Mitochondrial KATP channel opening initiate ischemic preconditioning in organotypic hippocampal slice cultures

Introduction

Ischemic preconditioning (IPC) is an intrinsic adaptive condition that results in tolerance in different organs when they are subjected to mild ischemic insults prior to a ‘lethal’ ischemic insult. In heart, the mitochondrial ATP dependent potassium (mtK+ATP) channel activation has been reported to mediate preconditioning protection. However, in brain role of mtK+ATP channel in the IPC induction is still not clearly understood. Additionally, our previous studies demonstrated that IPC induce neuronal survival in CA1 region of hippocampus required epsilon protein kinase C (ɛPKC) activation^{1, 2}. Thus, the goal of the present study was to define whether mtK+ATP channel activation is required during triggering phase of IPC. Further, we hypothesize that ɛPKC mediated neuroprotection require mtKATP channel activation.

Methods

Hippocampal slices were obtained from 9–11 days old Sprague Dawley rats and cultured for 14–15 days before experiments. Slices exposed to oxygen/glucose deprivation (OGD) for 40 min (‘test’ ischemia) were used as the ischemia group. Slices exposed to OGD for 15 min (or a pharmacological agent), 48 h prior to ‘test’ ischemia were used as the IPC/pharmacological preconditioned (PPC) groups. Propidium Iodide (PI) fluorescence images were obtained using a SPOT CCD camera and were digitized using SPOT advanced software. Percentage of relative optical intensity was used as an index of cell death. Results are expressed, as mean ± SD. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.

Results

We confirm our previous findings that IPC induce neuroprotection in CA1 region of hippocampus in organotypic slice cultures. PI fluorescence of ‘test’ ischemic and IPC groups were 54.8 ± 14.8% (n = 12) and 28.5 ± 13.0 % (n = 8), respectively (p<0.001). To test the hypothesis that mtK+ATP channel opening mimic IPC induced neuroprotection, we exposed slices to the mtK+ATP channel agonists (diazoxide 50 μM/pinacidil 10 μm). PI fluorescence of diazoxide and pinacidil treated groups were 16.8 ± 7.6 % (n = 8) and 31.9 ± 5.1% (n = 5), respectively (p < 0.001, compared to ischemia). To further characterize role of mtK+ATP channel in IPC and after ɛPKC agonist (ψɛRACK peptide 0.02 μM) mediated PC, slices were treated with the mtK+ATP channel antagonist 5-hydroxydecanoic acid (HD, 100 ìM). This treatment abolished IPC/ɛPKC mediated neuroprotection. PI fluorescence of HD treated and IPC groups were 58.13 ± 15.9 % (n =12) and 29.7 ± 15.7 % (n =8), respectively (p < 0.001). Similarly, PI fluorescence values of ɛPKC agonist and ɛPKC agonist plus HD treated groups were 33.5 ± 6.7 % (n = 5), and 54.3 ± 3.3 % (n = 5), respectively.

Conclusion

The signal transduction pathway that ensues following IPC/ PPC require opening of mtK+ATP channel. It is possible however, that the plasma K+ATP may also play a role in the IPC-induced neuroprotection.