RÉSUMÉ
Insomnia is one of the most common sleep disorders, affecting 10-15% of the global population. Because classical remedies used to treat insomnia have various side effects, new therapeutics for insomnia are attracting attention. In the present study, we found that N2-Ethyl-N4-(furan-2-ylmethyl) quinazoline-2,4-diamine (AR-001) has adenosine A1 receptor agonistic activity and exhibits hypnotic efficacy by decreasing sleep onset latency and increasing total sleep time in a pentobarbital-induced sleep model. This hypnotic effect of AR-001 was significantly inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). As a result of immunohistochemistry, AR-001 was shown to increase neural activity in the sleep-promoting region, ventrolateral preoptic nucleus (VLPO), and decrease neural activity in the wake-promoting region, basal forebrain (BF), and lateral hypothalamus (LH), and that these effects of AR-001 were significantly inhibited by DPCPX treatment. In addition, AR-001 increased adenosine A1 receptor mRNA levels in the hypothalamus. In conclusion, this study suggests that AR-001 has a hypnotic effect, at least partially, through adenosine A1 receptor and may have therapeutic potential for insomnia.
RÉSUMÉ
Thioredoxin 1 (Trx1) serves a central role in redox homeostasis. It is involved in numerous other processes, including oxidative stress and apoptosis. However, to the best of our knowledge, the role of Trx1 in inflammation remains to be explored. The present study investigated the function and mechanism of cell permeable fused Tat-Trx1 protein in macrophages and a mouse model. Transduction levels of Tat-Trx1 were determined via western blotting. Cellular distribution of transduced Tat-Trx1 was determined by fluorescence microscopy. 2',7'-Dichlorofluorescein diacetate and TUNEL staining were performed to determine the production of reactive oxygen species and DNA fragmentation. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR (RT-qPCR), respectively. Effects of skin inflammation were determined using hematoxylin and eosin staining, changes in ear weight and ear thickness, and RT-qPCR in ear edema animal models. Transduced Tat-Trx1 inhibited lipopolysaccharide-induced cytotoxicity and activation of NF-κB, MAPK and Akt. Additionally, Tat-Trx1 markedly reduced the production of inducible nitric oxide synthase, cyclooxygenase-2, IL-1ß, IL-6 and TNF-α in macrophages. In a 12-O-tetradecanoylphorbol-13-acetate-induced mouse model, Tat-Trx1 reduced inflammatory damage by inhibiting inflammatory mediator and cytokine production. Collectively, these results demonstrated that Tat-Trx1 could exert anti-inflammatory effects by inhibiting the production of pro-inflammatory mediators and cytokines and by modulating MAPK signaling. Therefore, Tat-Trx1 may be a useful therapeutic agent for diseases induced by inflammatory damage.
RÉSUMÉ
FK506-binding proteins (FKBPs) belong to the immunophilin family and are linked to various disease states, including the inflammatory response. The inhibition of cytokine and chemokine expression in addition to positive effects of FKBPs on corneal inflammation in animal models suggests that they may be used for ophthalmic delivery in the treatment of dry eye disease. To pass the effective barriers protecting eye tissues, testing the transduction domains of FKBPs is essential. However, monitoring their transduction efficiencies is not a simple task. The quantitative measurement of FKBP interactions was performed using a cell model with a specific G protein-coupled receptor, as FKBPs had been known to act at the inositol 1,4,5-trisphosphate receptor (IP3R) leading to the inhibition of intracellular calcium mobilization. Because of its luminescence amplitude and stability, human urotensin II receptor was expressed in aequorin parental cells to measure the action of selected FKBPs. This luminescence-based functional assay platform exhibited a high signal-to-background ratio of more than 100 and a Z' factor at 0.6204. As expected, changes in the sequence of the transduction domain affected the function of the FKBPs. The intracellular calcium mobilization assay with selected FKBPs represented a robust and reliable platform to screen initial candidates. Although the precise nature of the control that FKBPs exert on the IP3R is uncertain, this approach can be used to develop innovative anti-inflammatory treatments for dry eye disease by optimizing protein transduction domain sequences.
Sujet(s)
Protéines de liaison au tacrolimus , Tacrolimus , Séquence d'acides aminés , Animaux , Calcium , Protéines de transport , Humains , Tacrolimus/pharmacologie , Protéines de liaison au tacrolimus/génétique , Protéines de liaison au tacrolimus/métabolismeRÉSUMÉ
BACKGROUND: Stress is an important cause of skin disease, including hair loss. The hormonal response to stress is due to the HPA axis, which comprises hormones such as corticotropin releasing factor (CRF), adrenocorticotropic hormone (ACTH), and cortisol. Many reports have shown that CRF, a crucial stress hormone, inhibits hair growth and induces hair loss. However, the underlying mechanisms are still unclear. The aim of this study was to examine the effect of CRF on human dermal papilla cells (DPCs) as well as hair follicles and to investigate whether the HPA axis was established in cultured human DPCs. RESULTS: CRF inhibited hair shaft elongation and induced early catagen transition in human hair follicles. Hair follicle cells, both human DPCs and human ORSCs, expressed CRF and its receptors and responded to CRF. CRF inhibited the proliferation of human DPCs through cell cycle arrest at G2/M phase and induced the accumulation of reactive oxygen species (ROS). Anagen-related cytokine levels were downregulated in CRF-treated human DPCs. Interestingly, increases in proopiomelanocortin (POMC), ACTH, and cortisol were induced by CRF in human DPCs, and antagonists for the CRF receptor blocked the effects of this hormone. CONCLUSION: The results of this study showed that stress can cause hair loss by acting through stress hormones. Additionally, these results suggested that a fully functional HPA axis exists in human DPCs and that CRF directly affects human DPCs as well as human hair follicles under stress conditions.
Sujet(s)
Alopécie/métabolisme , Corticolibérine/métabolisme , Axe hypothalamohypophysaire , Axe hypophyso-surrénalien/métabolisme , Hormone corticotrope/métabolisme , Cellules cultivées/métabolisme , Derme/cytologie , Poils/croissance et développement , Follicule pileux/cytologie , Humains , Hydrocortisone/métabolisme , Axe hypothalamohypophysaire/métabolisme , Récepteur CRH/métabolismeRÉSUMÉ
BACKGROUND: There is a growing interest in the relationship among stress hormones, neuroendocrine signaling, and skin diseases, including hair loss. Previous reports showed that stress hormones inhibit human hair growth and induce early catagen transition. Moreover, a CRH receptor antagonist reversed CRH-induced alopecia in a mouse model, suggesting that antagonization of the CRH receptor is a key clinical strategy to treat stress-induced hair loss. OBJECTIVES: The aim of this study was to investigate the protective effect of CRH receptor antagonists from Pulsatilla chinensis on human hair follicles (hHFs) and human dermal papilla cells (hDPCs). METHODS: hHFs were observed and scored by hair cycle. The levels of cAMP, a second messenger, were measured in each group. In addition, the mRNA and protein levels of factors related to the hair cycle were measured. Furthermore, the expression levels of various members of the mitogen-activated protein kinase (MAPK) signaling pathway related to stress were measured. RESULTS: CRH induced early catagen transition in an ex vivo hair organ culture model. In addition, CRH downregulated the levels of alkaline phosphatase (ALP) and hair anagen-related cytokines in cultured hDPCs. Moreover, CRH induced the phosphorylation of JNK, c-Jun, p38, ERK, and Akt in cultured hDPCs. CRH receptor antagonists isolated from P chinensis reversed these CRH-induced modulations in both ex vivo hair follicles (HFs) and cultured hDPCs. CONCLUSIONS: These results indicate that P chinensis effectively blocks CRH receptor function and that saponin derivatives from P chinensis could be a pharmaceutical and cosmetic approach to treat stress-induced hair loss.
Sujet(s)
Préparations pharmaceutiques , Pulsatilla , Poils , Follicule pileux , Humains , Récepteur CRHRÉSUMÉ
Antioxidant 1 (ATOX1) protein has been reported to exhibit various protective functions, including antioxidant and chaperone. However, the effects of ATOX1 on the inflammatory response has not been fully elucidated. Thus, we prepared cell permeable Tat-ATOX1 and studied the effects on lipopolysaccharide (LPS)- and 12-O-tetradecanoyl phorbol-13- acetate (TPA)-induced inflammation. Experimental results showed that transduced Tat-ATOX1 protein significantly suppressed LPS-induced intracellular reactive oxygen species (ROS). Also, Tat-ATOX1 protein markedly inhibited LPS- and TPA-induced inflammatory responses by decreasing cyclooxygenase- 2 (COX-2) and inducible nitric oxide synthase (iNOS) and further inhibited phosphorylation of mitogen activated protein kinases (MAPKs; JNK, ERK and p38) and the nuclear factor-kappaB (NF-κB) signaling pathway. These results indicate that the Tat-ATOX1 protein has a pivotal role in inflammation via inhibition of inflammatory responses, suggesting Tat-ATOX1 protein may offer a therapeutic strategy for inflammation. [BMB Reports 2018; 51(12): 654-659].
Sujet(s)
Transporteurs de cations/métabolisme , Mitogen-Activated Protein Kinases/métabolisme , Chaperons moléculaires/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Animaux , Transporteurs de cations/génétique , Protéines de transport du cuivre , Cyclooxygenase 2/métabolisme , Modèles animaux de maladie humaine , Régulation négative/effets des médicaments et des substances chimiques , Oedème/induit chimiquement , Oedème/anatomopathologie , Lipopolysaccharides/pharmacologie , Mâle , Souris , Souris de lignée ICR , Chaperons moléculaires/génétique , Nitric oxide synthase type II/métabolisme , Cellules RAW 264.7 , Espèces réactives de l'oxygène/métabolisme , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Transduction du signal/effets des médicaments et des substances chimiques , 12-Myristate-13-acétate de phorbol/toxicitéRÉSUMÉ
The popular screening method for urotensin (UT) receptor antagonists is to measure the intracellular calcium concentration with a calcium-sensitive fluorescent dye. This assay format has an inherent limitation on the problem related to the fluorescence interference as it involves fluorescent dyes. In the present study, a label-free assay for the screening of UT receptor antagonists was developed by using dynamic mass redistribution (DMR) assay based on label-free optical biosensor. The addition of urotensin II (UII) stimulated a DMR profile to HEK293 cells stably expressing the human UT receptor (HEK293UT cells) but not on parental cells. The EC50 value of UII in label-free assay was 4.58 nM, which is very similar to that in conventional calcium mobilization assay (4.15 nM). Compared with the calcium mobilization assay for UII (Z' factor, 0.77), the current label-free assay presented improved Z' factor (0.81), with a relatively similar S/B ratio (28.0 and 25.6, respectively). The known high-affinity UT receptor antagonists, SB657510, GSK562590, and urantide, exhibited comparable IC50 values but rather less potent in the DMR assay than in calcium mobilization. Our DMR assay was able to present various functional responses, including inverse agonism in SB657510 and GSK1562590 as well as partial agonism in urantide. Moreover, the DMR assay exerted the stable antagonist window upon the minimal agonist stimulus. These results suggest that the label-free cell-based UT receptor assay can be applicable to evaluate the various functional activities of UT receptor-related drug candidates.
Sujet(s)
Calcium/métabolisme , Récepteurs couplés aux protéines G/analyse , Récepteurs couplés aux protéines G/métabolisme , Spectrométrie de fluorescence/méthodes , Colorants fluorescents/analyse , Cellules HEK293 , Humains , Récepteurs couplés aux protéines G/agonistes , Urotensines/analyse , Urotensines/métabolismeRÉSUMÉ
Recent advances in basic and clinical studies have identified Rho kinase (ROCK) as an important target potentially implicated in a variety of cardiovascular diseases and ROCK inhibitors were considered as a pharmacological strategy to prevent and treat cardiovascular diseases. To screen the small molecule compound library against ROCK, a high throughput screening (HTS) campaign was carried out using immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Z' value and signal to background (S/B) ratio were achieved at 0.76 and 5.27 for the pilot library screening of the most diverse set consisting of 15,040 compounds with a reasonable reconfirmation rate. From this screening campaign, four novel scaffolds, such as 3- nitropyridine, 4-methoxy-1,3,5,-triazine, naphthalene-1,4-dione, and 2,3-dihydro-1H-pyrrolo[2,3-b]quinoxaline, were yielded. Particularly, we found that 3-nitropyridine derivatives possess potent inhibitory activity and selectivity for ROCK. Our findings provide important information for the design of novel ROCK inhibitor.
Sujet(s)
Évaluation préclinique de médicament/méthodes , Tests de criblage à haut débit/méthodes , Inhibiteurs de protéines kinases/composition chimique , Inhibiteurs de protéines kinases/pharmacologie , Bibliothèques de petites molécules/composition chimique , Bibliothèques de petites molécules/pharmacologie , rho-Associated Kinases/antagonistes et inhibiteurs , Conception de médicament , Humains , Modèles moléculaires , rho-Associated Kinases/métabolismeRÉSUMÉ
The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.
Sujet(s)
Évaluation préclinique de médicament , Europium/composition chimique , Polarisation de fluorescence/méthodes , Tests de criblage à haut débit/méthodes , Sulfonamides/pharmacologie , Urotensines/antagonistes et inhibiteurs , Animaux , Fixation compétitive , Dosage biologique , Découverte de médicament , Fluorescence , Cellules HEK293 , Humains , Concentration inhibitrice 50 , Ligands , Souris , Thérapie moléculaire ciblée , Liaison aux protéines , Radio-isotopes , Dosage par compétition , Rats , Reproductibilité des résultats , Sensibilité et spécificité , Sulfonamides/analyse , Sulfonamides/synthèse chimique , Facteurs temps , Urotensines/génétique , Urotensines/métabolismeRÉSUMÉ
Control of NF-κB release through the inhibition of IκB kinase ß (IKKß) has been identified as a potential target for the treatment of inflammatory and autoimmune diseases. To screen the small molecule compound library against IKKß, a high-throughput screening (HTS) campaign was carried out using immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Through serial optimization of assay conditions, the Z' value was achieved at 0.88 from the pilot library screening of the most diverse 7,243 compounds with reconfirmation rate of 63%. The results from this HTS campaign identified three novel scaffolds for the prospective IKKß inhibitor, such as 7-benzoyl-4-phenylcyclopenta[1,2] oxazine, 1-(thiophen or furan)-2,3-dihydroimidazo[1,5] pyridine and 2-phenyloxazolo[5,4] pyridine. Particularly, 7-benzoyl-4-phenylcyclopenta[1,2] oxazine derivatives presented potent inhibitory activity and selectivity for IKKß. These findings suggest that the current TR-FRET assay system for IKKß was successful to identify hits for novel IKKß inhibitors as a robust, reproducible and sensitive HTS system.
Sujet(s)
Antienzymes/analyse , Transfert d'énergie par résonance de fluorescence , Tests de criblage à haut débit , I-kappa B Kinase/antagonistes et inhibiteurs , Antienzymes/composition chimique , HumainsRÉSUMÉ
ETHNOPHARMACOLOGICAL RELEVANCE: In Korea, Morus alba leaves have been traditionally administered as natural therapeutic agent for the alleviating dropsy and diabetes. AIM OF THE STUDY: The present study was performed to evaluate melanin-concentrating hormone receptor subtype 1 (MCH1) antagonism of the ethanol extract of Morus alba leaves (EMA) and its anti-obesity effect in diet-induced obese (DIO) mice. MATERIALS AND METHODS: The binding affinity of EMA for the MCH1 receptor with europium-labeled MCH (Eu-MCH), the function of recombinant MCH1 receptors expressed in CHO cells, and the anti-obesity effects in DIO mice were evaluated. RESULTS: MCH1 receptor binding studies showed, EMA exhibited a potent inhibitory activity with IC50 value of 2.3+/-1.0 microg/ml. EMA (10-100 microg/ml) also inhibited the intracellular calcium mobilization with the recombinant MCH1 receptors expressed in CHO cells. In an anti-obesity study with DIO mice, longterm oral administrations of EMA for 32 consecutive days produced a dose-dependent decrease in body weight and hepatic lipid accumulation. CONCLUSIONS: These results suggest that chronic treatment with EMA exerts an anti-obesity effect in DIO mice, and its direct MCH1 receptor antagonism may contribute to decrease body weight.
Sujet(s)
Agents antiobésité/usage thérapeutique , Morus , Obésité/traitement médicamenteux , Phytothérapie , Extraits de plantes/usage thérapeutique , Récepteur hormone hypophysaire/antagonistes et inhibiteurs , Tissu adipeux/effets des médicaments et des substances chimiques , Animaux , Agents antiobésité/pharmacocinétique , Agents antiobésité/pharmacologie , Poids/effets des médicaments et des substances chimiques , Cellules CHO , Calcium/métabolisme , Cricetinae , Cricetulus , Cyclobutanes/pharmacologie , Cyclobutanes/usage thérapeutique , Régime alimentaire , Ration calorique/effets des médicaments et des substances chimiques , Humains , Métabolisme lipidique/effets des médicaments et des substances chimiques , Foie/effets des médicaments et des substances chimiques , Souris , Souris de lignée C57BL , Extraits de plantes/pharmacocinétique , Extraits de plantes/pharmacologieRÉSUMÉ
Abnormal phenotypes resulting from haploinsufficiency (HI) are due to the loss of one allele. Recent studies in budding yeast have shown that HI originates from insufficient protein levels or from a stoichiometric imbalance between subunits of protein complexes. In humans, however, HI often involves transcription factors. Therefore, the species differences in HI and the molecular mechanisms of species-specific HI remain under investigation. In this study, HI in fission yeast was systematically surveyed. HI in fission yeast affected genes related to signaling and to basic cellular processes, as observed in budding yeast. These results suggest that there are species differences in HI and that the HI that occurs in fission yeast is intermediate to and HI in budding yeast and humans.
Sujet(s)
Délétion de gène , Génome fongique , Schizosaccharomyces/génétique , Allèles , Protéines fongiques/génétique , Dosage génique , Gènes fongiques , Phénotype , Schizosaccharomyces/croissance et développement , Spécificité d'espèceRÉSUMÉ
Hydrazinocurcumin (HC), a synthetic derivative of curcumin, has been reported to inhibit angiogenesis via unknown mechanisms. Understanding the molecular mechanisms of the drug's action is important for the development of improved compounds with better pharmacological properties. A genomewide drug-induced haploinsufficiency screening of fission yeast gene deletion mutants has been applied to identify drug targets of HC. As a first step, the 50% inhibition concentration (IC50) of HC was determined to be 2.2 microM. The initial screening of 4,158 mutants in 384-well plates using robotics was performed at concentrations of 2, 3, and 4 microM. A second screening was performed to detect sensitivity to HC on the plates. The first screening revealed 178 candidates, and the second screening resulted in 13 candidates, following the elimination of 165 false positives. Final filtering of the condition-dependent haploinsufficient genes gave eight target genes. Analysis of the specific targets of HC has shown that they are related to septum formation and the general transcription processes, which may be related to histone acetyl transferase. The target mutants showed 65% growth inhibition in response to HC compared with wild-type controls, as shown by liquid culture assay.
Sujet(s)
Curcumine/analogues et dérivés , Délétion de gène , Génome fongique , Hydrazines/pharmacologie , Protéines de Schizosaccharomyces pombe/composition chimique , Schizosaccharomyces/effets des médicaments et des substances chimiques , Curcumine/pharmacologie , Évaluation préclinique de médicament , Haploïdie , Hétérozygote , Concentration inhibitrice 50 , Schizosaccharomyces/génétique , Schizosaccharomyces/croissance et développement , Schizosaccharomyces/métabolisme , Protéines de Schizosaccharomyces pombe/antagonistes et inhibiteurs , Protéines de Schizosaccharomyces pombe/génétique , Protéines de Schizosaccharomyces pombe/métabolismeRÉSUMÉ
INTRODUCTION: Filter plates are available from many vendors reflecting the growth of their scientific applications in various fields, however, the heterogeneous nature of those applications are the major factor to block the expansion of various filter plate options in a research scale operation. The development of an automatic vacuum filtration system for a conventional plate washer was presented as a possible solution for the filtration process in filter plate applications, especially in the melanin concentrating hormone receptor subtype 1 (MCH1) receptor binding assay with the time resolved fluorescence technology. METHODS: The pilot modification was done in the Embla 96/384 well washer by replacing the original plate carriage with a new carriage mimicking the conventional vacuum manifold to add the function of flow-through vacuum filtration for filter plates. The performance of new vacuum filtration system was evaluated with MCH1 receptor binding assay and ligand washout experiments. RESULTS: The mean background values from ligand washout experiments in AcroWell filter plates were 6406+/-502.9 with a manual vacuum manifold and 5563+/-585.8 with the vacuum filtration system. Z' factors were calculated as 0.6101+/-0.095 for the MCH1 receptor binding assay with the vacuum filtration system. DISCUSSION: The new plate carriage for a conventional plate washer was developed for filter plate applications to enable its use in a flow-through vacuum filtration application in addition to the conventional plate washing by an aspiration. The results from ligand washout and receptor binding assay suggest that the vacuum filtration system can provide a cost-effective solution for filter plate applications and may alleviate the most common problems of those heterogeneous assays to develop as high throughput operations without major investments for the professional workstations.
Sujet(s)
Conception d'appareillage/instrumentation , Conception d'appareillage/méthodes , Animaux , Fixation compétitive , Dosage biologique/instrumentation , Dosage biologique/méthodes , Études d'évaluation comme sujet , Filtration/instrumentation , Filtration/méthodes , Humains , Ligands , Souris , Rats , Récepteur somatostatine/analyse , Récepteur somatostatine/métabolisme , VideRÉSUMÉ
A series of dinaphtho[1,2-b;2',3'-d]furan-7,12-dione derivatives were synthesized and evaluated for inhibitory activities against receptor tyrosine kinases. The naphthofuroquinone compounds with dialkylaminoethoxy group at C(5)-position (7, 8, 10, and 11) manifested strong inhibitory activities against epidermal growth factor receptor and vascular endothelial growth factor receptor. Docking study of 11 with EGFR was also performed.
Sujet(s)
Antienzymes/synthèse chimique , Furanes/synthèse chimique , Naphtoquinones/synthèse chimique , Quinazolines/synthèse chimique , Récepteurs à activité tyrosine kinase/antagonistes et inhibiteurs , Antienzymes/pharmacologie , Récepteurs ErbB/antagonistes et inhibiteurs , Furanes/pharmacologie , Naphtoquinones/pharmacologie , Quinazolines/pharmacologie , Récepteurs aux facteurs de croissance endothéliale vasculaire/antagonistes et inhibiteurs , Relation structure-activitéRÉSUMÉ
The current study examined angiotensin receptor (ATR) regulation in proliferating rat aortic vascular smooth muscle cells (VSMCs) in culture. Radioligand competition analysis coupled with RNase protection assays (RPAs) revealed that angiotensin type 1a receptor (AT(1a)R) densities (B(max)) increased by 30% between 5 and 7 days in culture [B(max) (fmol/mg protein): day 5, 379 +/- 8.4 vs. day 7, 481 +/- 12, n = 3, P < 0.05] under conditions in which no significant changes in AT(1a)R mRNA expression occurred [in RPA arbitrary units (AU): day 5, 0.23 +/- 0.01 vs. day 7, 0.24 +/- 0.04, n = 4] or in mRNA synthesis determined by nuclear run-on assays [AU: day 5, 0.35 +/- 0.14 vs. day 7, 0.33 +/- 0.11, n = 5]. In contrast, polysome distribution analysis indicated that AT(1a)R mRNA was more efficiently translated in day 7 cells compared with day 5 [% of AT(1a)R mRNA in fraction 2 out of total AT1R mRNA recovered from the sucrose gradient: day 5, 20.9 +/- 9.9 vs. day 7, 56.8 +/- 5.6, n = 3, P < 0.001]. Accompanying the polysome shift was 50% less RNA-protein complex (RPC) formation between VSMC cytosolic RNA binding proteins in day 7 cells compared with 5-day cultures and the 5' leader sequence (5'LS) of the AT(1a)R [5'LS RPC (AU): day 5, 0.62 +/- 0.15 vs. day 7, 0.23 +/- 0.03; n = 4, P < 0.05] and also with exon 2 [Exon 2 RPC (AU): day 5, 35.0 +/- 5.7 vs. day 7, 17.2 +/- 3.6; n = 4, P < 0.05]. Taken together, these results suggest that AT(1a)R expression is regulated by translation during VSMC proliferation in part by RNA binding proteins that interact within exon 2 in the 5'LS of the AT(1a)R mRNA.
Sujet(s)
Muscles lisses vasculaires/cytologie , Biosynthèse des protéines , Récepteur de type 1 à l'angiotensine-II/biosynthèse , Animaux , Aorte/cytologie , Aorte/métabolisme , Prolifération cellulaire , Cellules cultivées , Régulation de l'expression des gènes/physiologie , Muscles lisses vasculaires/métabolisme , Liaison aux protéines , ARN messager/métabolisme , Rats , Rats de lignée F344 , Récepteur de type 1 à l'angiotensine-II/génétiqueRÉSUMÉ
Previously, we showed that ANG II receptors in cultured rat renomedullary interstitial cells (RMICs) are osmotically regulated (19). The current study examined the mechanisms underlying this osmotic regulation in RMICs cultured in isoosmotic (300 mosmol/kgH2O) and hyperosmotic (600 mosmol/kgH2O) conditions. Radioligand competition analysis coupled with RNase protection assays (RPA) and ligand-mediated receptor internalization studies revealed that RMICs primarily express the type 1a angiotensin receptor (AT(1a)R). When cultured under hyperosmotic conditions, the density (B(max)) of AT1R in RMIC membranes decreased by 31% [B(max) (pmol/mg protein): 300 mosmol/kgH2O, 6.44 +/- 0.46 vs. 600 mosmol/kgH2O, 4.42 +/- 0.37, n = 8, P < 0.01], under conditions in which no detectable changes in AT(1a)R mRNA expression or in the kinetics of ligand-mediated AT1R internalization were observed. RNA electromobility shift assays showed that RNA protein complex (RPC) formation between RMIC cytosolic RNA binding proteins and the 5' leader sequence (5'LS) of the AT(1a)R was increased 1.5-fold under hyperosmotic conditions [5'LS RPC (arbitrary units): 300 mosmol/kgH2O, 0.79 +/- 0.08 vs. 600 mosmol/kgH2O, 1.17 +/- 0.07, n = 4, P < 0.01]. These results suggest that the downregulation of AT(1a)R expression in RMICs cultured under hyperosmotic conditions is regulated at the posttranscriptional level by RNA binding proteins that interact within the 5'LS of the AT(1a)R mRNA. The downregulation of AT(1a)R expression under hyperosmotic conditions may be an important mechanism by which the activity of ANG II is regulated in the hyperosmotic renal medulla.
Sujet(s)
Régulation de l'expression des gènes , Médulla rénale/cytologie , Médulla rénale/métabolisme , Récepteur de type 1 à l'angiotensine-II/métabolisme , Équilibre hydroélectrolytique/physiologie , Animaux , Cellules cultivées , Liaison aux protéines , ARN messager/génétique , ARN messager/métabolisme , Rats , Récepteur de type 1 à l'angiotensine-II/génétique , Transcription génétiqueRÉSUMÉ
INTRODUCTION: Melanin concentrating hormone is an orexigenic hypothalamic neuropeptide, which plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. METHODS: Although radioligand receptor binding assay has been one of the most powerful tools for receptor research and drug discovery, the limitations of radioisotopes and the problems related to safety and waste disposal limits their application in high throughput screening and has led to a growing interest in alternative, nonradioactive technologies. To develop a sensitive and reproducible assay system for MCH1, the time-resolved fluorescence (TRF) receptor binding assay with AcroWell filter plates was tested and validated. RESULTS: Comparing to the radioligand receptor binding assay for MCH1, the TRF assay presented higher Z/Z' factors with the lower signal-to-noise ratio. The known high-affinity MCH1 receptor antagonist, SNAP-7941, exhibited an IC50 value of 1.66+/-0.10 nM that is very similar to the IC50 value of MCH in a radioligand binding assay with an excellent correlation coefficient (0.9884). DISCUSSION: These results suggest that our TRF receptor binding assay for MCH1 can achieve the desired sensitivity and reproducibility to replace the radioligand receptor assay in a fluorometric system that can be developed for high throughput screening.
Sujet(s)
Fluorimétrie/méthodes , Mélanines/antagonistes et inhibiteurs , Pipéridines/pharmacologie , Pyrimidines/pharmacologie , Récepteur hormone hypophysaire/antagonistes et inhibiteurs , Récepteur hormone hypophysaire/analyse , Animaux , Dosage biologique , Relation dose-effet des médicaments , Humains , Concentration inhibitrice 50 , Ligands , Mélanines/génétique , Mélanines/physiologie , Souris , Dosage par compétition , Rats , Récepteur hormone hypophysaire/génétique , Récepteur hormone hypophysaire/métabolisme , Reproductibilité des résultats , Sensibilité et spécificité , Facteurs tempsRÉSUMÉ
The present study was performed to evaluate the cardioprotective effects of [5-(2-methoxy-5-chloro-5-phenyl)furan-2-ylcarbonyl]guanidine (KR-32570) in rat and dog models of coronary artery occlusion and reperfusion. In addition, we sought to clarify the efficacy of KR-32570 on reperfusion-induced fatal ventricular arrhythmia. In anesthetized rats subjected to 45-min coronary occlusion and 90-min reperfusion, KR-32570 (i.v. bolus) dose-dependently reduced myocardial infarct size from 58.0% to 50.7%, 35.3%, 33.5% and 27.0% for 0.03, 0.1, 0.3 and 1.0 mg/kg, respectively (P<0.05). In anesthetized beagle dogs that underwent 1.2-h occlusion followed by 3.0-h reperfusion, KR-32570 (3 mg/kg, i.v. bolus) markedly decreased infarct size from 28.9% in vehicle-treated group to 8.0% (P<0.05), and reduced the reperfusion-induced release in creatine kinase isoenzyme MB, lactate dehydrogenase, Troponin-I and glutamic-oxaloacetic transaminase. KR-32570 dose-dependently decreased the incidence of premature ventricular contraction, ventricular tachycardia or ventricular fibrillation induced by ischemia and reperfusion in rats. Similar results were obtained in dogs with reperfusion-induced arrhythmia. In separate experiments to assess the effects of timing of treatment, KR-32570 given 10 min before or at reperfusion in rat models also significantly reduced the myocardial infarct size (40.9% and 46.1%, respectively) compared with vehicle-treated group. In all studies, KR-32570 caused no significant changes in any hemodynamic profiles. Taken together, these results indicate that KR-32570 significantly reduced the myocardial infarction and incidence of arrhythmias induced by ischemia and reperfusion in rats and dogs, without affecting hemodynamic profiles. Thus, it could be potentially useful in the prevention and treatment of myocardial injuries and lethal ventricular arrhythmias.
Sujet(s)
Troubles du rythme cardiaque/prévention et contrôle , Cardiotoniques/pharmacologie , Guanidines/pharmacologie , Infarctus du myocarde/prévention et contrôle , Lésion de reperfusion myocardique/prévention et contrôle , Antiport des ions sodium-hydrogène/antagonistes et inhibiteurs , Animaux , Troubles du rythme cardiaque/étiologie , Troubles du rythme cardiaque/physiopathologie , Cardiotoniques/administration et posologie , MB Creatine kinase/sang , Modèles animaux de maladie humaine , Chiens , Relation dose-effet des médicaments , Guanidines/administration et posologie , Ventricules cardiaques/effets des médicaments et des substances chimiques , Ventricules cardiaques/physiopathologie , L-Lactate dehydrogenase/sang , Mâle , Contraction myocardique/effets des médicaments et des substances chimiques , Contraction myocardique/physiologie , Infarctus du myocarde/étiologie , Infarctus du myocarde/physiopathologie , Lésion de reperfusion myocardique/complications , Lésion de reperfusion myocardique/physiopathologie , Rats , Rat Sprague-Dawley , Tachycardie ventriculaire/physiopathologie , Tachycardie ventriculaire/prévention et contrôle , Facteurs temps , Troponine I/sang , Fibrillation ventriculaire/physiopathologie , Fibrillation ventriculaire/prévention et contrôleRÉSUMÉ
The present study was performed to evaluate the cardioprotective effects of [5-(2-methoxy-5-chloro-5-phenyl)furan-2-ylcarbonyl]guanidine (KR-32570) on ischemia/reperfusion-induced mechanical and metabolic dysfunction in isolated rat hearts. In addition, the effects of KR-32570 on the Na(+)/H(+)-exchanger (NHE) and lipid peroxidation were also evaluated. KR-32570 strongly inhibited the recovery from acidosis induced by an NH(4)Cl prepulse in PS120 fibroblast cells expressing the human NHE-1 isoform (IC(50): 0.05 and 1.16 microM for KR-32570 and cariporide, respectively). In isolated perfused rat hearts subjected to 30-min ischemia/30-min reperfusion, KR-32570 (1-10 microM) significantly and concentration dependently improved cardiac contractile function and severe contracture in conjunction with causing a marked reduction in lactate dehydrogenase release. Additionally, it (1-10 microM) significantly increased the content of ATP, creatine phosphate and glycogen as well as decreased the tissue lactate content in heart homogenates following ischemia and reperfusion. KR-32570 (1-10 microM) significantly decreased the concentration of 8-iso-prostaglandin F(2 alpha), a reliable marker for oxidant stress, in perfusates from rat hearts subjected to ischemia and reperfusion. In separate experiments, KR-32570 significantly lowered the concentration of malondialdehyde in rat liver homogenate and inhibited Cu(2+)-induced peroxidation of low-density lipoprotein. Taken together, these results suggest that KR-32570 possesses potent cardioprotective effects in perfused rat hearts, and its effects may be mediated by inhibition of NHE-1, preservation of high-energy phosphates, and inhibition of lipid peroxidation.