RESUMO
Degeneration or survival of cerebral tissue after ischemic injury depends on the source, intensity, and duration of the insult. In the model of focal ischemia, reduced blood flow results in a cascade of pathophysiologic events, including inflammation, excitotoxicity, and platelet activation at the site of injury. One serine protease that is associated closely with and produced in response to central nervous system (CNS) injury is thrombin. Thrombin enters the injury cascade in brain either via a compromised blood-brain barrier or possibly from endogenous prothrombin. Thrombin mediates its action through the protease-activated receptor family (PAR-1, -3, and -4). PARs belong to the superfamily of G protein-coupled receptors with a 7-transmembrane domain structure and are activated by proteolytic cleavage of their N-terminus. We showed that thrombin can be neuroprotective or deleterious when present at different concentrations before and during oxygen-glucose deprivation, an in vitro model of ischemia. We examined the change in mRNA expression levels of PAR-1 to 4 as a result of transient focal ischemia in rat brain, induced by microinjection of endothelin near the middle cerebral artery. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, after ischemic insult on the ipsilesional side, PAR-1 was found to be downregulated significantly, whereas PAR-2 mRNA levels decreased only moderately. PAR-3 was upregulated transiently and then downregulated, and PAR-4 mRNA levels showed the most striking (2.5-fold) increase 12 hr after ischemia, in the injured side. In the contralateral hemisphere, mRNA expression was also affected, where decreased mRNA levels were observed for PAR-1, -2, and -3, whereas PAR-4 levels were reduced only after 7 days. Taken together, these data suggest involvement of the thrombin receptors PAR-1, PAR-3, and PAR-4 in the pathophysiology of brain ischemia.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , RNA Mensageiro/metabolismo , Receptores Ativados por Proteinase/genética , Traumatismo por Reperfusão/metabolismo , Trombina/metabolismo , Animais , Encéfalo/fisiopatologia , Infarto Encefálico/genética , Infarto Encefálico/metabolismo , Infarto Encefálico/fisiopatologia , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Regulação para Baixo/genética , Encefalite/genética , Encefalite/metabolismo , Encefalite/fisiopatologia , Endotelinas , Lateralidade Funcional/genética , Expressão Gênica/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Tempo de Reação/genética , Receptor PAR-1/genética , Receptores de Trombina/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Regulação para Cima/genéticaRESUMO
Protease-activated receptors (PARs), 7-transmembrane domain G protein-coupled receptors, are involved in tissue degeneration and repair upon injury. We demonstrate the expression of all four PAR subtypes in the postnatal eye and in retina of the adult rat by reverse transcription-polymerase chain reaction (RT-PCR). PAR-1 is regulated developmentally in the eye, with a decrease from P1, P9, to P16, whereas levels for PAR-2, PAR-3, and PAR-4 remain unchanged throughout. In the retina of the adult rat, PAR-1 is highly expressed, whereas PAR-2 and PAR-3 are moderately expressed, compared to low PAR-4 expression. To elucidate possible roles of PARs after trauma, we carried out semiquantitative RT-PCR analysis of expression of all 4 PAR subtypes, beginning 6 hr after partial optic nerve crush (ONC) in the adult rat until 3 weeks after the mild trauma. Levels of PAR mRNA for all four subtypes were upregulated as early as 6 hr after unilateral ONC, except PAR-3, which showed a delayed upregulation. PAR-1, PAR-3, and PAR-4 mRNA levels returned to almost basal levels at 3 weeks post-crush, whereas PAR-2 mRNA level was still high by the end of 3 weeks after crush. Although the lesion was unilateral, PAR mRNA expression in the contralateral, uninjured side was affected to levels almost comparable to those in the injured side. Previous studies have shown an increase in thrombin levels at the site of injury, retinal ganglion cell degeneration by necrosis and apoptosis, and PAR activation as consequences of nerve crush. PAR upregulation because of nerve crush in the mild trauma model could act as an effector of early cell death. Eventual return of receptor mRNA to basal levels is consistent with neuroprotection.
Assuntos
Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Traumatismos do Nervo Óptico/metabolismo , Receptores de Trombina/biossíntese , Retina/metabolismo , Animais , Olho/crescimento & desenvolvimento , Masculino , RNA Mensageiro/biossíntese , Ratos , Receptor PAR-1 , Receptor PAR-2 , Retina/crescimento & desenvolvimentoRESUMO
The expression of the genes for the alpha-subunit of AChR (AChR alpha), for the myogenic factors myogenin and MyoD, for the calcium-binding protein parvalbumin (PV), and for the muscular chloride channel CIC-1 was studied in the three mouse spinal muscular atrophies (SMAs). These were the mutants "wobbler" (WR), "muscle deficient" (MDF) and "progressive motor neuronopathy" (PMN). Murine myopathies "muscular dystrophy with myositis" (MDM) and "X-linked muscular dystrophy" (MDX) were used as controls. AChR alpha and myogenin mRNA levels were strongly elevated in muscles affected by SMAs (reflecting denervation), whereas only myogenin mRNA was moderately elevated in MDX and MDM muscles, probably due to fiber regeneration. As in denervated muscle, CIC-1 and PV mRNA levels were lowered in SMAs. No changes were seen in muscles of up to 222-day-old symptomless ciliary neurotrophic factor (CNTF) knockout mice. The patterns of gene expression were characteristic for the type of muscle disease, indicating their possible usefulness for clinical diagnosis.
