RESUMO
The innate immune system has been implicated in both AKI and CKD. Damaged mitochondria release danger molecules, such as reactive oxygen species, DNA, and cardiolipin, which can cause NLRP3 inflammasome activation and upregulation of IL-18 and IL-1ß It is not known if mitochondrial damage persists long after ischemia to sustain chronic inflammasome activation. We conducted a 9-month study in Sprague-Dawley rats after 45 minutes of bilateral renal ischemia. We detected glomerular and peritubular capillary rarefaction, macrophage infiltration, and fibrosis at 1 month. Transmission electron microscopy revealed mitochondrial degeneration, mitophagy, and deformed foot processes in podocytes. These changes progressed over the study period, with a persistent increase in renal cortical expression of IL-18, IL-1ß, and TGF-ß, despite a gradual decline in TNF-α expression and macrophage infiltration. Treatment with a mitoprotective agent (SS-31; elamipretide) for 6 weeks, starting 1 month after ischemia, preserved mitochondrial integrity, ameliorated expression levels of all inflammatory markers, restored glomerular capillaries and podocyte structure, and arrested glomerulosclerosis and interstitial fibrosis. Further, helium ion microscopy vividly demonstrated the restoration of podocyte structure by SS-31. The protection by SS-31 was sustained for ≥6 months after treatment ended, with normalization of IL-18 and IL-1ß expression. These results support a role for mitochondrial damage in inflammasome activation and CKD and suggest mitochondrial protection as a novel therapeutic approach that can arrest the progression of CKD. Notably, SS-31 is effective when given long after AKI and provides persistent protection after termination of drug treatment.
Assuntos
Interleucina-18/fisiologia , Interleucina-1beta/fisiologia , Isquemia/complicações , Rim/irrigação sanguínea , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/prevenção & controle , Regulação para Cima/efeitos dos fármacos , Doença Aguda , Animais , Masculino , Podócitos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity. METHODS: SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. RESULTS: Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. CONCLUSION: Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe(2+) that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. GENERAL SIGNIFICANCE: Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery.
RESUMO
Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-ß) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Glomerulonefrite/prevenção & controle , Túbulos Renais Proximais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/uso terapêutico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Capilares/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Glomerulonefrite/etiologia , Túbulos Renais Proximais/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Obesidade/complicações , Oligopeptídeos/farmacologia , Podócitos/efeitos dos fármacosRESUMO
Ischemia time during partial nephrectomy is strongly associated with acute and chronic renal injury. ATP depletion during warm ischemia inhibits ATP-dependent processes, resulting in cell swelling, cytoskeletal breakdown, and cell death. The duration of ischemia tolerated by the kidney depends on the amount of ATP that can be produced with residual substrates and oxygen in the tissue to sustain cell function. We previously reported that the rat can tolerate 30-min ischemia quite well but 45-min ischemia results in acute kidney injury and progressive interstitial fibrosis. Here, we report that pretreatment with SS-20 30 min before warm ischemia in the rat increased ischemia tolerance from 30 to 45 min. Histological examination of kidney tissues revealed that SS-20 reduced cytoskeletal breakdown and cell swelling after 45-min ischemia. Electron microscopy showed that SS-20 reduced mitochondrial matrix swelling and preserved cristae membranes, suggesting that SS-20 enhanced mitochondrial ATP synthesis under ischemic conditions. Studies with isolated kidney mitochondria showed dramatic reduction in state 3 respiration and respiratory control ratio after 45-min ischemia, and this was significantly improved by SS-20 treatment. These results suggest that SS-20 increases efficiency of the electron transport chain and improves coupling of oxidative phosphorylation. SS-20 treatment after ischemia also significantly reduced interstitial fibrosis. These new findings reveal that enhancing mitochondrial bioenergetics may be an important target for improving ischemia tolerance, and SS-20 may serve well for minimizing acute kidney injury and chronic kidney disease following surgical procedures such as partial nephrectomy and transplantation.
Assuntos
Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Isquemia Quente , Trifosfato de Adenosina/biossíntese , Animais , Avaliação Pré-Clínica de Medicamentos , Rim/irrigação sanguínea , Rim/metabolismo , Masculino , Mitocôndrias/metabolismo , Oligopeptídeos/uso terapêutico , Distribuição Aleatória , Ratos Sprague-DawleyRESUMO
Microvascular rarefaction, or loss of microvascular density, is increasingly implicated in the progression from acute ischemic kidney injury to chronic kidney disease. Microvascular dropout results in chronic tissue hypoxia, interstitial inflammation, and fibrosis. There is currently no therapeutic intervention for microvascular rarefaction. We hypothesize that capillary dropout begins with ischemic damage to endothelial mitochondria due to cardiolipin peroxidation, resulting in loss of cristae and the failure to regenerate ATP upon reperfusion. SS-31 is a cell-permeable peptide that targets the inner mitochondrial membrane and binds selectively to cardiolipin. It was recently shown to inhibit cardiolipin peroxidation by cytochrome c peroxidase activity, and it has been shown to protect mitochondrial cristae in proximal tubular cells during ischemia, and accelerated ATP recovery upon reperfusion. We found mitochondrial swelling and loss of cristae membranes in endothelial and medullary tubular epithelial cells after 45-min ischemia in the rat. The loss of cristae membranes limited the ability of these cells to regenerate ATP upon reperfusion and led to loss of vascular integrity and to tubular cell swelling. SS-31 prevented mitochondria swelling and protected cristae membranes in both endothelial and epithelial cells. By minimizing endothelial and epithelial cell injury, SS-31 prevented "no-reflow" after ischemia and significantly reduced the loss of peritubular capillaries and cortical arterioles, interstitial inflammation, and fibrosis at 4 wk after ischemia. These results suggest that mitochondria protection represents an upstream target for pharmacological intervention in microvascular rarefaction and fibrosis.
Assuntos
Injúria Renal Aguda/prevenção & controle , Cardiolipinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Nefrite/prevenção & controle , Oligopeptídeos/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Trifosfato de Adenosina/metabolismo , Animais , Citoproteção , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Fibrose , Rim/metabolismo , Rim/fisiopatologia , Rim/ultraestrutura , Masculino , Microvasos/metabolismo , Microvasos/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Dilatação Mitocondrial/efeitos dos fármacos , Nefrite/metabolismo , Nefrite/patologia , Nefrite/fisiopatologia , Oligopeptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Fatores de TempoRESUMO
Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.
Assuntos
Cardiolipinas/metabolismo , Citocromo-c Peroxidase/metabolismo , Isquemia/metabolismo , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oligopeptídeos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
The burst of reactive oxygen species (ROS) during reperfusion of ischemic tissues can trigger the opening of the mitochondrial permeability transition (MPT) pore, resulting in mitochondrial depolarization, decreased ATP synthesis, and increased ROS production. Rapid recovery of ATP upon reperfusion is essential for survival of tubular cells, and inhibition of oxidative damage can limit inflammation. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge mitochondrial ROS and inhibit MPT, suggesting that it may protect against ischemic renal injury. Here, in a rat model of ischemia-reperfusion (IR) injury, treatment with SS-31 protected mitochondrial structure and respiration during early reperfusion, accelerated recovery of ATP, reduced apoptosis and necrosis of tubular cells, and abrogated tubular dysfunction. In addition, SS-31 reduced medullary vascular congestion, decreased IR-mediated oxidative stress and the inflammatory response, and accelerated the proliferation of surviving tubular cells as early as 1 day after reperfusion. In summary, these results support MPT as an upstream target for pharmacologic intervention in IR injury and support early protection of mitochondrial function as a therapeutic maneuver to prevent tubular apoptosis and necrosis, reduce oxidative stress, and reduce inflammation. SS-31 holds promise for the prevention and treatment of acute kidney injury.
Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Trifosfato de Adenosina/metabolismo , Antioxidantes/uso terapêutico , Oligopeptídeos/uso terapêutico , Traumatismo por Reperfusão/complicações , Injúria Renal Aguda/fisiopatologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/fisiologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Modelos Animais , Oligopeptídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologiaRESUMO
OBJECTIVE: Previously, we demonstrated that a novel opiate peptide, 2',6'-dimethyl-tyrosine-D-Arg-Phe-Lys-NH2, provided cardioprotection against myocardial stunning in vivo. We subsequently showed that this peptide targeted mitochondria and can scavenge reactive oxygen species. The objective of this study was to determine the role of opioid versus antioxidant activity in cardioprotection. METHODS: We compared two mitochondria-targeted peptide analogs that lacked opioid activity: SS-31 (D-Arg-2',6'-dimethyl-tyrosine-Lys-Phe-NH2) and SS-20 (Phe-D-Arg-Phe-Lys-NH2). They differ in that only SS-31 has scavenging ability. Rats (n=8/group) were randomized to SS-31, SS-20 or placebo. The drugs (3 mg/kg) or saline was administered intraperitoneally 30 min before ligation of the left anterior descending artery for 60 min, and another dose given intraperitoneally 5 min before reperfusion for 60 min. Study endpoints included myocardial infarct size, cardiac arrhythmia and myocardial lipid peroxidation. RESULTS: The area at risk was similar among the groups. The infarct area/area at risk, however, was significantly smaller in the treatment groups (53.9+/-1.1% in SS-31 group, 47.1+/-1.4% in SS-20 group, versus 59.9+/-1% in the controls, P<0.01). Lipid peroxidation was significantly reduced by both SS-31 and SS-20 treatment. Arrhythmia occurred only during the early period of coronary occlusion and was less frequent and less severe in the peptide treatment groups than in the controls (Lambeth score 5 points, 3 points, versus 13 points in the controls, P<0.05). CONCLUSIONS: This study shows that pretreatment with both SS-31 and SS-20 significantly reduced myocardial lipid peroxidation and infarct size in ischemia-reperfusion injury, and suggests that the cardioprotective properties of 2',6'-dimethyl-tyrosine-D-Arg-Phe-Lys-NH2 was primarily mediated by its antioxidant properties. As SS-20 does not scavenge reactive oxygen species, it most likely reduces reactive oxygen species production during ischemia-reperfusion.
Assuntos
Antioxidantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Oligopeptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Reperfusão Miocárdica , Ratos , Ratos Sprague-DawleyRESUMO
The aim of this study was to assess whether the GAITRite gait analysis system an effective tool in evaluating parkinsonian bradykinesia. In their best ON and worst OFF states, 13 parkinsonian patients were asked to perform walking trail at their fastest velocity, and to submit to traditional timed tests. Significant correlations existed between OFF-ON improvement in gait parameters and in UPDRS III score (r(2)=0.59-0.78) except cadence. Among the gait parameters, stride length is the most effective indicator of timed test and UPDRS III score improvements. The GAITRite system is an efficient, yet effective device in evaluating parkinsonian bradykinesia and can be used as a substitute for the traditional timed test.
Assuntos
Diagnóstico por Computador/instrumentação , Avaliação da Deficiência , Marcha , Hipocinesia/diagnóstico , Doença de Parkinson/complicações , Adulto , Antiparkinsonianos/uso terapêutico , Humanos , Hipocinesia/tratamento farmacológico , Hipocinesia/etiologia , Levodopa/uso terapêutico , Pessoa de Meia-Idade , Doença de Parkinson/tratamento farmacológicoRESUMO
A method in layering dressings with a superficial active layer of sub-micrometer scaled fibrous structures is demonstrated. For this, polyvinylpyrolidone (PVP)-indomethacin (INDO) fibres (5% w/v PVP, 5% w/w indomethacin, using a 50:50 ethanol-methanol solvent system) were produced at different flow rates (50 µL/min and 100 µL/min) via a modified electrospinning device head (applied voltage varied between 15 ± 2 kV). We further assessed these structures for their morphological, physical and chemical properties using SEM, AFM, DSC, XRD, FTIR and HPLC-UV. The average diameter of the resulting 3D (ca. 500 nm in height) PVP-INDO fibres produced at 50 µL/min flow rate was 2.58 ± 0.30 µm, while an almost two-fold increase in the diameter was observed (5.22 ± 0.83 µm) when the flow rate was doubled. However, both of these diameters were appreciably smaller than the existing dressing fibres (ca. 30 µm), which were visible even when layered with the active spun fibres. Indomethacin was incorporated in the amorphous state. The encapsulation efficiency was 75% w/w, with complete drug release in 45 min. The advantages are the ease of fabrication and deposition onto any existing normal or functionalised dressing (retaining the original fabric functionality), elimination of topical product issues (application, storage and transport), rapid release of active and controlled loading of drug content (fibre layer).
Assuntos
Anti-Inflamatórios não Esteroides/química , Bandagens , Sistemas de Liberação de Medicamentos , Indometacina/química , Povidona/química , Administração Cutânea , Anti-Inflamatórios não Esteroides/administração & dosagem , Varredura Diferencial de Calorimetria , Composição de Medicamentos/métodos , Indometacina/administração & dosagem , Microscopia Eletrônica de Varredura , Povidona/administração & dosagem , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Dynorphin A (Dyn A) stimulates the release of ACTH in fetal sheep, a response that involves N-methyl-D-aspartate (NMDA) receptors but not the secretogogues corticotropin-releasing hormone or arginine vasopressin. We now find that neither Dyn A-(1-13) (0.5 mg/kg, i.v.) nor NMDA (4 mg/kg, i.v.) elicits ACTH release in postnatal lambs. This led us to hypothesize that Dyn A-(1-13) and NMDA might act to release placental ACTH. However, the ability of Dyn A-(1-13), NMDA, and the kappa-opioid receptor agonist U-50488H (1 mg/kg, i.v.) to release ACTH was lost after either fetal hypophysectomy (n = 4) or hypothalamo-pituitary disconnection (n = 4). These results indicate that neither the placenta nor the fetal pituitary is the site of action for these agonists and suggest a hypothalamic or suprahypothalamic site of action. Furthermore, the release of ACTH by Dyn A-(1-13) and NMDA was abolished after pretreatment with indomethacin, suggesting that they might cause the release of a prostanoid, possibly from the placenta, that subsequently acts at the hypothalamus or serves as a permissive factor in the action of Dyn A-(1-13) and NMDA at the hypothalamus.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Dinorfinas/farmacologia , Feto/fisiologia , N-Metilaspartato/farmacologia , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/embriologia , Hipotálamo/metabolismo , Indometacina/farmacologia , Fragmentos de Peptídeos/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/embriologia , Hipófise/metabolismo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Receptores Opioides kappa/agonistas , OvinosRESUMO
[Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) is a dermorphin analog that shows high affinity and selectivity for the mu opioid receptor. The intrathecal potency of [Dmt(1)]DALDA far exceeded its affinity at mu receptors and suggests that other mechanisms must be involved in its action in the spinal cord. The affinity and selectivity of [Dmt(1)]DALDA was determined using cell membranes expressing cloned human mu, delta, and kappa opioid receptors. Competitive displacement binding with [(3)H][Dmt(1)]DALDA, [(3)H]DPDPE (H-Tyr-d-Pen-Gly-Phe-d-Pen), and [(3)H]U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide] revealed K(i) of 156 +/- 26 pM for mu opioid receptor (MOR), 1.67 +/- 0.04 microM for delta opioid receptor (DOR), and K(i) of 4.4 +/- 1.7 nM for kappa opioid receptor (KOR), respectively. [Dmt(1)]DALDA increased guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding in MOR, DOR, and KOR membranes, with EC(50) being 17 (8.8-33) nM, 2 (1.2-3.2) microM, and 124 (15-1000) nM, respectively. Intrathecal [Dmt(1)]DALDA inhibited the tail-flick response in mice with ED(50) = 1.22 (0.59-2.34) pmol. Intrathecal administration of an antiserum against dynorphin A(1-17) or [Met(5)]enkephalin significantly attenuated the response to i.t. [Dmt(1)]DALDA, resulting in ED(50) of 6.2 (3.6-12.6) pmol and 6.6 (3.5-19.6) pmol, respectively. Neither antisera had any effect on the response to i.t. morphine. Intracerebroventricular (i.c.v.) [Dmt(1)]DALDA was not affected by previous i.c.v. administration of anti-Dyn or anti-ME. Pretreatment with norbinaltorphimine or naltriben also attenuated the antinociceptive response to i.t., but not i.c.v., [Dmt(1)]DALDA. These data suggest that i.t. [Dmt(1)]DALDA causes the release of dynorphin and [Met(5)]enkephalin-like substances that act at kappa and delta receptors, respectively, to contribute to the extraordinary potency of [Dmt(1)]DALDA.
Assuntos
Analgésicos Opioides/farmacologia , Endorfinas/fisiologia , Naloxona/análogos & derivados , Naltrexona/análogos & derivados , Oligopeptídeos/farmacologia , Analgésicos Opioides/administração & dosagem , Animais , Anticorpos Bloqueadores/farmacologia , Clonagem Molecular , Dinorfinas/antagonistas & inibidores , Dinorfinas/imunologia , Dinorfinas/fisiologia , Encefalina Metionina/antagonistas & inibidores , Encefalina Metionina/imunologia , Encefalina Metionina/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Temperatura Alta , Injeções Intraventriculares , Injeções Espinhais , Masculino , Camundongos , Naloxona/farmacologia , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Oligopeptídeos/administração & dosagem , Medição da Dor/efeitos dos fármacos , Ensaio Radioligante , Tempo de Reação , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacosRESUMO
We recently discovered an opioid peptide analgesic, 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt(1)]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt(1)]DALDA inhibited [(3)H]NE uptake into cardiac synaptosomes in vitro (IC(50) = 3.9 microM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt(1)]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [(3)H][Dmt(1)]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt(1)]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt(1)]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.
Assuntos
Analgésicos/farmacologia , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio Atordoado/prevenção & controle , Oligopeptídeos/farmacologia , Analgésicos/metabolismo , Animais , Cobaias , Técnicas In Vitro , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Norepinefrina/metabolismo , Oligopeptídeos/metabolismo , Sinaptossomos/metabolismoRESUMO
Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N(2)A cells (EC(50) in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca(2+) in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.
Assuntos
Antioxidantes/farmacologia , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Peptídeos/química , Tirosina/análogos & derivados , Motivos de Aminoácidos , Animais , Antioxidantes/química , Células CACO-2 , Cálcio/metabolismo , Cardiolipinas/metabolismo , Morte Celular , Sobrevivência Celular , Citocromos c/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Mitocôndrias Hepáticas/metabolismo , Nitrocompostos , Estresse Oxidativo , Oxigênio/metabolismo , Consumo de Oxigênio , Propionatos/química , Espécies Reativas de Oxigênio , Reperfusão , Traumatismo por Reperfusão , Fatores de Tempo , Tirosina/química , terc-Butil Hidroperóxido/químicaRESUMO
Recent studies suggest that delta-opioid receptors play a role in the development of opioid tolerance and led us to hypothesize that highly selective mu-opioid agonists may produce less tolerance. H-2',6'-dimethyltyrosine-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA) has extraordinary selectivity for mu-receptors (K(i)(delta)/K(i)(mu) > 14,000). Daily administration of [Dmt(1)]DALDA (5 times ED(50); s.c.) for 7 days increased ED(50) 3.6-fold from 0.16 to 0.58 micromol/kg. A higher dose of [Dmt(1)]DALDA (10 times ED(50), every 12 h) for 2.5 days resulted in a 11.7 times increase in the ED(50) (1.9 micromol/kg). Complete cross-tolerance to morphine was observed, with a 3.4- and 15.1-fold shift in the morphine ED(50), respectively. We also compared the extent of spinal versus supraspinal tolerance after repeated s.c. [Dmt(1)]DALDA administration. Five doses of [Dmt(1)]DALDA (10 times ED(50), every 12 h) resulted in a 3.4 times shift in the i.c.v. ED(50) (15.4 versus 4.6 pmol/mouse) but a 44 times shift in the i.t. ED(50) (52.9 versus 1.2 pmol/mouse). Tolerance to [Dmt(1)]DALDA was associated with 30 to 35% reduction in [(3)H][Dmt(1)]DALDA binding in brain and spinal cord. Coadministration of [Dmt(1)]DALDA with delta-antagonist naltriben (NTB) reduced spinal tolerance by 50%. Even after spinal tolerance had been established, addition of a delta-antagonist (NTB or H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH) significantly enhanced the potency of i.t. [Dmt(1)]DALDA 2- to 4-fold. These results suggest that agonist activation of delta-receptors is not necessary for the development of opioid tolerance; however, delta-receptors play a modulatory role in the maintenance of the tolerant state.