Gertrud Haeseler

Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol.

BACKGROUND AND OBJECTIVE Thymol is a naturally occurring phenol derivative used in anaesthetic practice as a stabilizer and preservative of halothane, usually at a concentration of 0.01%. Although analgesic effects have long been described for thymol and its structural homologue menthol, a molecular basis for these effects is still lacking. We studied the blocking effects of thymol and menthol on voltage-activated sodium currents in vitro as possible molecular target sites. METHODS Whole cell sodium inward currents via heterologously (HEK293 cells) expressed rat neuronal (rat type IIA) and human skeletal muscle (hSkM1) sodium channels were recorded in the absence and presence of definite concentrations of either thymol or menthol. RESULTS When depolarizing pulses to 0 mV were started from a holding potential of -70 mV, half-maximum blocking concentrations (IC50) for the skeletal muscle and the neuronal sodium channel were 104 and 149 mumol for thymol and 376 and 571 mumol for menthol. The blocking potency of both compounds increased at depolarized holding potentials with the fraction of inactivated channels. The estimated dissociation constant Kd for thymol and menthol from the inactivated state was 22 and 106 mumol for the neuronal and 23 and 97 mumol for the skeletal muscle sodium channel, respectively. CONCLUSIONS The results suggest that antinociceptive and local anaesthetic effects of thymol and menthol might be mediated via blockade of voltage-operated sodium channels with the phenol derivative thymol being as potent as the local anaesthetic lidocaine.

Structural requirements of phenol derivatives for direct activation of chloride currents via GABAA receptors

Propofol directly activates gamma-aminobutyric acid (GABA(A)) receptors in the absence of the natural agonist. This mechanism is supposed to contribute to its sedative-hypnotic actions. We studied the effects of seven structurally related phenol derivatives on chloride inward currents via rat alpha1beta2gamma2 GABA(A) receptors, heterologously expressed in HEK 293 cells in order to find structural determinants for this direct agonistic action. Only compounds with the phenolic hydroxyl attached directly to the benzene ring and with aliphatic substituents in ortho position to the phenolic hydroxyl activated chloride currents in the absence of GABA. Concentrations required for half-maximum effect were 980 microM for 2-methylphenol, 230 microM for 2,6-dimethylphenol, 200 microM for thymol, and 23 microM for propofol. Drug-induced chloride currents showed no desensitisation during the 2-s application. These results show that the position of the aliphatic substituents with respect to the phenolic hydroxyl group is the crucial structural feature for direct GABA(A) activation by phenol derivatives.

Propofol Blocks Human Skeletal Muscle Sodium Channels in a Voltage-Dependent Manner

Propofol decreases muscle tone in the absence of neuromuscular blocking drugs. This effect probably cannot be attributed solely to central nervous depression. We studied the effects of propofol on heterologously expressed skeletal muscle sodium channels. Our hypothesis was that the decrease in muscle tone may partly be attributed to an interaction of propofol with sarcolemmal sodium channels. Cells were voltage clamped and whole-cell sodium inward currents were recorded in the absence and presence of propofol. When depolarizing pulses to 0 mV were started from a holding potential close to the normal resting potential of muscle (−70 mV), or when a 2.5-s prepulse inducing slow inactivation was applied before the test pulse at −100 mV, a significant reduction in the peak current amplitude was achieved by 10 and 5 &mgr;M propofol, respectively (P < 0.001). Half-maximum blocking concentrations with these protocols were 23 and 22 &mgr;M. Blocking potency increased at depolarized membrane potentials with the fraction of inactivated channels; the estimated dissociation constant Kd from the inactivated state was 4.6 &mgr;M. These results suggest that propofol significantly blocks sarcolemmal sodium channels at clinically relevant concentrations while maintaining potentials close to the physiological resting potential.

Tramadol, fentanyl and sufentanil but not morphine block voltage-operated sodium channels

&NA; Lidocaine‐like sodium channel blocking drugs provide pain relief either by interrupting impulse conduction in neurons when applied locally in high concentrations or, when given systemically, by suppressing high‐frequency ectopic discharges due to preferential drug binding to inactivated channel states. Lidocaine‐like actions of opioids have frequently been demonstrated clinically. However, drug binding to resting and inactivated channel conformations has been studied systematically only in the case of meperidine. The aim of this in vitro study was to investigate the effects of four currently used opioids on heterologously expressed neuronal (NaV1.2) voltage‐gated sodium channels. Block of sodium currents was studied at hyperpolarized holding potentials and at depolarized potentials inducing either fast‐ or slow‐inactivation. Sufentanil, fentanyl and tramadol but not morphine reversibly suppressed sodium inward currents at high concentrations (half‐maximum blocking concentrations (IC50) 49 ± 4, 141 ± 6 and 103 ± 8 &mgr;M) when depolarizations were started from hyperpolarized holding potentials. Short depolarizations inducing fast‐inactivation and long prepulses inducing slow‐inactivation significantly (*p ≤ 0.001) increased the blocking potency for these opioids. 15% slow inactivated channels reduced the respective IC50 values to 5 ± 3, 12 ± 2 and 21 ± 2 &mgr;M. These results show that: (1) Sufentanil, fentanyl and tramadol block voltage‐gated sodium channels with half‐maximum inhibitory concentrations similar to the IC50 reported for meperidine. (2) Slow inactivation – a physiological mechanism to suppress ectopic activity in response to slow shifts in membrane potential – increases binding affinity for sufentanil, fentanyl and tramadol. (3) Morphine has no such effects.

The Nonpsychotropic Cannabinoid Cannabidiol Modulates and Directly Activates Alpha-1 and Alpha-1-Beta Glycine Receptor Function

Loss of inhibitory synaptic transmission within the dorsal horn of the spinal cord plays a key role in the development of chronic pain following inflammation or nerve injury. Inhibitory postsynaptic transmission in the adult spinal cord involves mainly glycine. Cannabidiol is a nonpsychotropic plant constituent of Cannabis sativa. As we hypothesized that non-CB receptor mechanisms of cannabidiol might contribute to its anti-inflammatory and neuroprotective effects, we investigated the interaction of cannabidiol with strychnine-sensitive α1 and α1β glycine receptors by using the whole-cell patch clamp technique. Cannabidiol showed a positive allosteric modulating effect in a low micromolar concentration range (EC50 values: α1 = 12.3 ± 3.8 μmol/l and α1β = 18.1 ± 6.2 μmol/l). Direct activation of glycine receptors was observed at higher concentrations above 100 μmol/l (EC50 values: α1 = 132.4 ± 12.3 μmol/l and α1β = 144.3 ± 22.7 μmol/l). These in vitro results suggest that strychnine-sensitive glycine receptors may be a target for cannabidiol mediating some of its anti-inflammatory and neuroprotective properties.

High‐affinity blockade of voltage‐operated skeletal muscle and neuronal sodium channels by halogenated propofol analogues

Voltage‐operated sodium channels constitute major target sites for local anaesthetic‐like action. The clinical use of local anaesthetics is still limited by severe side effects, in particular, arrhythmias and convulsions. These side effects render the search for new local anaesthetics a matter of high interest.

Endotoxin reduces availability of voltage-gated human skeletal muscle sodium channels at depolarized membrane potentials.

Objective:Critical illness myopathy is a common cause for difficulties in weaning from the respirator and prolonged rehabilitation of patients recovering from sepsis. Several studies have shown that the primary cause of acute generalized muscle weakness is loss of muscle membrane excitability. This study was designed to investigate a potential direct interaction of lipopolysaccharides from Escherichia coli with voltage-gated human skeletal muscle sodium channels (NaV1.4) in vitro. Design:In vitro laboratory investigation. Setting:University laboratory. Subjects:NaV1.4 sodium channel &agr;-subunits stably expressed in human embryonic kidney (HEK293) cells. Interventions:We investigated the effect of lipopolysaccharide on voltage-dependent sodium channel gating by using two distinct modes of application: 1) acute perfusion (pharmacologic lipopolysaccharide concentrations between 5 ng/mL and 50 &mgr;g/mL) in order to establish a concentration-effect relationship; and 2) incubation with a clinically relevant concentration of lipopolysaccharide (300 pg/mL). Measurements and Main Results:Lipopolysaccharide did not alter the kinetics of sodium current activation or inactivation when depolarizations were started from hyperpolarized holding potentials. However, when either fast or slow inactivation was induced by membrane depolarization before the test pulse, lipopolysaccharide reversibly reduced channel availability during the test pulse at concentrations of ≥50 ng/mL revealed by a maximum hyperpolarizing shift of −25 mV in the voltage dependence of fast and slow inactivation, respectively. Incubation with a lipopolysaccharide concentration of 300 pg/mL for 1 hr reproduced the effects on slow but not on fast inactivation. After 20 hrs of low-dose lipopolysaccharide, the peak sodium current was significantly reduced. Conclusions:Our results show that lipopolysaccharide interacts with voltage-gated sodium channels, reducing channel availability at depolarized membrane potentials during acute application, independent of the membrane potential after chronic exposure. These effects may contribute to reduced muscle membrane excitability in sepsis.

Structural requirements for voltage-dependent block of muscle sodium channels by phenol derivatives

We have studied the effects of four different phenol derivatives, with methyl and halogen substituents, on heterologously expressed human skeletal muscle sodium channels, in order to find structural determinants of blocking potency. All compounds blocked skeletal muscle sodium channels in a concentration‐dependent manner. The methylated phenol 3‐methylphenol and the halogenated phenol 4‐chlorophenol blocked sodium currents on depolarization from −100 mV to 0 mV with IC50 values of 2161 and 666 μM respectively. Methylation of the halogenated compound further increased potency, reducing the IC50 to 268 μM in 2‐methyl‐4‐chlorophenol and to 150 μM in 3,5‐dimethyl‐4‐chlorophenol. Membrane depolarization before the test depolarization increased sodium channel blockade. When depolarizations were started from −70 mV or when a 2.5 s prepulse was introduced before the test pulse inducing slow inactivation, the IC50 was reduced more than 3 fold in all compounds. The values of KD for the fast‐inactivated state derived from drug‐induced shifts in steady‐state availability curves were 14 μM for 3,5‐dimethyl‐4‐chlorophenol, 19 μM for 2‐methyl‐4‐chlorophenol, 26 μM for 4‐chlorophenol and 115 μM for 3‐methylphenol. All compounds accelerated the current decay during depolarization and slowed recovery from fast inactivation. No relevant frequency‐dependent block after depolarizing pulses applied at 10, 50 and 100 Hz was detected for any of the compounds. All the phenol derivatives that we examined are effective blockers of skeletal muscle sodium channels, especially in conditions that are associated with membrane depolarization. Blocking potency is increased by halogenation and by methylation with increasing numbers of methyl groups.

Anaesthesia with midazolam and S‐(+)‐ketamine in spontaneously breathing paediatric patients during magnetic resonance imaging

We evaluated safety and efficacy of a sedation technique based on rectal and intravenous S‐(+)‐ketamine and midazolam to achieve immobilization during Magnetic Resonance Imaging (MRI). Thirty‐four paediatric patients were randomly assigned to undergo either the sedation protocol (study group) or general anaesthesia (control group). Imaging was successfully completed in all children. Children in the study group received a rectal bolus (0.5 mg·kg−1 midazolam and 5 mg·kg–1 S‐(+)‐ketamine) and required additional i.v. supplementation (20 ± 10 μg·kg–1·min–1 S‐(+)‐ketamine and 4 ± 2 μg· kg−1· min−1 midazolam), spontaneous ventilation was maintained. Transient desaturation occurred once during sedation and four times in the control group (P=0.34). P ECO2 was 5.3 ± 0.5 kPa (40 ± 4 mmHg) in the study group and 4.1 ± 0.6 kPa (31 ± 5 mmHg) in the control group (P < 0.001). Induction and discharge times were shorter in the study group (P < 0.001), recovery times did not differ significantly between the groups. Our study confirms that a combination of rectal and supplemental intravenous S‐(+)‐ketamine plus midazolam is a safe and useful alternative to general anaesthesia for MRI in selected paediatric patients.

An improved model for the binding of lidocaine and structurally related local anaesthetics to fast-inactivated voltage-operated sodium channels, showing evidence of cooperativity.

The interaction of lidocaine‐like local anaesthetics with voltage‐operated sodium channels is traditionally assumed to be characterized by tighter binding of the drugs to depolarized channels. As inactivated and drug‐bound channels are both unavailable on depolarization, an indirect approach is required to yield estimates for the dissociation constants from channels in inactivated states. The established model, originally described by Bean et al., describes the difference in affinity between resting and inactivated states in terms of the concentration dependence of the voltage shift in the availability curve. We have tested the hypothesis that this model, which assumes a simple Langmuir relationship, could be improved by introducing a Hill‐type exponent, which would take into account potential sources of cooperativity. Steady‐state block by lidocaine was studied in heterologously (HEK 293) expressed human skeletal muscle sodium channels and compared with experimental data previously obtained for 2,6‐dimethylphenol, 3,5‐dimethyl‐4‐chlorophenol, and 4‐chlorophenol. Cells were clamped to membrane potentials from −150 to −5 mV, and a subsequent test pulse was used to assess the number of channels available to open. All compounds shifted the voltage dependence of channel availability in the direction of negative prepulse potentials. Prediction of the concentration dependence of the voltage shift in the availability curve was improved by the modified model, as shown by a marked reduction in the residual sum of squares. For all compounds, the Hill‐type exponent was significantly greater than one. These results could be interpreted in the light of the contemporary hypothesis that lidocaine functions as an allosteric gating effector to enhance sodium channel inactivation by strengthening the latch mechanism of inactivation, which is considered to be a particle‐binding process allosterically coupled to activation. Alternatively, they could be interpreted by postulating additional binding sites for lidocaine on fast‐inactivated sodium channels.