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OtherNEUROPHARMACOLOGY

Barbiturate-Like Actions of the Propanediol Dicarbamates Felbamate and Meprobamate

Jong M. Rho, Sean D. Donevan and Michael A. Rogawski
Journal of Pharmacology and Experimental Therapeutics March 1997, 280 (3) 1383-1391;

Abstract

Felbamate and meprobamate are structurally related propanediol dicarbamates that possess distinct pharmacological profiles. Felbamate is a minimally sedative, broad-spectrum anticonvulsant, whereas meprobamate is a strong sedative-anxiolytic agent. Previously, we reported that felbamate potentiates γ-aminobutyric acidA(GABAA) receptor Cl− currents and inhibits N-methyl-d-aspartate (NMDA) receptor currents. Here we further characterized the interaction of the two dicarbamates with GABAA receptors to determine the basis for their pharmacological differences. In whole-cell voltage-clamp recordings from cultured rat hippocampal neurons, meprobamate enhanced GABA-evoked responses in a concentration-dependent manner and, at high concentrations (>1 mM), exhibited a separate channel-blocking effect that limited the magnitude of GABAA receptor potentiation. At equivalent concentrations, meprobamate produced substantially greater potentiation than did felbamate. Furthermore, meprobamate (but not felbamate), in the absence of GABA, directly activated Cl− currents that could be attenuated by the GABAA receptor antagonists bicuculline and picrotoxin. The mean deactivation time constant of whole-cell currents evoked by 10 mM meprobamate (110 ms) or 1 and 3 μM GABA (180 ms) were faster than the deactivation time constant of 10 mM meprobamate (490 ms) or 3 mM felbamate (470 ms) in the presence of GABA. Meprobamate and felbamate prolonged the mean burst duration of GABA-activated unitary currents in excised outside-out membrane patches. In addition, at high (supratherapeutic) concentrations, meprobamate blocked NMDA-activated currents. We conclude that felbamate and meprobamate have barbiturate-like modulatory actions on GABAA receptors, but meprobamate has greater activity and, unlike felbamate, is able to directly activate the receptor.

The propanediol felbamate (2-phenyl-1,3-propanediol dicarbamate) is a novel broad-spectrum antiepileptic compound that produces little sedation (Leppik et al., 1991; Theodore et al., 1991; Ritter et al., 1993), whereas its analog meprobamate (2-methyl-2-propyl-1,3-propanediol dicarbamate) is an anxiolytic and sedative-hypnotic agent (Haefely et al., 1981) (fig.1). Recently, we demonstrated that felbamate potentiates GABA responses via its interaction with a site on the GABAA receptor that is distinct from the benzodiazepine recognition site (Rho et al., 1994). We also observed that felbamate can inhibit NMDA receptors via a channel-blocking action and also possibly by distinct effects on channel gating (Rhoet al., 1994; Subramaniam et al., 1995). The ability of felbamate to potentiate GABA-evoked Cl−currents has been confirmed in electrophysiological recordings from cultured fetal murine cortical neurons (Kume et al., 1996). In the same study, felbamate was also found to allosterically inhibit binding of the picrotoxin ligandt-[3H]butylbicycloorthobenzoate to rat brain slices (IC50, 250 μM), further confirming an interaction with GABAA receptors.

Figure 1
Figure 1

Chemical structures of the propanediol dicarbamates felbamate and meprobamate.

Although felbamate’s positive modulatory effects on the GABAA receptor are now relatively well established, the situation for meprobamate, a drug that was introduced into clinical use in the mid-1950s, is less clear. An early report indicated that GABAergic transmission was not affected by meprobamate (Haefelyet al., 1978). Subsequently, two reports suggested that meprobamate may bind to the benzodiazepine recognition site on GABAA receptors (Olsen, 1981a; Paul et al., 1981), although this was disputed in other studies (Squires and Braestrup, 1977; Mackerer et al., 1978; Polc et al., 1982). More recently, meprobamate was found to allosterically enhance benzodiazepine binding in a manner similar to barbiturates (Koeet al., 1986); in line with a barbiturate-like action, the drug inhibitst-[35S]butylbicyclophosphorothionate binding (Squires et al., 1983). Indeed, meprobamate was found to have behavioral actions distinct from benzodiazepines and more characteristic of barbiturates (Roache and Griffiths, 1987).

Despite the evidence that felbamate and meprobamate act as positive modulators of the GABAA receptor, the basis for their functional differences is unclear. Moreover, confirmation of a possible interaction of the drugs at the barbiturate recognition site on GABAA receptors has been hampered by the absence of a selective antagonist at this site. We have addressed these issues by comparing the effects of the two dicarbamates on GABAAreceptor responses in cultured rat hippocampal neurons with whole-cell voltage-clamp and single-channel recording techniques. We also sought to determine whether meprobamate could exert its behavioral actions in part through blockade of NMDA-type excitatory amino acid receptors, as is the case with felbamate. Our results confirm that meprobamate interacts with GABAA receptors in a barbiturate-like fashion and that it can inhibit NMDA receptors. Although felbamate has similar actions, there are significant differences between the drugs that could account for their distinctive pharmacological profiles.

Materials and Methods

Cell culture.

Hippocampal neurons were grown in monolayer culture as described previously (Segal, 1983; Donevan et al., 1992). In brief, hippocampi were dissected from 19-day-old Sprague-Dawley rat embryos (Harlan, Indianapolis, IN) and triturated in Modified Minimal Essential Medium with Earle’s salt (Advanced Biotechnologies, Columbia, MD). The resulting suspension was then plated as a monolayer onto 35-mm polystyrene Petri dishes (Falcon 3001; Becton Dickinson Labware, Oxnard, CA) precoated with Matrigel (Collaborative Biomedical Products, Bedford, MA). The plating medium was supplemented with N3 (composed of 20 mg/ml transferrin, 200 μM putrescine, 60 nM sodium selenite, 20 ng/ml triiodothyronine, 10 mg/ml insulin, 40 nM progesterone and 40 ng/ml corticosterone), 10% horse serum (GIBCO, Grand Island, NY), 10% fetal calf serum and 1% glutamine (Bottenstein, 1985; Guthrie et al., 1987). Cell cultures were incubated at 37°C in a humidified atmosphere for 6 to 12 days before use. Fresh growth medium that did not contain fetal calf serum or N3 was added after 6 days in culture.

Solutions.

At the beginning of each recording session, the culture medium was replaced with bathing solution containing 145 mM NaCl, 10 mM HEPES, 2.5 mM KCl, 0.1 mM CaCl2, 10 mM glucose, 1 μM tetrodotoxin (to block voltage-activated Na+channels) and 1 μM strychnine (to block glycine-activated Cl− currents). The bathing solution was adjusted to an osmolality of 315 to 325 mOsm/kg H20 with sucrose and to a pH of 7.4 with NaOH. For experiments in which the extracellular concentration of Cl− was varied, NaCl was replaced with Na gluconate. GABAA receptor Cl− currents were activated by 1 μM GABA dissolved in bathing solution. NMDA receptor currents were evoked by 10 μM NMDA dissolved in bathing solution containing 10 μM glycine to saturate the glycine site on NMDA receptors and 100 μM picrotoxin to block GABAA receptor currents directly activated by meprobamate.

Recording micropipettes were filled with intracellular solution containing 145 mM CsCl, 0.1 mM CaCl2, 10 mM HEPES and 1 mM EGTA (adjusted to an osmolality of 305–310 mOsm/kg H20 and to a pH of 7.2). In patch recordings, 2 mM Mg-ATP was included in the intracellular solution to retard run down of the GABA activated Cl− currents.

Meprobamate and felbamate were slowly dissolved in warm bathing solution (65–70°C) with rapid stirring and allowed to cool to room temperature before use.

Electrophysiology.

All electrophysiological recordings were conducted on the stage of a Nikon Diaphot inverted phase contrast microscope at room temperature (23–25°C). Currents were monitored with either an Axopatch 1B or 200A patch clamp amplifier (Axon Instruments, Burlingame, CA). Voltages corresponding to the currents were acquired with a high-speed chart recorder (Gould Electronics, Cleveland, OH), and digitized for off-line analysis with the Axotape software package (Axon Instruments). The holding potential for whole-cell recordings was −60 mV unless otherwise noted.

Unitary GABAA receptor currents were recorded in excised outside-out membrane patches using pipettes containing intracellular solution. The currents were filtered at 1 kHz (−3 dB; four-pole, low-pass Bessel filter) and digitally sampled at 10 kHz. The holding potential was −80 mV.

Pipettes.

Patch pipettes (4–8 megohm) were prepared from filament-containing thin-wall glass capillary tubes (1.5-mm outer diameter; World Precision Instruments, Sarasota, FL) with a four-stage horizontal pipette puller (model P-80/PC Flaming Brown, Sutter Instrument, Novato, CA). Micropipette tips were routinely fire polished, and, for single-channel recordings, were coated with Sylgard (Dow Corning, Midland, MI).

Drug perfusion.

Drugs were dissolved in buffer on the day of use and applied via a nine-barrel rapid perfusion system (modified from Tang et al., 1989) in which all barrels (320 μm outer diameter quartz tubes; J & W Scientific, Folsom, CA) emptiedvia a common tip positioned within 200 μm from the tip of the patch electrode in excised patch recordings and 400 μm from the cell under study in whole-cell recordings. Flow through each barrel was gravity fed and regulated by high-speed solenoid microvalves (The Lee Co., Westbrook, CT) operated by a programmable microprocessor-based controller. Switching between solutions occurred within <10 ms (seeDonevan et al., 1992). One barrel contained buffer and the others were filled with various drugs alone and in combination. Only one valve was open at a time, and the buffer solution was applied continuously between drug applications. In the single-channel recordings, drugs were applied for 15- to 60-s epochs, separated by 30- to 60-s wash periods.

Data analysis in whole-cell recordings.

Whole-cell currents were analyzed off-line with the pCLAMP and Axotape software packages (Axon Instruments). Concentration-response curves were fit using a nonlinear least-squares program (NFIT, Island Products, Galveston, TX). Concentration-response data for the dicarbamate potentiation of GABA responses were fit to the logistic equation100×I/Io=Pmax/{1+(EC50/[D])nH}, where I is the maximal GABAA receptor current in the presence of a dicarbamate at concentration [D], I o is the current evoked by 1 μM GABA alone, P max is the maximum percent potentiation, EC50 is the concentration of [D] producing one-half maximal potentiation and n His a slope factor. NMDA inhibition curves were fit to the logistic equation 100 × I/I o = 100/{1 + IC50/[D]} (assuming 100% block at high concentrations of meprobamate and felbamate), where [D] is the dicarbamate concentration and IC50 is the concentration producing 50% block. Whole-cell deactivation time constants were estimated by fits of the current trajectory to single-exponential decay functions.

Data analysis in single-channel recordings.

Patch currents were analyzed using the FETCHAN and pSTAT modules of pCLAMP. Bursts of single-channel openings were determined by detecting current level changes exceeding a 50% threshold criterion. Only patches demonstrating infrequent multiple openings (no more than three simultaneous openings apparent) were used for analysis. Because of the difficulty in ascertaining precise open times within bursts of openings that exhibited prominent fast flickering and transitions to subconductance levels (Hamill et al., 1983; Bormann et al., 1987), only burst durations could be reliably measured. Bursts were defined as an opening or a series of closely spaced openings separated by relatively long closed periods (Colquhoun and Hawkes, 1982). Operationally, bursts were taken to be openings in which closed intervals briefer than 5 ms were ignored (Macdonald et al., 1989a). Due to variability in burst frequencies presumably related to receptor desensitization and patch rundown (see Twyman and Macdonald, 1992; Porter et al., 1992), channel open probabilities were not compared among drug treatments.

Mean burst durations were determined from the pooled data of at least three separate patches. The pooled burst duration data sets were displayed in the form of logarithmically binned histograms, with the ordinate representing the square root of the normalized counts per bin, similar to the method of Sigworth and Sine (1987). The histogram data were fitted with second-order Gaussian functions, yielding two time constants.

Drugs.

Felbamate was kindly supplied by Dr. R. Duane Sofia (Carter-Wallace Laboratories, Cranbury, NJ). NMDA was obtained from Research Biochemicals (Natick, MA). All other drugs and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Aldrich (Milwaukee, WI).

Results

Meprobamate potentiates GABA-activated currents.

In whole-cell voltage-clamp recordings from cultured hippocampal neurons, application of 1 μM GABA evoked an inward current response (150–350 pA) that failed to desensitize during perfusion periods of up to 30 s. Coapplication of meprobamate produced a concentration-dependent potentiation of the GABA-evoked current (fig. 2A). At high concentrations (>1 mM), there was a slow decline in the current during the meprobamate coapplication followed by a transient rebound in the current upon termination of the drug application (“off-effect”). Concentration-response data for the peak current (either during the application of meprobamate or after its termination) are shown in figure 2B (filled circles). The estimated EC50and Hill coefficient values, derived from a logistic fit to the data, were 2.4 and 1.8 mM, respectively. For 3 and 10 mM meprobamate, the percent potentiation values during the drug application (open circles) were less than the corresponding values during the rebound. One explanation for the rebound phenomenon is that block occurs during the drug coapplication which is then relieved upon termination of the meprobamate perfusion (because the potentiated current decays more slowly than block is relieved). Assuming that this is the case, a crude estimate of the IC50 for meprobamate block of the GABAA receptor current can be derived from a logistic fit to the percent block data (assuming that the open receptor channel can be blocked by only one meprobamate molecule and that complete block can be achieved). This value was 12.6 mM.

Figure 2
Figure 2

Dicarbamate potentiation of GABA-evoked currents. A, Representative current traces demonstrating the potentiation of 1 μM GABA-evoked currents by increasing concentrations of meprobamate (MBM). At high concentrations, the current is partially blocked during the MBM + GABA perfusion and rebound occurs after termination of the perfusion. B, Concentration-response curve for MBM and felbamate potentiation of GABA-activated currents. Solid circles represent peak currents obtained either during MBM application or during the rebound; open circles reflect the maximum currents measured during perfusion with 3 and 10 mM MBM. Solid squares represent peak currents obtained during felbamate application. Each data point represents the mean of data from 8 to 11 cells; error bars indicate S.E. and where not shown were smaller than the size of the symbols. Maximal responses for meprobamate and felbamate could not be measured because of the limited solubility of the dicarbamates under the assay conditions.

Concentration-response data for felbamate potentiation of currents activated by 1 μM GABA are also shown in figure 2B (filled squares). As with meprobamate, the threshold concentration of felbamate producing minimal potentiation was ∼100 μM. However, at equivalent concentrations, meprobamate produced substantially greater peak potentiation than did felbamate. Although an off-effect was not evident with felbamate in a previous study (Rho et al., 1994; same protocol as fig. 2), we found that rebound could be obtained when a 3 mM felbamate perfusion was terminated during the continuous application of 3 μM GABA (fig. 3). The crude IC50value for felbamate estimated from the data of figure 3 was 14.4 mM.

Figure 3
Figure 3

Felbamate potentiation of GABA-evoked currents. A, Representative whole-cell current trace demonstrating the potentiation of GABA current by 3 mM felbamate (FBM). Note the rebound in the current upon termination of the FBM application (“off-effect”). B, Percent potentiation (mean ± S.E.) for the plateau and peak response in nine experiments similar to that depicted in A. The peak current potentiation was significantly greater than the plateau potentiation (P < .01, paired t-test).

Meprobamate activates currents in the absence of GABA.

As shown in figure 4, high concentrations of meprobamate (>300 μM) in the absence of GABA activated an inward current response similar to that obtained with GABA. The magnitude of the current response increased in a concentration-dependent fashion. In fact, 10 mM meprobamate-activated currents were substantially larger in magnitude than the current activated by 1 μM GABA. As shown in the graph of figure 4, the concentration of meprobamate estimated to evoke a current of magnitude similar to that produced by 1 μM GABA was ∼5 mM. Thus, meprobamate was 5,000-fold less potent than GABA.

Figure 4
Figure 4

Currents evoked by meprobamate (MBM) in the absence of GABA. Top, Representative current traces showing responses to 1, 3 and 10 mM MBM; for comparison, the current evoked by 1 μM GABA alone in the same cell is shown (left). An off-effect in this cell was not prominent. Bottom, Concentration-response curve for currents directly activated by MBM (solid circles). Open circle indicates the current evoked by 1 μM GABA alone. Each data point represents the mean ± S.E. of 3 to 10 cells tested.

Currents activated by meprobamate are carried by Cl−.

Cl− is well known to carry the current obtained upon activation of GABAAreceptors by GABA. To examine the hypothesis that the current directly evoked by meprobamate is also carried by Cl−, the reversal potential of currents evoked by 10 mM meprobamate were determined in a series of experiments conducted at three different extracellular Cl− concentrations. In the sample recording shown in figure 5, the current evoked in 148 mM external Cl− exhibited a null potential of +5.5 mV. The mean null potential values for experiments carried out with 33, 93 and 148 mM external Cl− (plotted in fig. 5B) closely matched the theoretical values predicted by the Nernst equation (line), indicating that meprobamate activates a Cl− current.

Figure 5
Figure 5

Currents directly activated by meprobamate (MBM) are carried by Cl−. A, Representative whole-cell currents activated by 10 mM MBM in the presence of 148 mM external Cl−. The holding potential was set at various levels between −40 mV and +60 mV. The null (reversal) potential was +5.5 mV. All traces are from the same cell. Note the rebound current upon termination of the meprobamate perfusion. B, Reversal potentials for currents evoked by 10 mM MBM at three different extracellular Cl− concentrations ([Cl−]e). Each point indicates the mean ± S.E. of data from three to four cells. The asterisk identifies the value for 148 mM Cl− as in A. The line represents the theoretical dependence of the equilibrium potential on [Cl−]e from the Nernst equation.

Currents directly activated by meprobamate are blocked by GABAA receptor antagonists.

To determine whether the Cl− current activated by meprobamate is carried by GABAA receptors, we examined whether the GABAA receptor antagonists bicuculline and picrotoxin could block meprobamate responses. As illustrated in figure 6, both picrotoxin and bicuculline reduced meprobamate-activated currents. The onset of the picrotoxin block required several seconds; both 10 and 100 μM picrotoxin produced nearly complete block. Partial recovery occurred during the 30- to 60-s period after termination of the picrotoxin perfusion. Because of the slow onset and recovery, experiments with picrotoxin used a protocol in which the antagonist was applied in a pulse during the continuous application of meprobamate as in figure 6 (left). The failure of full recovery may be caused, at least in part, by desensitization or rundown because, in a separate series of control experiments in which cells were perfused continuously with 10 mM meprobamate for 3 to 4 min, there was a gradual diminution in the whole-cell current (data not shown). In contrast, the bicuculline block occurred more rapidly, but even at a concentration of 100 μM the current was not completely blocked.

Figure 6
Figure 6

Currents evoked by 10 mM meprobamate (MBM) are blocked by the GABAA receptor antagonists picrotoxin (PTX) and bicuculline (BIC). Top, Representative whole-cell current traces demonstrating block of MBM-activated currents by PTX and BIC. The trace to the left shows the slow onset and recovery with PTX. Bottom, Fraction of 10 mM MBM-evoked current obtained in the presence of 10 and 100 μM PTX and BIC. Each bar represents the mean ± S.E. of four to eight cells tested.

Felbamate and meprobamate prolong the deactivation time constants of GABA-activated whole-cell currents.

The deactivation of whole-cell currents evoked by application of GABA or GABA in combination with meprobamate or felbamate were fit to single-exponential functions (ignoring the period of rebound, when present) (fig. 7A). As summarized in figure 7B, the mean deactivation time constants for currents evoked by 10 mM meprobamate + 1 μM GABA and 3 mM felbamate + 3 μM GABA were substantially longer (by more than 2½-fold) than the mean deactivation time constants of currents evoked by 10 mM meprobamate or by 1 and 3 μM GABA.

Figure 7
Figure 7

Deactivation of whole-cell currents evoked by meprobamate (MBM), GABA and felbamate (FBM) or MBM plus GABA. A, Representative deactivation currents scaled to the peak of the best single exponential fits (solid curves). Legend to treatments (indicated by letters) is given in B. B, Mean ± S.E. time constant values from 4–9 experiments similar to that illustrated in A; **P < .01 with respect to the means for both GABA data sets.

Felbamate and meprobamate prolong the burst duration of GABA-activated single-channel currents.

The dicarbamate potentiation of GABAA receptor responses was further characterized in single-channel recordings from excised outside-out membrane patches. GABA-evoked unitary current had a principal conductance level of ∼30 pS. Representative unitary currents induced by 2 μM GABA, and by 3 mM felbamate and 3 mM meprobamate in the presence of 2 μM GABA are illustrated in figure 8. In the presence of the two dicarbamates, burst durations were prolonged (see below) and the unitary currents exhibited flickering, suggesting channel block.

Figure 8
Figure 8

Single-channel currents evoked by 2 μM GABA, and 3 mM felbamate (FBM) and 3 mM meprobamate (MBM) in the presence of 2 μM GABA. The top and middle traces are from the same outside-out patch; the bottom trace is from a separate patch. Boxed areas are shown on an expanded time scale. In the absence of agonist no spontaneous openings were observed.

Burst durations for GABA-activated single-channel currents in the presence or absence of felbamate and meprobamate are summarized in the logarithmically binned frequency distribution histograms of figure9. Each histogram was derived from pooled data from four to seven patches (1,680–2,404 bursts). Data were fit to second-order Gaussian functions. The short (τ1, ∼0.1 ms) and long (τ2, ∼6 ms) time constants derived from these fits are summarized in table 1. Felbamate and meprobamate increased τ2 without substantially affecting τ1.

Figure 9
Figure 9

Felbamate (FBM) and meprobamate (MBM) increase the burst duration of single GABAA receptor currents in outside-out patch recordings. Data are presented as logarithmically binned burst duration frequency distribution histograms. The control 2 μM (left) and 1 μM (right) GABA distributions are represented with thin lines and the distributions in the presence of 3 mM FBM + 2 μM GABA and 3 mM MBM + 1 μM GABA are represented by thick lines. The distributions are fit to a second-order Gaussian function; parameters of the fits are presented in table 1. The total number of bursts represented in the left and right histograms are, respectively, 1687 (control) and 2355 (+ FBM), and 2404 (control) and 1680 (+ MBM).

View this table:
Table 1

Parameters of fits to burst duration distributions of unitary currents activated by GABA and GABA plus felbamate or meprobamate

Meprobamate inhibits NMDA-activated currents.

Previously, we reported that clinically relevant concentrations of felbamate block NMDA receptors (Rho et al., 1994; Subramaniam et al., 1995). In the present study, we sought to determine whether meprobamate had a similar effect. As illustrated in figure10A, meprobamate produced a concentration-dependent block of currents activated by 10 μM NMDA (+ 10 μM glycine). The meprobamate block occurred rapidly and there was also rapid recovery. The concentration-response relationship for meprobamate block of NMDA receptor current is shown in figure 10B (closed circles). For comparison, we carried out a series of parallel experiments with felbamate (open circles). The IC50 values obtained from the best logistic fits to the concentration-response data for meprobamate and felbamate were 4.0 and 3.1 mM, respectively.

Figure 10
Figure 10

Meprobamate (MBM) and felbamate (FBM) inhibition of NMDA-evoked currents. Left, Representative current traces showing inhibition by 1, 3 and 10 mM MBM of currents activated by 10 μM NMDA (+ 10 μM glycine). Right,Concentration-response curve for inhibition of NMDA-evoked currents by MBM (closed circles) and FBM (open circles). Each point represents mean ± S.E. of data from five to six cells.

Discussion

Previously, we obtained evidence that felbamate can potentiate GABAA receptors and inhibit NMDA receptors, and we proposed that this combination of actions could, at least in part, account for the clinical antiseizure activity of the drug (Rho et al., 1994; Subramaniam et al., 1995). The aim of the present study was to characterize in more detail the actions of felbamate and the related propanediol dicarbamate meprobamate on GABAAreceptors, and also to determine whether meprobamate blocks NMDA receptors as does felbamate. In whole-cell voltage-clamp recordings, we confirmed that both propanediol dicarbamates potentiate GABAA receptor currents in a concentration-dependent manner. Furthermore, our results indicate several similarities between the actions of the dicarbamates and the previously reported actions of the barbiturates phenobarbital and pentobarbital. Like barbiturates, the dicarbamates produced a rapid and reversible enhancement of GABA responses. For both felbamate and meprobamate, the threshold for this GABA-potentiating effect was 100 μM, but at equivalent concentrations the magnitude of the potentiation produced by meprobamate was greater than that of felbamate. At high concentrations (>3 mM), meprobamate caused a gradual decline in the current during the drug application, and upon termination of the perfusion there was a rebound that we have termed the off-effect. Rebound also occurred in experiments with 3 mM felbamate, but was less prominent. A similar off-effect has been observed with phenobarbital and pentobarbital (Rho et. al., 1996). We have proposed that this phenomenon reflects rapid relief of channel block superimposed on a slower decay of the potentiated current. Such a model requires the off-rate for channel unblock to be much more rapid than drug dissociation from the site on the GABAA receptor mediating potentiation (and also of GABA from its binding site in experiments where both GABA and a dicarbamate or barbiturate are used).

What is the pharmacological significance of the channel-blocking action of barbiturates and dicarbamates? Channel block occurring at concentrations close to or in the same range as GABAAreceptor potentiation would be expected to limit the extent of positive modulation (resulting in a partial agonist-like effect), and could contribute to the reduced tendency of drugs like phenobarbital and felbamate to produce sedation at anticonvulsant doses (see alsoffrench-Mullen et al., 1993; Rho et al., 1996).

In addition to potentiating GABA-evoked currents, meprobamate activated inward currents in the absence of GABA. It is well recognized that barbiturates, such as pentobarbital, can directly activate GABAA receptor currents (Mathers and Barker, 1980; Suzdaket al., 1986; Yang and Olsen, 1987; Robertson, 1989). Thus, this action of meprobamate is in line with its other barbiturate-like properties. Meprobamate was 5000-fold less potent than GABA as an agonist of the Cl− current, but it had high efficacy, producing currents comparable in amplitude to those activated by GABA. Because we used a perfusion system that continuously exchanges the solution bathing the cell under study, it is unlikely that the currents in these experiments reflect meprobamate potentiation of trace amounts of GABA in the bath solution. Moreover, this possibility can be unequivocally excluded by noting that the mean deactivation time constant for meprobamate-activated currents was significantly different from that of meprobamate in the presence of GABA (fig. 7).

Reversal potential measurements in experiments in which we varied the extracellular Cl− concentration confirmed that the meprobamate-activated inward current is carried by Cl−. Strychnine was added to all perfusion solutions so that the Cl− current is not caused by activation of glycine receptors. However, the GABAA receptor antagonists picrotoxin and bicuculline blocked the Cl− current, which indicated that it is probably carried by GABAA receptors. We have previously observed that picrotoxin was more potent than bicuculline as an antagonist of Cl− currents directly activated by the barbiturate pentobarbital, whereas bicuculline was more potent in blocking GABA. (100 μM bicuculline completely blocked the response to 1 μM GABA; see Rho et al., 1996.) Similarly, meprobamate-activated currents were more potently blocked by picrotoxin than bicuculline (fig. 6). Picrotoxin is an allosteric inhibitor of the GABAA receptor that acts at a site distinct from the GABA recognition site (Olsen, 1981b; Yoon et al., 1993). It has been proposed that barbiturates and picrotoxin act in a functionally reciprocal fashion, with barbiturates prolonging the time spent in a long-duration open state and picrotoxin having the opposite effect (Twyman et al., 1989b). Whether this functional interaction reflects a direct interaction at a common site on the GABAA receptor is not yet established. However, as for picrotoxin block of pentobarbital-activated current (Rho et al., 1996), picrotoxin block of meprobamate-activated current occurred more slowly than block of GABA-activated current (see Rhoet al., 1996), which possibly indicated a requirement for the unbinding of meprobamate in order for picrotoxin block to occur. This would be consistent with binding of meprobamate and picrotoxin to the same or adjacent sites.

It is well recognized that pentobarbital does not bind to the GABA recognition site on GABAA receptors. To explain how the GABA recognition site antagonist bicuculline blocks pentobarbital-activated currents, Rho et al. (1996) proposed that bicuculline acts as an allosteric antagonist. Presumably the partial bicuculline block of meprobamate-activated currents occurs in a similar fashion.

In contrast to meprobamate, felbamate does not directly activate GABAA receptors in the absence of GABA (Rho et al., 1994). It is unclear whether this difference is caused by intrinsic differences in the activity of felbamate or reflects the inability to test sufficiently high concentrations because of felbamate’s limited solubility. Nevertheless, as summarized in table2, there are differences among the barbiturates and dicarbamates in their relative potencies as potentiators of GABA (P) and direct GABAA receptor agonists (A). Whereas the sedative-hypnotic compounds pentobarbital and meprobamate have low A/P ratios (≤3), the less sedative drug phenobarbital has a higher ratio (6). Moreover, felbamate, which is not generally sedating but is frequently associated with insomnia (Leppik and Wolff, 1995), had no measurable agonist activity (A/P ratio >30). We therefore propose that GABAA receptor positive modulators that produce direct receptor activation at concentrations closer to the concentrations producing GABA modulation (low A/P ratio as in table 2) have greater sedative activity. This implies that direct GABAAreceptor activation is important to the powerful sedative-anesthetic effects of barbiturates such as pentobarbital and the dicarbamate meprobamate. In fact, at serum meprobamate concentrations associated with coma in man [120 mg/l (549 μM); Bailey, 1981], the drug would be expected to have such a direct agonist activity.

View this table:
Table 2

Comparison of estimated threshold concentrations of pentobarbital, phenobarbital, meprobamate and felbamate for potentiation and direct activation of GABAA receptor currents

The deactivation time constants of currents activated by felbamate and meprobamate in the presence of GABA were substantially longer than the deactivation time constants of currents activated by GABA or the dicarbamates alone. Previously, we reported that phenobarbital markedly prolongs the decay of GABAA receptor currents, implying that pentobarbital and GABA can mutually stabilize binding to their respective recognition sites (Rho et al., 1996). In fact, the prolongation of the GABAA receptor current deactivation rate by felbamate and meprobamate was similar to that previously observed for pentobarbital (time constant, 480 ms; Rho et al., 1996). Thus, felbamate and meprobamate appear to share with pentobarbital the property of mutual stabilization of GABA binding.

Our data from single-channel recordings provide additional evidence for the barbiturate-like nature of the GABAA receptor potentiation produced by the dicarbamates. Both felbamate and meprobamate prolonged the mean burst duration of control GABA currents in a similar fashion to barbiturates (Macdonald et al., 1989b; Twyman et al., 1989a; Macdonald and Twyman, 1992; Rhoet al., 1996). In addition, at the high concentrations (3 mM) used in our experiments, both felbamate and meprobamate induced flickering of the GABAA receptor currents, compatible with the rapid channel block we propose as a mechanism to explain the off-effect in the whole-cell recording experiments. A similar flickery block was observed previously with high concentrations of phenobarbital and pentobarbital (Rho et al., 1996). If entry of the dicarbamates into the pore of the GABAA receptor prevents channel closure, this could, in part, contribute to their prolongation of burst duration. Although flickering in the single channel recordings is most easily explained by a pore blocking mechanism, allosteric effects on channel gating could also produce flickering and the rebound observed in the macroscopic recordings.

Recently, Kume et al. (1996) reported that felbamate produced a complex inhibition of the binding oft-[3H]butylbicycloorthobenzoate, a picrotoxin-like antagonist, to thick frozen sections from rat brain. In electrophysiological recordings, the same investigators observed that felbamate modestly enhanced whole-cell GABA-activated Cl−currents, confirming our earlier observation (Rho et al., 1994). Furthermore, pentobarbital potentiation and picrotoxin inhibition of these Cl− currents was unaffected by felbamate. It was concluded that felbamate acts at a site on the GABAA receptor complex that is allosterically coupled to the picrotoxin recognition site and distinct from other known sites, including the barbiturate binding site. In the absence of an antagonist for the barbiturate site, it is difficult to pharmacologically evaluate the possibility that the dicarbamates act at the same site as barbiturates. As did Kume et al. (1996), we also found that 3 mM felbamate failed to affect whole-cell GABA-activated currents potentiated by 30 μM to 1 mM pentobarbital (unpublished observations). However, in a parallel series of experiments, 3 mM meprobamate did reduce the potentiation of 1 μM GABA currents by 1 mM pentobarbital [control, 2100 ± 330% potentiation (n = 11); + 3 mM meprobamate, 530 ± 100% (n = 5); P < .01, grouped t-test]. This result is compatible with the possibility that meprobamate competes for binding with pentobarbital (and has lower intrinsic efficacy), but may also reflect greater channel-blocking activity of meprobamate in comparison with pentobarbital. Thus, at present, although it can be stated that the actions of felbamate and meprobamate on GABAA receptors are similar to those of barbiturates, further studies are required to determine whether the dicarbamates bind to the same or a distinct domain of the GABAA receptor complex as barbiturates.

Having established previously that felbamate inhibits NMDA-receptor-mediated responses (Rho et al., 1994;Subramaniam et al., 1995), it was of interest to determine whether meprobamate might have a similar action. Our results demonstrate that the drug can indeed produce a concentration-dependent block of NMDA receptors (threshold, 300 μM). Meprobamate was slightly less potent than felbamate in this regard, and, more importantly, for meprobamate, the NMDA receptor blocking effect occurred at concentrations beyond the therapeutic range: therapeutic serum concentrations for meprobamate have been reported to be 10 to 40 mg/l (46–183 μM) (Baselt, 1982). Indeed, the concentrations of meprobamate producing GABA potentiation (threshold, 100 μM) are well within this range. In contrast, low therapeutic (anticonvulsant) serum concentrations of felbamate are 20 to 80 mg/l (100–300 μM) (Leppiket al., 1991; Theodore et al., 1991; Ritteret al., 1993). Thus, at therapeutic concentrations, felbamate would be expected to have effects on both GABAA(threshold, 100 μM) and NMDA receptors (threshold, 100–300 μM). As was the case with felbamate (Rho et al., 1994), we found that glycine (at concentrations up to 100 μM) did not fully reverse the meprobamate block of NMDA receptors (unpublished observations). Thus it is unlikely that meprobamate exerts its NMDA receptor blocking activity by an action at the glycine modulatory site on NMDA receptors (see Subramaniam et al., 1995).

In summary, our present results indicate that felbamate and meprobamate act as barbiturate-like positive modulators of GABAAreceptors, and that they also inhibit NMDA receptors. Whereas the action of felbamate on the two receptor systems occurs in the same range of concentrations, meprobamate is a more active potentiator of GABAA receptors, and it also produces a direct agonist action (although relatively less potently than the sedative-anesthetic barbiturate pentobarbital). For both dicarbamates, channel block of the GABAA receptor limits the extent of potentiation, so that neither compound is as potent a central nervous system depressant as pentobarbital. The various differences in the actions of felbamate and meprobamate on GABAA receptors could account for their distinct pharmacological profiles.

Footnotes

  • Send reprint requests to: Michael A. Rogawski, M.D., Ph.D., Neuronal Excitability Section, NINDS, NIH, Building 10, Room 5N-250, 10 Center Drive MSC 1408, Bethesda, MD 20892-1408.

  • ↵1 Present address: Department of Neurology, Children’s Hospital and Medical Center, University of Washington School of Medicine, Seattle, WA 98105, U.S.A.

  • ↵2 Present address: Department of Neurology, University of Utah School of Medicine, Salt Lake City, UT 84112, U.S.A.

  • Abbreviations:
    GABA
    γ-aminobutyric acid
    NMDA
    N-methyl-d-aspartate
    HEPES
    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
    EGTA
    ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid..
    • Received August 23, 1996.
    • Accepted November 26, 1996.

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OtherNEUROPHARMACOLOGY

Barbiturate-Like Actions of the Propanediol Dicarbamates Felbamate and Meprobamate

Jong M. Rho, Sean D. Donevan and Michael A. Rogawski
Journal of Pharmacology and Experimental Therapeutics March 1, 1997, 280 (3) 1383-1391;
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