RESUMO
Type 1 inositol 1,4,5-trisphosphate receptors are phosphorylated by cyclic-AMP-dependent protein kinase A at serines 1589 and 1755, with serine 1755 phosphorylation greatly predominating in the brain. Inositol 1,4,5-trisphosphate receptor protein kinase A phosphorylation augments Ca(2+) release. To assess type 1 protein kinase A phosphorylation dynamics in the intact organism, we developed antibodies selective for either serine 1755 phosphorylated or unphosphorylated species. Immunohistochemical studies reveal marked variation in localization. For example, in the hippocampus the phosphorylated type 1 inositol 1,4,5-trisphosphate receptor is restricted to CA1, while the unphosphorylated receptor occurs ubiquitously in CA1-CA3 and dentate gyrus granule cells. Throughout the brain the phosphorylated type 1 inositol 1,4,5-trisphosphate receptor is selectively enriched in dendrites, while the unphosphorylated receptor predominates in cell bodies. Focal cerebral ischemia in rats and humans is associated with dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors, and glutamatergic excitation of cerebellar Purkinje cells mediated by ibogaine elicits dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors that precedes evidence of excitotoxic neuronal degeneration. We have demonstrated striking variations in regional and subcellular distribution of inositol 1,4,5-trisphosphate receptor phosphorylation that may influence normal physiological intracellular Ca(2+) signaling in rat and human brain. We have further shown that the subcellular distribution of inositol 1,4,5-trisphosphate receptor phosphorylation in neurons is regulated by excitatory neurotransmission, as well as excitotoxic insult and neuronal ischemia-reperfusion. Phosphorylation dynamics of type 1 inositol 1,4,5-trisphosphate receptors may modulate intracellular Ca(2+) release and influence the cellular response to neurotoxic insults.
Assuntos
Isquemia Encefálica/metabolismo , Canais de Cálcio/metabolismo , Neurônios/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adulto , Animais , Especificidade de Anticorpos , Western Blotting , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/patologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dendritos/metabolismo , Feminino , Humanos , Ibogaína/toxicidade , Imuno-Histoquímica , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Neurônios/citologia , Especificidade de Órgãos , Células PC12 , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Células de Purkinje/citologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Poly(ADP-ribose) polymerase (PARP) transfers ADP ribose groups from NAD(+) to nuclear proteins after activation by DNA strand breaks. PARP overactivation by massive DNA damage causes cell death via NAD(+) and ATP depletion. Heretofore, PARP has been thought to be inactive under basal physiologic conditions. We now report high basal levels of PARP activity and DNA strand breaks in discrete neuronal populations of the brain, in ventricular ependymal and subependymal cells and in peripheral tissues. In some peripheral tissues, such as skeletal muscle, spleen, heart, and kidney, PARP activity is reduced only partially in mice with PARP-1 gene deletion (PARP-1(-/-)), implicating activity of alternative forms of PARP. Glutamate neurotransmission involving N-methyl-d-aspartate (NMDA) receptors and neuronal nitric oxide synthase (nNOS) activity in part mediates neuronal DNA strand breaks and PARP activity, which are diminished by NMDA antagonists and NOS inhibitors and also diminished in mice with targeted deletion of nNOS gene (nNOS(-/-)). An increase in NAD(+) levels after treatment with NMDA antagonists or NOS inhibitors, as well as in nNOS(-/-) mice, indicates that basal glutamate-PARP activity regulates neuronal energy dynamics.
Assuntos
Encéfalo/metabolismo , Dano ao DNA/genética , Ácido Glutâmico/metabolismo , Óxido Nítrico/metabolismo , Proteínas/genética , Animais , Autorradiografia , Células Cultivadas , Ativação Enzimática , Imuno-Histoquímica , Rim/metabolismo , Camundongos , Camundongos Knockout , N-Metilaspartato/metabolismo , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases , Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Streptozotocin (STZ) selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme whose overactivation by DNA strand breaks depletes its substrate NAD+ and then ATP, leading to cellular death from energy depletion. We demonstrate DNA damage and a major activation of PARP in pancreatic islets of STZ-treated mice. These mice display a 500% increase in blood glucose and major pancreatic islet damage. In mice with homozygous targeted deletion of PARP (PARP -/-), blood glucose and pancreatic islet structure are normal, indicating virtually total protection from STZ diabetes. Partial protection occurs in PARP +/- animals. Thus, PARP activation may participate in the pathophysiology of type I diabetes, for which PARP inhibitors might afford therapeutic benefit.
