RESUMO
Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Droga/genética , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Sítios de Ligação , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Glibureto/farmacologia , Glibureto/uso terapêutico , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fator 1 Induzível por Hipóxia/genética , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/prevenção & controle , Imuno-Histoquímica , Canais KATP/genética , Canais KATP/metabolismo , Luciferases/genética , Masculino , Microvasos/citologia , Microvasos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Wistar , Receptores de Droga/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Receptores de Sulfonilureias , TransfecçãoRESUMO
Acute spinal cord injury (SCI) causes progressive hemorrhagic necrosis (PHN), a poorly understood pathological process characterized by hemorrhage and necrosis that leads to devastating loss of spinal cord tissue, cystic cavitation of the cord, and debilitating neurological dysfunction. Using a rodent model of severe cervical SCI, we tested the hypothesis that sulfonylurea receptor 1-regulated (SUR1-regulated) Ca(2+)-activated, [ATP](i)-sensitive nonspecific cation (NC(Ca-ATP)) channels are involved in PHN. In control rats, SCI caused a progressively expansive lesion with fragmentation of capillaries, hemorrhage that doubled in volume over 12 hours, tissue necrosis, and severe neurological dysfunction. SUR1 expression was upregulated in capillaries and neurons surrounding necrotic lesions. Patch clamp of cultured endothelial cells exposed to hypoxia showed that upregulation of SUR1 was associated with expression of functional SUR1-regulated NC(Ca-ATP) channels. Following SCI, block of SUR1 by glibenclamide or repaglinide or suppression of Abcc8, which encodes for SUR1 by phosphorothioated antisense oligodeoxynucleotide essentially eliminated capillary fragmentation and progressive accumulation of blood, was associated with significant sparing of white matter tracts and a 3-fold reduction in lesion volume, and resulted in marked neurobehavioral functional improvement compared with controls. We conclude that SUR1-regulated NC(Ca-ATP) channels in capillary endothelium are critical to development of PHN and constitute a major target for therapy in SCI.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Canais de Cálcio/metabolismo , Células Endoteliais/metabolismo , Hemorragia/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Canais de Sódio/metabolismo , Traumatismos da Medula Espinal/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Comportamento Animal/efeitos dos fármacos , Capilares/metabolismo , Capilares/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glibureto/farmacologia , Hemorragia/tratamento farmacológico , Hemorragia/etiologia , Hemorragia/patologia , Hipoglicemiantes/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Necrose/tratamento farmacológico , Necrose/etiologia , Necrose/metabolismo , Necrose/patologia , Oligodesoxirribonucleotídeos Antissenso , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização , Ratos , Ratos Long-Evans , Receptores de Droga , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Receptores de Sulfonilureias , Fatores de TempoRESUMO
Pathological conditions in the central nervous system, including stroke and trauma, are often exacerbated by cerebral edema. We recently identified a nonselective cation channel, the NC(Ca-ATP) channel, in ischemic astrocytes that is regulated by sulfonylurea receptor 1 (SUR1), is opened by depletion of ATP and, when opened, causes cytotoxic edema. Here, we evaluated involvement of this channel in rodent models of stroke. SUR1 protein and mRNA were newly expressed in ischemic neurons, astrocytes and capillaries. Upregulation of SUR1 was linked to activation of the transcription factor Sp1 and was associated with expression of functional NC(Ca-ATP) but not K(ATP) channels. Block of SUR1 with low-dose glibenclamide reduced cerebral edema, infarct volume and mortality by 50%, with the reduction in infarct volume being associated with cortical sparing. Our findings indicate that the NC(Ca-ATP) channel is crucially involved in development of cerebral edema, and that targeting SUR1 may provide a new therapeutic approach to stroke.