Jörg Ahrens

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.

Role of Cannabinoids in the Treatment of Pain and (Painful) Spasticity

Both the discovery of the endocannabinoid system (ECS) and its role in the control of pain and habituation to stress, as well as the significant analgesic and antihyperalgesic effects in animal studies, suggest the usefulness of cannabinoids in pain conditions. However, in human experimental or clinical trials, no convincing reduction of acute pain, which may be caused by a pronociceptive, ECS-triggered mechanism on the level of the spinal cord, has been demonstrated. In contrast, in chronic pain and (painful) spasticity, an increasing number of randomized, double-blind, placebo-controlled studies have shown the efficacy of cannabinoids, which is combined with a narrow therapeutic index. Patients with unsatisfactory response to other methods of pain therapy and who were characterized by failed stress adaptation particularly benefited from treatment with cannabinoids. None of the attempts to overcome the disadvantage of the narrow therapeutic index, either by changing the route of application or by formulating balanced cannabinoid preparations, have resulted in a major breakthrough. Therefore, different methods of administration and other types of cannabinoids, such as endocannabinoid modulators, should be tested in future trials.

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.

Anti-leucine rich glioma inactivated 1 protein and anti-N-methyl-D-aspartate receptor encephalitis show distinct patterns of brain glucose metabolism in 18F-fluoro-2-deoxy-d-glucose positron emission tomography.

BackgroundPathogenic autoantibodies targeting the recently identified leucine rich glioma inactivated 1 protein and the subunit 1 of the N-methyl-D-aspartate receptor induce autoimmune encephalitis. A comparison of brain metabolic patterns in 18F-fluoro-2-deoxy-d-glucose positron emission tomography of anti-leucine rich glioma inactivated 1 protein and anti-N-methyl-D-aspartate receptor encephalitis patients has not been performed yet and shall be helpful in differentiating these two most common forms of autoimmune encephalitis.MethodsThe brain 18F-fluoro-2-deoxy-d-glucose uptake from whole-body positron emission tomography of six anti-N-methyl-D-aspartate receptor encephalitis patients and four patients with anti-leucine rich glioma inactivated 1 protein encephalitis admitted to Hannover Medical School between 2008 and 2012 was retrospectively analyzed and compared to matched controls.ResultsGroup analysis of anti-N-methyl-D-aspartate encephalitis patients demonstrated regionally limited hypermetabolism in frontotemporal areas contrasting an extensive hypometabolism in parietal lobes, whereas the anti-leucine rich glioma inactivated 1 protein syndrome was characterized by hypermetabolism in cerebellar, basal ganglia, occipital and precentral areas and minor frontomesial hypometabolism.ConclusionsThis retrospective 18F-fluoro-2-deoxy-d-glucose positron emission tomography study provides novel evidence for distinct brain metabolic patterns in patients with anti-leucine rich glioma inactivated 1 protein and anti-N-methyl-D-aspartate receptor encephalitis.

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.

A transmembrane residue influences the interaction of propofol with the strychnine-sensitive glycine alpha1 and alpha1beta receptor.

BACKGROUND:Propofol, well known for its anesthetic effects, acts as a positive allosteric modulator of the α-aminobutyric acid type A (GABAA) receptor but also enhances the function of the glycine receptor. The GABA modulatory effects of propofol are influenced by an amino acid residue located within the second transmembrane domain (TM2) of the GABAA receptor β subunit. In glycine α1 subunits, the homologous residue (serine 267) affects the glycine modulatory actions of alcohols and alkane anesthetics. In the present study we investigated the role of this residue on the interaction of propofol with the glycine α1 and α1β receptor. METHODS:The influence of propofol on wild type and mutant (α1S267M, α1S267I, α1S267Mβ, α1S267Iβ) glycine receptors expressed in human embryonic kidney 293 cells was investigated by using the whole-cell clamp technique. RESULTS:Mutation of the α1 subunit TM2 serine residue to either isoleucine or methionine decreased the sensitivity of the receptor to glycine, and abolished the direct activation of the glycine receptor by propofol. Additionally, the methionine and particularly the isoleucine mutation decreased the glycine-enhancing actions of propofol. CONCLUSIONS:The nature of the TM2 residue (267) of the glycine α1 subunit influences the glycine modulatory effect of propofol and direct activation of the receptor by this anesthetic. A comparison of the impact of such complementary mutations on the interaction of propofol with glycine and GABAA receptors should permit a better understanding of the molecular determinants of action of propofol on these structurally related receptors and may aid in the development of selective glycine receptor modulators.

The distinct effects of lipid emulsions used for "lipid resuscitation" on gating and bupivacaine-induced inhibition of the cardiac sodium channel Nav1.5.

BACKGROUND: Systemic administration of lipid emulsions is an established treatment for local anesthetic intoxication. However, it is unclear by which mechanisms lipids achieve this function. The high cardiac toxicity of the lipophilic local anesthetic bupivacaine probably results from a long-lasting inhibition of the cardiac Na+ channel Nav1.5. In this study, we sought to determine whether lipid emulsions functionally interact with Nav1.5 or counteract inhibition by bupivacaine. METHODS: Human embryonic kidney cells expressing human Nav1.5 were investigated by whole-cell patch clamp. The effects of Intralipid® and Lipofundin® were explored on functional properties and on bupivacaine-induced inhibition. RESULTS: Intralipid and Lipofundin did not affect the voltage dependency of activation, but induced a small hyperpolarizing shift of the steady-state fast inactivation and impaired the recovery from fast inactivation. Lipofundin, but not Intralipid, induced a concentration-dependent but voltage-independent tonic block (42% ± 4% by 3% Lipofundin). The half-maximal inhibitory concentration (IC50) values for tonic block by bupivacaine (50 ± 4 µM) were significantly increased when lipids were coapplied (5% Intralipid: 196 ± 22 µM and 5% Lipofundin: 103 ± 8 µM). Use-dependent block by bupivacaine at 10 Hz was also reduced by both lipid emulsions. Moreover, the recovery of inactivated channels from bupivacaine-induced block was faster in the presence of lipids. CONCLUSIONS: Our data indicate that lipid emulsions reduce rather than increase availability of Nav1.5. However, both Intralipid and Lipofundin partly relieve Nav1.5 from block by bupivacaine. These effects are likely to involve not only a direct interaction of lipids with Nav1.5 but also the ability of lipid emulsions to absorb bupivacaine and thus reduce its effective concentration.

Structural features of phenol derivatives determining potency for activation of chloride currents via α1 homomeric and α1β heteromeric glycine receptors

1 Phenol derivatives constitute a family of neuroactive compounds. The aim of our study was to identify structural features that determine their modulatory effects at glycine receptors. 2 We investigated the effects of four methylated phenol derivatives and two halogenated analogues on chloride inward currents via rat α1 and α1β glycine receptors, heterologously expressed in HEK 293. 3 All compounds potentiated the effect of a submaximal glycine concentration in both α1 homomeric and α1β glycine receptors. While the degree of maximum potentiation of the glycine 10 μM effect in α1β receptors was not different between the compounds, the halogenated compounds achieved half‐maximum potentiating effects in the low μM range – at more than 20‐fold lower concentrations compared with their nonhalogenated analogues (P<0.0001). The coactivating effect was over‐ridden by inhibitory effects at concentrations >300 μM in the halogenated compounds. Neither the number nor the position of the methyl groups significantly affected the EC50 for coactivation. 4 Only the bimethylated compounds 2,6 and 3,5 dimethylphenol (at concentrations >1000 μM) directly activated both α1 and α1β receptors up to 30% of the maximum response evoked by 1000 μM glycine. 5 These results show that halogenation in the para position is a crucial structural feature for the potency of a phenolic compound to positively modulate glycine receptor function, while direct activation is only seen with high concentrations of compounds that carry at least two methyl groups. The presence of the β subunit is not required for both effects.

Methadone is a local anaesthetic-like inhibitor of neuronal Na+ channels and blocks excitability of mouse peripheral nerves

BACKGROUND Opioids enhance and prolong analgesia when applied as adjuvants to local anaesthetics (LAs). A possible molecular mechanism for this property is a direct inhibition of voltage-gated Na(+) channels which was reported for some opioids. Methadone is an effective adjuvant to LA and was recently reported to inhibit cardiac Na(+) channels. Here, we explore and compare LA properties of methadone and bupivacaine on neuronal Na(+) channels, excitability of peripheral nerves, and cell viability. METHODS Effects of methadone were explored on compound action potentials (CAP) of isolated mouse saphenous nerves. Patch clamp recordings were performed on Na(+) channels in ND7/23 cells, the α-subunits Nav1.2, Nav1.3, Nav1.7, and Nav1.8, and the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2). Cytotoxicity was determined using flow cytometry. RESULTS Methadone (IC50 86-119 µM) is a state-dependent and unselective blocker on Nav1.2, Nav1.3, Nav1.7, and Nav1.8 with a potency comparable with that of bupivacaine (IC50 177 µM). Both bupivacaine and methadone also inhibit C- and A-fibre CAPs in saphenous nerves in a concentration-dependent manner. Tonic block of Nav1.7 revealed a discrete stereo-selectivity with a higher potency for levomethadone than for dextromethadone. Methadone is also a weak blocker of HCN2 channels. Both methadone and bupivacaine induce a pronounced cytotoxicity at concentrations required for LA effects. CONCLUSIONS Methadone induces typical LA effects by inhibiting Na(+) channels with a potency similar to that of bupivacaine. This hitherto unknown property of methadone might contribute to its high efficacy when applied as an adjuvant to LA.

Lack of positive allosteric modulation of mutated α1S267I glycine receptors by cannabinoids

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. Ajulemic acid and HU210 are non-psychotropic, synthetic cannabinoids. Cannabidiol is a non-psychotropic plant constituent of cannabis sativa. There are hints that non-cannabinoid receptor mechanisms of these cannabinoids might be mediated via glycine receptors. In this study, we investigated the impact of the amino acid residue serine at position 267 on the glycine-modulatory effects of ajulemic acid, cannabidiol and HU210. Mutated α1S267I glycine receptors transiently expressed in HEK293 cells were studied by utilising the whole-cell clamp technique. The mutation of the α1 subunit TM2 serine residue to isoleucine abolished the co-activation and the direct activation of the glycine receptor by the investigated cannabinoids. The nature of the TM2 (267) residue of the glycine α1 subunit is crucial for the glycine-modulatory effect of ajulemic acid, cannabidiol and HU210. An investigation of the impact of such mutations on the in vivo interaction of cannabinoids with glycine receptors should permit a better understanding of the molecular determinants of action of cannabinoids.