Accidental administration of local anaesthetics into the systemic circulation may lead to seizures, dysrhythmias and cardiovascular depression . These side-effects are reasons to search for alternative compounds with local anaesthetic properties that do not exhibit toxic effects when given systemically.
Ketamine is a general anaesthetic agent that is also employed in regional anaesthesia. Successful use in intravenous (i.v.) regional anaesthesia, caudal anaesthesia and epidural anaesthesia has been reported [2-4]. Similar doses of ketamine are used in regional anaesthesia as in general anaesthesia. Accidental systemic application of local anaesthetic doses of ketamine, therefore, will not result in seizures, dysrhythmias or cardiovascular depression. Ketamine may, thus, constitute a safe alternative to local anaesthetics. However, there is some doubt about the quality of local anaesthesia produced by ketamine. In patients undergoing transurethral resection of the bladder, spinal anaesthesia with ketamine failed to completely suppress sensation of painful stimuli .
Inhibition of neuronal sodium channels is regarded as the most important molecular mechanism underlying local anaesthesia . Sodium channels are inhibited by ketamine [6-9]. However, the effects of ketamine on human neuronal sodium channels have only partially been established . This study was designed, therefore, to elucidate the concentration-dependent effects of ketamine on the conductance, steady-state activation and steady-state inactivation of human sodium channels natively expressed in a human neuronal cell line. In view of conflicting clinical data, this information is important to evaluate further the local anaesthetic characteristics of ketamine.
SH-SY5Y cells were grown in non-confluent monolayer using RPMI medium (Biochrom, Berlin, Germany) at 37°C with 95% air and 5% CO2. Growth medium contained 10% fetal calf serum, glutamine (2 mmol), penicillin (100 U mL−1) and streptomycin (100 μg mL−1). Neuronal differentiation was induced by exposure to retinoic acid (10 μmol) for 3-7 days. Treatment of these cells with retinoic acid results in a reduction in cell division and neurite extension [10,11].
Patch-clamp whole-cell recording
The recording pipettes (3.3 ± 0.5 MΩ) were fabricated from borosilicate glass capillaries (o.d., 1.5 mm; i.d., 1 mm; Science Products, Hofheim, Germany) on a puller (Narashige, Inc., Tokyo, Japan) and were filled with a solution containing (mmol): caesium methanesulphonate, 80; CaCl2, 1; MgCl2, 5; TEA, 20; HEPES, 10; EGTA, 11; adenosine 5′-triphosphate (disodium salt), 10; and guanosine 5′-triphosphate (lithium salt), 0.5 (pH 7.3 with CsOH). The osmotic pressure was adjusted with sucrose to 294 ± 7 mOsmol L−1. A liquid junction potential of 7 ± 1 mV (n = 3) was measured between the internal and external solutions . Membrane potentials were not corrected for this liquid junction potential . Tight-seal whole-cell recordings were obtained according to the method of Hamill and colleagues  (uncompensated series resistance 5.6 ± 1.4 MΩ). Measurements of cells with a capacitance of >15 pF (read from the dial of the Axoclamp 200A patch-clamp amplifier® (Axon Instruments, Foster City, CA, USA), mean 11 ± 2 pF) were excluded from further evaluation. To improve the voltage control during activation of the Na+ current, series resistance and capacitance compensation (66% ± 6%) were employed. The maximal residual voltage error did not exceed 3 mV. Voltage commands were delivered via the Axoclamp® amplifier and collected online with the pCLAMP acquisition and analysis program. Current signals were filtered at 5 kHz (−3 dB, four-pole low-pass Bessel filter) and sampled at ≥20 kHz by a digital interface (DigiData2000®; Axon Instruments). Residual capacitance transients and leak conductance were subtracted by means of P/4 protocol .
All experiments were carried out at room temperature (21-23°C). Different concentrations of ketamine in the extracellular solution were applied with a seven-barrelled superfusion pipette placed 100-250 μm from the cell body.
Stimulation protocols and data analysis
Na+ currents were stimulated by activation and inactivation protocols. The holding potential during all experiments was −80 mV. The activation protocol consisted of a 100 ms hyperpolarizing prepulse to −100 mV followed by a 50 ms depolarizing test pulse to various membrane potentials (−80, −70, −60, −50, −40, −30, −20, −10, 0, 20, 30, 40 mV). The current-voltage curves for the peak Na+ currents elicited by this activation protocol were fitted with the following equation: Equation (1)
where V is the test potential, VNa is the Na+ reversal potential and G(V) is the conductance, defined by the following Boltzmann equation: Equation (2)
where Gmax is the maximum Na+ conductance, Vm50 is the voltage where G(V) is half of Gmax and km is the slope of the relationship between channel activation and membrane voltage.
The inactivation protocol consisted of a 250 ms prepulse to various membrane potentials (−140, −130, −120, −110, −100, −90, −80, −70, −60, −50, −40, −20 mV) followed by a 10 ms depolarizing test pulse to −10 mV. The current-voltage curves of the peak Na+ currents elicited by this inactivation protocol were fitted by the Boltzmann function (equation 2) with Vm50 and km substituted by Vh50 and kh.
The concentration-dependent action of ketamine on the reduction of maximal Na+ conductance (block JOURNAL/ejanet/04.02/00003643-200209000-00003/ENTITY_OV0435/v/2017-07-27T035905Z/r/image-png) was fitted with the Hill function: Equation (3)
where c is the concentration of ketamine, blockmaxJOURNAL/ejanet/04.02/00003643-200209000-00003/ENTITY_OV0435/v/2017-07-27T035905Z/r/image-png is the maximal ketamine block of the Na+ conductance, EC50 is the concentration of 50% blockmaxJOURNAL/ejanet/04.02/00003643-200209000-00003/ENTITY_OV0435/v/2017-07-27T035905Z/r/image-png and γ is the Hill coefficient. All fits were done using the NFit® software (University of Texas, Galveston, TX, USA). The statistical comparison of the concentration dependent block of activation and inactivation was performed with a two-tailed t-test or F-test  at P = 0.05. All results are means ± SEM.
Before performing the pharmacological experiments with ketamine, the basic voltage-dependent properties of Na+ currents in human SH-SY5Y cells were established (Fig. 1). For this purpose, SH-SY5Y cells were stimulated with activation and inactivation protocols (see Methods). After characterizing the basic activation and inactivation properties, the effects of ketamine on the activation and inactivation of these human Na+ currents were investigated (Figs 2-4).
Basic properties of Na+ currents in SH-SY5Y cells
Stimulation of SH-SY5Y cells with successive depolarizing pulses (test potentials from −80 to +40 mV in 10 mV steps) activated a family of inward currents, which are illustrated as a series of superimposed traces in Figure 1a. The cells were maintained in solution containing 4-AP, TEA, Cs+ and Cd2+ to block K+ and Ca2+ currents, respectively. The observed inward currents evoked by the activation protocol could be blocked by TTX (data not shown), indicating that they were due to the opening of voltage-dependent Na+ channels. The Na+ currents exhibited a typical voltage-dependent activation. The threshold of detectable currents was between −60 and −50 mV. The maximal peak inward current was at a voltage of about −10 mV (Fig. 1b). The Na+ currents depended on the prepulse potential (Fig. 1c). The more positive the prepulse potential, the smaller the Na+ conductance evoked by the depolarization, consistent with classical steady-state inactivation (Fig. 1d, squares).
The data points of maximal current amplitude resulting from the activation as well as the inactivation protocol could be fitted with a Boltzmann function equivalent to equation (2). This procedure allowed the determination of the membrane potential where half of the ion channels are in the activated state (activation protocol) as well as the membrane potential where half of the ion channels are in the inactivated state (inactivation protocol). The potential of half-maximal activation was −19 mV (Fig. 1d, circles) and the potential of half-maximal inactivation was −78 mV (Fig. 1d, squares).
Ketamine effects on the current-voltage relationship of activation
Na+ currents were elicited by the same activation protocol as in Figure 1a under control conditions (without ketamine), under the influence of increasing concentrations (0.25, 0.5, 1, 2, 4 mmol) of ketamine, and after ketamine was washed out from the cell bath. Ketamine reversibly inhibited the amplitudes of the Na+ currents as depicted as superimposed traces in Figure 2a. The amplitudes of the peak Na+ currents under each condition (control, drug, washout) were plotted against the test potential (Fig. 2b). After the addition of ketamine, the Na+ currents were reduced throughout the whole range of test potentials (Fig. 2b). The amplitudes of the peak Na+ currents were fitted by equation (1). This allowed one to distinguish the effects of ketamine on different activation parameters of the Na+ current (Gmax, VNA, Vm50) evoked by the activation protocols (Fig. 2b, solid lines). Ketamine 2 mmol reduced the conductance of the Na+ currents Gmax to <50% without changing the equilibrium potential VNa (estimated by the fit in equation 1). The effect of ketamine on the voltage of half-maximal activation of the Na+ currents Vm50 could be visualized after calculating the normalized conductance (Fig. 2c): the voltage dependence of activation of the Na+ current was shifted in a hyperpolarizing direction (to the left; Fig. 2c, squares). Ketamine thus exerted two effects on the activation parameters of sodium channels: it inhibited the ion channel conductance and it shifted the mid-point of current activation to more negative potentials.
Ketamine effects on the current-voltage relationship of the steady-state inactivation
The effects of ketamine on the steady-state inactivation curves were investigated in more detail by determining complete steady-state inactivation curves in the absence and presence of ketamine (Fig. 3a). The same protocols were used as described above (Fig. 1c). Again, two separate effects of ketamine could be differentiated. First, ketamine reduced the maximal amplitude of Na+ currents elicited by the inactivation protocol (Fig. 3b). Second, it shifted the voltage dependence of the steady-state Na+ inactivation to more hyperpolarized membrane potentials. To show more clearly this leftward shift, the normalized conductance of the Na+ current amplitudes was calculated (Fig. 3c, squares). The inactivation behaviour of the Na+ channels was, therefore, influenced by ketamine in a similar manner as the voltage dependence of channel activation.
Concentration dependence of ketamine effects
Next, we analysed whether the effects of ketamine on the activation and inactivation of Na+ channels were concentration-dependent. Both experiments with the activation protocol (Fig. 2) as well as those with the inactivation protocol (Fig. 3) showed a reduction of the Na+ current conductance. The concentration dependence of this conductance block (block JOURNAL/ejanet/04.02/00003643-200209000-00003/ENTITY_OV0435/v/2017-07-27T035905Z/r/image-png) is shown in Figure 4a. The data points were fitted by the Hill function (equation (3)) yielding concentrations of the half-maximal effect EC50 of 1120 μmol (activation protocol) and of 1440 μmol (inactivation protocol). For every concentration, the conductance data points derived from activation or inactivation protocols were not statistically different.
Beside the reduction of the Na+ current conductance, ketamine caused a shift of the voltage dependence of the activation and inactivation in the hyperpolarizing direction. The concentration dependence of this shift was quantified. The hyperpolarizing shift of the mid-point of current activation and the mid-point of current inactivation became evident at a concentration of 0.25 mmol ketamine and increased up to 4 mV with rising concentrations. For every concentration, the data points of activation and inactivation were not statistically different from each other.
In conclusion, ketamine inhibited the conductance of human Na+ channels and altered the voltage-dependent activation and inactivation of these ion channels in a concentration-dependent and reversible manner. The concentration-response curves for the effects of ketamine on the activation and inactivation of these human ion channels were not statistically different.
Ketamine is an i.v. anaesthetic that is also used clinically as a local anaesthetic agent. It has been reported to inhibit sodium channels in a variety of preparations [6-9], but information about the effects on human neuronal sodium channels was limited . The present investigation established the concentration-dependent effects of ketamine on conductance, steady-state activation and steady-state inactivation of sodium channels natively expressed in human neuronal SH-SY5Y cells. These effects of ketamine are compatible with local anaesthetic action.
Ketamine blocked the sodium conductance at concentrations supposed to occur during regional anaesthesia . Effects on both steady-state activation and inactivation also begin to occur at clinically relevant concentrations. The hyperpolarizing shifts of both activation and inactivation accounted for <5 mV. Even such small shifts of steady-state inactivation will contribute to a reduction of sodium currents by decreasing the number of channels that can be activated by membrane depolarization. However, in the case of ketamine, this shift would be counterbalanced by a shift of channel activation into the same direction. As this hyperpolarizing shift would lower the action potential threshold, it would increase neuronal excitability. From these results, it may be concluded that the blockade of neuronal impulse conduction by ketamine  in human neurons will rather result from the inhibition of the sodium channel conductance than from changes in the voltage dependence of channel activation or inactivation.
The Hill coefficients of the concentration-response curves for inhibition of the sodium channels elicited by both the activation and inactivation protocol are both close to unity. This result is consistent with the concept of a single site of interaction by which ketamine exhibits the observed inhibitory effects. This view is supported by results obtained with human brain sodium channels activated by batrachotoxin . Although these channels did not inactivate, they were inhibited by ketamine with an identical IC50 as the inactivating sodium channels in SH-SY5Y cells.
The inhibitory effects of ketamine on sodium channels in an amphibian myelinated nerve membrane preparation have been described as mainly a reduction of the permeability constants . The inhibitory action was not altered by channel inactivation and the effects were described by first-order binding to receptors within the ion channels. The similarity of action on sodium channels in SH-SY5Y cells and the amphibian axonal preparation suggests that little variation may exist between the action of ketamine on sodium channels from different species. The similarity, furthermore, indicates that SH-SY5Y cells constitute a valid model to investigate local anaesthetic action.
In summary, the molecular effects of ketamine investigated on human neuronal sodium channels as well as the results of several other laboratory studies [6-9] are consistent with the hypothesis that ketamine possess local anaesthetic properties. Whether ketamine alone or in combination with other anaesthetic agents [17,19] will help do decrease the incidence of severe side-effects during regional anaesthesia needs further clinical evaluation.
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Keywords:© 2002 European Academy of Anaesthesiology
ANAESTHESIA, CONDUCTION; ANAESTHETICS, INTRAVENOUS; anaesthetics, dissociative, ketamine; ANAESTHETICS, LOCAL; ION CHANNELS, sodium channels