RESUMO
Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.
Assuntos
Animais , Ratos , Traumatismo por Reperfusão/prevenção & controle , Isquemia Encefálica , Proteína Quinase C/metabolismo , Proteína Quinase C/farmacologia , Ataque Isquêmico Transitório , Estresse Oxidativo , Fármacos Neuroprotetores/farmacologia , Dexmedetomidina/farmacologia , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/farmacologia , Heme Oxigenase (Desciclizante)/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologiaRESUMO
BACKGROUND: The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. METHODS: Thirty-eight rats were randomly assigned to five groups: sham (n...=...6), ischemic (n...=...8), chelerythrine (a PKC inhibitor; 5...mg.kg-1 IV administered 30...min before cerebral ischemia) (n...=...8), dexmedetomidine (100.....g.kg-1 IP administered 30...min before cerebral ischemia (n...=...8), and dexmedetomidine...+...chelerythrine (n...=...8). Global transient cerebral ischemia (10...min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24...hours after ischemia insult. RESULTS: We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p...<...0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p...<...0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p...<...0.05 and p...<...0.01, respectively) and diminished its beneficial neuroprotective effects. CONCLUSION: Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.
Assuntos
Isquemia Encefálica , Dexmedetomidina , Ataque Isquêmico Transitório , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Ratos , Animais , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/farmacologia , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/farmacologia , Dexmedetomidina/farmacologia , Ratos Sprague-Dawley , Estresse Oxidativo , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controleRESUMO
BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)-induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.
Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Proteína Quinase C/farmacologia , Células-Tronco/efeitos dos fármacos , Western Blotting , Adesão Celular , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Sobrevivência Celular , Condrócitos/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)- induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.
Assuntos
Humanos , Células-Tronco/efeitos dos fármacos , Proteína Quinase C/farmacologia , Cartilagem Articular/citologia , Condrócitos/citologia , Adesão Celular , Comunicação Celular , Diferenciação Celular , Sobrevivência Celular , Western Blotting , Técnicas de Cultura de Células , Condrócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoKATP channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.
Assuntos
Animais , Masculino , Precondicionamento Isquêmico Miocárdico/métodos , Éteres Metílicos/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Inibidores da Agregação Plaquetária/farmacologia , Canais de Potássio/farmacologia , Proteína Quinase C/farmacologia , Antiarrítmicos/farmacologia , Western Blotting , /análise , Ácidos Decanoicos/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Hidroxiácidos/farmacologia , Isquemia/prevenção & controle , Substâncias Protetoras/farmacologia , Distribuição Aleatória , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fatores de Tempo , Troponina I/análiseRESUMO
This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoKATP channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.
Assuntos
Precondicionamento Isquêmico Miocárdico/métodos , Éteres Metílicos/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Inibidores da Agregação Plaquetária/farmacologia , Canais de Potássio/farmacologia , Proteína Quinase C/farmacologia , Animais , Antiarrítmicos/farmacologia , Western Blotting , Caspase 8/análise , Ácidos Decanoicos/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Hidroxiácidos/farmacologia , Isquemia/prevenção & controle , Masculino , Substâncias Protetoras/farmacologia , Distribuição Aleatória , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sevoflurano , Fatores de Tempo , Troponina I/análiseRESUMO
Osmolarity reduction (20%) elicited 3H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na+-dependent and La3+-, Gd3+- and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca2+ ([Ca2+]i) rise with contributions from external Ca2+ influx and internal Ca2+ release, probably through the mitochondrial Na+-Ca2+ exchanger; and (iii) activation of a [Ca2+]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca2+]i rise by preventing depolarization with La3+, and by the inhibitory effects of Ca2+-free medium (EGTA; 50%), CGP37157 (the mitochondrial Na+-Ca2+ exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na+ omission, 75% by La3+, 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca2+ release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K+-stimulated NE release, studied in parallel, was unaffected by Na+ omission, or by La3+, Gd3+ or ruthenium red. It was fully dependent on external Ca2+, insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K+-evoked depolarization and [Ca2+]i rise mechanisms, are able to trigger a depolarization-dependent, Ca2+-dependent and TeTX-sensitive mechanism for neurotransmitter release.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/citologia , Exocitose/fisiologia , Norepinefrina/metabolismo , Sinaptossomos/fisiologia , Animais , Bário/farmacologia , Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carbazóis/farmacologia , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Interações Medicamentosas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Indóis/farmacologia , Lantânio/farmacologia , Concentração Osmolar , Ésteres de Forbol/farmacologia , Cloreto de Potássio/farmacologia , Proteína Quinase C/farmacologia , Ratos , Ratos Wistar , Rutênio Vermelho/farmacologia , Sódio/farmacologia , Trocador de Sódio e Cálcio/farmacologia , Espectrometria de Fluorescência/métodos , Sinaptossomos/efeitos dos fármacos , Toxina Tetânica/farmacologia , Tiazepinas/farmacologia , Fatores de Tempo , Trítio/metabolismo , ômega-Conotoxinas/farmacologiaRESUMO
The nucleus accumbens (NAcc) has been shown to play a role in motor and spatial learning. Protein kinase C (PKC) has been implicated in the mechanisms of initiation and maintenance of long-term potentiation that is thought to be involved in the storage of long-term memory. In the present study, the importance of de novo synthesis of PKC-gamma within the NAcc in the acquisition and retention of spatial discrimination learning was assessed using an antisense knockdown approach. Separate groups of Long-Evans rats were exposed to acute microinfusions (6microg/microl) of PKC-gamma antisense oligodeoxynucleotide (AS-ODN), control oligodeoxynucleotide (C-ODN) or vehicle into the NAcc at 24 and 3h before each training session. Behavioral findings showed that the blockade of NAcc-PKC-gamma translation caused impairments in the early phase of learning and retention of spatial information. Biochemical experiments showed that PKC-gamma expression was reduced and Ca(2+)/phospholipid-dependent protein kinase C (PKC) activity was blocked significantly in the AS-ODN-treated rats in comparison with control rats. The present findings suggest that NAcc-PKC-gamma plays a role during the early acquisition of spatial learning. Also, retention test results suggest that NAcc-PKC-gamma may be working as an intermediate factor involved in the onset of molecular mechanisms necessary for spatial memory consolidation within the NAcc.
Assuntos
Aprendizagem/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteína Quinase C/farmacologia , Percepção Espacial/efeitos dos fármacos , Animais , Transtornos Cognitivos/induzido quimicamente , Immunoblotting , Masculino , Microinjeções , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/efeitos adversos , Proteína Quinase C/administração & dosagem , Proteína Quinase C/efeitos adversos , Ratos , Ratos Long-EvansRESUMO
Bradykinin caused graded contractions of rings of rabbit aorta and jugular vein with EC50 values of 1.3 microM and 2.2 nM. In denuded preparations, responses of bradykinin in jugular vein but not in aorta were potentiated 1,000-fold. Both preparations bathed in calcium-free solution showed markedly depressed responses to bradykinin, but addition of 1 mM EGTA further inhibited bradykinin responses only in aorta. Time-course experiments carried out in calcium-free solution plus EGTA revealed that bradykinin contractions in rabbit aorta were very sensitive to extracellular calcium, whereas responses of the jugular vein depended on both extracellular and intracellular calcium sources. Responses to bradykinin in both tissues were unaffected by nicardipine (1 microM) but were partially antagonized by NiCl2 (0.1-0.3 mM). Ryanodine (30 microM) incubated in calcium-free medium markedly inhibited jugular vein responses to bradykinin but had no effect on aortic responses. Phorbol ester (1 microM) caused a slow tonic contraction in jugular vein but not in aorta and inhibited bradykinin responses in the former preparation. Staurosporine (1-100 nM) and 1-(5-isoquinolinesulfonyl)-2-methylpiperizine (H-7, 3 and 10 microM) caused a dose-dependent inhibition of bradykinin-induced contractions in jugular vein but were less effective in aorta.(ABSTRACT TRUNCATED AT 250 WORDS)