RESUMO
This study aimed to determine whether electrical stimulation-based twitch exercise is effective in inhibiting the progression of immobilization-induced muscle fibrosis. 19 Wistar rats were randomly divided into a control group (n=6), an immobilization group (n=6; with immobilization only), and a Belt group (n=7; with immobilization and twitch exercise through the belt electrode device, beginning 2 weeks after immobilization). The bilateral soleus muscles were harvested after the experimental period. The right soleus muscles were used for histological analysis, and the left soleus muscles were used for biochemical and molecular biological analysis. As a result, in the picrosirius red images, the perimysium and endomysium were thicker in both the immobilization and Belt groups compared to the control group. However, the perimysium and endomysium thickening were suppressed in the Belt group. The hydroxyproline content and alpha-SMA, TGF-beta1, and HIF-1alpha mRNA expressions were significantly higher in the immobilization and belt groups than in the control group. These expressions were significantly lower in the Belt group than in the immobilization group. The capillary-to-myofiber ratio and the mRNA expressions of VEGF and PGC-1alpha were significantly lower in the immobilization and belt groups than in the control group, these were significantly higher in the Belt group than in the immobilization group. From these results, Electrical stimulation-based twitch exercise using the belt electrode device may prevent the progression of immobilization-induced muscle fibrosis caused by downregulating PGC-1alpha/VEGF pathway, we surmised that this intervention strategy might be effective against the progression of muscle contracture. Keywords: Immobilization, Skeletal muscle, Fibrosis, Electrical stimulation-based twitch exercise, PGC-1alpha/VEGF pathway.
Assuntos
Regulação para Baixo , Fibrose , Músculo Esquelético , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fator A de Crescimento do Endotélio Vascular , Animais , Masculino , Ratos , Progressão da Doença , Estimulação Elétrica , Terapia por Estimulação Elétrica/métodos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Doenças Musculares/prevenção & controle , Doenças Musculares/etiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Condicionamento Físico Animal/fisiologia , Ratos Wistar , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Although electrical muscle stimulation (EMS) of skeletal muscle effectively prevents muscle atrophy, its effect on the breakdown of muscle component proteins is unknown. In this study, we investigated the biological mechanisms by which EMS-induced muscle contraction inhibits disuse muscle atrophy progression. Experimental animals were divided into a control group and three experimental groups: immobilized (Im; immobilization treatment), low-frequency (LF; immobilization treatment and low-frequency muscle contraction exercise), and high-frequency (HF; immobilization treatment and high-frequency muscle contraction exercise). Following the experimental period, bilateral soleus muscles were collected and analyzed. Atrogin-1 and Muscle RING finger 1 (MuRF-1) mRNA expression levels were significantly higher for the experimental groups than for the control group but were significantly lower for the HF group than for the Im group. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) mRNA and protein expression levels in the HF group were significantly higher than those in the Im group, with no significant differences compared to the Con group. Both the Forkhead box O (FoxO)/phosphorylated FoxO and protein kinase B (AKT)/phosphorylated AKT ratios were significantly lower for the Im group than for the control group and significantly higher for the HF group than for the Im group. These results, the suppression of atrogin-1 and MuRF-1 expression for the HF group may be due to decreased nuclear expression of FoxO by AKT phosphorylation and suppression of FoxO transcriptional activity by PGC-1alpha. Furthermore, the number of muscle contractions might be important for effective EMS.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Fatores de Transcrição , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , PPAR gama/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/prevenção & controle , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Proteínas Musculares/metabolismo , RNA Mensageiro/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismoRESUMO
This study investigated the effects of wheel-running using the upper limbs following immobilization after inducing arthritis in the knees of rats. Forty male Wistar rats (aged 8 weeks) divided into four groups randomly: arthritis (AR), immobilization after arthritis (Im), wheel-running exercise with the upper limbs following immobilization after arthritis induction (Im+Ex) and sham arthritis induction (Con). The knee joints of the Im and Im+Ex groups were immobilized with a cast for 4 weeks. In the Im+Ex group, wheel-running exercise was administered for 60 min/day (5 times/week). The swelling and the pressure pain threshold (PPT) of the knee joint were evaluated for observing the condition of inflammatory symptoms in affected area, and the paw withdraw response (PWR) was evaluated for observing the condition of secondary hyperalgesia in distant area. Especially, in order to evaluate histological inflammation in the knee joint, the number of macrophage (CD68-positive cells) in the synovium was examined. The expression of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn (L2-3 and L4-5) was examined to evaluate central sensitization. The Im+Ex group showed a significantly better recovery than the Im group in the swelling, PPTs, and PWRs. Additionally, CGRP expression of the spinal dorsal horn (L2-3 and L4-5) in the Im+Ex group was significantly decreased compared with the Im group. According to the results, upper limb exercise can decrease pain in the affected area, reduce hyperalgesia in distant areas, and suppress the central sensitization in the spinal dorsal horn by triggering exercise-induced hypoalgesia (EIH).
Assuntos
Artrite/patologia , Imobilização/métodos , Inflamação/prevenção & controle , Articulação do Joelho/fisiopatologia , Dor/prevenção & controle , Condicionamento Físico Animal/métodos , Extremidade Superior/fisiologia , Animais , Artrite/etiologia , Artrite/reabilitação , Modelos Animais de Doenças , Inflamação/patologia , Masculino , Dor/patologia , Ratos , Ratos Wistar , Corno Dorsal da Medula Espinal/patologiaRESUMO
BACKGROUND: Although researchers have recommended exercise training and psychosocial intervention to manage chronic pain, an effective intervention for Japanese community-dwelling older adults with chronic pain has not been established. This randomized controlled trial examined whether exercise training combined with psychosocial intervention more effectively improves pain, psychological status and physical activity than does exercise training alone in this population. METHODS: We randomized 128 older adults with chronic pain to either an intervention group (n = 64) involving exercise training combined with psychosocial intervention, or a control group (n = 64) involving only exercise training. Exercise training comprised weekly 60-min sessions for 12 weeks. Psychosocial intervention involved changing participants' focus on pain using self-management education and cognitive behavioural therapy, and participants recorded their daily pain intensity and step counts. Pain intensity, psychological status and physical activity were assessed before and 12 weeks after the intervention. RESULTS: A time-by-group interaction emerged for psychological status (p = 0.003) and physical activity (p < 0.001), both favouring the intervention group. The intervention group also showed greater improvement in pain intensity at 12 weeks than did the control group (p = 0.007). CONCLUSIONS: Exercise training combined with psychosocial intervention improves key outcome indicators more effectively than does exercise training alone in older adults with chronic pain. SIGNIFICANCE: Although research has shown that combined exercise and psychosocial intervention is optimal for managing chronic pain, our study is the first, to the best of our knowledge, to test a specific intervention of this type in community-dwelling older adults with chronic pain in Japan.
Assuntos
Dor Crônica/terapia , Terapia Cognitivo-Comportamental/métodos , Terapia por Exercício/métodos , Autogestão/métodos , Idoso , Idoso de 80 Anos ou mais , Exercício Físico , Feminino , Humanos , Vida Independente , Japão , Masculino , Saúde Mental , Medição da DorRESUMO
This study examined the aging effect on disuse muscle atrophy prevention using heat stress. Wistar rats aged 7 and 60 weeks were divided into three groups as follows: control, immobilized (Im), and immobilized and heat stressed (ImH). Heat stress was given by immersing the hindlimbs in hot water (42 °C) for 60 min, once in every 3 days and the gastrocnemius (GAS) and soleus (SOL) muscles were extracted after 14 days. Muscle-fiber types were classified using ATPase staining. Heat shock protein 70 (HSP70) was assessed through Western blotting. In GAS muscle of both groups and SOL muscle of 7-week-old rats, the fiber diameter of each muscle type in the ImH group significantly increased compared with that in the Im group. However, this could not be observed in the SOL muscle of the 60-week-old rats. The increased percentage of type-I fibers and variability of types I and II muscle-fiber diameter were evident in the SOL muscle of the 60-week rats. HSP70 was significantly elevated in the ImH group compared with in the Im group in both muscle types of both age groups. Thus, effectiveness of heat stress in the prevention of disuse muscle atrophy appears unsatisfactory in aging muscle fibers.
Assuntos
Envelhecimento , Proteínas de Choque Térmico HSP70/metabolismo , Hipertermia Induzida/métodos , Músculo Esquelético/fisiopatologia , Transtornos Musculares Atróficos/prevenção & controle , Transtornos Musculares Atróficos/fisiopatologia , Animais , Resposta ao Choque Térmico , Masculino , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Transtornos Musculares Atróficos/diagnóstico , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
This study investigated the effect of continuous passive motion (CPM) initiated after the onset of arthritis in rats. Rats were injected with 3 % kaolin/carrageenan in the knee joint and randomized to the control, immobilization (IM), or CPM group. The knee joints of the IM and CPM groups were immobilized with a cast for 56 days. In the CPM group, CPM exercise was administered for 60 min/day (6 times/week). Joint transverse diameter and pressure pain threshold (PPT) were assessed as indicators of inflammation, and paw withdrawal response (PWR) was assessed as indicator of secondary hyperalgesia. Central sensitization was analyzed by measuring calcitonin gene-related peptide (CGRP) expression levels in the spinal dorsal horn. In the CPM group, the PPT was significantly increased compared with the IM group from 14 to 35 days, and PWR was significantly decreased from 14 to 56 days. Additionally, CGRP expression in the super facial layer (I-II) of the spinal dorsal horn (L4-5) in the CPM group was significantly decreased compared with the IM group. Our study found the CPM initiated after the onset of arthritis promoted the recovery of inflammation and mitigated secondary hyperalgesia.
Assuntos
Artrite/complicações , Hiperalgesia/prevenção & controle , Inflamação/terapia , Terapia Passiva Contínua de Movimento , Dor/prevenção & controle , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Hiperalgesia/etiologia , Inflamação/etiologia , Masculino , Dor/etiologia , Limiar da Dor , Distribuição Aleatória , Amplitude de Movimento Articular , Ratos Wistar , Restrição Física , Corno Dorsal da Medula Espinal/metabolismoRESUMO
AIM: The effects of heat shock transcription factor 1 (HSF1) deficiency on the fibre type composition and the expression level of nuclear factor of activated T cells (NFAT) family members (NFATc1, NFATc2, NFATc3 and NFATc4), phosphorylated glycogen synthase kinase 3α (p-GSK3α) and p-GSK3ß, microRNA-208b (miR-208b), miR-499 and slow myosin heavy chain (MyHC) mRNAs (Myh7 and Myh7b) of antigravitational soleus muscle in response to unloading with or without reloading were investigated. METHODS: HSF1-null and wild-type mice were subjected to continuous 2-week hindlimb suspension followed by 2- or 4-week ambulation recovery. RESULTS: In wild-type mice, the relative population of slow type I fibres, the expression level of NFATc2, p-GSK3 (α and ß), miR-208b, miR-499 and slow MyHC mRNAs (Myh7 and Myh7b) were all decreased with hindlimb suspension, but recovered after it. Significant interactions between train and time (the relative population of slow type I fibres; P = 0.01, the expression level of NFATc2; P = 0.001, p-GSKß; P = 0.009, miR-208b; P = 0.002, miR-499; P = 0.04) suggested that these responses were suppressed in HSF1-null mice. CONCLUSION: HSF1 may be a molecule in the regulation of the expression of slow MyHC as well as miR-208b, miR-499, NFATc2 and p-GSK3 (α and ß) in mouse soleus muscle.
Assuntos
Fatores de Transcrição de Choque Térmico/biossíntese , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Cadeias Pesadas de Miosina/biossíntese , Animais , Peso Corporal/fisiologia , Quinase 3 da Glicogênio Sintase/biossíntese , Quinase 3 da Glicogênio Sintase/genética , Gravitação , Fatores de Transcrição de Choque Térmico/genética , Elevação dos Membros Posteriores , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , MicroRNAs/genética , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/citologia , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/genética , Tamanho do Órgão/fisiologia , Recuperação de Função FisiológicaRESUMO
Four monoclonal antibodies (mAbs) against 4-aminobenzoate hydroxylase (EC 1.14.13.27) have been produced (H. Tsuji et al., J. Biol. Chem. 265 (1990) 16064; T. Ogawa et al., Biochim. Biophys. Acta 1115 (1992) 220). Of the mAbs, three mAbs (mAb-A, -B1 and -B2) recognize the FAD-binding domain of the enzyme. In the present study, the epitopes of the mAbs on the enzyme have been examined using pGEX-2T expression systems for DNA fragments encoding various partial amino acid sequences of 4-aminobenzoate hydroxylase. The epitopes for mAb-A, -B1 and -B2 were shown to be on sequences 413-434, 435-460 and 380-413, respectively. These findings suggest that these epitopes for mAb-A, -B1 and -B2 may be close to the isoalloxazine moiety of FAD, which plays a central role in the catalysis of the enzyme.
Assuntos
Agaricus/enzimologia , Anticorpos Monoclonais/imunologia , Epitopos/análise , Oxigenases de Função Mista/imunologia , Proteínas Recombinantes de Fusão/imunologia , Anticorpos Monoclonais/química , DNA/biossíntese , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/imunologia , Glutationa Transferase/química , Glutationa Transferase/imunologia , Oxigenases de Função Mista/química , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/químicaRESUMO
A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.
Assuntos
Alérgenos/genética , Glicoproteínas/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Proteínas de Soja , Glycine maxRESUMO
We had previously found that a perchloric acid-soluble protein (PSP1) occurs in rat liver, and that this novel protein inhibits protein synthesis in a rabbit reticulocyte lysate system (T. Oka, H. Tsuji, C. Noda, K. Sakai, Y.-H. Hong, I. Suzuki, S. Muñoz, Y. Natori, J. Biol. Chem. 270 (1995) 30060-30067). In the present study, we analyzed lipid components bound to PSP1. Native PSP1 was purified from rat liver using Sephadex G-75, DE-52 cellulose and IgGPSP-affinity chromatography, and the lipid components were extracted. The components obtained from the purified PSP1 were shown to be free fatty acids by thin-layer chromatography. By GC-MS, six major fatty acids were identified as 14:0, 16:0, 18:0, 18:1, 18:2 and 20:4. 1 mol of PSP1 contained 1.26 mol of total fatty acid components. The fatty acid-binding assay of PSP1 showed that the Bmax was 1.25 mol fatty acid/mol PSP1 and the Kd value for palmitic acid was 6.03 microM. The concentration of PSP1 mRNA in rat liver increased 2.3-fold by the administration of peroxisome proliferator, bezafibrate. These findings show that PSP1 is a fatty acid-binding protein-like protein, which is involved in the intracellular metabolism of fatty acid and is quite different from the known fatty acid-binding proteins.
Assuntos
Ácidos Graxos/análise , Proteínas de Choque Térmico/química , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Ribonucleases , Animais , Proteínas de Transporte/química , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/isolamento & purificação , Fígado/metabolismo , Masculino , Proteína P2 de Mielina/química , Miocárdio/metabolismo , Inibidores da Síntese de Proteínas/química , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Proteínas Recombinantes/químicaRESUMO
Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. Previously, we developed NPY-overexpressing mice by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but we failed to observe the obese phenotype in the heterozygote. Here we report that in the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibited significantly increased body weight gain with transiently increased food intake after 50% sucrose--loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of our observation period.
Assuntos
Arginina/análogos & derivados , Encéfalo/fisiologia , Sacarose Alimentar/farmacologia , Neuropeptídeo Y/fisiologia , Obesidade/fisiopatologia , Envelhecimento , Animais , Arginina/farmacologia , Cicloexanos/farmacologia , Ingestão de Energia/efeitos dos fármacos , Homozigoto , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuropeptídeo Y/genética , Obesidade/induzido quimicamente , Obesidade/genética , Fenótipo , Regiões Promotoras Genéticas , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Valores de Referência , Antígenos Thy-1/genética , Xantenos/farmacologiaRESUMO
The purpose of this study was to investigate the influence of heat treatment on glucocorticoid (GC)-induced myopathy. Eight-week-old Wistar rats were randomly assigned to the control, Dex, and Dex + Heat groups. Dexamethasone (2 mg/kg) was injected subcutaneously 6 days per week for 2 weeks in the Dex and Dex + Heat group. In the Dex + Heat group, heat treatment was performed by immersing hindlimbs in water at 42 °C for 60 min, once every 3 days for 2 weeks. The extensor digitorum longus muscle was extracted following 2 weeks of experimentation. In the Dex + Heat group, muscle fiber diameter, capillary/muscle fiber ratio, and level of heat shock protein 72 were significantly higher and atrogene expression levels were significantly lower than in the Dex group. Our results suggest that heat treatment inhibits the development of GC-induced myopathy by decreasing atrogene expression and increasing angiogenesis.
Assuntos
Dexametasona/efeitos adversos , Glucocorticoides/efeitos adversos , Temperatura Alta/uso terapêutico , Atrofia Muscular/prevenção & controle , Doenças Musculares/induzido quimicamente , Doenças Musculares/prevenção & controle , Animais , Proteínas de Choque Térmico HSP72/metabolismo , Masculino , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Doenças Musculares/complicações , Doenças Musculares/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Distribuição Aleatória , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.
Assuntos
Crista Neural/química , Peptídeos/metabolismo , Receptores de Neuropeptídeo Y/análise , Receptores de Neurotransmissores/análise , Animais , Glioma/química , Glioma/patologia , Glioma/ultraestrutura , Humanos , Radioisótopos do Iodo , Ligantes , Crista Neural/patologia , Crista Neural/ultraestrutura , Neuroblastoma/química , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Células PC12 , Peptídeo YY , Ratos , Células Tumorais CultivadasRESUMO
We have shown previously that peptide YY (PYY) receptors are uniquely distributed in various mammalian brains and also have identified the receptor from porcine hippocampal membranes as a protein of 50,000 mol wt. To extend these observations, both the characteristics of PYY-receptor interaction and the structure of the receptor have been examined and compared with those of its sister peptide, neuropeptide Y (NPY), in the brains of various vertebrates including mammals (human, dog, guinea pig, rat, and mouse), birds (chicken), reptiles (snapping turtle), amphibians (bullfrog), and fish (yellowtail fish). The affinities and relative potencies of PYY as well as NPY receptors for pancreatic polypeptide (PP) family peptides were about the same in all species examined except for chickens. PYY and NPY bound to both the PYY and NPY receptors with high affinities, but porcine and avian PPs did not. In chicken brain, however, PYY, NPY, porcine PP, and avian PP all bound to the receptors with high affinity. Analysis of the equilibrium binding data for PYY receptors produced curvilinear Scatchard plots in all of the species, suggesting the existence of high and low affinity binding sites. Affinity cross-linking using disuccinimidyl suberate followed by electrophoretic analysis of ligand-receptor complexes characterized the molecular size of PYY and NPY receptors. [125I]PYY was cross-linked to a protein of 50,000 mol wt without sulfhydryl-bonded subunits on mammalian hippocampal membranes. A receptor protein with the same mol wt was identified in other brain areas, including hypothalamus and pituitary, PYY receptors in other vertebrate brains were similar in size to those of mammalian species except in chicken brain, where a receptor protein of 67,000 mol wt was observed. In addition, we also have demonstrated that the NPY receptor is a monomeric 50,000 and 55,000 mol wt protein in mammalian and fish brains, respectively. These findings indicate that brain PYY and NPY receptors in most vertebrate species from fish to man are pharmacologically and structurally similar and have been well conserved over a period of evolution of 400 million yr. The divergence of the receptors observed in chicken brain may reflect some change in their function.
Assuntos
Evolução Biológica , Encéfalo/metabolismo , Receptores de Neurotransmissores/metabolismo , Vertebrados/metabolismo , Animais , Reagentes de Ligações Cruzadas , Humanos , Masculino , Peso Molecular , Neuropeptídeo Y/metabolismo , Peptídeo YY , Peptídeos/metabolismo , Receptores de Neuropeptídeo Y , Receptores de Neurotransmissores/química , Succinimidas/metabolismoRESUMO
We have previously identified the peptide-YY (PYY) receptor on porcine brain membranes as a 50-kDa protein after chemical cross-linking. PYY receptors are discretely distributed in the brain of various mammals, to which neuropeptide-Y (NPY), but not pancreatic polypeptide (PP), bind with great specificity. The present study was carried out in order 1) to identify and characterize the PYY receptor in the avian brain, 2) to compare it with the APP receptor that had been demonstrated in the cerebellum, and 3) to examine [125I]APP-binding activity in the porcine brain. [125I]PYY was bound to chicken brain membranes via high affinity (Kd = 2.19 x 10(-10) M) and low affinity (Kd = 1.93 x 10(-7) M) components. The binding sites were highly specific for PYY and APP as well as for NPY and PPP, coupled to a guanine nucleotide regulatory protein, and distributed in various brain areas, including the cerebellum. The C-terminal fragments of PYY, PYY-(17-36) and PYY-(24-36), exhibited low potency in inhibiting binding, but behaved like full agonists. Porcine brain membranes, on the other hand, possessed two orders of the APP-binding sites, a high affinity component (Kd = 4.24 x 10(-9) M) and a low affinity component (Kd = 3.08 x 10(-7) M). APP binding showed a high specificity for APP, but not for PPP, NPY, or PYY. The binding activity was highest in the pituitary gland, followed by the hippocampus, amygdala, cerebral cortex, hypothalamus, and cerebellum. Guanosine 5'-O-thiotriphosphate, a nonhydrolyzable GTP analog, did not inhibit the binding of [125I]APP to porcine or chicken brain membranes, which ran counter to the results of PYY receptors in both species. Cross-linking studies have demonstrated that receptor-bound [125I]APP is cross-linked to a protein of 67 kDa without disulfide-linked subunits in both porcine and chicken brain membranes. In the latter species, [125I]PYY and [125I]NPY were also cross-linked to the same 67-kDa proteins, which were different from the receptor proteins (50 kDa) in mammalian species. These results indicate that chicken brain has receptors specific for PYY and NPY, as was found in mammalian brains, and that PYY, NPY, and PP act in the brain through interaction at multiple receptor sites, which are similar to and shared by other members of the PP family. Furthermore, the finding that APP-binding sites in porcine brain are more specific than those in avian brain suggests that an endogenous peptide similar to APP may exist in porcine brain.
Assuntos
Encéfalo/metabolismo , Neuropeptídeo Y/metabolismo , Polipeptídeo Pancreático/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Neurotransmissores/metabolismo , Animais , Ligação Competitiva , Galinhas , Cinética , Especificidade de Órgãos , Receptores de Neuropeptídeo Y , Especificidade da Espécie , SuínosRESUMO
We investigated the penetration of plasma pancreatic polypeptide (PP) into the third cerebral ventricular fluid (CSF) of dogs. Plasma and CSF levels of PP were measured by RIA during the iv infusion of PP and during such stimuli as eating, insulin-induced hypoglycemia, and physical exercise. Plasma and CSF levels of insulin and glucose were also measured and compared during eating and insulin-induced hypoglycemia. Plasma glucose increased after feeding and decreased after insulin injection, followed by a corresponding change in CSF glucose without an apparent time lag. CSF insulin insignificantly increased after feeding and the injection of insulin, while CSF PP did not increase despite the marked elevation of plasma PP in response to these stimuli. CSF PP did not increase after the infusion of exogenous PP. Strenuous exercise, however, evoked an increase in both plasma and CSF PP levels; the CSF response was prompt, but more prolonged than that of plasma, suggesting the slow removal of PP from CSF. We conclude that 1) PP and insulin in CSF do not appear to play a major role in the short term regulation of food intake and acute changes in energy metabolism; and 2) PP, probably after entering the brain, may modulate brain function in such physiological situations as strenuous exercise.
Assuntos
Ventrículos Cerebrais/fisiologia , Ingestão de Alimentos , Hipoglicemia/metabolismo , Insulina/farmacologia , Polipeptídeo Pancreático/metabolismo , Condicionamento Físico Animal , Animais , Glicemia/metabolismo , Temperatura Corporal , Ventrículos Cerebrais/efeitos dos fármacos , Cães , Hidrocortisona/sangue , Hipoglicemia/induzido quimicamente , Insulina/sangue , Insulina Regular de Porco , Cinética , Polipeptídeo Pancreático/sangue , Polipeptídeo Pancreático/líquido cefalorraquidiano , Fatores de TempoRESUMO
We studied the pharmacological and structural characteristics of peptide YY (PYY) receptors in porcine brain. PYY and neuropeptide Y (NPY) bound with high affinity to crude membrane preparations from the hippocampus, but their C-terminal fragments bound with 30- to 450-fold lower affinity Guanine nucleotides, especially the nonhydrolyzable GTP analog GTP gammas inhibited the binding of [125I]PYY, but other transmitters, hormones, and central nervous system-acting drugs did not, except for gangliosides at high concentrations. These results suggest that PYY receptor binding resides in the N-terminal part of the PYY molecule and is coupled to a guanine nucleotide regulatory protein. To elucidate PYY receptor structure, we used the cross-linking reagent disuccinimidyl suberate to covalently attach [125I]PYY to its receptors in the hippocampus membrane. Gel electrophoresis followed by autoradiography revealed a band centered at the mol wt of 50,000. Competitive inhibition of binding by unlabeled PYY resulted in a parallel inhibition of labeling of the 50,000 mol wt protein. The reducing agent 2-mercaptoethanol did not affect the appearance of this band, suggesting that the PYY receptor does not have subunits connected by sulfhydryl bonds. Furthermore, cross-linking [125I]NPY to its receptors in the porcine hippocampus produced the same 50,000 mol wt band regardless of 2-mercaptoethanol. The identity in the size of NPY and PYY receptors together with their similarities in binding properties including regional distribution and peptide specificity, indicate that NPY and PYY regulate brain function through interaction at a common receptor site.
Assuntos
Encéfalo/metabolismo , Receptores de Neurotransmissores/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Nucleotídeos de Guanina/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Hipocampo/metabolismo , Mercaptoetanol/farmacologia , Peso Molecular , Neuropeptídeo Y/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeo YY , Peptídeos/metabolismo , Receptores de Neuropeptídeo Y , Receptores de Neurotransmissores/efeitos dos fármacos , Succinimidas , Suínos , Tionucleotídeos/farmacologia , Distribuição TecidualRESUMO
We have previously characterized peptide-YY (PYY) receptors in porcine hippocampal membranes. We demonstrate here that brain PYY receptors can be extracted in the active state using digitonin. Among several detergents tested for their suitability to extract active PYY receptors, digitonin gave the most favorable results, as judged by specific binding of [125I]PYY to the solubilized receptors. The binding of [125I]PYY to digitonin extract was dependent on incubation time, temperature, and protein and magnesium ion concentrations and had a pH optimum of 6-7. Solubilized PYY receptors maintained the rank order of potencies for various related peptides and PYY fragments characteristic of the membrane PYY receptor: PYY greater than neuropeptide Y (NPY) much much greater than avian and porcine pancreatic polypeptide, and PYY greater than PYY-(22-36) much much greater than PYY-(1-22) and PYY-(22-28), respectively. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract; the high affinity component had affinities and binding capacities similar to those of the membrane PYY receptor. Solubilized PYY receptors also retained their sensitivity to guanine nucleotides. PYY was cross-linked to its receptors with disuccinimidyl suberate, solubilized with digitonin, and cross-linked to digitonin-solubilized receptors. The resulting complexes were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Using these procedures, we identified a PYY receptor species with a molecular size of 50,000, which was the same size as the labeled protein in native membrane homogenates. Solubilized NPY receptors were also the same size. The solubilized cross-linked PYY receptor was adsorbed by wheat germ agglutinin-agarose and Concanavalin-A, suggesting its glycoprotein nature. These data suggest that the specific binding properties of the PYY receptor are inherent in the solubilized glycoprotein molecules. The solubilization in digitonin of PYY receptors from membranes should allow a more complete molecular and functional characterization of PYY-mediated events and purification of the receptor.
Assuntos
Hipocampo/química , Peptídeos/metabolismo , Receptores de Neurotransmissores/isolamento & purificação , Adsorção , Animais , Cromatografia em Gel , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Técnicas In Vitro , Lectinas/metabolismo , Peso Molecular , Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/farmacologia , Peptídeo YY , Receptores de Neuropeptídeo Y , Receptores de Neurotransmissores/efeitos dos fármacos , Solubilidade , SuínosRESUMO
When n-octyl-beta-D-glucoside was used in several detergents to extract active avian pancreatic polypeptide (APP) receptors, a specific binding of [125I]APP to the solubilized chicken cerebellar and porcine hippocampal membranes was found. The binding of [125I]APP to the solubilized receptors was dependent on incubation time, temperature, and protein concentrations and appeared to have a slightly acidic optimal pH. APP binding to chicken and porcine brain extracts showed a high specificity for APP, although the chicken receptors do not discriminate well between APP and its related peptides, neuropeptide Y and peptide YY. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the brain extracts as in membrane-bound receptors; however, the high affinity component of the chicken receptor showed a decreased affinity after extraction. APP receptors in chicken and porcine brain extracts retained their insensitivity to the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate). Cross-linking studies were performed with the homobifunctional cross-linker disuccinimidyl suberate and brain membrane receptors solubilized with n-octyl-beta-D-glucoside. An APP receptor species with a M(r) of 67,000, the same size as that of the labeled protein in native membrane homogenates of chicken and pig brains, was identified. However, in the canine brain we observed a M(r) 85,000 receptor protein, suggesting that species differences exist among the structures of brain APP receptors. The solubilized cross-linked APP receptors in these species were adsorbed by wheat germ agglutinin-agarose and by concanavalin A, indicating that they are glycoprotein in nature. The availability of the solubilized receptors from vertebrate brains with n-octyl-beta-D-glucoside represents an important step toward the purification and molecular characterization of the APP receptors.
Assuntos
Encéfalo/metabolismo , Polipeptídeo Pancreático/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Membrana Celular/metabolismo , Galinhas , Detergentes , Cães , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Hipocampo/metabolismo , Radioisótopos do Iodo , Cinética , Lectinas , Magnésio/farmacologia , Receptores dos Hormônios Gastrointestinais/efeitos dos fármacos , Receptores dos Hormônios Gastrointestinais/isolamento & purificação , Solubilidade , Especificidade da Espécie , SuínosRESUMO
A new series of 3-(4-hydroxy-1-naphthalenyl)-2-propenoic acids was prepared and the inhibitory activities of its members on IL-1 generation were evaluated both by in vitro systems using human monocytes and/or rat exudated macrophages stimulated with LPS, and by an in vivo system using the rat CMC-LPS air-pouch model. Many compounds in this series were found to be potent inhibitors of IL-1 generation both in vitro and in vivo. Structure-activity relationships indicated that in the rat CMC-LPS air-pouch model by oral administration the (Z)-2-substituted propenoic acids with 3-alkoxy, 5-alkyl, and 4-hydroxy substituents on the naphthalene ring exhibit optimal inhibition. Among the compounds evaluated, (Z)-3-(5-ethyl-4-hydroxy-3-methoxy-1-naphthalenyl)-2-methyl-2-propeno ic acid (20a), which inhibited IL-1 generation from human monocytes with an IC50 value of 3.0 microM and had an IC50 value of 1.4 microM for rat exudated macrophages, showed the most potent inhibitory activity in the rat CMC-LPS model by oral administration. Compound 20a also showed antiinflammatory effects in animal models of inflammation.