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Comparative Study
. 2009 May;110(5):986-95.
doi: 10.1097/ALN.0b013e31819dadc7.

Neuronal preconditioning by inhalational anesthetics: evidence for the role of plasmalemmal adenosine triphosphate-sensitive potassium channels

Carsten Bantel  1 , Mervyn Maze, Stefan Trapp
Affiliations
Comparative Study

Neuronal preconditioning by inhalational anesthetics: evidence for the role of plasmalemmal adenosine triphosphate-sensitive potassium channels

Carsten Bantel et al. Anesthesiology. 2009 May.

Abstract

Background: Ischemic preconditioning is an important intrinsic mechanism for neuroprotection. Preconditioning can also be achieved by exposure of neurons to K+ channel-opening drugs that act on adenosine triphosphate-sensitive K+ (K(ATP)) channels. However, these agents do not readily cross the blood-brain barrier. Inhalational anesthetics which easily partition into brain have been shown to precondition various tissues. Here, the authors explore the neuronal preconditioning effect of modern inhalational anesthetics and investigate their effects on K(ATP) channels.

Methods: Neuronal-glial cocultures were exposed to inhalational anesthetics in a preconditioning paradigm, followed by oxygen-glucose deprivation. Increased cell survival due to preconditioning was quantified with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction test. Recombinant plasmalemmal K(ATP) channels of the main neuronal type (Kir6.2/SUR1) were expressed in HEK293 cells, and the effects of anesthetics were evaluated in whole cell patch clamp recordings.

Results: Both sevoflurane and the noble gas xenon preconditioned neurons at clinically used concentrations. The effect of sevoflurane was independent of K(ATP) channel activation, whereas the effect of xenon required the opening of plasmalemmal K(ATP) channels. Recombinant K(ATP) channels were activated by xenon but inhibited by halogenated volatiles. Modulation of mitochondrial K-ATP channels did not affect the activity of K(ATP) channels, thus ruling out an indirect effect of volatiles via mitochondrial channels.

Conclusions: The preconditioning properties of halogenated volatiles cannot be explained by their effect on K(ATP) channels, whereas xenon preconditioning clearly involves the activation of these channels. Therefore, xenon might mimic the intrinsic mechanism of ischemic preconditioning most closely. This, together with its good safety profile, might suggest xenon as a viable neuroprotective agent in the clinical setting.

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Figures

Fig. 1
Fig. 1
Preconditioning by xenon but not sevoflurane involves adenosine triphosphate–sensitive K+ (KATP) channels. A depicts the experimental timeframe adhered to. Neuronal and glial cells were cocultured for 2 weeks before a 2-h preconditioning stimulus with the anesthetics ± antagonists. After a further 24 h, cultures were exposed to oxygen–glucose deprivation (OGD) for 75 min, and a further 24 h later, damage was assessed using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. B, C, and D show the mean ± SEM data for preconditioning experiments. Cell viability was measured with MTT as optical density at 570 nm. The “normalized effect” describes the relative viability compared with maximal damage (OGD only, no preconditioning) and maximal survival (no OGD, naive cultures). Therefore, 100% cell viability represented the viability observed in naive cultures at the end of the protocol, whereas 0% cell viability indicated the viability found in cultures exposed to OGD only. B demonstrates that xenon preconditioning (Xe, 1 minimum alveolar concentration [MAC]) is effectively abolished by the KATP channel inhibitor tolbutamide (Xe + Tb, 0.1 mm), but not the mitochondrial K-ATP channel blocker 5-hydroxy-decanoic acid (5-HD, 0.5 mm). (C) In contrast, while sevoflurane (Sev, 2 MAC) effectively preconditions the cultures, this effect is not significantly reduced by tolbutamide (Tb) or 5-HD. (D) Preconditioning by the KATP channel opener diazoxide (Dz, 0.01 mm) is effectively blocked by either tolbutamide or 5-HD. Numbers (n) are given above the bars. *P < 0.05; **P < 0.01.
Fig. 2
Fig. 2
Adenosine triphosphate–sensitive K+ (KATP) channels expressed in HEK293 cells are mildly inhibited by sevoflurane. (A) Whole cell recording at a holding potential of −20 mV from an HEK293 cell transfected with Kir6.2 and SUR1. Sevoflurane (Sev) and tolbutamide (Tb) were bath applied as indicated by the horizontal bars. The dashed line indicates the zero-current level. Vertical deflections indicate the current response to a 700-ms voltage ramp from −20 mV to −120 mV. These current responses at points i and ii were plotted against the command voltage to obtain current–voltage relations (B). These demonstrate that the washout current has a reversal potential near the potassium equilibrium potential. (C) Current–voltage relations taken from A showing the concentration-dependent small inhibition of the KATP current by sevoflurane at clinically relevant concentrations, compared with the block by tolbutamide.
Fig. 3
Fig. 3
Halogenated volatile anesthetics inhibit adenosine triphosphate–sensitive K+ (KATP) channels. (A) Whole cell recording at a holding potential of −20 mV from an HEK293 cell transfected with Kir6.2 and SUR1. The dashed line indicates the zero-current level. Halothane, isoflurane, and sevoflurane (1 mm each) were bath applied as indicated by the horizontal bars. Each drug reversibly inhibited the whole cell KATP current. (B) Current–voltage relations taken from a recording as that shown in A demonstrating that the current inhibited by the volatile anesthetics has a reversal potential near EK. (C) Concentration–response curves of the inhibitory effect of halothane (n = 4–8), isoflurane (n = 3–5), and sevoflurane (n = 3–8) on whole cell KATP currents. The current in presence of the drug (I) is expressed as a fraction of the current in its absence (IC). The solid lines describe the best fit with a Hill equation using Ki = 1.32 mm and Hill coefficient (h) = 1.75 for halothane, Ki = 2.08 mm and h = 1.39 for isoflurane, and Ki = 47 mm and h = 0.34 for sevoflurane. In each case, it is assumed that complete inhibition of the KATP current could theoretically be achieved with high enough concentrations of the anesthetic. One minimum alveolar concentration is indicated by the vertical dashed line in each graph.
Fig. 4
Fig. 4
Xenon activates adenosine triphosphate–sensitive K+ (KATP) channels. (A) Whole cell recording from HEK293 cell transfected with Kir6.2 and SUR1 demonstrating reversible activation of the current by 80% xenon. (B) Mean effects of the two test concentrations of xenon on the holding current at −20 mV from KATP channel–transfected HEK293 cells. (C) Current–voltage relations in the absence and presence of 80% xenon and xenon + 0.1 mm tolbutamide. Xenon potentiated the KATP current, whereas tolbutamide completely inhibited the current, even in the presence of xenon. (D) Mean data from experiments as depicted in C. Mean holding current at −20 mV in the presence of the drug expressed as a fraction of that in the absence of any drug. Xenon led to an increase in holding current and tolbutamide blocked both the xenon-potentiated current and the current in the absence of xenon (dashed line). Numbers (n) are given above the bars. *P < 0.05.
Fig. 5
Fig. 5
Adenosine triphosphate–sensitive K+ (KATP) currents in whole cell recordings are not affected by specific blockers of mitochondrial KATP (mito K-ATP) channels. (A) Whole cell recording demonstrating the lack of effect of the mito K-ATP channel blocker 5-hydroxy-decanoic acid (5-HD, 0.5 mm) on KATP channels made up of Kir6.2 and SUR1. In contrast, tolbutamide (Tb) strongly and reversibly inhibited the whole cell current. The drugs were applied as indicated by the bars above the recording. (B) Current–voltage relations in the absence (control) and presence of 0.5 mm 5-HD or 0.1 mm tolbutamide. (C) Mean data from experiments as that shown in A. *P < 0.05.
Fig. 6
Fig. 6
Whole cell adenosine triphosphate–sensitive K+ (KATP) currents are not affected by specific openers of mito K-ATP channel openers. (A) Whole cell recording from HEK293 cell expressing Kir6.2 and SUR1. Plotted is the mean holding current at −20 mV every 15 s. The SUR1-specific K+ channel opener diazoxide (Dz) is applied as indicated by the solid bars and concentration-dependently potentiated the whole cell current. (B) Current–voltage relations demonstrating the lack of effect of the cardiac K+ channel opener pinacidil (Pin, 0.1 mm) on Kir6.2/SUR1 currents in HEK293 cells. (C) Mean effects of the various K+ channel openers on Kir6.2/SUR1 currents. (D) Diazoxide at 10 μm increases the plasma KATP current independently of its potential effect on mitochondrial KATP channels, as demonstrated by the lack of effect of the mitochondrial KATP channel inhibitor 5-hydroxy-decanoic acid (5-HD) on the diazoxide activation of the whole cell current. Numbers (n) are given above the bars. *P < 0.05.
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