RESUMEN
An antisense (AS) oligodeoxynucleotide based on a conserved sequence in the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) was used to decrease expression of this Ca(2+) transporter in primary neuronal cultures. Two AS oligo applications decreased NCX activity by approximately 40% within 12-24 h, and neither sense (S) or missense (MS) oligos altered NCX activity. The reduced NCX expression was confirmed by immunoblots and enzyme-linked immunosorbent assays (ELISAs). Resting [Ca(2+)](i) levels were 20% higher in AS-treated neurons and showed a slower return to baseline levels following activation of Ca(2+) influx by N-methyl-D-aspartate (NMDA). These results suggest that NCX plays a significant role in maintaining neuronal Ca(2+) homeostasis and in restoring baseline Ca(2+) levels following depolarization.
Asunto(s)
Encéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Animales , Encéfalo/citología , Encéfalo/metabolismo , Calcio/metabolismo , Células Cultivadas , Secuencia Conservada/genética , Ensayo de Inmunoadsorción Enzimática , Colorantes Fluorescentes , Fura-2 , Homeostasis/efectos de los fármacos , Immunoblotting , Líquido Intracelular/metabolismo , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacología , Neuronas/citología , Neuronas/metabolismo , Oligonucleótidos Antisentido/genética , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
We have assessed the functional properties of both calmodulin (CaM) and the plasma membrane Ca(2+)-ATPase in brains of young, middle aged, and old Fisher 344 rats. Under optimal conditions of saturating Ca2+ and ATP, the CaM-activated Ca(2+)-ATPase activity was decreased with increasing age, particularly when CaM isolated from the brains of aged rats was used to stimulate the enzyme. In the case of CaM, structural modifications within the primary sequence of the protein from aged brains were identified. We found that during normal biological aging approximately 6 methionine residues were modified to their corresonding sulfoxide per CaM, and no other amino acids were modified. Some aspects of the age-related decline in the effectiveness of CaM as an activator of Ca(2+)-ATPase could be simulated using a range of reactive oxygen species (including hydrogen peroxide and oxoperoxynitrite) and, in the latter case, the extent of oxidative modification of specific methionine residues was directly related to their surface accessibility. The pattern of oxidative modification of the methionines in the aged CaM was less straightforward, though both in vitro oxidation of CaM and aging within the brain markedly decreased the functional properties of this important Ca(2+)-regulating protein.