Endogenous phosphorylation in vitro: differential effects of brain state (anesthesia, post-mortem) on...

In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [32P]Labelled phosphoproteins were separated by SDS-slab gel electrophoresis, and the autoradiographs were analyzed by densitometry. We report here that Band F of our previous reports can be separated into two components, F1 and F2, using an improved gel system. This separation is particularly relevant in this report since these components appear to be differentially sensitive to the manipulations used. Specifically, we found that while F1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F2 was relatively stable. While phosphorylation of F2 was also stable 24 h post-mortem, Band F1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F2 somewhat, it eliminated the in vitro phosphorylation of Band F1. We found that under light barbiturate anesthesia, just at the time when the animals lost the righting reflex, the in vitro phosphorylation of Bands D (MR 78,000--80,000 daltons), F1 (MR 47,000--49,000) and F2 (MR 40,000--45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F1 was depressed in tissue prepared from animals that were deeply comatose. These effects of pentobarbital were more pronounced when animals were sacrificed by liquid nitrogen immersion, rather than by decapitation. Cyclic AMP-dependent phosphorylation of Band D exhibited remarkable stability 24 h post-morten (7 days in one case), even when brain tissue was left at room temperature (21--23 degrees C). Phosphorylation of Band F1, however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Band F1 is: (3) increased in phosphorylation in liquid nitrogen P2 preparations, and may be (4) cAMP-independent, (5) rapidly turning over its phosphate in vivo, and (6) altered by a training experience. Other evidence suggests that: (7) Band F1 phosphorylation may be Ca2+-dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. The results suggest an important and perhaps unique role for Band F1 in neuronal function.