Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit.

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 µM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 µM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.

Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission.

The term 'Ca2+ channel alpha2delta ligands' has recently been applied to an evolving drug class that includes gabapentin (Neurontin) and pregabalin (Lyrica), and reflects significant progress over the past decade in elucidating the mechanism of action of these drugs: a novel, specific action at one of the subunits constituting voltage-sensitive Ca2+ channels. Binding of these ligands to the alpha2delta subunit is considered to explain their usefulness in treating several clinical disorders, including epilepsy, pain from diabetic neuropathy, postherpetic neuralgia and fibromyalgia, and generalized anxiety disorder. The evidence indicates a relationship between alpha2delta subunit binding and the modulation of processes that subserve neurotransmission. This modulation is characterized by a reduction of the excessive neurotransmitter release that is observed in certain neurological and psychiatric disorders.

Carboxylate bioisosteres of gabapentin.

A series of carboxylate bioisosteres of structures related to gabapentin 1 have been prepared. When the carboxylate was replaced by a tetrazole, this group was recognized by the alpha2-delta protein. Further characterization of alpha2-delta binding compounds 14a and 14b revealed a similar pattern of functional in vitro and in vivo activity to gabapentin 1.

Dual effects of gabapentin and pregabalin on glutamate release at rat entorhinal synapses in vitro.

We have recently shown that the anticonvulsant drugs phenytoin, lamotrigine and sodium valproate all reduce the release of glutamate at synapses in the entorhinal cortex in vitro. In the present investigation we determined whether this property was shared by gabapentin and pregabalin, using whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) in layer V neurons in slices of rat entorhinal cortex. Both drugs reduced the amplitude and increased the paired-pulse ratio of EPSCs evoked by electrical stimulation of afferent inputs, suggesting a presynaptic effect to reduce glutamate release. The frequency of spontaneous EPSCs (sEPSCs) was concurrently reduced by GBP, further supporting a presynaptic action. There was no significant change in amplitude although a slight reduction was seen, particularly with gabapentin, which may reflect a reduction in the number of larger amplitude sEPSCs. When activity-independent miniature EPSCs were recorded in the presence of tetrodotoxin, both drugs continued to reduce the frequency of events with no change in amplitude. The reduction in frequency induced by gabapentin or pregabalin was blocked by application of the l-amino acid transporter substrate l-isoleucine. The results show that gabapentin and pregabalin, like other anticonvulsants, reduce glutamate release at cortical synapses. It is possible that this reduction is a combination of two effects: a reduction of activity-dependent release possibly via interaction with P/Q-type voltage-gated Ca channels, and a second action, as yet unidentified, occurring downstream of Ca influx into the presynaptic terminals.

Preferential action of gabapentin and pregabalin at P/Q-type voltage-sensitive calcium channels: inhibition of K+-evoked [3H]-norepinephrine release from rat neocortical slices.

Gabapentin (GBP; Neurontin) and pregabalin (PGB; CI-1008), efficacious drugs in several neurological and psychiatric disorders, inhibit neurotransmitter release from mammalian brain slices at therapeutically relevant concentrations. A detailed investigation, exploring the basis for this in vitro phenomenon, focused on norepinephrine (NE) and rat neocortical tissue in complementary assays of neurotransmitter release and radioligand binding. The results are consistent with the hypothesis that GBP, PGB, and related substances decrease neocortical NE release by acting at the alpha2delta subunit of presynaptic P/Q-type voltage-sensitive Ca2+ channels (VSCC) subserving Ca2+ influx in noradrenergic terminals. The inhibitory action appears competitive with [Ca2+]o and preferential to those neurons undergoing prolonged depolarization. Other results indicate that the reduction of exocytotic NE release is independent of L- and N-type VSCC, classical drug/peptide binding sites on VSCC, Na+ channels, alpha2-adrenoceptors, NE transporter, and system L amino acid transporter. These findings suggest a selective modulation of P/Q-type VSCC that are implicated in neurotransmission and several GBP-responsive pathologies.

Comparison of the ORL1 receptor-mediated inhibition of noradrenaline release in human and rat neocortical slices.

The effects of nociceptin/orphanin (N/OFQ) and the selective ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) were studied on electrically-evoked release of [(3)H]-noradrenaline ([(3)H]-NA) from human and rat neocortical slices. Specimens of human tissue were obtained during neurosurgery. Slices were preincubated with 0.1 microM [(3)H]-NA, superfused in the presence of desipramine, idazoxan, and naloxone (1 microM each), and stimulated electrically up to three times under conditions (4 pulses, 100 Hz, 2 ms, 60 mA) that prevent inhibition of evoked [(3)H]-NA release by endogenous modulators accumulating during ongoing stimulation. N/OFQ decreased electrically-evoked [(3)H]-NA release in both human and rat neocortical slices in a concentration-dependent manner. The respective pEC(50) values were 7.74 [CI(95): 7.47, 8.04] and 7.64 [CI(95): 7.48, 7.77], and the maximal inhibitions were 36.9% [CI(95): 32.4%, 41.8%] and 66.4% [CI(95): 61.7%, 72.7%]. N/OFQ (1 microM) inhibited K(+) (15 mM)-evoked [(3)H]-NA release from neocortical slices of both species by a similar magnitude, either in the presence or absence of tetrodotoxin. The nonpeptide ORL1 antagonist J-113397 competitively attenuated, with similar potency, the inhibition of electrically-evoked [(3)H]-NA release by N/OFQ in both species (pA(2) values: human, 8.16 [CI(95): 7.64, 8.64]; rat, 8.47 [CI(95): 8.27, 8.67]). J-113397 (0.1 microM) by itself did not alter either the evoked or spontaneous [(3)H]-NA release, suggesting that presynaptic ORL1 receptors are not activated by endogenous N/OFQ under the stimulation conditions employed. This study provides the first evidence that N/OFQ modulates [(3)H]-NA release in human neocortex via specific ORL1 receptors most likely located on noradrenergic axon terminals.

Inhibition of neuronal Ca(2+) influx by gabapentin and pregabalin in the human neocortex.

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.