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One site of action of the anticonvulsant, analgesic, and anxiolytic drugs gabapentin and pregabalin is the alpha(2)delta-subunit of voltage-sensitive Ca(2+) channels (VSCC). We therefore analyzed the effects of gabapentin and pregabalin on K(+)-evoked release of (3)H-gamma-aminobutyric acid (GABA) and (3)H-glutamate from superfused human neocortical synaptosomes. These neurotransmitters are released by Ca(2+)-dependent exocytosis and by Ca(2+)-independent uptake reversal. When a GABA transport inhibitor was present throughout superfusion to isolate exocytotic conditions, gabapentin and pregabalin (100 microM each) reduced K(+)-evoked (3)H-GABA release by 39% and 47%, respectively. These effects were antagonized by the alpha(2)delta-ligand L: -isoleucine (1 microM) suggesting the alpha(2)delta-subunit of terminal VSCC to mediate the reduction of exocytosis. Both drugs had no effect on exocytotic (3)H-glutamate release and also failed to modulate the release of (3)H-GABA and (3)H-glutamate caused by reversed uptake in the absence of external Ca(2+). Thus, an inhibition of glutamate release by gabapentin and pregabalin as main anticonvulsant principle is not supported by our experiments. An anticonvulsant mode of action of both drugs may be the reduction of a proconvulsant exocytotic GABA release.

Anticonvulsant, analgesic, and anxiolytic effects have been observed both in preclinical and clinical studies with gabapentin (GBP) and pregabalin (PGB). These drugs appear to act by binding to the alpha(2)delta subunit of voltage-sensitive Ca(2+) channels (VSCC), resulting in the inhibition of neurotransmitter release. In this study, we examined the effects of GBP and PGB (mostly 100 microM, corresponding to relatively high preclinical/clinical plasma levels) on the release of neurotransmitters in human neocortical slices. These slices were prelabeled with (3)H-dopamine ((3)H-DA), (3)H-choline (to release (3)H-acetylcholine ((3)H-ACh)), (3)H-noradrenaline ((3)H-NA), and (3)H-serotonin ((3)H-5-HT), and stimulated twice in superfusion experiments by elevation of extracellular K(+) in the presence and absence of GBP and PGB. The alpha(2)delta ligands produced significant inhibitions of K(+)-evoked (3)H-ACh, (3)H-NA, and (3)H-5-HT release between 22% and 56% without affecting (3)H-DA release. Neither drug reduced (3)H-NA release in the presence of L: -isoleucine, a putative alpha(2)delta antagonist. Interestingly, this antagonism did not occur using the enantiomer, D: -isoleucine. These results suggest that GBP and PGB are not general inhibitors of VSCC and neurotransmitter release. Such alpha(2)delta ligands appear to be selective modulators of the release of certain, but not all, neurotransmitters. This differential modulation of neurotransmission presumably contributes to their clinical profile.

Temperature-induced changes of affinity and efficacy of the alpha2-adrenoceptor full agonist UK14,304 and the partial agonists clonidine and guanfacine were investigated to elucidate the mechanism of partial agonism at the terminal alpha2-autoreceptor. The effect of temperature on the efficacy of the substances was tested in 3H-noradrenaline release experiments at 37 degrees C and at room temperature. Human neocortical slices were prelabeled with 3H-noradrenaline, superfused, and stimulated electrically under autoinhibition-free conditions. Furthermore, saturation binding experiments with human neocortical synaptosomes were performed at 37 degrees C and 17 degrees C to evaluate the influence of temperature on the affinity of 3H-clonidine and 3H-UK14,304. Temperature-induced changes of the association and dissociation rate constants of 3H-UK14,304 and 3H-clonidine were assessed in corresponding kinetic binding experiments. Our experiments reveal that clonidine and guanfacine lose efficacy when the temperature is lowered, whereas no change was noted for the full agonist UK14,304. Moreover, the affinity of clonidine and guanfacine was shown to decrease at lower temperature. Kinetic experiments indicated that the loss of affinity observed for 3H-clonidine at 17 degrees C is due to a marked reduction of the association rate. The loss of efficacy of the partial agonists is most likely related to the short binding duration; partial agonists do not bind long enough to the receptor to mediate a maximum response. The discrepancy between the time required to elicit a maximum response and the actual binding time may be greater for partial agonists at lower temperatures, thus, causing the intrinsic activity to decline.

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