RESUMO
OBJECTIVE: We conducted a systematic review in order to understand the relationship between imaging-visualised meniscus pathologies, hyaline cartilage, joint replacement and pain in knee osteoarthritis (OA). DESIGN: A search of the Medline, Excerpta Medica database (EMBASE) and Cochrane library databases was performed for original publications reporting association between imaging-detected meniscal pathology (extrusion or tear/damage) and longitudinal and cross-sectional assessments of hyaline articular cartilage loss [assessed on magnetic resonance imaging (MRI)], incident joint replacement and pain (longitudinal and cross-sectional) in knee OA. Each association was qualitatively characterised by a synthesis of data from each analysis, based upon study design and quality scoring (including risk of bias assessment and adequacy of covariate adjustment using Cochrane recommended methodology). RESULTS: In total 4,878 abstracts were screened and 82 publications were included (comprising 72 longitudinal analyses and 49 cross-sectional). Using high quality, well-adjusted data, meniscal extrusion and meniscal tear/damage were associated with longitudinal progression of cartilage loss, cross-sectional cartilage loss severity and joint replacement, independently of age, sex and body mass index (BMI). Medial and lateral meniscal tears were associated with cartilage loss when they occurred in the body and posterior horns, but not the anterior horns. There was a lack of high quality, well-adjusted meniscal pathology and pain publications and no clear independent association between meniscal extrusion or tear/damage with pain severity, progression in pain or incident frequent knee symptoms. CONCLUSION: Meniscal features have strong associations with cartilage loss and joint replacement in knee OA, but weak associations with knee pain. Systematic review PROSPERO registration number: CRD 42020210910.
Assuntos
Artroplastia de Substituição , Cartilagem Articular , Osteoartrite do Joelho , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Estudos Transversais , Humanos , Articulação do Joelho/patologia , Imageamento por Ressonância Magnética/métodos , Meniscos Tibiais/diagnóstico por imagem , Meniscos Tibiais/patologia , Meniscos Tibiais/cirurgia , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/cirurgia , Dor/patologiaRESUMO
BACKGROUND: Radiographic measures of osteoarthritis (OA) are based upon two dimensional projection images. Active appearance modelling (AAM) of knee magnetic resonance imaging (MRI) enables accurate, 3D quantification of joint structures in large cohorts. This cross-sectional study explored the relationship between clinical characteristics, radiographic measures of OA and 3D bone area (tAB). METHODS: Clinical data and baseline paired radiographic and MRI data, from the medial compartment of one knee of 2588 participants were obtained from the NIH Osteoarthritis Initiative (OAI). The medial femur (MF) and tibia (MT) tAB were calculated using AAM. 'OA-attributable' tAB (OA-tAB) was calculated using data from regression models of tAB of knees without OA. Associations between OA-tAB and radiographic measures of OA were investigated using linear regression. RESULTS: In univariable analyses, height, weight, and age in female knees without OA explained 43.1%, 32.1% and 0.1% of the MF tAB variance individually and 54.4% when included simultaneously in a multivariable model. Joint space width (JSW), osteophytes and sclerosis explained just 5.3%, 14.9% and 10.1% of the variance of MF OA-tAB individually and 17.4% when combined. Kellgren Lawrence (KL) grade explained approximately 20% of MF OA-tAB individually. Similar results were seen for MT OA-tAB. CONCLUSION: Height explained the majority of variance in tAB, confirming an allometric relationship between body and joint size. Radiographic measures of OA, derived from a single radiographic projection, accounted for only a small amount of variation in 3D knee OA-tAB. The additional structural information provided by 3D bone area may explain the lack of a substantive relationship with these radiographic OA measures.
Assuntos
Fêmur/patologia , Osteoartrite do Joelho/patologia , Tíbia/patologia , Fatores Etários , Idoso , Estatura , Peso Corporal , Feminino , Fêmur/diagnóstico por imagem , Humanos , Imageamento Tridimensional , Modelos Lineares , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Obesidade/complicações , Tamanho do Órgão , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/diagnóstico por imagem , Osteófito/diagnóstico por imagem , Osteófito/etiologia , Osteófito/patologia , Radiografia , Tíbia/diagnóstico por imagemRESUMO
BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance. METHODS AND RESULTS: The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed significantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins. CONCLUSION: Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity.
RESUMO
SUMMARY BACKGROUND: We have previously shown that the receptor-like protein tyrosine phosphatase (PTP) CD148 is essential for initiating glycoprotein VI (GPVI) signaling in platelets. We proposed that CD148 does so by dephosphorylating the C-terminal inhibitory tyrosine of Src family kinases (SFKs). However, this mechanism is complicated by CD148-deficient mouse platelets having a concomitant reduction in GPVI expression. OBJECTIVES: To investigate the effect of CD148 on GPVI signaling independent of the decrease in GPVI expression and to further establish the molecular basis of the activatory effect of CD148 and downregulation of GPVI. METHODS: CD148-deficient mouse platelets were investigated for functional and biochemical defects. The DT40/NFAT-lucifierase reporter assay was used to analyze the effect of CD148 on GPVI signaling. CD148-SFK interactions and dephosphorylation were quantified using biochemical assays. RESULTS: CD148-deficient mouse platelets exhibited reduced collagen-mediated aggregation, secretion and spreading in association with reduced expression of GPVI and FcR gamma-chain and reduced tyrosine phosphorylation. The phosphorylation status of SFKs suggested a global reduction in SFK activity in resting CD148-deficient platelets. Studies in a cell model confirmed that CD148 inhibits GPVI signaling independent of a change in receptor expression and through a mechanism dependent on tyrosine dephosphorylation. Recombinant CD148 dephosphorylated the inhibitory tyrosines of Fyn, Lyn and Src in vitro, although paradoxically it also dephosphorylated the activation loop of SFKs. CONCLUSIONS: CD148 plays a critical role in regulating GPVI/FcR gamma-chain expression and maintains a pool of active SFKs in platelets by directly dephosphorylating the C-terminal inhibitory tyrosines of SFKs that is essential for platelet activation.
Assuntos
Colágeno/farmacologia , Ativação Plaquetária/genética , Quinases da Família src/metabolismo , Animais , Plaquetas , Células Cultivadas , Camundongos , Camundongos Knockout , Fosforilação , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/fisiologia , Receptores FcRESUMO
To define the role of regulators of G-protein signaling (RGS) in chemoattractant-mediated responses, RGS4 and the receptors for platelet-activating factor (PAFR), formylated peptides (FR), or interleukin-8 (CXCR1) were stably coexpressed in a rat basophilic leukemia (RBL-2H3) cell line. The data demonstrate that RGS4 inhibited responses by PAFR (i.e., phosphoinositide (PI) hydrolysis, Ca2+ mobilization) but not by FR or CXCR1. An N-terminal 33 amino acid deletion mutant of RGS4 (DeltaRGS4), deficient in GAP (GTPase activating protein) activity and plasma membrane localization, had no effect on either PAFR, FR, or CXCR1. RGS4, but not DeltaRGS4, also blocked phosphorylation of PAFR by platelet-activating factor (PAF) and, unexpectedly, by phorbol 12-myristate 13-acetate (PMA); it also blocked cross-phosphorylation by formylmethionylleucylphenylalanine (fMLP). A point mutant of RGS4 (N88S), deficient in GAP activity but not membrane localization, partially blocked PAFR phosphorylation but had no effect on PAFR-mediated PI hydrolysis and Ca2+ mobilization. Truncation of the cytoplasmic tail of PAFR (mPAFR) resulted in a loss of its susceptibility to inhibition by RGS4. Taken together, the data indicate that of the receptors studied, RGS4 selectively inhibited responses to PAFR, which preferentially couples to Gq. At the level of expression studied, RGS4 did not inhibit FR or CXCR1 which activates Gi to transduce cellular signals. Since the tail-deleted mutant of PAFR was not affected by RGS4, and RGS4 blocked homologous as well as heterologous phosphorylation of this receptor, it is possible that RGS4 interferes sterically with the cytoplasmic tail of PAFR. Thus, in addition to stimulating the GTPase activity of Galpha, RGS4 prevents G protein activation by PAFR and the homologous and heterologous phosphorylation of this receptor.
Assuntos
Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas RGS/fisiologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Animais , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Sinalização do Cálcio , Citoplasma/genética , Citoplasma/fisiologia , Vetores Genéticos/síntese química , Hidrólise , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosfatidilinositóis/metabolismo , Fosforilação , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/fisiologia , Proteínas RGS/biossíntese , Proteínas RGS/química , Proteínas RGS/genética , Ratos , Receptores de Formil Peptídeo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.
Assuntos
Carboxiliases/química , Carboxiliases/fisiologia , Ácidos Graxos/metabolismo , Fígado/enzimologia , Oxigênio/metabolismo , Fosfatase Alcalina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Glicemia/metabolismo , Western Blotting , Células COS , Cromatografia em Agarose , Clonagem Molecular , DNA Complementar/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ácidos Graxos/sangue , Privação de Alimentos , Insulina/sangue , Fígado/metabolismo , Masculino , Metionina/química , Dados de Sequência Molecular , Miocárdio/metabolismo , Fosforilação , Biossíntese de Proteínas , Isoformas de Proteínas , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNA , Estreptozocina , Distribuição Tecidual , TransfecçãoRESUMO
Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Ligação Competitiva/genética , Ativação Enzimática/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/fisiologia , Glutationa Transferase/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fosfolipase C beta , Mutação Puntual , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genéticaRESUMO
Phagocyte migration and activation at sites of inflammation is mediated through chemoattractant receptors that are coupled to G-proteins. Early studies from our laboratory demonstrated G-protein-mediated phospholipase C activation by chemoattractants. Recently, this laboratory developed cellular and animal models to allow biochemical, cell biological and molecular genetic approaches to be used in determining the mechanisms of chemoattractant receptor function, regulation, and cross regulation. These studies provided evidence that chemoattractant receptors activate distinct pathways for chemotaxis and exocytosis and cross-regulate each other's function at multiple levels. A major site of regulation is through phosphorylation of receptors by G-protein-coupled receptor kinases and by protein kinase C. In addition, the activation of phospholipase C by chemoattractants is also regulated at additional sites distal to receptor phosphorylation. These may include modulation of G-protein activation by regulators of G-protein signaling (RGS) and modification of phospholipase C. Phosphorylation of phospholipase Cbeta3 by both protein kinase A and protein kinase C has been demonstrated. The function and regulation of chemoattractant receptors are also being examined in mouse models. In these studies, mice deficient in leukotriene B4 receptors have been generated by targeted gene disruption. These mice displayed reduced neutrophil accumulation in certain inflammation models and sex-related differences in platelet-activating-factor induced anaphylaxis.
Assuntos
Quimiotaxia de Leucócito , Fagócitos/imunologia , Receptores Imunológicos/fisiologia , Receptores de Peptídeos/fisiologia , Animais , Proteínas de Ligação ao GTP , Humanos , Fosforilação , Transdução de SinaisRESUMO
Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [(35)S]GTPgammaS binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G(i) family, and not G(s), G(q), G(12), or G(13). We additionally established that Edg-1 activates G(i) in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G(13) as well as G(i). Because several of the biological actions of S1P are assumed to proceed through the G(12/13) family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G(i) but also with G(q) and G(13). These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas I-kappa B , Proteínas Imediatamente Precoces/metabolismo , Lisofosfolipídeos , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Animais , Linhagem Celular , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Inibidor de NF-kappaB alfa , Receptores de Lisofosfolipídeos , Proteínas Recombinantes/metabolismo , Esfingosina/metabolismo , Spodoptera , Radioisótopos de Enxofre , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Acetyl-CoA carboxylase (ACC) is regarded in liver and adipose tissue to be the rate-limiting enzyme for fatty acid biosynthesis; however, in heart tissue it functions as a regulator of fatty acid oxidation. Because the control of fatty acid oxidation is important to the functioning myocardium, the regulation of ACC is a key issue. Two cardiac isoforms of ACC exist, with molecular masses of 265 kDa and 280 kDa (ACC265 and ACC280). In this study, these proteins were purified from rat heart and used in subsequent phosphorylation and immunoprecipitation experiments. Our results demonstrate that 5' AMP-activated protein kinase (AMPK) is able to phosphorylate both ACC265 and ACC280, resulting in an almost complete loss of ACC activity. Although cAMP-dependent protein kinase phosphorylated only ACC280, a dramatic loss of ACC activity was still observed, suggesting that ACC280 contributes most, if not all, of the total heart ACC activity. ACC280 and ACC265 copurified under all experimental conditions, and purification of heart ACC also resulted in the specific copurification of the alpha2 isoform of the catalytic subunit of AMPK. Although both catalytic subunits of AMPK were expressed in crude heart homogenates, our results suggest that alpha2, and not alpha1, is the dominant isoform of AMPK catalytic subunit regulating ACC in the heart. Immunoprecipitation studies demonstrated that specific antibodies for both ACC265 and ACC280 were able to coimmunoprecipitate the alternate isoform along with the alpha2 isoform of AMPK. Taken together, the immunoprecipitation and the purification studies suggest that the two isoforms of ACC in the heart exist in a heterodimeric structure, and that this structure is tightly associated with the alpha2 subunit of AMPK.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoenzimas/metabolismo , Complexos Multienzimáticos/metabolismo , Miocárdio/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Western Blotting , Masculino , Fosforilação , Testes de Precipitina , Ratos , Ratos Sprague-DawleyRESUMO
Malonyl-CoA is a potent inhibitor of fatty acid uptake into the mitochondria. Although the synthesis of malonyl-CoA in the heart by acetyl-CoA carboxylase (ACC) has been well characterized, no information is available as to how malonyl-CoA is degraded. We demonstrate that malonyl-CoA decarboxylase (MCD) activity is present in the heart. Partial purification revealed a protein of approximately 50 kDa. The role of MCD in regulating fatty acid oxidation was also studied using isolated, perfused hearts from newborn rabbits and adult rats. Fatty acid oxidation in rabbit hearts increased dramatically between 1 day and 7 days after birth, which was accompanied by a decrease in both ACC activity and malonyl-CoA levels and a parallel increase in MCD activity. When adult rat hearts were aerobically reperfused after a 30-min period of no-flow ischemia, levels of malonyl-CoA decreased dramatically, which was accompanied by a decrease in ACC activity, a maintained MCD activity, and an increase in fatty acid oxidation rates. Taken together, our data suggest that the heart has an active MCD that has an important role in regulating fatty acid oxidation rates.
Assuntos
Carboxiliases/fisiologia , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Animais Recém-Nascidos , Carboxiliases/química , Carboxiliases/metabolismo , Masculino , Miocárdio/enzimologia , Oxirredução , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCbeta3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCbeta3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCbeta3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCbeta3 and appears to block the ability of Gbetagamma to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCbeta3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoenzimas/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Fosfolipases Tipo C/metabolismo , Linhagem Celular , AMP Cíclico/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Hidrólise , Fosfatidilinositóis/metabolismo , Fosfolipase C beta , FosforilaçãoRESUMO
The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.
Assuntos
Proteínas de Ligação ao GTP/química , Animais , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores da Neurocinina-1/metabolismo , Receptores de Serotonina/metabolismo , Receptores 5-HT1 de Serotonina , Receptores de Trombina/metabolismo , SpodopteraRESUMO
The human 5-hydroxytryptamine1A receptor, when expressed in Spodoptera frugiperda (Sf9) cells, facilitates the binding of [35S]GTPgammaS to a co-expressed GTP-binding regulatory protein, Gz, consistent with constitutive activity. The antagonists 4-(2'-methoxyphenyl)-1-[2'(n-2"-pyridinyl)-p-iodobenzamido]ethyl-p ipe razine (p-MPPI) and the related fluorobenzamido analogue p-MPPF had little (p-MPPI) or no (p-MPPF) effect on this activity. In contrast, a third antagonist, the neuroleptic spiperone, produced an almost complete suppression. Thus, using G protein activation as an index of receptor activity, p-MPPF was classified as a neutral antagonist, p-MPPI as a partial inverse agonist, and spiperone as essentially a full inverse agonist. As predicted, spiperone displayed properties consistent with a special form of noncompetitive antagonism when used to displace the agonist [125I]R-(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl)amin o]tetralin. Our data profile Sf9 cells as a unique vehicle for the characterization of inverse agonists, as these cells support a systematic pairing of mammalian receptors and G proteins, quantitative assays of G protein activation, and unambiguously labeled populations of coupled and uncoupled receptors.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/metabolismo , Aminopiridinas/metabolismo , Animais , Inibidores Enzimáticos/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Ligantes , Piperazinas/metabolismo , Receptores 5-HT1 de Serotonina , Espiperona/metabolismo , Spodoptera , Tetra-Hidronaftalenos/metabolismoRESUMO
We determined whether high fatty acid oxidation rates during aerobic reperfusion of ischemic hearts could be explained by a decrease in malonyl-CoA levels, which would relieve inhibition of carnitine palmitoyl-transferase 1, the rate-limiting enzyme involved in mitochondrial uptake of fatty acids. Isolated working rat hearts perfused with 1.2 mM palmitate were subjected to 30 min of global ischemia, followed by 60 min of aerobic reperfusion. Fatty acid oxidation rates during reperfusion were 136% higher than rates seen in aerobically perfused control hearts, despite the fact that cardiac work recovered to only 16% of pre-ischemic values. Neither the activity of carnitine palmitoyltransferase 1, or the IC50 value of malonyl-CoA for carnitine palmitoyl-transferase 1 were altered in mitochondria isolated from aerobic, ischemic, or reperfused ischemic hearts. Levels of malonyl-CoA were extremely low at the end of reperfusion compared to levels seen in aerobic controls, as was the activity of acetyl-CoA carboxylase, the enzyme which produces malonyl-CoA. The activity of 5'-AMP-activated protein kinase, which has been shown to phosphorylate and inactivate acetyl-CoA carboxylase in other tissues, was significantly increased at the end of ischemia, and remained elevated throughout reperfusion. These results suggest that accumulation of 5'-AMP during ischemia results in an activation of AMP-activated protein kinase, which phosphorylates and inactivates ACC during reperfusion. The subsequent decrease in malonyl-CoA levels wil result in accelerated fatty acid oxidation rates during reperfusion of ischemic hearts.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Ácidos Graxos/metabolismo , Malonil Coenzima A/metabolismo , Complexos Multienzimáticos/fisiologia , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Quinases Ativadas por AMP , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Masculino , Malonil Coenzima A/análise , Reperfusão Miocárdica , Oxirredução , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Perfusão , Fosforilação , Ratos , Ratos WistarRESUMO
Human UC11 astrocytoma cells were used to investigate the role of protein kinase C (PKC) and other kinases in neurokinin (NK)1 receptor desensitization. The selective NK1 receptor agonist [Sar9,Met(O2)11]-substance P stimulated a biphasic accumulation of [3H]inositol phosphates ([3H]IPs) in the presence of 10 mM LiCl in cells that had been prelabeled with [3H]inositol. An initial rapid phase of [3H]IP accumulation during the first 1 min was followed by a slower sustained phase for up to 90 min. These results demonstrate that the human NK1 receptor desensitizes rapidly but only partially. The selective PKC inhibitor Ro31-8220 did not prevent rapid NK1 receptor desensitization but after a longer incubation significantly potentiated human NK1 receptor agonist-stimulated accumulation of [3H]IPs. These results suggest that, although PKC does not mediate the process of rapid desensitization, it does have an inhibitory role at later times. This conclusion is supported by studies with staurosporine, phorbol dibutyrate, and the protein phosphatase inhibitor okadaic acid. Studies using AlF4-, an agent that can directly activate G proteins, and Ro31-8220 suggested that PKC can exert inhibitory effects 'downstream' of receptor activation, although immunoprecipitation of the G proteins alpha q/alpha 11 demonstrated that they do not undergo phosphorylation in UC11 cells and are unlikely to be the target of PKC-mediated inhibitory feedback. Delayed inhibitory feedback by PKC may be mediated by phosphorylation of phospholipase C, although an additional site of action on the NK1 receptor cannot be ruled out.
Assuntos
Proteína Quinase C/metabolismo , Receptores da Neurocinina-1/metabolismo , Fosfolipases Tipo C/metabolismo , Ativação Enzimática , Éteres Cíclicos/farmacologia , Retroalimentação , Humanos , Indóis/farmacologia , Cinética , Antagonistas dos Receptores de Neurocinina-1 , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Substância P/análogos & derivados , Substância P/farmacologia , Especificidade por Substrato , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
1. The potency of the non-peptide antagonists CP-96,345 and RP 67580 on NK1 receptor-stimulated [3H]-inositol phosphate accumulation in cell lines or tissue from three different species has been examined. 2. We have used: UC11 cells, derived from a human astrocytoma, and rat LRM55 glial cells, both of which express large numbers of functional NK1 receptors, and the well characterized guinea-pig ileum which expresses both NK1 and NK3 receptors. 3. RP 67580 has an approximately 25 fold lower affinity for NK1 receptors in human UC11 cells (Kd = 194 nM) than in rat LRM55 cells (Kd = 7.9 nM), in contrast CP-96,345 has an approximately 200 fold lower affinity in rat LRM55 cells (Kd = 210 nM) relative to human UC11 cells (Kd = 0.99 nM). The pharmacological profile of CP-96,345 and RP 67580 in guinea-pig ileum was similar to that observed in human UC11 cells. 4. In conclusion, we have demonstrated that previously reported species differences in binding affinities for the non-peptide NK1 antagonists, CP-96,345 and RP 67580, are also observed in inhibition of NK1 receptor stimulated hydrolysis of inositol phospholipids.
Assuntos
Compostos de Bifenilo/farmacologia , Íleo/efeitos dos fármacos , Indóis/farmacologia , Fosfatos de Inositol/metabolismo , Receptores de Neurotransmissores/antagonistas & inibidores , Animais , Sítios de Ligação , Carbacol/farmacologia , Linhagem Celular , Feminino , Cobaias , Humanos , Hidrólise , Íleo/metabolismo , Isoindóis , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Neurocinina B/metabolismo , Receptores de Neurotransmissores/metabolismo , Receptores de Taquicininas , Substância P/análogos & derivados , Substância P/farmacologia , Células Tumorais CultivadasRESUMO
The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified.