodies had a neonate without protective levels.

Conclusions

Incidence of NT in Kilifi County has significantly reduced, with reductions following immunisation campaigns. Our results suggest immunisation efforts are effective if sustained and efforts should continue to expand coverage.     

Characterization of beta-lactotensin,a bioactive peptide derived from bovine beta-lactoglobulin,as a neurotensin agonist

beta-Lactotensin (beta-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine beta-lactoglobulin. The ileum-contracting activity of beta-LT was blocked by the NT1 antagonist SR48692. beta-LT was selective for the neurotensin NT2 receptor while neurotensin was selective for the NT1 receptor. beta-LT is the first natural ligand showing selectivity for the NT2 receptor. beta-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of beta-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT2 antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of beta-LT. These results suggest that the hypertensive activity of beta-LT is mediated by the NT2 receptor. It was concluded that the NT1 and NT2 receptors mediate the opposite effect on blood pressure.     

Neurotensin: dual roles in psychostimulant and antipsychotic drug responses

Central administration of neurotensin (NT) results in a variety of neurobehavioral effects which, depending upon the administration site, resemble the effects of antipsychotic drugs (APDs) and psychostimulants. All clinically effective APDs exhibit significant affinities for dopamine D(2) receptors, supporting the hypothesis that an increase in dopaminergic tone contributes to schizophrenic symptoms. Psychostimulants increase extracellular dopamine (DA) levels and chronics administration can produce psychotic symptoms over time. APDs and psychostimulants induce Fos and NT expression in distinct striatal subregions, suggesting that changes in gene expression underlie some of their effects. To gain insight into the functions of NT, we analyzed APD and psychostimulant induction of Fos in NT knockout mice and rats pretreated with the NT antagonist SR 48692. In both NT knockout mice and rats pretreated with SR 48692, haloperidol-induced Fos expression was markedly attenuated in the dorsolateral striatum; amphetamine-induced Fos expression was reduced in the medial striatum. These results indicate that NT is required for the activation of specific subpopulations of striatal neurons in distinct striatal subregions in response to both APDs and psychostimulants. This review integrates these new findings with previous evidence implicating NT in both APD and psychostimulant responses.     

Compound 48/80 inhibits neurotensin-induced hypotension in rats

The intravenous injection of neurotensin (NT) (0.4 and 1.1 nmoles/kg) produced dose-dependent hypotensive effects in pentobarbital anesthetized rats. The acute or chronic administration of compound 48/80, a well known mast cell depletor, completely abolished the hypotensive effect of low to medium doses of NT and unmasked the previously unknown hypertensive effect of high doses (4.0 nmoles/kg) of NT. This hypertensive effect was significantly reduced by infusing the animals with [D-Trp¹¹]-NT a selective antagonist of NT. The hypotensive action of NT in control rats was also significantly reduced by pretreating the animals with disodium cromoglycate, an antiallergic drug which is believed to stabilize mast cells membranes, or with a mixture of azatadine and methysergide. The results suggest the participation of histamine, serotonin and possibly other endogenous vasoactive substances, to the hypotensive action of NT in rats. The possible origin of these mediators is discussed.     

Neurotensin-induced antinociception and hypothermia in mice: antagonism by TRH and structural analogs of TRH

Intracisternal (IC) administration of neurotensin (NT) in a dose of 10 micrograms produced a significant hypothermia and antinociception in the hot-plate test in mice. Both of these effects of IC NT were completely antagonized by concomitant administration of equimolar doses of thyrotropin-releasing hormone (TRH) and several TRH congeners including 3-methyl-His-TRH (pGlu-3-methyl-His-Pro-NH2), MK-771 (pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide), beta-ala-TRH (pGlu-His-Pro-beta-ala-NH2), and RX-77368 (pGlu-His-dimethyl-Pro-NH2). The antagonism by TRH and TRH analogs on NT-induced hypothermia and antinociception was dose-dependent. Of particular interest was the finding that RX-77368 not only blocked the effects of NT but also produced hyperalgesia. It appears that TRH analogs that are more resistant to biologic degradation are, like TRH, capable of blocking NT-induced behaviors.     

Growth and yield of lentil and wheat inoculated with three Glomus isolates from Saskatchewan soils

The vesicular-arbuscular mycorrhizal fungi (VAMF) Glomus clarum (Nicol. and Schenck) isolate NT4, G. mosseae (Nicol. and Gerd.) Gerd. and Trappe isolate NT6 and G. versiforme (Karst.) Berch isolate NT7 coexist in wheat field soils in Saskatchewan. This study assessed the response of lentil (Lens esculenta L.) and wheat (Triticum aestivum L.) to monospecific and mixed cultures of these VAMF isolates. Seedlings were inoculated with 100 spores of a VAMF isolate, or an equal mixture of spores of two isolates, and grown in a sterile soil mix in a growth chamber. Both crops responded differently to these different VAMF isolates. In the case of lentil, G. clarum NT4 was more effective than G. mosseae NT6 and G. versiforme NT7, and significantly increased (P<0.05) the shoot dry weight (43%) and grain yield (57%) compared with the uninoculated control. There was a significant positive correlation between the percentage of VAMF colonized roots and shoot dry weight (r=0.672^***) and shoot phosphorus concentration (r=0.608^***) of lentil. In the case of wheat, G. clarum NT4 had no effect on shoot dry weight, but produced significant (P<0.08) increases in grain yield (12%) and the phosphorus concentration of the shoot and grain. Although G. clarum NT4 and G. mosseae NT6 both produced similar levels of VAM colonization in wheat, the only response of wheat to isolate NT6 was an increase in plant height at harvest. The efficacy of G. clarum NT4 on both crops appeared to be related to its ability to produce more arbuscular colonization than G. mosseae NT6. Dual inoculation of seedlings with G. clarum NT4 and G. mosseae NT6 resulted in competition between these two isolates. This was evident from a comparison of plant shoot dry weight and grain yield, and VAMF spore production on the two crops inoculated either with isolate NT4 alone or in combination with NT6. G. mosseae NT6 reduced the efficacy of G. clarum NT4 by 16% when dual inoculated on lentil, but had no effect when the host was wheat. Based on spore production, it was found that G. clarum NT4 was more competitive than G. mosseae NT6 when dual inoculated on lentil or wheat. Isolate NT4 produced ca. 2000 and 500 spores/ 100 g substrate, respectively, in the lentil and wheat pots, which was approximately 2–3 times more spores than those produced by isolate NT6 with either crop. When the plants were dual inoculated, there was a 15–19% reduction in spore production by G. clarum NT4 and a 50–70% decrease in spore production by G. mosseae NT6. Our results show that G. clarum NT4 was more competitive and effective in its ability to colonize and increase the growth and yield of lentil and wheat than G. mosseae NT6 or G. versiforme NT7. The relative performance of isolate NT4 with different host plants suggests that this VAMF isolate exhibits a host preference for lentil.     

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor–stroma interaction

Background

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.

Methods

EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.

Results

The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.

Conclusion and general significance

The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Isolation and primary structure of an amphibian neurotensin.

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.