RESUMEN
Oxidative stress has a central role in aging and in several age-linked diseases such as neurodegenerative diseases, diabetes and cancer. Mitochondria, as the main cellular source and target of reactive oxygen species (ROS) in aging, are recognized as very important players in the above reported diseases. Impaired mitochondrial oxidative phosphorylation has been reported in several aging tissues. Defective mitochondria are not only responsible of bioenergetically less efficient cells but also increase ROS production further contributing to tissues oxidative stress. Acetyl-L-carnitine (ALCAR) is a biomolecule able to limit age-linked mitochondrial decay in brain, liver, heart and skeletal muscles by increasing mitochondrial efficiency. Here the global changes induced by aging and by ALCAR supplementation to old rat on the mitochondrial proteome of rat liver has been analyzed by means of the two-dimensional polyacrylamide gel electrophoresis. Mass spectrometry has been used to identify the differentially expressed proteins. A significant age-related change occurred in 31 proteins involved in several metabolisms. ALCAR supplementation altered the levels of 26 proteins. In particular, ALCAR reversed the age-related alterations of 10 mitochondrial proteins relative to mitochondrial cristae morphology, to the oxidative phosphorylation and antioxidant systems, to urea cycle, to purine biosynthesis.
Asunto(s)
Acetilcarnitina/farmacología , Envejecimiento/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteoma/efectos de los fármacos , Factores de Edad , Envejecimiento/fisiología , Animales , Electroforesis en Gel Bidimensional , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Proteoma/análisis , Proteoma/metabolismo , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H(2)O(2) scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Acetilcarnitina/farmacología , Envejecimiento , Animales , Cisteína/química , Cisteína/metabolismo , Electroforesis en Gel Bidimensional , Masculino , Espectrometría de Masas , Peso Molecular , Nootrópicos/farmacología , Oxidación-Reducción , Peroxirredoxinas/genética , Ratas , Ratas Endogámicas F344 , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Several pathologies related to nervous tissue alterations are characterized by a chronic pain syndrome defined by persistent or paroxysmal pain independent or dependent on a stimulus. Pathophysiological mechanisms related to neuropathic disease are associated with mitochondrial dysfunctions that lead to an activation of the apoptotic cascade. In a model of peripheral neuropathy obtained by the loose ligation of the rat sciatic nerve, acetyl-L-Carnitine (ALCAR; 100 mg/kg intraperitoneally [i.p.] twice daily for 14 days) was able to reduce hyperalgesia and apoptosis. In the present study, different mechanisms for the analgesic and the antineuropathic effect of ALCAR are described. The muscarinic blocker atropine (5 mg/kg i.p.) injected simultaneously with ALCAR did not antagonize the ALCAR antihyperalgesic effect on the paw-pressure test but significantly reduced the analgesic effect of ALCAR. Conversely, the antineuropathic effect of ALCAR was prevented by cotreatment with the nicotinic antagonist mecamylamine (2 mg/kg i.p. twice daily for 14 days). A pharmacological silencing of the nicotinic receptors significantly reduced the X-linked inhibitor of apoptosis protein-related protective effect of ALCAR on the apoptosis induced by ligation of the sciatic nerve. Taken together, these data highlight the relevance of nicotinic modulation in neuropathy treatment.
Asunto(s)
Acetilcarnitina/uso terapéutico , Apoptosis/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Receptores Nicotínicos/metabolismo , Ciática , Animales , Atropina/farmacología , Caspasa 3/metabolismo , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Etiquetado Corte-Fin in Situ/métodos , Masculino , Mecamilamina/farmacología , Antagonistas Muscarínicos/farmacología , Antagonistas Nicotínicos/farmacología , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Ratas Sprague-Dawley , Ciática/tratamiento farmacológico , Ciática/patología , Ciática/fisiopatología , Factores de Tiempo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
INTRODUCTION: This study was designed to determine the short-term effect of acetyl-l-carnitine (ALC) on symptoms of withdrawal in opiate-dependent subjects and animals and, in particular, on pain, given the efficacy of ALC in other typologies of pain. The study consists of 2 branches: a clinical study and a preclinical one, both with a randomized placebo-controlled design. METHODS: Thirty subjects meeting clinical criteria for methadone dependence were consecutively recruited and treated with ALC 2 g/d or placebo for a 3-week detoxification period. Withdrawal symptoms and pain were evaluated through the Short Opiate Withdrawal Syndrome scale, and the Huskisson's analogue scale for pain. In the preclinical study, mice previously received a pretreatment (saline solution or morphine), and subsequently, each group was randomly divided in 4 subgroups that received a treatment of saline, methadone, ALC, or amitriptyline, respectively. Hot plate test and Writhing test were used to evaluate pain intensity. RESULTS: Average Short Opiate Withdrawal Syndrome total scores during the first 5 days of treatment resulted significantly higher in controls than in the ALC group (P < 0.05). Pain scores in the Huskisson's analogue scale were considerably lower in the group of patients taking ALC than in the control group after 1 week of ALC treatment until the end of the study. Results of the preclinical study show that the administration of methadone for 7 days in morphine-tolerant mice did not produce any modification of the pain threshold. By contrast, the 7-day coadministration of methadone and ALC in morphine-tolerant mice induced an analgesic effect evaluated 3 hours after the last injection. DISCUSSION: Acetyl-L-carnitine acted as an effective antihyperalgesic agent for relieving opiate-withdrawal hyperalgesia in animals and displayed clinical efficacy on other withdrawal symptoms such as muscular tension, muscular cramps, and insomnia. Considering its tolerability, the excellent side effect profile, the absence of significant interactions, and the lack of abuse potential, ALC can be considered as a useful pharmacological adjunct in the treatment of opiate withdrawal.
Asunto(s)
Acetilcarnitina/uso terapéutico , Analgésicos Opioides/efectos adversos , Metadona/efectos adversos , Nootrópicos/uso terapéutico , Dolor/tratamiento farmacológico , Dolor/etiología , Síndrome de Abstinencia a Sustancias/complicaciones , Adulto , Amitriptilina/uso terapéutico , Analgésicos no Narcóticos/uso terapéutico , Analgésicos Opioides/administración & dosificación , Animales , Modelos Animales de Enfermedad , Esquema de Medicación , Femenino , Humanos , Masculino , Metadona/administración & dosificación , Ratones , Morfina/administración & dosificación , Trastornos Relacionados con Opioides/complicaciones , Trastornos Relacionados con Opioides/tratamiento farmacológico , Dimensión del Dolor , Factores de Tiempo , Adulto JovenRESUMEN
Literature data indicate L-carnitine (LC), a trans-mitochondrial carrier of acetyl and long chain groups, as an agent possessing protective effects against oxidative stress in mammalian cells. However, the major factor involved in the protective mechanism is not known. The protection activity exerted by this agent against reactive oxygen species induced by hydrogen peroxide (H2O2) and t-butylhydroperoxide (t-butyl-OOH) treatment in isolated human peripheral blood lymphocytes (PBLs) has been studied. Human lymphocytes cells were isolated and pre-incubated with 5 mM LC before H2O2 (100 microM) and t-butyl-OOH (400 microM) treatment. The protective effect of LC on treated PBLs was measured by single cell gel electrophoresis and the analysis of chromosomal aberrations. Results show that lc treated cells exhibited a significant decrease in the number of oxidative induced single-strand breaks and chromosomal aberrations.
Asunto(s)
Carnitina/farmacología , Aberraciones Cromosómicas/efectos de los fármacos , Linfocitos/efectos de los fármacos , Oxidantes/farmacología , Sustancias Protectoras/farmacología , Complejo Vitamínico B/farmacología , Ensayo Cometa , Humanos , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , terc-Butilhidroperóxido/farmacologíaRESUMEN
Acetyl-L-carnitine (ALC) is a molecule widely present in the central nervous system (CNS) formed by the reversible acetylation of carnitine. It acts by stimulating energy metabolism. Reported neurobiological effects of this substance include modulation of brain energy and phospholipid metabolism; cellular macromolecules (including neurotrophic factors and neurohormones); synaptic transmission of multiple neurotransmitters. ALC is of considerable interest for its clinical application in Alzheimer's disease and in the treatment of painful neuropathies. There are experimental data that it affects attention and antagonizes deterioration of ability to learn, improving long-term memory. Moreover, ALC influences nonassociative learning of sensitization type in Hirudo medicinalis. These findings are suggesting that ALC might exert its effects by means of new protein synthesis. ALC or saline solution was injected intraperitoneally each day for 21 days in rats. Poly(A)+ RNAs were isolated from control and treated rat brain. Suppression subtractive hybridisation (SSH) method was applied for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatments. The technique generates an equalized representation of differentially expressed genes irrespective of their relative abundance, and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are regulated or switched off/on after ALC treatment. We identified two modulated genes, the isoform gamma of 14-3-3 protein and a precursor of ATP synthase lipid-binding protein, and one gene switched on by the treatment, the heat shock protein hsp72.
Asunto(s)
Acetilcarnitina/farmacología , Encéfalo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas 14-3-3/biosíntesis , Proteínas 14-3-3/genética , Acetilcarnitina/metabolismo , Animales , Encéfalo/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Biblioteca de Genes , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Masculino , ATPasas de Translocación de Protón Mitocondriales/biosíntesis , ATPasas de Translocación de Protón Mitocondriales/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Hibridación de Ácido Nucleico/métodos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The analgesic activity of acetyl-L-carnitine (ALCAR) in neuropathic pain is well established. By contrast, its potential efficacy in the relief of acute pain has not been reported. The antinociceptive effect of ALCAR was, therefore, examined in the mouse hot-plate and abdominal constriction tests, and in the rat paw-pressure test. ALCAR (100 mg kg(-1) s.c. twice daily for seven days) produced an increase of the pain threshold in both mice and rats. ALCAR was also able to reverse hyperalgesia induced by kainic acid and NMDA administration in the mouse hot-plate test. The antinociception produced by ALCAR was prevented by the unselective muscarinic antagonist atropine, the M(1) selective antagonists pirenzepine and S-(-)-ET126, and by the choline uptake inhibitor hemicholinium-3 (HC-3). By contrast the analgesic effect of ALCAR was not prevented by the opioid antagonist naloxone, the GABA(B) antagonist CGP 35348, the monoamine synthesis inhibitor (alpha)-methyl-p-tyrosine, and the Gi-protein inactivator pertussis toxin. Moreover, ALCAR antinociception was abolished by pretreament with an antisense oligonucleotide (aODN) against the M(1) receptor subtype, administered at the dose of 2 nmol per single i.c.v injection. On the basis of the above data, it can be postulated that ALCAR exerted an antinociceptive effect mediated by a central indirect cholinergic mechanism. In the antinociceptive dose-range, ALCAR did not impair mouse performance evaluated by the rota-rod and hole-board tests.
Asunto(s)
Acetilcarnitina/farmacología , Analgésicos/farmacología , Umbral del Dolor/efectos de los fármacos , Receptores Muscarínicos/fisiología , Animales , Ratones , Antagonistas Muscarínicos/farmacología , Dimensión del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Ratas , Ratas WistarRESUMEN
Skeletal muscle in congestive heart failure is responsible for increased fatigability and decreased exercise capacity. A specific myopathy with increased expression of fast-type myosins, myocyte atrophy, secondary to myocyte apoptosis triggered by high levels of circulating tumor necrosis factor-alpha (TNF-alpha) has been described. In an animal model of heart failure, the monocrotaline-treated rat, we have observed an increase of apoptotic skeletal muscle nuclei. Proapoptotic agents, caspase-3 and -9, were increased, as well as serum levels of TNF-alpha and its second messenger sphingosine. Treatment of rats with L-carnitine, known for its protective effect on muscle metabolism injuries, was found to inhibit caspases and to decrease the levels of TNF-alpha and sphingosine, as well as the number of apoptotic myonuclei. Staurosporine was used in in vitro experiments to induce apoptosis in skeletal muscle cells in culture. When L-carnitine was applied to skeletal muscle cells, before staurosporine treatment, we observed a reduction in apoptosis. These findings show that L-carnitine can prevent apoptosis of skeletal muscles cells and has a role in the treatment of congestive heart failure-associated myopathy.