This study focused on the effects of status epilepticus on the activity of calcineurin, a neuronally enriched, calcium-dependent phosphatase. Calcineurin is an important modulator of many neuronal processes, including learning and memory, induction of apoptosis, receptor function and neuronal excitability. Therefore, a status epilepticus-induced alteration of the activity of this important phosphatase would have signi®cant physiological implications. Status epilepticus was induced by pilocarpine injection and allowed to continue for 60 min. Brain region homogenates were then assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A signi®cant status epilepticus-dependent increase in both basal and Mn 21 -dependent calcineurin activity was observed in homogenates isolated from the cortex and hippocampus, but not the cerebellum. This increase was resistant to 150 nM okadaic acid, but sensitive to 50 mM okadaic acid. The increase in basal activity was also resistant to 100 mM sodium orthovanadate. Both maximal dephosphorylation rate and substrate af®nity were increased following status epilepticus. However, the increase in calcineurin activity was not found to be due to an increase in calcineurin enzyme levels. Finally, increase in calcineurin activity was found to be NMDAreceptor activation dependent. The data demonstrate that status epilepticus resulted in a signi®cant increase in both basal and maximal calcineurin activity.

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. This study examined the effects of moderate, central fluid percussion injury on the activity of this important neuronal enzyme. Animals were sacrificed at several time-points postinjury and cortical, hippocampal, and cerebellar homogenates were assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant brain injury-dependent increase was observed in both hippocampal and cortical homogenates under both basal and maximally-stimulated reaction conditions. This increase persisted 2-3 weeks post-injury. Brain injury did not alter substrate affinity, but did induce a significant increase in the apparent maximal dephosphorylation rate. Unlike the other brain regions, no change in calcineurin activity was observed in the cerebellum following brain injury. No brain region tested displayed a significant change in calcineurin enzyme levels as determined by Western blot, demonstrating that increased enzyme synthesis was not responsible for the observed increase in activity. The data support the conclusion that fluid percussion injury results in increased calcineurin activity in the rat forebrain. This increased activity has broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

c-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABA A R function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABA A receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABA A receptors were subsequently analyzed for both allosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABA A R was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABA A receptor a1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABA A receptor a1 subunit modulated allosteric modulator binding to the GABA A receptor.