Nilufar Foadi

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.

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.

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.

Modulation of Glycine Receptor Function by the Synthetic Cannabinoid HU210

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. HU210 is a non-psychotropic, synthetic cannabinoid. As we hypothesized that non-CB receptor mechanisms of HU210 might contribute to its anti-inflammatory and anti-nociceptive effects we investigated the interaction of HU210 with strychnine-sensitive α1 glycine receptors by using the whole-cell patch clamp technique. HU210 showed a positive allosteric modulating effect in a low micromolar concentration range (EC50: 5.1 ± 2.6μmol/l). Direct activation of glycine receptors was observed at higher concentrations above 100 μmol/l (EC50: 188.7 ± 46.2μmol/l). These in vitro results suggest that strychnine-sensitive glycine receptors may be a target for HU210 mediating some of its anti-inflammatory and anti-nociceptive properties.

The opioid methadone induces a local anaesthetic‐like inhibition of the cardiac Na+ channel, Nav1.5

Treatment with methadone is associated with severe cardiac arrhythmias, a side effect that seems to result from an inhibition of cardiac hERG K+ channels. However, several other opioids are inhibitors of voltage‐gated Na+ channels. Considering the common assumption that an inhibition of the cardiac Na+ channel Nav1.5, is the primary mechanism for local anaesthetic (LA)‐induced cardiotoxicity, we hypothesized that methadone has LA‐like properties leading to a modulation of Nav1.5 channels.

Defective polysialylation and sialylation induce opposite effects on gating of the skeletal Na+ channel NaV1.4 in Chinese hamster ovary cells.

Polysialic acid (polySia) is a large, negatively charged homopolymer of 2,8-linked N-acetylneuraminic acid residues resulting from remodeling and extension of protein-bound sialic acid (Sia) residues and seems to have a key role in regulating neural cell development and function. The aim of this study was to explore and compare the effects of polySia and sialylation on gating of voltage-gated sodium channels. The skeletal muscle α-subunit NaV1.4 was transiently expressed in wild-type Chinese hamster ovary (CHO) cells or in mutant CHO cells with deficits in their capacity to produce sialylated or polysialylated membrane components. Expression in both mutant cell lines resulted in larger peak current amplitudes as compared to wild-type CHO cells. Loss of Sia and polySia also resulted in significant shifts of voltage-dependent activation and steady-state inactivation, however, in opposite directions. Furthermore, only the loss of Sia had a significant effect on recovery from fast inactivation. Our data demonstrate for the first time that gating of voltage-gated sodium channels seems to be differentially regulated by polySia and Sia.