Charles P. Taylor

A summary of mechanistic hypotheses of gabapentin pharmacology

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

Na+ channels as targets for neuroprotective drugs

Drugs that block voltage-dependent Na+ channels are well known as local anaesthetics, antiarrhythmics and anticonvulsants. Recent studies show that these compounds also provide a powerful mechanism of cytoprotection in animal models of cerebral ischaemia, hypoxia or head trauma. In this article Charles Taylor and Brian Meldrum review evidence indicating that Na+ channel modulators are neuroprotective and describe recent ideas for the molecular sites of action of voltage-dependent Na+ channel blockers. Clinical trials with several compounds are now in progress for stroke and traumatic head injury, and the therapeutic potential for this group of compounds is discussed.

Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of α‐ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the α‐amino group to glutamate. Measurements of synthesis of 14C‐glutamate and of 14C‐glutamine from H14CO3 have shown that 14C‐amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mm. The specific radioactivity of 14C‐glutamine indicated that ∼30% of glutamine was derived from 14CO2 fixation. Using gabapentin, an inhibitor of the cytosolic branched‐chain aminotransferase, synthesis of 14C‐glutamate and 14C‐glutamine from H14CO3− was inhibited by 31%. These results suggest that transamination of α‐ketoglutarate to glutamate in Müller cells is slow, the supply of branched‐chain amino acids may limit flux, and that branched‐chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H14CO3−. 14C‐metabolites in brain extracts were measured to determine net incorporation of 14CO2 and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14CO2 fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ∼60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.

Na+ currents that fail to inactivate

Textbook accounts give the impression that Na+ channels are short-acting binary switches: depolarization opens them, but only for about one millisecond. In contrast to this simplified view, a small but significant fraction of the total Na+ current in neurons occurs because channels open after long delays or in long-duration bursts of openings. Such non-inactivating Na+ current acts physiologically in neurons to amplify synaptic potentials and enhance endogenous rhythmicity, and also to aid repetitive firing of action potentials. In glial cells it also may regulate Na(+)-K+ ATPase activity. The evidence for non-inactivating Na+ current in a variety of neurons and glia is reviewed, along with a brief discussion of its ion channel substrate and its relevance for neurological diseases and drug therapy.

Potent and stereospecific anticonvulsant activity of 3-isobutyl GABA relates to in vitro binding at a novel site labeled by tritiated gabapentin.

3-Isobutyl GABA is a derivative of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and is also structurally related to the novel anticonvulsant gabapentin. The S(+) enantiomer of 3-isobutyl GABA blocks maximal electroshock seizures in mice and also potently displaces tritiated gabapentin from a novel high-affinity binding site in rat brain membrane fractions. The R(-) enantiomer is much less active in both assays, suggesting that the gabapentin binding site is involved in the anticonvulsant activity of 3-isobutyl GABA.

Effects of anticonvulsant drug gabapentin on the enzymes in metabolic pathways of glutamate and GABA

Gabapentin is a novel anticonvulsant drug. The anticonvulsant mechanism of gabapentin is not known. Based on the amino acid structure of gabapentin we explored its possible effects on glutamate and gamma-aminobutyric acid (GABA) metabolism in brain as they may relate to its anticonvulsant mechanisms of action. Gabapentin was tested for its effects on seven enzymes in the metabolic pathways of these two neurotransmitters: alanine aminotransferase (AL-T), aspartate aminotransferase (AS-T), GABA aminotransferase (GABA-T), branched-chain amino acid aminotransferase (BCAA-T), glutamine synthetase (Gln-S), glutaminase (GLNase), and glutamate dehydrogenase (GDH). In the presence of 10 mM gabapentin, only GABA-T, BCAA-T, and GDH activities were affected by this drug. Inhibition of GABA-T by gabapentin was weak (33%). The Ki values for inhibition of cytosolic and mitochondrial forms of GABA-T (17-20 mM) were much higher than the Km values for GABA (1.5-1.9 mM). It is, therefore, unlikely that inhibition of GABA-T by gabapentin is clinically relevant. As with leucine, gabapentin stimulated GDH activity. The GDH activity in rat brain synaptosomes was activated 6-fold and 3.4-fold, respectively, at saturating concentrations (10 mM) of leucine and gabapentin. The half-maximal stimulation by gabapentin was observed at approximately 1.5 mM. Gabapentin is not a substrate of BCAA-T, but it exhibited a potent competitive inhibition of both cytosolic and mitochondrial forms of brain BCAA-T. Inhibition of BCAA-T by this drug was reversible. The Ki values (0.8-1.4 mM) for inhibition of transamination by gabapentin were close to the apparent Km values for the branched-chain amino acids (BCAA) L-leucine, L-isoleucine, and L-valine (0.6-1.2 mM), suggesting that gabapentin may significantly reduce synthesis of glutamate from BCAA in brain by acting on BCAA-T.

Gabapentin anticonvulsant action in rats: disequilibrium with peak drug concentrations in plasma and brain microdialysate

The concentration-time profile of [14C]gabapentin (GBP) in plasma and brain interstitial fluid (ISF) was determined following a single 15 mg/kg intravenous bolus dose to rats. Brain ISF was sampled with a microdialysis probe in striatum. Blood was also collected serially to 4 h postdose. At termination, brain was sectioned into regions and [14C]GBP concentrations were determined. Anticonvulsant effects were determined by maximal electroshock in rats with identical dosing. Plasma [14C]GBP declined linearly after dosing while brain ISF [14C]GBP concentration peaked at approximately 1 h and then declined in parallel with plasma concentration. Throughout, brain ISF [14C]GBP concentration was approximately 3-6% of [14C]GBP concentration in plasma. However, at 4 h postdose, whole brain tissue [14C]GBP concentration was equal to or greater than the concentration of [14C]GBP in plasma. Maximal anticonvulsant effect lagged behind both plasma and brain ISF GBP concentrations. The anticonvulsant effect of GBP is delayed by time-dependent events other than distribution from blood to brain.

Damage from oxygen and glucose deprivation in hippocampal slices is prevented by tetrodotoxin, lidocaine and phenytoin without blockade of action potentials

In vitro ischemia (IVI) was simulated with rat hippocampal slices in medium lacking D-glucose, equilibrated with 95% nitrogen, 5% carbon dioxide. Within 5-8 min, synaptic potentials disappeared and a DC negative shift (5-15 mV) occurred. Prolonged application of 95% oxygen and D-glucose 12 min later did not allow synaptic potentials to recover. Slices pretreated with sodium channel blocking drugs allowed synaptic potentials to recover after IVI. Tetrodotoxin (TTX, 100-600 nM), the anticonvulsant phenytoin (5.0 to 100 microM) and the local anesthetic lidocaine (2.0 to 200 microM) each delayed or prevented negative DC shifts from IVI. Histological examination showed that drug treatments also prevented CA1 pyramidal cell damage from IVI. Neuroprotection occurred without blocking synaptic potentials or presynaptic fiber volleys, suggesting relevance for treatment of brain ischemia.

Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD 810, CI 912), a novel anticonvulsant.

Zonisamide is a novel anticonvulsant that prevents seizures in laboratory animals and in man. Zonisamide (3 micrograms/ml and above) blocked the sustained firing of action potentials induced by depolarizing steps of current injected across the membrane of intracellularly recorded spinal cord neurons. Responses to GABA and glutamate were not altered by zonisamide, and spontaneous synaptically evoked activity was not reduced until higher concentrations of zonisamide (10 micrograms/ml) were applied.

SIMULATION OF HIPPOCAMPAL AFTERDISCHARGES SYNCHRONIZED BY ELECTRICAL INTERACTIONS

Recent experiments have shown that hippocampal pyramidal cells can generate synchronized action potentials even when chemical synapses are blocked. The computer simulations reported here showed that communication between cells by extracellular currents could cause this synchrony, provided that (1) individual neurons were sufficiently excitable and that (2) the resistivity of the extracellular medium was sufficiently high. Synchronization was enhanced if electronic junctions were also present.