A significant increase in both basal and maximal calcineurin activity in the rat pilocarpine model of status epilepticus

Abstract

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.

AMPA/Kainate Receptor-Mediated Downregulation of GABAergic Synaptic Transmission by Calcineurin after Seizures in the Developing Rat Brain

“…As shown in Figure 3B, CaN-A␣ expression was unchanged at 1 h after hypoxia compared with control levels (n ϭ 5 animals per group). These data indicate that the acute increase in CaN activity measured 1 h after hypoxia-induced seizures is not likely a result of a rapid increase in CaN expression but rather from an increase CaN activation state or kinetics, similar to that reported in adult status epilepticus models (Kurz et al, 2001). At 6 h after hypoxia, we observed a modest but nonsignificant increase (10%) in CaN-A␣ expression (n ϭ 5 per group) (data not shown).…”

“…These data suggested that CaN kinetics were altered by hypoxia. Notably, an early increase in basal and maximal CaN activity also has been reported in the adult brain after experimental status epilepticus (Kurz et al, 2001). Ca 2ϩ influx through GluRs regulates Ca 2ϩ -dependent intracellular enzymes such as CaN (Snyder et al, 1998b;Morioka et al, 1999;Vignes, 2001;Shibasaki et al, 2002) and calcium/calmodulindependent protein kinase II (CaMKII) , and CaN can be activated by increased intracellular Ca 2ϩ specifically through AMPAR activation (Nishi et al, 2002).…”

“…Homogenates were then normalized for protein, separated into aliquots, and stored at Ϫ80°C until use. CaN activity was then assayed using the procedure detailed by Kurz et al (2001). All reaction tubes were prepared on ice and contained the following: 25 mM morpholinopropanesulfonic acid, pH 7.0, 1 mM DTT, 2 mM p-nitrophenol phosphate (pNPP), and 150 nM okadaic acid.…”

“…Inhibitory GABA receptor (GABAR)-mediated synaptic transmission can be negatively modulated by the endogenous phosphatase calcineurin-A (CaN-A) (Huang and Dillon, 1998), and CaN-A can be activated by seizures in vivo (Kurz et al, 2001). Because CaN-A activation is Ca 2ϩ dependent, we hypothesized that AMPAR-mediated activation of CaN-A during and after hy-poxic seizures may contribute to hippocampal hyperexcitability by acutely downregulating inhibitory synaptic transmission.…”

Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death

“…High levels of Cn activity predispose thymocytes, T cells, Jurkat cells, and neuronal cells to apoptosis induced by T cell receptor activation, calcium ionophores, growth-factor deprivation, L-glutamate, and status epilepticus (18)(19)(20)(21). In our screen to identify new calpain substrates by using an in vitro small pool assay, we have identified mouse cain͞cabin1 as a putative calpain substrate.…”