RESUMEN
Protein kinase C (PKC) is an important intracellular signalling enzyme. Numerous studies have suggested that alterations in this enzyme occur in aging and dementia. The objective of this study was to examine PKC in the cerebral microcirculation in aging and Alzheimer's disease. PKC activity, amount, and isoform distribution were analyzed in microvessels from adult and aged rodents as well as from Alzheimer patients and nondemented elderly controls. PKC activity was lower in Alzheimer vessels than in vessels from control brains, despite the presence of similar levels of PKC enzyme. In contrast, both activity and enzyme levels in young and aged rats were comparable. The beta-isoform was present in both rat and human microvessels and there were no age- or disease-related alterations. The loss in activity in cerebromicrovascular PKC in Alzheimer's suggest that perturbations in phosphorylation signalling cascades may exist at the Alzheimer blood-brain barrier.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Encéfalo/irrigación sanguínea , Proteína Quinasa C/metabolismo , Anciano , Envejecimiento/metabolismo , Animales , Barrera Hematoencefálica , Estudios de Casos y Controles , Citosol/enzimología , Humanos , Immunoblotting , Isoenzimas/metabolismo , Microcirculación/enzimología , Ratas , Ratas Endogámicas F344 , Transducción de Señal , Acetato de Tetradecanoilforbol/metabolismoRESUMEN
The cerebral vasculature is central to the maintenance of the neuronal microenvironment. We have previously demonstrated that brain microvessels in Alzheimer's disease produce high, potentially toxic, levels of nitric oxide. It is our hypothesis that neuronal injury in Alzheimer's disease occurs because an abnormal endothelium secretes factors that are toxic to neurons. In this study, we report that inhibition of protein kinase C in endothelial cells causes release of a factor that is toxic to neurons in vitro. Our results demonstrate that this endothelium-derived toxic factor is soluble, heat-labile, susceptible to proteolysis, and loses activity with repeated freeze-thawing. The molecular weight of this putative protein is between 10 and 50 kDa, and 8 hours are required after protein kinase C inhibition to detect the endothelium-derived toxic factor in the media. Finally, the endothelium-derived toxic factor kills neurons within 2 hours, suggesting that cell death occurs via necrosis, not apoptosis. These data support the notion that endothelial cells can create an injurious microenvironment for neurons by producing molecules with noxious properties. Altered/dysfunctional endothelial cells in the cerebral microcirculation could be a novel, unexplored source of neurotoxic factors in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Endotelio Vascular/metabolismo , Neurotoxinas/biosíntesis , Proteína Quinasa C/antagonistas & inhibidores , Enfermedad de Alzheimer/enzimología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/citología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/enzimología , Humanos , Microcirculación , Peso Molecular , Degeneración Nerviosa , Neuronas/citología , RatasRESUMEN
Despite the close morphological association of beta-amyloid and vascular cells, the functional effects of amyloid in cerebral endothelial cells in Alzheimer's disease have not been assessed. In this study, effects of amyloid fractions purified from senile plaques of AD brains were compared to synthetic amyloid peptides for their ability to affect brain endothelial cells in vitro. Our results indicate that plaque-derived amyloid inhibit brain endothelial cell proliferation in vitro by 40%. This inhibition was specific for plaque-derived amyloid, was not evoked by synthetic A beta 1-40, and was not mediated by alterations in intracellular calcium levels. Amyloid fractions from AD brains, although not directly toxic to brain endothelial cells, inhibit endothelial replication in vitro and therefore could alter the ability of vessels to repair and regenerate after injury.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/farmacología , Encéfalo/efectos de los fármacos , División Celular/efectos de los fármacos , Endotelio/citología , Encéfalo/metabolismo , Calcio/metabolismo , Recuento de Células , Células Cultivadas , Endotelio/efectos de los fármacos , Humanos , Técnicas In VitroRESUMEN
Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.