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High-affinity block of voltage-operated rat IIA neuronal sodium channels by 2,6 di-tert-butylphenol, a propofol analogue

Haeseler, G.*; Leuwer, M.

European Journal of Anaesthesiology (EJA): March 2003 - Volume 20 - Issue 3 - p 220-224
Original Article
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Background and objective: Propofol is a phenol derivative (2,6 di-isopropylphenol) with a unique effect profile including activating effects on GABAA and blocking effects on voltage-operated sodium channels. If the substituents in the 2- and the 6-positions are replaced by tert-butyl groups, the resulting phenol derivative, 2,6 di-tert-butylphenol, despite being a close structural propofol analogue, completely lacks GABAA receptor effects. The aim of this in vitro study was to investigate the effects of 2,6 di-tert-butylphenol on voltage-operated neuronal sodium channels in order to determine whether and, if so, how these structural changes alter the sodium channel-blocking effect seen with propofol.

Methods: Whole-cell sodium inward currents through heterologously expressed rat type IIA sodium channels were recorded in the absence and presence of definite concentrations of 2,6 di-tert-butylphenol and propofol.

Results: When applied at concentrations ≥30 μmol, 2,6 di-tert-butylphenol completely and irreversibly blocked sodium inward currents. The blockade equilibrium time was about 2 min. A partial washout was possible only if the application was stopped before the equilibrium of the blockade was achieved.

Conclusions: 2,6 Di-tert-butylphenol exerts a high-affinity block of neuronal sodium channels. Apparently, the slight structural differences of 2,6 di-tert-butylphenol in comparison with propofol - which account for the lack of GABAA receptor effects - enhance its voltage-operated sodium channel-blocking effects. As 2,6 di-tert-butylphenol is much more potent than most sodium channel blockers in clinical use, it might be of interest in the development of local anaesthetics.

*Hannover Medical School, Department of Anesthesiology, Hannover, Germany;University of Liverpool, University Department of Anaesthesia, Liverpool, UK

Correspondence to: Gertrud Haeseler, Department of Anaesthesiology; OE8050, Hannover Medical School, D-30623 Hannover, Germany. E-mail: Haeseler.Gertrud@MH-Hannover.de; Tel: +49 511 532 2070; Fax: +49 511 532 5649

Accepted for publication June 2002 EJA 902

Voltage-gated sodium channels are responsible for the increase in sodium permeability during the phase of rapid rise of the action potential in nerve, skeletal muscle, neuroendocrine and heart cells, thereby mediating tissue excitability. They have been described as target sites, not only for local anaesthetic and antidysrrhythmic drugs, but also for general anaesthetics [1-4]. The role of sodium channel blockade in the production of the anaesthetic state in vivo is considered to be minimal [5], but it is worth-while noting that propofol, and a series of structural analogues, exert effects both on voltage-operated sodium channels and on GABAA receptors at similar concentrations in vitro[4,6-10]. Replacing the isopropyl groups in positions 2 and 6 of the propofol molecule by tert-butyl groups yields 2,6 di-tert-butylphenol (Fig. 1), a compound that lacks activity at GABAA receptors [7]. 2,6 Di-tert-butylphenol does produce anaesthesia, but more slowly than propofol and at a much higher concentration of 80-100 mg kg−1 compared with 5-10 mg kg−1 in the case of propofol. The lethal dose of 2,6 di-tert-butylphenol in mice was 120 mg kg−1[11].

Figure 1

Figure 1

We wanted to investigate how the structural differences between propofol and 2,6 di-tert-butylphenol, which apparently abolish the activity at GABAA receptors, affect the ability of the compound to block voltage-operated sodium channels.

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Methods

The methods have been already been described in detail [4,6]. The effects of 2,6 di-tert-butylphenol and propofol were studied in vitro on heterologously expressed α-subunits of rat brain IIa sodium channels. Stably transfected HEK 293 cell lines, expressing the α-subunit of rat brain IIa sodium channels, were a gift from Professor Lehmann-Horn, Ulm, Germany. Successful channel expression was verified electrophysiologically. The clone has been used in previous investigations [12]. 2,6 Di-tert-butylphenol (Sigma Chemicals Co., Deisenhofen, Germany) and propofol (Zeneca GmbH, Plankstadt, Germany) were prepared as a 1 mol stock solution in ethanol, which was light-protected and stored in glass vessels at −20°C. The stock solution was dissolved directly in bath solution immediately before the experiments. Drug-containing vials were vigorously vortexed for 120 min. The solution was applied via a glass-polytetrafluoroethylene perfusion system and a stainless steel superfusion pipette. The bath solution contained (mmol): NaCl 140, MgCl2 1, KCl 4, CaCl2 2, Hepes 5 and dextrose 5. Patch electrodes contained (mmol): CsCl2 130, MgCl2 2, EGTA 5 and Hepes 10. Cells were voltage-clamped [13], and whole-cell sodium inward currents following single pulses from −100 to 0 mV were recorded using the EPC9 digitally controlled amplifier in combination with Pulse and Pulse Fit software (HEKA Electronics, Lambrecht, Germany). Each experiment consisted of test recordings with the drug present at only one concentration, and of drug-free control recordings before and after the test. Each cell was exposed to one test concentration only. The effects of the diluent ethanol are relevant only in drug concentrations >1000 μmol [4].

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Results

2,6 Di-tert-butylphenol completely blocked the sodium inward current following infrequent depolarizing pulses from −100 to 0 mV at concentrations ≥30 μmol. Three experiments were performed for each drug concentration, and the representative current traces derived from four different experiments are shown in Figure 2. When applied for >1 min, blocking effects were irreversible in our preparation during 5 min of washout (Fig. 2, first, second and fourth rows). When the application of 30 μmol 2,6 di-tert-butylphenol was stopped before the equilibrium of the blockade was obtained, a partial washout of the blocking effect was possible (Fig. 2, third row).

Figure 2

Figure 2

To determine the equilibrium time for the blockade induced by 2,6 di-tert-butylphenol, submaximal blocking concentrations of this agent (either 10 or 20 μmol) were applied to the patch as long as the seal resistance remained in the range of several GΩ (>4 min following the start of drug application). The representative current traces shown in Figure 3 reveal that the amount of block achieved - by either 20 μmol (upper traces) or 10 μmol (traces in rows 2-5) - of 2,6 di-tert-butylphenol reached a steady-state value 2 min after application of about 80% for the 20 μmol dose and of 30-50% for the 10 μmol dose.

Figure 3

Figure 3

Figure 4 shows representative current traces derived from four different propofol experiments using the same preparation. The equilibrium time of block by propofol was rapid (within 60 s of application), and >80% of the effect was reversible during washout. Threshold concentrations for the block of sodium inward currents by propofol in these experiments were in the range 10-30 μmol; 300 μmol propofol achieved a 64% block.

Figure 4

Figure 4

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Discussion

Our data show that 2,6 di-tert-butylphenol is a potent sodium channel blocker. The blocking effects of 2,6 di-tert-butylphenol on voltage-operated neuronal sodium channels were complete in concentrations as low as 30 μmol. The compound is more potent than propofol, whose threshold concentration for blockade of sodium inward currents was about 30 μmol. In our preparation, propofol was slightly less potent than recently described in the literature [3]. This is most probably related to a difference in the experimental protocol: in our study, each cell was exposed to one test concentration only, while in the study mentioned above, four concentrations were tested at the same cell. We conclude that the effects on voltage-operated neuronal sodium channels do not explain the difference in anaesthetic potency between propofol and 2,6 di-tert-butylphenol [11]. As 2,6 di-tert-butylphenol has no activity at GABAA receptors [7] - whereas 80% of the propofol effects on rat hippocampal neurons were sensitive to the GABA-antagonist bicuculline in vitro[9] - it is reasonable to assume that the effects of propofol on GABAA receptors account, to a large extent, for the difference in anaesthetic potency between the compounds. Moreover, the fact that the propofol analogue 2,6 di-tert-butylphenol has anaesthetic effects only in very high concentrations, despite being a potent sodium channels blocker, suggests that the sodium channel blocking properties of propofol do not contribute to a large extent to its anaesthetic potency. In view of the small therapeutic range of 2,6 di-tert-butylphenol, when it is tentatively used as an anaesthetic [11], it may be that its sodium channel blocking effect rather determines its side-effects than the anaesthetic effect itself.

The effects of 2,6 di-tert-butylphenol were only partially reversed during several minutes of washout in our preparation, whereas blocking effects of propofol were readily reversible. At present, we cannot exclude the possibility that a longer washout would have led to a fuller recovery from 2,6 di-tert-butylphenol-induced sodium channel blockade. However, this question could only be addressed in a system that offers stable conditions for longer recording times (i.e. ≥1 h). When working with expression systems, several problems - such as voltage shifts [14] or run-down of channels - are related to longer recording times. Thus, we did not take into account results obtained after 10 min of recording time.

The potency of 2,6 di-tert-butylphenol to block sodium inward currents in vitro is more than one order of magnitude higher than the blocking potency of the local anaesthetic lidocaine. For lidocaine, half-maximum blocking concentrations on heterologously expressed rat brain α-subunits were 935 ± 100 μmol [15]. With regard to the blocking potency and the incomplete reversibility of the block during washout, our 2,6 di-tert-butylphenol data are analogous to the results previously obtained for a bulkier lidocaine analogue (N-β-phenyletyl-lidocaine) [16], which has emerged as a long-acting local anaesthetic in vivo[17]. Apparently, steric constraints like the presumed 'shielding' of the hydrogen bond-donating phenolic hydroxyl group by the bulkier side chains - which is supposed to account for the lack of activity at GABAA receptors seen with 2,6 di-tert-butylphenol [7] - do not hold for voltage-operated sodium channels. There are two postulated hydrophobic local anaesthetic binding domains in the sodium channel, and each binding domain can accommodate up to a 12-hydrocarbon chain [18,19]. We speculate that once 2,6 di-tert-butylphenol has reached the receptor site, the compound is retained by strong hydrophobic interactions within this hydrophobic pocket.

In conclusion, our results show that 2,6 di-tert-butylphenol exerts high-affinity blockade of voltage-operated neuronal sodium channels. In vivo studies might reveal a potential long-lasting local anaesthetic effect of 2,6 di-tert-butylphenol, analogous to the effect described for the lidocaine derivative tonicaine (N-β-phenyletyl-lidocaine).

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Acknowledgement

We are indebted to Professor Lehmann-Horn, Ulm, Germany, for providing the transfected cells, to Dr Hans-Peter Reiffen, Department of Anaesthesiology, Hannover, Germany, for help with software problems, to Jobst Kilian and Andreas Niesel, Department of Neurology, Hannover, for technical support, to Birgitt Nentwig, Department of Anaesthesiology, Hannover, for taking care of the cell culture, and to Wolfgang Heyde, Clinical Pharmacy, Hannover, for providing stock solutions of the compound.

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Keywords:

ANAESTHETICS, INTRAVENOUS, propofol; BIOCHEMICAL PHENOMENA, binding sites; MEMBRANE TRANSPORT PROTEINS, ion channels, sodium channels; PHENOLS, 2,6 di-tert-butylphenol

© 2003 European Society of Anaesthesiology