Effect of ɛPKC translocation on respiration in synaptosomal mitochondria after ischemic preconditioning

Introduction

Recently we showed that protein kinase c epsilon (ɛPKC) played a key role in the signaling pathway of ischemic preconditioning (IPC) ¹ . In the same study we demonstrated that ɛPKC is translocated from cytosolic to the particulate compartment following IPC ¹ . However, the exact subcellular localization of the translocated ɛPKC after IPC has not been defined. The goals of the present study were to define the subcellular localization of translocated ɛPKC at different reperfusion periods after IPC and to define whether this translocation is accompanied by a physiological response. Since we showed that IPC protected brain against mitochondrial dysfunction ² , and other groups showed that activated ɛPKC exerts its anti-apoptotic effect by acting on mitochondria ³ , we concentrated our studies on brain mitochondria after IPC.

Method

IPC was produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 min in the rat ² . The hippocampus was dissected out at different reperfusion times and subjected to the subcellular fractionation to separate synaptosomes, non-synaptic mitochondria, and cytosol. The level of ɛPKC was determined by Western blotting followed by densitometric analysis. The effect of ɛPKC activation on respiration of synaptosomal mitochondria was determined polarographically using ɛPKC specific peptide activator (ψɛRACK)^{1, 2}. For respiration studies synaptosomes were permeabilized with 0.007% digitonin.

Results

We confirmed our previous findings that the amount of ɛPKC in soluble fraction of hippocampus was lower by 54% (n=3;p<0. 05) and 57% (n=3;p<0.05) at 0.5 h and 3 h of reperfusion after IPC, respectively when compared with the corresponding sham group. Concomitantly, we found increases in levels of ɛPKC in non-synaptic mitochondrial fraction by 40% (n=3) only at 0.5 h of reperfusion after IPC when compared with the sham group (n=2). Synaptosomal levels of ɛPKC increased by 32% (n=3), 76% (n=3), and 91% (n=3) at 0.5, 3, and 24 h of reperfusion after IPC, respectively when compared with the corresponding sham group (0.5 h, n=3; 1 h, n=2; 3 h, n=3). Since we found higher levels of ɛPKC in the synaptosomal fraction after IPC, in the next study we determined the effect of ɛPKC activation on mitochondrial functions in synaptosomes. The rate of oxygen consumption was higher by 39% (p<0.05) in ψɛRACK treated synaptosomes (n=8) compared to carrier peptide (Tat) treated synaptosomes (n=8), when succinate+glycerol-3-phosphate were used as the substrates. Similarly the rate of oxygen consumption was higher by 25% (p<0.05) in ψɛRACK treated synaptosomes (n=8) as compared to Tat treated synaptosomes (n=8), when ascorbate+TMPD were used as substrates.

Conclusion

Increase in the level of synaptosomal ɛPKC after IPC and the effect of its activation on synaptosomal mitochondria suggest that higher levels of this anti-apoptotic PKC in synaptosomes may protect synaptosomal mitochondria from the excitotoxicity that results from cerebral ischemia. This suggests that a key mechanism by which ɛPKC is neuroprotective is by translocating to mitochondria.