RESUMEN
Death-associated protein kinase (DAP-kinase) is Ca(2+)/calmodulin-dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon-gamma-induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP-kinase, the tissue distribution and developmental changes in mRNA expression of DAP-kinase were investigated by Northern blot and in situ hybridization analyses. DAP-kinase mRNA was predominantly expressed in brain and lung. In brain, DAP-kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP-kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP-kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP-kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP-kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP-kinase and neuronal cell death.
Asunto(s)
Apoptosis/fisiología , Encéfalo/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , ARN Mensajero/análisis , Animales , Proteínas Reguladoras de la Apoptosis , Isquemia Encefálica/fisiopatología , Proteínas Quinasas Asociadas a Muerte Celular , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hibridación in Situ , Prosencéfalo/irrigación sanguínea , ARN Complementario , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABAA, nACh, 5-HT3, P2X, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D, L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate.