Neuropeptide FF receptor antagonist, RF9, attenuates the fever induced by central injection of LPS in mice

The endogenous opioid system has been found to be involved in the fever caused by lipopolysaccharide (LPS). Neuropeptide FF (NPFF, FLFQPQRF-NH₂) is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system. Therefore, those data suggested a link between LPS-induced fever and NPFF systems. Using a model of acute neuroinflammation, we sought to determine the effects of NPFF systems on the fever induced by i.c.v. injection of LPS. Coinjected with different doses of NPFF (10 and 30 nmol), the fever of LPS (125 ng) was not modified. Interestingly, the selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it decreased the fever of LPS (125 ng) after cerebral administration in mice. These results suggest that NPFF receptors activation is required for LPS to produce fever. This interaction is the first evidence that NPFF systems participate in the control of acute neuroinflammation in conscious animals.     

Inhibition of neuropeptide FF (NPFF)-induced hypothermia and anti-morphine analgesia by RF9, a new selective NPFF receptors antagonist

Neuropeptide FF (NPFF) belongs to a neuropeptide family including two precursors (pro-NPFF_A and pro-NPFF_B) and two receptors (NPFF₁ and NPFF₂). Very recently, the novel compound RF9 was reported as the truly selective antagonist on NPFF receptors. The present study examined the effects of RF9 on the hypothermia and anti-morphine action induced by NPFF in mice. (1) RF9 injected into the third ventricle was devoid of any residual agonist activity, but it completely antagonized the hypothermic effects of NPFF (30 or 45 nmol) after cerebral administration in mice; (2) RF9 did not alter the tail-flick latency and morphine analgesia in nociceptive test, however, co-administration of RF9 prevented the anti-morphine action of intracerebroventricularly applied NPFF (10 nmol, i.c.v.) in the mouse tail-flick assay. Collectively, our data indicate that RF9, behaving as a truly pure NPFF receptors antagonist, prevents NPFF-induced drops of the body temperature and morphine analgesia in mice. In addition, it further confirms that the hypothermia and anti-morphine action of NPFF are mediated directly by NPFF receptors.     

神经肽FF的生物活性研究进展

神经肽FF(neuropeptide FF,NPFF)最初于1985年从牛脑中分离得到,是一种哺乳动物体内普遍存在的八肽。NPFF最早因具有调节阿片镇痛活性而引起关注,而后陆续发现NPFF具有多种生理功能,包括调节体温、心血管、神经内分泌、胃肠运动、摄食、抗炎、免疫调节、以及神经保护等。现在认为NPFF是一种具有激素样活性的神经递质,因此对其生理功能的深入研究有助于理解NPFF在多种生理系统中的作用机制及其潜在多肽药物的研发。本文综述了近年来NPFF生物活性的最新研究进展,结合本实验室已有研究基础,重点介绍了NPFF在神经内分泌、免疫调节、抗炎、神经保护、及信号通路方面的进展,并展望了NPFF类多肽药物今后的发展方向。     

Crypteins derived from the mouse neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice

NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests.     

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe⁴→(pF)Phe⁴; (b) Ala⁷, Ala¹¹→Aib⁷, Aib¹¹; (c) Leu¹⁴, Ala¹⁵→Arg¹⁴, Lys¹⁵. Compared with the first series, N-terminus of the second series was changed from Phe¹ to Nphe¹. All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK_i = 9.33; pEC₅₀ = 7.50). In particular, [(pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was found to be a highly potent agonist with pK_i = 10.78 in binding studies and pEC₅₀ = 9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe¹, (pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was the best antagonist with pA₂ = 8.39 and showed high binding affinity with pK_i = 9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.     

Substance P N-terminal fragment SP(1-7) attenuates chronic morphine tolerance and affects dynorphin B and nociceptin in rats

The N-terminal substance P fragment SP_1-7 is known to modulate hyperalgesia and opioid withdrawal in animal models. This study examined the effects of intraperitoneal (i.p.) injections of SP_1-7 on chronic morphine tolerance and on the levels of dynorphin B (DYN B) and nociceptin/orphanin FQ (N/OFQ) in various brain areas of male Sprague-Dawley rats. Morphine tolerance was induced by subcutaneous injections of the opioid (10 mg/kg) twice daily for 7 days. SP_1-7 injected i.p. (185 nmol/kg) 30 min prior to morphine reduced the development of morphine tolerance. Immunoreactive (ir) DYN B and N/OFQ peptide levels were measured in several areas of the central nervous system. Levels of ir DYN B in rats treated with SP_1-7 and morphine were decreased in the nucleus accumbens, substantia nigra and ventral tegmental area and increased in the frontal cortex. The ir N/OFQ levels were increased in the periaqueductal gray and decreased in the nucleus accumbens. Since the concentration profiles of the two peptides were altered by SP_1-7 in the areas that are implicated in the modulation of opioid tolerance and analgesia, it is suggested that DYN B and N/OFQ systems may be involved in the effects of SP_1-7 on opioid tolerance.     

Neuropeptide FF (NPFF) reduces the expression of cocaine-induced conditioned place preference and cocaine-induced sensitization in animals

The endogenous brain opioid system is believed to play an important role in mediating reward mechanisms. Opioid innervation is high in many limbic regions and reinforcing actions of many drugs of abuse, including cocaine, are thought to be mediated via endogenous opioid system. The aim of the present study was to indicate whether the anti-opioid peptide, neuropeptide FF (NPFF; FLFQPQRF-NH₂) was able to modify the rewarding effect of cocaine (5 mg/kg) measured in the expression of conditioned place preference (CPP) test in rats and the expression of sensitization to hyperlocomotor effect of cocaine (10 mg/kg) in mice. Our results indicate that NPFF (5, 10, and 20 nmol) given intracerebroventricularly (i.c.v.) inhibited the expression of cocaine-induced CPP at the dose of 10 nmol (P < 0.01) and 20 nmol (P < 0.001). Moreover, NPFF inhibited the expression of cocaine-induced sensitization to its hyperlocomotor effect at the dose of 20 nmol (P < 0.05) and acute hyperlocomotor effect of cocaine at doses of 5 nmol (P < 0.01), 10 nmol (P < 0.01), and 20 nmol (P < 0.05). Our study suggests that NPFF may participate in a rewarding effect of cocaine measured in the CPP paradigm. On the other hand, our experiments indicate that NPFF is involved in the mechanism of expression of sensitization to cocaine hyperlocomotion but this effect seems to be non-specific because NPFF also inhibited the acute hyperlocomotor effect of cocaine.     

Functional expression of opioid receptor—like receptor and its endogenous specific agonist nociceptin／orphanin FQ during mouse embryogenesis

INTRODUCTIONOpioidreceptorsbelongtotheG-protein-coupledreceptorfamilythatischaracterizedbytheseventransmembranespanningdomainsinstructure.Threesubtypesoftheopioidreceptors(H,6,andK)havebeenclonedandcharacterizedthroughtheir1.CorrespondingauthorFunctionalexpressionofORLIandN/OFQduringmouseembryogenesisdistinctaffinitiesfordifferentopioidligands.TheseopioidreceptorsareallcoupledtotheinhibitoryGprotein(Gi)andnegativelyregulateadenylatecyclase[1].Opioidreceptor--likereceptor(ORLI),anew…     

Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1-13), (1-11) and (1-7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3-1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1-13) and (1-11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1-13) and 4.75 for N/OFQ (1-11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1-7) led to be less potent than N/OFQ and its fragments, (1-13) and (1-11). Antinociception induced by N/OFQ or N/OFQ (1-13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1-13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1-11) and (1-7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1-13) still possess antinociceptive activity through NOP receptors.     

Molecular characterization and functional expression of opioid receptor-libe_1 receptor

INTRODUCTIONOpioidactsinthecentralandperipheralnervoussystem(CNSandPNS)to'producenumerouspharmacologicaleffects.Repetitiveorcontinuoususeofopioids,however,causesdrugtoleranceanddependence.Threesubtypesofopioidreceptors,termedp)6,andK,havebeencloned.TheyarecoupledtotheinhibitoryGproteinandnegativelyregulateadenylylcyclasell].RecentlyanewG-proteincoupledreceptortermedopioidreceptor-likereceptor(ORLI)whichbelongstothenewlycharacterizedopioidreceptorsubfamilyhadbeenclonedfromhumanbrainst…     

Somatomedin C in the pancreas of young and adult,normal and obese,hyperinsulinemic mice

Summary Immunocytochemical, immunochemical and RNA-hybridization techniques were used to map the distribution of somatomedin C (Sm-C; insulin-like growth factor I; IGF-I) in the pancreas of young and adult lean and obese mice. The D cells in the islets of Langerhans showed intense cytoplasmic Sm-C immunoreactivity, extending into their processes. Only slight Sm-C immunoreactivity was seen in A and B cells, apparently confined to the plasma membranes. In the exocrine pancreas scattered duct cells were immunopositive. Starvation increased, while feeding decreased the Sm-C immunoreactivity in B cells. RNA-hybridization analyses revealed that roughly the same number of Sm-C mRNA molecules, as calculated per DNA amount in the pancreas, could be demonstrated in young and adult, lean and obese mice. Radioimmunoassay (RIA) determinations of total Sm-C showed that there were about equal concentrations in the pancreas of lean and obese mice. There were marked differences between the liver and the pancreas, in that the RIA Sm-C values for the former were twice those in the latter while, in contrast, the corresponding values for the Sm-C mRNA, i.e. the agent determining the synthesis of Sm-C, were about 100 times higher in the liver as compared to that in the pancreas. We interpret our results as follows: The D cells in the islets form and secrete Sm-C in both young and adult, lean and obese mice, while A and B cells bind, but do not necessarily synthesize this peptide. Our results obtained in vivo on mature pancreatic tissue are in contrast to those obtained in tissue-culture studies on fetal and neonatal islets, in which B cells synthesize Sm-C.     

Interrelationships in mice of antipyrine half-life, hepatic monooxygenase activities and liver S9-mediated mutagenicity of aflatoxin B1, benzo[alpha]pyrene 7,8-dihydrodiol, 2-acetylaminofluorene and N-nitrosomorpholine

To evaluate the predictive value of serum antipyrine half-life AP(T1/2) as an index of hepatic carcinogen metabolism, groups of C57BL/6 and DBA/2 mice were treated with various inducers and inhibitors of cytochrome P-450-dependent monooxygenases (pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), 5,6-benzoflavone (5,6-BF), 3-methylcholanthrene (MC), disulfiram (DIS), 7,8-BF). Groups of mice were also given ethanol (3% in drinking water) for 12 days. Within each group, mean serum AP-(T1/2) was compared with (i) the in vitro activity of hepatic microsomal benzo[alpha]pyrene (BP) 3-hydroxylase, 2-acetylaminofluorene (AAF)-N-hydroxylase and aldrin monooxygenase, and (ii) the liver S9-mediated mutagenicity of aflatoxin B1 (AFB), trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene (BP 7,8-diol), 2-acetylaminofluorene and N-nitrosomorpholine (NMOR) in Salmonella typhimurium strains. Serum AP(T1/2) was only correlated negatively with the activity of BP 3-hydroxylase (P less than 0.001) and aldrin monooxygenase (P less than 0.001). No statistically significant correlation was found between serum AP(T1/2) and liver S9-mediated mutagenicity for any of the four carcinogens. On the basis of these results, we conclude that serum AP(T1/2) may not be a reliable index of the capacity of liver to convert carcinogens into reactive intermediates.     

cis-9,trans-11 conjugated linoleic acid stimulates expression of angiopoietin like-4 in the placental extravillous trophoblast cells

A number of studies have been carried out to examine the biological function of conjugated linoleic acid (CLA) and its potential health benefits. However, not much is known about how CLA isomers mediate their effect on angiogenesis and vascularization during early placentation. In this paper we demonstrate that cis-9,trans-11(c9,t11)-CLA stimulated the expression of angiopoietin like-4 (ANGPTL4) mRNA and protein accompanied by tube formation in first trimester placental trophoblast cells, HTR8/SVneo whereas the other CLA isomer, trans-10,cis-12 (t10,c12)-CLA had no such effects. c9,t11-CLA however did not stimulate expression of the most potent angiogenic factor, vascular endothelial growth factor (VEGF) in these cells. Silencing ANGPTL4 in these cells significantly reduced the stimulatory effect of c9,t11-CLA on tube formation, indicating the involvement of ANGPTL4. In addition, c9,t11-CLA increased the mRNA expression of several pro-angiogenic factors such as fatty acid binding protein-4 (FABP4), cyclooxygenase-2 (COX-2) and adipose differentiation-related protein (ADRP) in HTR8/SVneo cells. c9,t11-CLA also induced the uptake of docosahexaenoic acid, 22:6n − 3 (DHA), a stimulator of tube formation in these cells. Triacsin C, an acylCoA synthetase inhibitor, attenuated c9,t11-CLA induced DHA uptake, tube formation and cellular proliferation in HTR8/SVneo cells.     

Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase in Escherichia coli and used in the preparative synthesis of drug metabolites. Low dissolved oxygen (DO) concentration (<1%) during expression was found to be critical for producing active P450s. Control of temperature, pH and glycerol supplementation in 10-L fermentations enhanced enzyme expression 31–86%. Additional improvements were obtained by altering media formulations, resulting in bicistronic expression levels of 890, 1,800 and 1,010 nmol/L for 3A4, 2C9 and 1A2, respectively. The P450 titers achieved in fermentors exceeded those in flask fermentations by 3- to 6-fold in this study and up to 10-fold when compared with previously reported literature [FEBS Lett (1996) 397:210–214, Arch Biochem Biophys (1996) 327:254–259, Biochem Pharmacol (1998) 55:1315–1325, Drug Metab Pharmacokinet (2003) 18:42–47, Nat Biotechnol (1997) 15:784–788; Metab Eng (2000) 2:115–125]. Intact cells and isolated membranes obtained from 10-L fermentations were used to establish an efficient bioconversion system for the generation of metabolites. To demonstrate the utility of this approach, known metabolites of the anabolic steroid testosterone, the anti-inflammatory agent diclofenac and the analgesic agent phenacetin, were generated using 3A4, 2C9 and 1A2, respectively. The reaction conditions were optimized for pH, temperature, DO concentration, use of co-solvent and glucose supplementation. Conversion yields of 29–93% were obtained from 1-L reactions, enabling isolation of 59 mg 6-hydroxytestosterone, 110 mg 4-hydroxydiclofenac and 88 mg acetaminophen.