Inhibition of [3H]batrachotoxinin A-20alpha-benzoate binding to sodium channels and sodium channel function by endocannabinoids.

A number of putative endocannabinoids were found to modify the binding of [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) to site 2 on voltage-gated sodium channels of mouse brain and achieve functional inhibition of sodium channels in vitro. 2-Arachidonoyl-glycerol (2-AG), arachidonoyl glycerol ether (AGE), N-arachidonoyl-dopamine (NADA) gave almost complete inhibition of [(3)H]BTX-B binding with IC(50) values of 90.4, 51.2 and 20.7 microM, respectively. The CB1 receptor antagonist AM251 (2 microM) had no effect on the displacement of radioligand by these endocanabinoids. Arachidonoyl-glycine (A-Gly) and arachidonoyl-GABA (A-GABA) were apparently less effective inhibitors of [(3)H]BTX-B binding giving 14.8+/-2.2 and 23.9+/-4.8% inhibition at 100 microM. Phenylmethanesulphonylfluoride (PMSF) did not alter the inhibitory effects of 2-AG, AGE, NADA and A-Gly on binding, but the efficacy of 100 microM A-GABA was increased by 60.3+/-6.3% (P<0.05). Scatchard analyses showed that 2-AG, AGE and NADA reduce the binding of [(3)H]BTX-B by lowering B(max) although increases in K(D) were also evident for AGE and NADA. Our kinetic experiments found that 2-AG, AGE and NADA increase the dissociation velocity of radioligand from site 2 on sodium channels demonstrating that these endocannabinoids operate as allosteric inhibitors of [(3)H]BTX-B binding. 2-AG, AGE and NADA inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes at low micromolar concentrations and again the capacities of A-Gly and A-GABA to inhibit this response were less pronounced. The three most effective endocannabinoids (2-AG, AGE and NADA) were then examined in a synaptosomal transmitter release assay where they were observed to inhibit sodium channel- (veratridine-dependent) release of l-glutamate and GABA in the low micromolar range. These effects also occurred through a mechanism that was not influenced by 2 microM AM251. It is concluded that direct inhibition of sodium channel function leading to reduced neuronal excitation and depression of presynaptic release of amino acid transmitters is a property shared by several endocannabinoids.

Sodium channel inhibition by anandamide and synthetic cannabimimetics in brain.

Anandamide is a prominent member of the endocannabinoids, a group of diffusible lipid molecules which influences neuronal excitability. In this context, endocannabinoids are known to modulate certain presynaptic Ca(2+) and K(+) channels, either through cannabinoid (CB1) receptor stimulation and second messenger pathway activation or by direct action. We investigated the susceptibility of voltage-sensitive sodium channels to anandamide and other cannibimimetics using both biochemical and electrophysiological approaches. Here we report that anandamide, AM 404 and WIN 55,212-2 inhibit veratridine-dependent depolarization of synaptoneurosomes (IC(50)s, respectively 21.8, 9.3 and 21.1 microM) and veratridine-dependent release of L-glutamic acid and GABA from purified synaptosomes [IC(50)s: 5.1 microM (L-glu) and 16.5 microM (GABA) for anandamide; 1.6 microM (L-glu) and 3.3 microM (GABA) for AM 404, and 12.2 (L-glu) and 14.4 microM (GABA) for WIN 55,212-2]. The binding of [3H]batrachotoxinin A 20-alpha-benzoate to voltage-sensitive sodium channels was also inhibited by low to mid micromolar concentrations of anandamide, AM 404 and WIN 55,212-2. In addition, anandamide (10 microM), AM 404 (10 microM) and WIN 55,212-2 (1 microM) were found to markedly block TTX-sensitive sustained repetitive firing in cortical neurones without altering primary spikes, consistent with a state-dependent mechanism. None of the inhibitory effects we demonstrate on voltage-sensitive sodium channels are attenuated by the potent CB1 antagonist AM 251 (1-2 microM). Anandamide's action is reversible and its effects are enhanced by fatty acid amidohydrolase inhibition. We propose that voltage-sensitive sodium channels may participate in a novel signaling pathway involving anandamide. This mechanism has potential to depress synaptic transmission in brain by damping neuronal capacity to support action potentials and reducing evoked release of both excitatory and inhibitory transmitters.

Reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels.

Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC50 of about 1 microM. In the presence of brevetoxin the IC50 increased to about 80 microM. Reserpine inhibited binding of batrachotoxinin-A [3H]benzoate ([3H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to 0.08 microM, then a rebound stimulation from 0.1 to 0.8 microM, followed by complete inhibition by 80 microM. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC50 of about 1 microM. Reserpine at 1 microM slightly reduced the off-rate of [3H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 microM it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3-10 microM elicited a partial inhibition of the binding of [3H]brevetoxin-3. The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited binding of [3H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as an inhibitor of batrachotoxin-elicited sodium influx from an IC50 of about 2 microM to an IC50 of about 50 microM. The results suggest that reserpine binds at both a local anesthetic site to cause allosteric inhibition of batrachotoxin-binding and action, but that it also binds to another site causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Local anesthetics also may bind to the brevetoxin site.

Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels.

Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the alpha subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either alpha-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2+ influx.

BIIR 561 CL: a novel combined antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and voltage-dependent sodium channels with anticonvulsive and neuroprotective properties.

Antagonists of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, as well as of voltage-gated sodium channels, exhibit anticonvulsive and neuroprotective properties in vivo. One can postulate that a compound that combines both principles might be useful for the treatment of disorders of the central nervous system, like focal or global ischemia. Here, we present data on the effects of dimethyl-(2-[2-(3-phenyl-[1,2, 4]oxadiazol-5-yl)-phenoxy]ethyl)-amine hydrochloride (BIIR 561 CL) on neuronal AMPA receptors and voltage-dependent sodium channels. BIIR 561 CL inhibited AMPA receptor-mediated membrane currents in cultured cortical neurons with an IC50 value of 8.5 microM. The inhibition was noncompetitive. In a cortical wedge preparation, BIIR 561 CL reduced AMPA-induced depolarizations with an IC50 value of 10.8 microM. In addition to the effects on the glutamatergic system, BIIR 561 CL inhibited binding of radiolabeled batrachotoxin to rat brain synaptosomal membranes with a Ki value of 1.2 microM. The compound reduced sodium currents in voltage-clamped cortical neurons with an IC50 value of 5.2 microM and inhibited the veratridine-induced release of glutamate from rat brain slices with an IC50 value of 2.3 microM. Thus, BIIR 561 CL inhibited AMPA receptors and voltage-gated sodium channels in a variety of preparations. BIIR 561 CL suppressed tonic seizures in a maximum electroshock model in mice with an ED50 value of 2.8 mg/kg after s.c. administration. In a model of focal ischemia in mice, i.p. administration of 6 or 60 mg/kg BIIR 561 CL reduced the area of the infarcted cortical surface. These data show that BIIR 561 CL is a combined antagonist of AMPA receptors and voltage-gated sodium channels with promising anticonvulsive and neuroprotective properties.

[3H]batrachotoxinin-A 20-alpha-benzoate binding to sodium channels in rat brain: sensitivity to tetrodotoxin and divalent cations.

The sodium channel blocker tetrodotoxin partially inhibited [3H]batrachotoxinin-A 20-alpha-benzoate binding to rat brain synaptosomes. This inhibition results from a decrease in the apparent affinity of the radioligand, which indicates that sites 1 and 2 of the sodium channel are not independent as thought previously. Computer-assisted data analysis allowed two binding sites for BTX-B to be distinguished. These sites could be differentiated by means of the divalent cations Mg2+ and Ca2+, that inhibit BTX-B binding completely. Tetrodotoxin diminished the inhibition by Mg2+ and vice versa, suggesting a common mechanism of action for the inhibition.