Stimulus-Dependent Modulation of [H]Norepinephrine Release from Rat Neocortical Slices by Gabapentin and Pregabalin

Gabapentin (GBP; Neurontin) has proven efficacy in several neurological and psychiatric disorders yet its mechanism of action remains elusive. This drug, and the related compounds pregabalin [PGB; CI-1008, S-(1)-3-isobutylgaba] and its enantiomer R-(2)-3-isobutylgaba, were tested in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [H]norepinephrine ([H]NE). The variables addressed were stimulus type (i.e., electrical, K, veratridine) and intensity, concentration dependence, onset and reversibility of action, and commonality of mechanism. Both GBP and PGB inhibited electrically and K-evoked [H]NE release, but not that induced by veratridine. Inhibition by these drugs was most pronounced with the K stimulus, allowing determination of concentration-effect relationships (viz., 25 mM K stimulus: GBP IC50 5 8.9 mM, PGB IC50 5 11.8 mM). R-(2)-3-Isobutylgaba was less effective than PGB to decrease stimulation-evoked [H]NE release. Other experiments with GBP demonstrated the dependence of [H]NE release inhibition on optimal stimulus intensity. The inhibitory effect of GBP increased with longer slice exposure time before stimulation, and reversed upon washout. Combination experiments with GBP and PGB indicated a similar mechanism of action to inhibit K-evoked [H]NE release. GBP and PGB are concluded to act in a comparable, if not identical, manner to preferentially attenuate [H]NE release evoked by stimuli effecting mild and prolonged depolarizations. This type of modulation of neurotransmitter release may be integral to the clinical pharmacology of these drugs. Gabapentin [GBP; Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] (Fig. 1) is efficacious in several neurological and psychiatric disorders, including epilepsy (Chadwick et al., 1998), neuropathic pain (Rosenberg et al., 1997), migraine (Mathew et al., 1999), tremor (Gironell et al., 1999), social phobia (Pande et al., 1999), bipolar depression (Young et al., 1999), and drug abuse/withdrawal (Myrick et al., 1998). The mechanism(s) of action that explains this broad spectrum of therapeutic utility is controversial (Taylor et al., 1998). One interesting hypothesis originates from the observation that [H]GBP binds to the auxiliary a2d subunit of voltage-sensitive calcium channels (VSCC) (Gee et al., 1996). Because the a2d subunit can modulate Ca 21 conductance of the a1 subunit (Walker and De Waard, 1998), there exists the possibility that the GBP-a2d subunit complex negatively modulates neuronal a1 VSCC subunits (viz., L-, N-, and P/Qtype VSCC) to decrease depolarization-induced Ca influx. In this regard, GBP has recently been shown to inhibit Ca currents in various neuronal cells (Stefani et al., 1998), and to attenuate K-induced Ca influx in synaptosomes (or presynaptic axon terminals) (Meder and Dooley, 2000). Decreases of depolarization-evoked Ca entry into neurons by GBP presumably translate into reductions of neuronal excitability and neurotransmitter release. Such changes offer a logical explanation for the normalization or attenuation of neuronal dysfunction seen in GBP-responsive central nervous system disorders. Previous investigations have, in fact, demonstrated that GBP can cause relatively small yet consistent inhibitions (i.e., #20% of control values at relevant physiological concentrations) of electrically evoked, calcium-dependent H-neurotransmitter release from mammalian brain slices (Reimann, 1983; Schlicker et al., 1985); these neurotransmitters included norepinephrine, dopamine, and 5-hydroxytryptamine. In the present study, we tested GBP for effects in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [H]norepinephrine (NE). The variables of interest were stimulus type (i.e., electrical, K, veratridine), stimulus intensity, concentration dependence, onset of action, and reversibility of action. In some experiments, pregabalin [PGB; CI-1008, S-(1)-3-isobutylgaba, S-(1)-4-amino-3-(2-methylReceived for publication June 20, 2000. 1 Preliminary reports of this work were presented at the 2nd Meeting of European Neuroscience, Strasbourg, France, September 24–28, 1996; the 26th Society for Neuroscience Congress, Washington, DC, November 16–21, 1996; and the 28th Society for Neuroscience Congress, Los Angeles, CA, November 7–12, 1998. ABBREVIATIONS: GBP, gabapentin; VSCC, voltage-sensitive calcium channels; NE, norepinephrine; PGB, pregabalin; R-IBG, R-(2)-3-isobutylgaba; N.S., not significant. 0022-3565/00/2953-1086$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 295, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 3032/864948 JPET 295:1086–1093, 2000 Printed in U.S.A. 1086 at A PE T Jornals on A ril 2, 2017 jpet.asjournals.org D ow nladed from propyl)butanoic acid] (Fig. 1)] and its enantiomer R-(2)-3isobutylgaba (R-IBG) were evaluated for comparison to GBP. PGB has a pharmacology similar to that of GBP, including nanomolar displacement of [H]GBP binding to rat neocortical membranes (Bryans and Wustrow, 1999) and clinical efficacy at lower doses in epilepsy, diabetic neuropathy, and anxiety disorders (Abou-Khalil et al., 1999; A. C. Pande and R. M. Poole, personal communication). The experiments were designed to characterize the modulatory effects of GBP and PGB on neurotransmitter release. The results presented here may offer a rationale for the clinical findings with these drugs, and facilitate the design of additional experiments directed at mechanism of action. Experimental Procedures Subjects. Male rats (Sprague-Dawley, 200–220 g; Charles River Laboratories, Wilmington, MA) were housed in an Association for the Assessment and Accreditation of Laboratory Animal Care International-accredited facility according to the standards outlined in the Guide for the Use and Care of Laboratory Animals. The macroenvironment was controlled to provide a temperature of 23 6 3°C, a relative humidity of 43%, and 12-h light/dark cycles. Animals had ad libitum access to food and water, and were maintained for a minimum of 5 days before sacrifice by decapitation. The brains were removed by blunt dissection and placed in ice-cold buffer until slice