RESUMO
Protein kinase Calpha (PKCalpha) plays a major role in the induction of long-term depression (LTD) in a cerebellar Purkinje cell (PC). The sequential activation model for classical PKC states that PKCalpha translocates to the plasma membrane by binding Ca(++) and then becomes fully activated by binding diacylglycerol (DAG), which enables estimation of the activity by monitoring its localization. Here, we performed simultaneous electrophysiological recording and fluorescence imaging in a cultured PC expressing GFP-tagged PKCalpha. When a PC was depolarized, PKCalpha transiently translocated to the plasma membrane in a Ca(++)-dependent manner. Application of membrane permeable DAG or the blocker of DAG lipase prolonged the translocation. These results suggest that the sequential activation model is applicable to PCs. Conjunctive applications of glutamate and depolarization pulse induced LTD, but did not prolong the translocation. Thus, our results imply that persistent activation of PKCalpha is not necessary for the expression of LTD.
Assuntos
Depressão Sináptica de Longo Prazo/fisiologia , Proteína Quinase C-alfa/metabolismo , Células de Purkinje/enzimologia , Animais , Cálcio/metabolismo , Membrana Celular/enzimologia , Células Cultivadas , Cerebelo/citologia , Diglicerídeos/metabolismo , Proteínas de Fluorescência Verde/genética , Técnicas de Patch-Clamp , Proteína Quinase C-alfa/genética , Células de Purkinje/citologia , Ratos , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in the peripheral blood leukocytes of dogs, rabbits and cats. Rabbits had significantly higher plasma glucose concentrations than dogs or cats. Feline leukocytes showed higher LDH and lower MDH activities than canine or rabbit leukocytes. The M/L ratio, defined as the MDH activity divided by the LDH activity in cytosolic fractions, was considered to be a good indicator with which to evaluate the metabolic state in animal tissues. The M/L ratio was highest in canine and lowest in feline leukocytes. LDH-2 and LDH-3 isoenzymes were dominant in canine leukocytes. LDH-1 and LDH-2 were dominant in rabbit leukocytes, whereas LDH-5 was dominant in feline leukocytes. It was evident that there were significant differences in energy metabolism between the leukocytes of dogs, rabbits and cats.