RESUMO
The IGF-II/mannose 6-phosphate (M6P) receptor is a single-transmembrane domain glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. The receptor may also mediate certain biological effects in response to IGF-II binding by interacting with G proteins. However, the nature of the IGF-II/M6P receptor's interaction with the G protein or with G protein-coupled receptor (GPCR) interacting proteins such as ß-arrestin remains unclear. Here we report that [(125)I]IGF-II receptor binding in the rat hippocampal formation is sensitive to guanosine-5'-[γ-thio]triphosphate, mastoparan, and Mas-7, which are known to interfere with the coupling of the classical GPCR with G protein. Monovalent and divalent cations also influenced [(125)I]IGF-II receptor binding. The IGF-II/M6P receptor, as observed for several GPCRs, was found to be associated with ß-arrestin 2, which exhibits sustained ubiquitination after stimulation with Leu(27)IGF-II, an IGF-II analog that binds rather selectively to the IGF-II/M6P receptor. Activation of the receptor by Leu(27)IGF-II induced stimulation of extracellular signal-related kinase 1/2 via a pertussis toxin-dependent pathway. Additionally, we have shown that IGF-II/M6P receptors under normal conditions are associated mostly with detergent-resistant membrane domains, but after stimulation with Leu(27)IGF-II, are translocated to the detergent-soluble fraction along with a portion of ß-arrestin 2. Collectively these results suggest that the IGF-II/M6P receptor may interact either directly or indirectly with G protein as well as ß-arrestin 2, and activation of the receptor by an agonist can lead to alteration in its subcellular distribution along with stimulation of an intracellular signaling cascade.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Hipocampo/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Arrestinas/genética , Arrestinas/metabolismo , Colesterol/metabolismo , Proteínas de Ligação ao GTP/genética , Hipocampo/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Peptídeos/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Venenos de Vespas/farmacologia , beta-Arrestina 2 , beta-ArrestinasRESUMO
BACKGROUND: Myocardial energy metabolism is a strong predictor of postoperative cardiac function. This study profiled the metabolites and metabolic changes in the myocardium exposed to sevoflurane, propofol, and Intralipid and investigated the underlying molecular mechanisms. METHODS: Sevoflurane (2 vol%) and propofol (10 and 100 microM) in the formulation of 1% Diprivan (AstraZeneca Inc., Mississauga, ON, Canada) were compared for their effects on oxidative energy metabolism and contractility in the isolated working rat heart model. Intralipid served as a control. Substrate flux through the major pathways for adenosine triphosphate generation in the heart, that is, fatty acid and glucose oxidation, was measured using [H]palmitate and [C]glucose. Biochemical analyses of nucleotides, acyl-CoAs, ceramides, and 32 acylcarnitine species were used to profile individual metabolites. Lipid rafts were isolated and used for Western blotting of the plasma membrane transporters CD36 and glucose transporter 4. RESULTS: Metabolic profiling of the hearts exposed to sevoflurane and propofol revealed distinct regulation of fatty acid and glucose oxidation. Sevoflurane selectively decreased fatty acid oxidation, which was closely related to a marked reduction in left ventricular work. In contrast, propofol at 100 microM but not 10 microM increased glucose oxidation without affecting cardiac work. Sevoflurane decreased fatty acid transporter CD36 in lipid rafts/caveolae, whereas high propofol increased pyruvate dehydrogenase activity without affecting glucose transporter 4, providing mechanisms for the fuel shifts in energy metabolism. Propofol increased ceramide formation, and Intralipid increased hydroxy acylcarnitine species. CONCLUSIONS: Anesthetics and their solvents elicit distinct metabolic profiles in the myocardium, which may have clinical implications for the already jeopardized diseased heart.
Assuntos
Antígenos CD36/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Coração/efeitos dos fármacos , Éteres Metílicos/farmacologia , Miocárdio/metabolismo , Propofol/farmacologia , Anestésicos Intravenosos/farmacologia , Animais , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Coração/fisiologia , Técnicas In Vitro , Masculino , Metaboloma/efeitos dos fármacos , Metaboloma/fisiologia , Miocárdio/enzimologia , Oxirredução/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , SevofluranoRESUMO
Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.
Assuntos
Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Regeneração Nervosa/fisiologia , Sistema Nervoso/citologia , Esfingolipídeos/fisiologia , Animais , Ceramidas/metabolismo , Células PC12 , RatosRESUMO
In sympathetic neurons, C6-ceramide, as well as endogenous ceramides, blocks apoptosis elicited by NGF (nerve growth factor) deprivation. The mechanism(s) involved in ceramide-induced neuronal survival are poorly understood. Few direct targets for the diverse cellular effects of ceramide have been identified. Amongst those proposed is PP-1c, the catalytic subunit of serine/threonine PP-1 (protein phosphatase-1). Here, we present the first evidence of PP-1c activation by ceramide in live cells, namely NGF-deprived sympathetic neurons. We first determined PP activity in cellular lysates from sympathetic neurons treated with exogenous ceramide and demonstrated a 2-3-fold increase in PP activity. PP activation was completely blocked by the addition of the specific type-1 PP inhibitor protein I-2 as well as by tautomycin, but unaffected by 2 nM okadaic acid, strongly indicating that the ceramide-activated phosphatase activity was PP-1c. Inhibition of PP activity by phosphatidic acid (which has been reported to be a selective inhibitor of PP-1c) and tautomycin (a PP-1 and PP-2A inhibitor), but not by 10 nM okadaic acid, abolished the anti-apoptotic effect of ceramide in NGF-deprived neurons, suggesting that activation of PP-1c is required for ceramide-induced neuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1c activation, suggesting that two consequences of NGF deprivation in sympathetic neurons are inhibition of PP-1c and subsequent hyperphosphorylation of pRb protein. These findings suggest a novel mechanism for ceramide-induced survival, and implicate the involvement of PPs in apoptosis induced by NGF deprivation.
Assuntos
Ceramidas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Sistema Nervoso Simpático/citologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator de Crescimento Neural/deficiência , Fator de Crescimento Neural/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1 , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
C6-ceramide protects sympathetic neurons from apoptosis caused by nerve growth factor (NGF) deprivation. Here, we report for the first time that ceramide generated "de novo" is also anti-apoptotic. Moreover, C6-ceramide is converted to long-chain ceramides in a process inhibited by fumonisin B1. The anti-apoptotic effect of C6-ceramide is due to the short analogue as to the long-chain ceramides. C6-ceramide shares mechanisms of action with NGF. C6-ceramide induces TrkA phosphorylation and selective activation of the phosphatidyl inositol 3-kinase (PI3-kinase)/Akt pathway but not the MAPK/ERK pathway. Importantly, the PI3-kinase inhibitor LY294002 abolishes the pro-survival effect of C6-ceramide. We identified a novel way to activate retrograde-mediated neuronal survival in the absence of NGF. Using compartmented cultures we show that addition of C6-ceramide exclusively to distal axons is sufficient to abort nuclear apoptosis. Our system offers a very unique alternative to understand the molecular bases of retrograde signaling in the absence of retrograde transport of neurotrophins. In search for a natural ligand that leads to ceramide generation we examined the activation of the sphingomyelin (SM) cycle downstream the p75 neurotrophin receptor (p75NTR). We found that in sympathetic neurons, selective activation of p75NTR by brain-derived neurotrophin factor or NGF plus K252a induces elevation of ceramide that correlates with SM hydrolysis. However, p75NTR activation does not generate sufficient ceramide to block apoptosis probably due to the rapid decrease in p75NTR expression that occurs upon NGF withdrawal.