Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase M synthesis and induce immediate preconditioning protection of rat hippocampal slices

Abstract

Anaesthetic preconditioning occurs when a volatile anaesthetic, such as sevoflurane, is administered before a hypoxic or ischaemic insult; this has been shown to improve neuronal recovery after the insult. We found that sevoflurane-induced preconditioning in the rat hippocampal slice enhances the hypoxic hyperpolarization of CA1 pyramidal neurons, delays and attenuates their hypoxic depolarization, and increases the number of neurons that recover their resting and action potentials after hypoxia. These altered electrophysiological effects and the improved recovery corresponded with an increase in the amount of a constitutively active, atypical protein kinase C isoform found in brain, protein kinase M zeta (PKMζ). A selective inhibitor of this kinase, zeta inhibitory peptide (ZIP), blocked the increase in the total amount of PKMζ protein and the amount of the activated form of this kinase, phospho-PKMζ (p-PKMζ); it also blocked the altered electrophysiological effects and the improved recovery. We found that both cycloheximide, a general protein synthesis inhibitor, and rapamycin, a selective inhibitor of the mTOR pathway for regulating protein synthesis, blocked the increase in p-PKMζ, the electrophysiological changes, and the improved recovery due to sevoflurane-induced preconditioning. Glibenclamide, a KATP channel blocker, when present only during the hypoxia, prevented the enhanced hyperpolarization, the delayed and attenuated hypoxic depolarization, and the improved recovery following sevoflurane-induced preconditioning. To examine the function of persistent PKMζ and KATP channel activity after the preconditioning was established, we administered 4% sevoflurane for 30 min and then discontinued it for 30 min before 10 min of hypoxia. When either tolbutamide, a KATP channel blocker, or ZIP were administered at least 15 min after the washout of sevoflurane, there was little recovery compared with sevoflurane alone. Thus, continuous KATP channel and PKMζ activity are required to maintain preconditioning protection. We conclude that sevoflurane induces activation of the mTOR pathway, increasing the new protein synthesis of PKMζ, which is constitutively phosphorylated to its active form, leading to an increased KATP channel-induced hyperpolarizaton. This hyperpolarization delays and attenuates the hypoxic depolarization, improving the recovery of neurons following hypoxia. Thus, sevoflurane acts via a metabotropic pathway to improve recovery following hypoxia.

Figure 1. Effect of zeta inhibitory peptide (ZIP) and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia

A, resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given via a calibrated vaporizer in the gas stream for 15 min and then washed out for 5 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. The figure shows the potential for 15 min after hypoxia; the data in the text and the statistics are from 60 min after the hypoxia. When 5 μm ZIP is applied 10 min before and during the sevoflurane treatment, the effects of sevoflurane on the electrophysiological changes are blocked. All points are the mean ± SEM (n = 8 slices and animals for each group, one cell was recorded from each slice). When a bar is not shown the size of the standard error is less than the size of the symbol for that point. B, a continuous recording from a single CA1 pyramidal cell from each group before, during and after hypoxia; the data in A come from 10 such recordings from each group. The 3 responses after hypoxia in the sevoflurane-alone hypoxia group are due to Schaffer collateral/commissural stimulation and do not represent spontaneous activity. The horizontal calibration is indicated by the 10 min hypoxia bar, the vertical calibration is ∼60 mV from before hypoxia to after hypoxia in the untreated and ZIP + sevo-treated groups (the scale is the same for the sevoflurane preconditioning group).

Figure 2. Effect of ZIP and sevoflurane-induced preconditioning on protein kinase M zeta (PKMζ) protein levels before, during and after 10 min of hypoxia

Slices are either untreated, subjected to sevoflurane-induced preconditioning or ZIP 10 min before sevoflurane-induced preconditioning. A and C, For each Western blot an untreated non-hypoxic control is run on the same gel and used as a normalization standard; its density is set to 1 and all other bands are a ratio of this density. Six CA1 regions from tissue treated in the same chamber are from a single animal and are microdissected and pooled to run in a single lane. GAPDH is not affected by hypoxia and used to normalize the values for total protein within each lane. Each bar represents the mean and standard error of the mean from 10 animals. Sevoflurane-induced preconditioning significantly increased the amount of PKMζ protein before, during and after hypoxia. ZIP reduced the amount of PKMζ protein at these time points. * P < 0.05 compared with untreated group. B, representative Western blots from each experiment measuring PKMζ and GAPDH. C, effect of ZIP and sevoflurane-induced preconditioning on phosphorylated protein kinase M zeta (p-PKMζ) protein levels before, during and after 10 min of hypoxia. p-PKMζ is the activated form of PKMζ; unlike other members of the PKC family, PKMζ is constitutively phosphorylated and activated. Treatment is as described above. Sevoflurane-induced preconditioning significantly increased the amount of p-PKMζ protein before, during and after hypoxia. Zeta inhibitiory peptide reduced the amount of p-PKMζ protein at these time points. D, representative Western blots from each experiment measuring p-PKMζ and GAPDH.

Figure 3. Effect of cycloheximide or rapamycin and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia

Resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia. In untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given in the gas stream for 15 min and then washed out for 5 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. The figure shows the potential for 20 min after hypoxia; the data in the text and the statistics are from the data at 60 min after the hypoxia. When either cycloheximide (200 μm), a general inhibitor of protein synthesis, or rapamycin (200 nm), a specific inhibitor of the mTOR pathway, is applied 15 min before and during the sevoflurane treatment, the effects of sevoflurane on the electrophysiological changes are blocked. All points are the mean ± SEM. When a bar is not shown, the size of the standard error is less than the size of the symbol for that point. N is equal to the number of slices and animals for each group; one cell was recorded from each slice. The number of animals for the untreated group is n = 8; the sevoflurane-induced preconditioning group n = 6; the sevoflurane plus cycloheximide group n = 8; the sevoflurane plus rapamycin group n = 8.

Figure 4. Effect of cycloheximide or rapamycin and sevoflurane-induced preconditioning on phosphorylated protein kinase M zeta (p-PKMζ) protein levels before, during and after 10 min of hypoxia

A, slices are either untreated, subjected to sevoflurane-induced preconditioning or either cycloheximide (200 μm) or rapamycin (200 nM) 15 min before and during sevoflurane application. For each Western blot an untreated non-hypoxic control is run on the same gel and used as a normalization standard; its density is set to 1 and all other bands are a ratio of this density. GAPDH is used to normalize the values for total protein within each lane. Six CA1 regions from tissue treated in the same chamber are from a single animal and are microdissected and pooled to run in a single lane. Each bar represents the mean and SEM from 10 animals. Sevoflurane-induced preconditioning significantly increased the amount of p-PKMζ protein before, during and after hypoxia. When either cycloheximide or rapamycin was present before the sevoflurane the amount of p-PKMζ protein was reduced, compared with sevoflurane-induced preconditioning alone. * P < 0.05 compared with untreated group. B, representative Western blots from each experiment.

Figure 5. Effect of glibenclamide and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia

A, resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane-induced preconditioning is given, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. When 5 μm glibenclamide, a blocker of the K_ATP channel, is applied only during the hypoxia, the effects of sevoflurane-induced preconditioning on the electrophysiological changes are blocked. All points are the mean ± SEM (n = 10 slices and animals for each group; one cell was recorded from each slice). When a bar is not shown, the size of the standard error is less than the size of the symbol for that point.

Figure 6. Effect of tolbutamide and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia

Resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given in the gas stream for 30 min and then washed out for 30 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. When tolbutamide (5 μm) is applied during the hypoxia, only after sevoflurane washout for 30 min, the effects of sevoflurane on the electrophysiological changes are blocked. The figure shows the potential for 25 min after hypoxia; the data in the text and the statistics are from the data at 60 min after the hypoxia. All points are the mean ± SEM. When a bar is not shown, the size of the standard error is less than the size of the symbol for that point. N is equal to the number of slices and animals for each group; one cell was recorded from each slice. The number of animals for the untreated group is n = 7; the sevoflurane-induced preconditioning group n = 6; the sevoflurane plus tolbutamide group n = 6.

Figure 7. Effect of PKMζ inhibitory peptide (ZIP) and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia

Resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given in the gas stream for 30 min and then washed out for 30 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. When ZIP (5 μm) is applied 15 min before and during the hypoxia, the effects of sevoflurane on the electrophysiological changes are blocked. The figure shows the potential for 25 min after hypoxia; the data in the text and the statistics are from the data at 60 min after the hypoxia. All points are the mean ± SEM. When a bar is not shown, the size of the standard error is less than the size of the symbol for that point. Each treatment group contained 10 slices from 10 different animals; one cell was recorded from each slice.

Figure 8. Potential mechanism of sevoflurane-induced preconditioning

The black arrows do not imply a direct action – there may be intermediate effects.