Interaction of high concentrations of riluzole with recombinant skeletal muscle sodium channels and adult-type nicotinic receptor channels

Riluzole is a neuroprotective drug that modulates glutamergic transmission but also blocks the inactivated state of voltage-gated neuronal sodium channels at very low concentrations (about 0.1 microM). After nausea, the most common adverse effect of riluzole is asthenia, which could be due to a block of muscle sodium channels or acetylcholine receptor channels. Using the patch-clamp technique, we applied riluzole on recombinant voltage-gated skeletal muscle sodium and adult nicotinic acetylcholine receptor channels expressed in a mammalian cell line (HEK 293). Riluzole blocked the inactivated state of voltage-gated skeletal muscle sodium channels, shifting the midpoint of the steady-state inactivation curve to more negative potentials, but only in comparatively high concentrations (> or = 0.1 mM). At these concentrations, riluzole also caused an open-channel block at acetylcholine receptor channels. We conclude that riluzole has only a mild blocking effect on the inactivated state of voltage-gated skeletal muscle sodium channels and nicotinic acetylcholine receptor channels. As the plasma concentration of riluzole in amyotrophic lateral sclerosis (ALS) patients approximates 2 microM, it seems unlikely that asthenia is caused by a block of skeletal muscle sodium channels or acetylcholine receptor channels by riluzole.     

Lamotrigine derivatives and riluzole inhibit INa,P in cortical neurons

The persistent, slowly inactivating fraction of the sodium current is involved in key functions in the CNS such as dendritic integration of synaptic inputs and cellular excitability. We have studied whether established anti-epileptic drugs and neuroprotective agents target the persistent sodium current. Two lamotrigine derivatives (sipatrigine and 202W92) and riluzole inhibited the persistent sodium current at low, therapeutic concentrations. In contrast, lamotrigine and the classical antiepileptic agents phenytoin and valproic acid blocked the fast-inactivating sodium channel but failed to affect the persistent fraction. The ability to influence either mode of channel activity may represent a defining feature of each drug subclass, changing profoundly their clinical indications. Given the damaging role of a sustained influx of sodium in both pharmaco-resistant seizures or excitotoxic insults, we suggest the utilization of drugs that suppress the persistent conductance.     

Noise of the slowly inactivating Na current in suprachiasmatic nucleus neurons

Slow depolarization induces a riluzole-sensitive persistent Na current (INa,P) in several types of neurons and a pharmacologically similar slowly inactivating Na current (INa,S) in rat suprachiasmatic nucleus neurons. In isolated suprachiasmatic nucleus neurons, INa,S fluctuations were analyzed to characterize the Na channel responsible for INa,S. The single-channel current near resting potential was -0.53+/-0.04 pA in an external solution containing 151 mM Na+ and 5 mM Na+ in the patch pipette. Tetrodotoxin completely blocked INa,S and single-channel current noise; 25 microM riluzole also suppressed INa,S and current noise by about 80% without a significant effect on mean single-channel current. The data suggest that single-channel noise is due to the opening of channels mediating INa,S with a conductance of about 3.4 pS.     

Is the persistent sodium current a specific target of anti-absence drugs?

The persistent Na+ current (INaP) has been proposed as the putative target of the anti-absence antiepileptic drugs. Accordingly, the effect of reference anti-absence drugs ethosuximide (ESM) and valproate (VPA), and of the new antiepileptic drug levetiracetam (LEV), on INaP have been tested in CA1 hippocampal neurons and compared to the classic anticonvulsant phenytoin (PHT) and the neuroprotective agent riluzole (RIL). Whole-cell patch-clamp recordings of the slowly inactivating current, fully characterized as INaP, were performed with a standard voltage-step protocol on thin hippocampal slices prepared from rat brain. Both PHT (100 microM) and RIL (10 microM) strongly depressed INaP, whereas ESM (1 mM) induced a slight decrease of INaP and VPA (1 mM) had no effect. Likewise, 60-min perfusion with relevant concentrations of LEV (10, 32 or 100 microM) did not modify INaP. In conclusion, these data question the impact of INaP depression as an anti-absence mechanism, and also disclaim the involvement of INaP in the antiepileptic mechanism of LEV.     

Kainate-induced currents in rat cortical neurons in culture are modulated by riluzole

The action of the neuroprotective and anticonvulsant agent riluzole on kainate-induced currents was studied in rat cortical neurons in primary culture by using the whole-cell configuration of the patch-clamp technique. Kainate elicited macroscopic, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive inward currents in all the patched cells and the amplitude of the current was concentration-dependent (EC50= 106 microM). Riluzole decreased the inward currents induced by 100 microM kainate at all holding potentials and the reduction was dose-dependent (IC50= 101 microM). The maximal response to kainate decreased in the presence of 50 microM riluzole, without changing its EC50, indicating a noncompetitive mechanism of inhibition. The amplitude of the responses induced by kainate under control conditions and during riluzole was a linear function of the membrane potential and the reversal potential of the currents was not significantly different in the two experimental conditions. Instead, the total conductance of the cell membrane for the currents induced by 100 microM kainate was significantly reduced in the presence of 50 microM riluzole (P < 0.05). The analysis of the kainate membrane current noise performed under control conditions and during perfusion of 100 microM riluzole revealed that riluzole reduced the probability of kainate-activated ionic channels to be in the open state. Conversely, the unitary conductance of channels, as well as their characteristic time constant, seemed to be unchanged. These results reveal an additional mechanism by which riluzole can interact with glutamatergic neurotransmission and provides further support for the idea that riluzole may prove beneficial in the treatment of central nervous system injuries involving the excitotoxic actions of glutamate.     

Riluzole reduces hyperactivity of subthalamic neurons induced by unilateral 6-OHDA lesion in the rat brain.

An abnormal increase in the activity of neurons of the subthalamic nucleus is a key pathophysiological feature of Parkinson's disease. We sought to determine whether riluzole, a sodium channel inhibitor that interferes with glutamatergic neurotransmission, affects neuronal activity in this brain region. Intravenous administration of riluzole reduced the discharge rate of subthalamic neurons in rats with 6-OHDA-induced lesions of the midbrain. By contrast, no effect was observed in nonlesioned control animals. This property may contribute to the neuroprotective effects of riluzole in animal models of PD through the modulation of the glutamatergic inputs these neurons feedback to nigral dopaminergic neurons.     

A novel neuroprotective mechanism of riluzole: direct inhibition of protein kinase C

In addition to its antiexcitotoxic action, the anti-amyotrophic lateral sclerosis (ALS) neuroprotectant riluzole protects against nonexcitotoxic oxidative neuronal injury. In light of evidence that protein kinase C (PKC) mediates oxidative stress in cortical culture, we examined the possibility that riluzole's antioxidative neuroprotection involves PKC inhibition. Riluzole (30 microM) blocked phorbol 12-myristate 13-acetate (PMA)-induced increases in membrane PKC activity in cultured cortical cells. Suggesting a direct action, riluzole also inhibited the activity of purified PKC. Consistently, both PKC depletion and oxidative neuronal death induced by PMA were markedly attenuated by riluzole. The site of action of riluzole on PKC was not likely the diacylglycerol binding site but the catalytic domain, since riluzole did not alter radiolabeled phorbol-12,13-dibutyrate binding, but inhibited PKM, the catalytic domain of PKC. However, increasing ATP concentrations did not alter the inhibition of PKC by riluzole, making it unlikely that riluzole is a competitive inhibitor of ATP binding at PKM. Present results have demonstrated that riluzole directly inhibits PKC, which action may contribute to its antioxidative neuroprotective effects. In addition, it appears possible that PKC inhibition may be able to explain some of its well-known channel inhibitory and neuroprotective effects. Combined with findings that PKC activity is increased in ALS, the present results suggest that PKC may be a potential therapeutic target in ALS.     

Cisplatin modulates voltage gated channel currents of dorsal root ganglion neurons of rats

The anticancer drug cis-diammindichloroplatin (CDDP, cisplatin) causes severe side effects like peripheral sensitive neuropathy. The toxicity of CDDP has been linked to changes in intracellular calcium homeostasis ([Ca2+]i). Voltage activated calcium channel currents (ICa(V)) are important for the regulation of [Ca2+]i; therefore, this study was designed to examine the effect of CDDP on ICa(V) in comparison to voltage activated potassium (IK(V)) and sodium (INa(V)) channel currents using the whole cell patch clamp method on dorsal root ganglion neurons of rats. In small neurons (or=?25 microm) were less sensitive to CDDP. The peak ICa(V) was reduced by 14.1+/-2.3% and IK(V) by 12.8+/-3.4% (100 microM). The sensitivity of INa(V) in large neurons to CDDP was not different compared to small neurons. We conclude that the reduction of ICa(V) in small cells may be responsible for the neurotoxic side effects CDDP causes in sensory neurons.     

Rasagiline alone and in combination with riluzole prolongs survival in an ALS mouse model

Abstract. Rasagiline is an antiapoptotic compound with neuroprotective potential. We examined its neuroprotective effect alone and in combination with the putative glutamate release blocker riluzole in the G93A model of familial amyotrophic lateral sclerosis (fALS). Endpoints of experimental treatment were survival and motor activity. The drug had a significant dose-dependent therapeutic effect on both preclinical and clinical motor function and survival of the animals. We also found that the combination of rasagiline with riluzole is safe and increases survival by about 20 % in a dosedependent manner. Therefore, we conclude that the combination of rasagiline and riluzole is a promising clinical combination for the improvement of current neuroprotective treatment strategies of ALS.     

Modulation of AMPA receptors in spinal motor neurons by the neuroprotective agent riluzole

We investigated the interaction of riluzole, a therapeutic agent used in amyotrophic lateral sclerosis (ALS), with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels in mouse spinal motor neurons in culture using whole-cell patch-clamp recording techniques. Kainate elicited concentration-dependent (EC(50) = 35 microM) inward currents in all the patched cells. These responses were mediated primarily through the activation of AMPA receptors with a negligible contribution from kainate receptors, because bath application of 100 microM GYKI53655, a potent noncompetitive AMPA receptor antagonist, completely blocked the kainate-induced currents. Riluzole (0.5-100 microM) reduced in a dose-dependent manner the kainate-induced currents with an IC(50) of 1.54 microM in all tested neurons (n = 25) and this effect was found to be reversible. The response to kainate decreased in the presence of 1 microM riluzole in all spinal motor neurons tested, without changing its EC(50), indicating a noncompetitive mechanism of inhibition. The amplitude of the responses induced by kainate under control condition and during riluzole was a linear function of the membrane potential. The reversal potential of the current was not significantly different in the two experimental conditions, whereas the total conductance of the motor neurons for the currents induced by 100 microM kainate was reduced significantly in the presence of 1 microM riluzole (P < 0.05). These results reveal an interaction of riluzole with glutamatergic neurotransmission in spinal cord motor neurons and can contribute to explain its beneficial effect in the ALS treatment.