Introduction

Abstract Introduction Materials & Methods Results Discussion References

Neuronal nicotinic receptors have attracted much interest during the past few years, largely due to the discovery that the Alzheimer's brain loses many of its nicotinic receptors by the time of death, whereas muscarinic receptors are much less affected (Kellar et al., 1987; Araujo et al., 1988). So far, therapeutic approaches directed toward cholinergic systems in the brain have focused on stimulation of postsyaptic muscarinic cholinergic receptors, either directly with muscarinic agonists or indirectly by cholinesterase inhibition. Unfortunately these two strategies have thus far yielded only modest improvements in the cognitive functions of Alzheimer's patients. Stimulation of brain nicotinic receptors has been shown to enhance cognitive function in lower mammals (Woodruff-Pak et al., 1994; Arendash et al., 1995a, b; Decker et al., 1995; Bjugstad et al., in press), consistent with the idea that these nicotinic receptors may be potential targets treatment of Alzheimer's and other dementias (Newhouse et al., 1993).

Molecular biological studies have revealed a plethora of nicotinic receptor subunits in the vertebrate brain (Papke, 1993; McGehee and Role, 1995; Lindstrom, 1996; Albuquerque et al., 1997). Although there is still little understanding of the functional consequences of this receptor multiplicity, several labs are investigating the pharmacological properties of the predominant nicotinic receptor subtypes occurring in the nervous system to provide a rational basis for the design of compounds selective for particular nicotinic receptor subtypes that influence particular mental or motor functions (Decker et al., 1995; de Fiebre et al., 1995). Flores et al. (1992) have shown that the major receptor subtype displaying high nicotine, cytisine and methylcarbamyl-choline affinity in the rat brain is the alpha4-beta2 combination. A major receptor subtype showing low affinity for nicotine but high affinity for BTX contains alpha7 subunits (Wonnacott, 1986; Luetje et al., 1990). Alpha7-containing receptors have been implicated in cognitive processes affected by hippocampal function, including sensory gating and spatial memory (Luntz-Leybman et al., 1992; Bjugstad et al., in press).

Pharmacological investigations of nicotinic receptors have been facilitated by the availability of many potent natural toxins, including curare, the erythrina alkaloids, the algal toxin anatoxin-a (Swanson and Albuquerque, 1992), the frog toxin epibatidine (Badio and Daly, 1994; Alkondon and Albuquerque, 1995), the flowering plant toxin methyllcaconitine (Ward et al., 1990), and of course, nicotine. The pharmacological properties of some other potent nicotinic toxins, including leptodactyline (Erspamer, 1959) and anabaseine, have not yet been reported in much detail.

Anabaseine (fig. 1) was initially isolated from a marine worm, but has subsequently been found in certain species of ants (Kem et al., 1971; Wheeler et al., 1981). It is as toxic as nicotine when injected in mice (Kem et al., 1976), stimulates acetylcholine release from rat brain cortical minces (Meyer et al., 1987) and elevates cortical ACh and norepinephrine levels in the intact rat (Summers et al., 1997). As with nicotine, anabaseine enhances passive avoidance behavior in nucleus basalis-lesioned rats (Meyer et al., 1994). At the molecular level anabaseine differs from nicotine and anabasine by having a tetrahydropyridyl ring whose imine double bond is electronically conjugated with the 3-pyridyl ring (fig. 1). This causes its two rings to be approximately coplanar in their relative orientation, whereas the two rings in the tobacco alkaloids nicotine and anabasine are almost perpendicular to each other (Whidby and Seeman, 1976; Seeman, 1984).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structures of the three nicotinic alkaloids. Anabaseine (A) is an animal toxin, whereas nicotine (C) and anabasine (D) are plant alkaloids. Under physiological conditions anabaseine occurs in three almost equally populated forms (Zoltewicz et al., 1989; Bloom, 1990): cyclic iminium (A), cyclic imine and ammonium-ketone (B). Only the cyclic iminium form of anabaseine possesses significant nicotinic agonist activity (Kem et al., 1994b; Kem et al., submitted).

Because some anabaseine derivatives have been shown to enhance a variety of cognitive behaviors (Meyer et al., 1994; Woodruff-Pak et al., 1994; Arendash et al., 1995b), we examined the pharmacological properties of anabaseine on a variety of vertebrate, mostly mammalian, nicotinic receptors. To quantitatively compare anabaseine with the tobacco alkaloids, we measured the potencies and binding affinities of all three compounds on the same receptors under identical experimental conditions. Several important pharmacological differences were found to exist between the three compounds. Each displays a unique spectrum of action upon the various nicotinic receptors. Our data indicate that these compounds will be useful molecular models to design agonists selective for particular nicotinic receptors. Portions of this study were previously reported in abstract form (Kem and Papke, 1992; Kem et al., 1994a).

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

Chemicals. Anabaseine was synthesized according to Bloom (1990). The fully ionized, synthetic ammonium-ketone dihydrochloride salt (MW 251) was used in all of our experiments. DMAB-anabaseine dihydrochloride was synthesized as previously described (Kem, 1971; Zoltewicz et al., 1989). Stock solutions of anabaseine, anabasine, nicotine and DMAB-anabaseine were kept in the dark at 5°C for a maximum of 1 wk to avoid deterioration of the alkaloids. (S)-Anabasine free base and reagents used to synthesize anabaseine were obtained from Aldrich (Nukwayjee, WI); (S)-nicotine free base, mecamylamine and other experimental drugs from Sigma Chemical Co. (St. Louis, MO); BTX and TTX from Boehringer-Mannheim (Indianapolis, IN). Radioisotopically labeled compounds, ³H-MCC, ¹²⁵ICl and ⁸⁶RbCl were purchased from Du Pont-New England Nuclear (Boston, MA).

Frog skeletal muscle contractility. The two symmetrical rectus abdominis muscles of each frog (Rana pipiens, purchased from Nasco, Ft. Atkinson, WI) were used so that anabaseine potency relative to nicotine or anabasine could be measured on muscles from the same animal. Each muscle was mounted in a 10-ml tissue bath containing frog saline (115 mM NaCl, 5.0 mM KCl, 7.0 mM CaCl₂ and 2.0 mM sodium phosphate buffer, pH 7.2) which was continuously bubbled with oxygen at room temperature. The resting tension was initially adjusted to 1.0 g. After 30 min the muscles were briefly (20 sec) contracted with isotonic KCl saline (NaCl replaced with KCl) to measure the maximum isometric force of contracture. After complete recovery, each muscle was challenged with a sequence of 9 or 10 increasing concentrations of agonist until a maximum contractural force was observed. After each application the muscles were washed twice with normal saline and allowed to recover at least 30 min before the next contracture, due to the relatively slow relaxation after exposure to the three alkaloids. After the various agonist concentrations were tested, the final contractility of each muscle was again measured with isotonic KCl saline. A concentration-response curve for each muscle was constructed and expressed as a percentage of the average KCl-induced contracture force. The concentration-response data for each compound was fitted to the Hill equation using SigmaPlot and the EC₅₀ and its S.E. were calculated from the computer-fitted curve.

Patch clamp experiments with neuromuscular type nicotinic receptors. BC3H-1 cells were cultured according to Covarrubias et al. (1989). During single channel recordings they were bathed in a saline containing 140 mM NaCl, 5.4 KCl, 10 mM NaHEPES, 1.8 mM CaCl₂ and 2.0 mM MgCl₂ titrated to pH 7.4. Single channels were recorded from cell-attached patches. The pipette saline containing agonist was otherwise identical to the bath saline. In most cases, cells were incubated for 5 to 12 min with 48 nM BTX before recordings to reduce the number of available channels in a patch. Single channel records were stored on videotape using a digital audioprocesser (20 kHz bandwidth). For computer analysis of single channel records, recordings were replayed and digitized at 50 kHz with analog filtering to yield a bandwidth of 5 kHz. Single channel events were analyzed with standard half amplitude threshold crossing methods (Auerbach and Lingle, 1987).

Xenopus oocyte expression and functional analysis of rat brain nicotinic receptors. Preparation of in vitro synthesized cRNA transcripts and oocyte injection have been described previously (de Fiebre et al., 1995). Recordings were made 2 to 7 days after injections. Oocytes were placed in a Lucite recording chamber with a 0.6 ml total volume and perfused at room temperature with frog saline (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl_2, 10 mM HEPES, pH 7.3) containing 1 µM atropine to block potential muscarinic responses. Calcium-activated chloride channels were not suppressed in our experiments, because their functional presence does not affect the agonist concentration-response relation (Papke et al., 1977a). Drugs were diluted in perfusion solution and then applied after preloading of a 2.0 ml length of tubing at the terminus of the perfusion system. A Mariotte flask filled with Ringer saline was used to maintain a constant hydrostatic pressure for drug deliveries and washes. The rate (6 ml/min) of drug delivery was constant for all compound concentrations and receptor subtypes. Current responses to drug administration were measured using a two electrode voltage clamp with a holding potential of -50 mV. Recordings were made using a Warner Instruments oocyte amplifier interfaced with National Instruments LabView software.

Radioligand binding to nicotinic receptors. The steady-state binding of the three nicotinic compounds to neuromuscular type receptors was measured indirectly by assessing their ability to inhibit the rate of ¹²⁵I-BTX binding to Torpedo californica membranes prepared according to Eldefrawi et al. (1980).

Measurement of ganglionic nicotinic receptor activation. Rat pheochromocytoma (PC 12) cells grown in the absence of nerve growth factor on polylysine-coated plastic culture dishes were loaded overnight with ⁸⁶Rb preceding the efflux assays, which were carried out essentially as described by Lukas and Cullen (1988) at pH 7.4. After washing away extracellular rubidium three times, the agonist (in saline containing 10 µM atropine sulfate to inhibit muscarinic receptors and 0.5 mM ouabain to prevent rubidium reuptake by active transport) was added and 1 min later the released rubidium was removed for gamma counting. The rubidium efflux during agonist stimulation was expressed as a percent of total cellular rubidium released by 1 mM carbachol during the same time. All efflux estimates were corrected for spontaneous efflux in the absence of agonist. The amount of ⁸⁶Rb remaining after the 1 min test period in each cell sample was determined after exposure to 1.0 M NaOH for at least 1 hr. All measurements were done in quadruplicate.

i.c.v. administration of anabaseine. When injected i.c.v. with a nicotinic agonist, rats rapidly become immobile with extended legs (Abood et al., 1981). This prostration response was used to compare the central activity of anabaseine relative to nicotine. After implantation of a metal cannula into the third ventricle, 5 days were allowed for recovery from surgery. The rat received injections with 2 or 4 µl of the experimental compound dissolved in sterile 0.9% NaCl solution, pH 6.5. Prostration was judged to have occurred when all four legs of the Sprague-Dawley male rat (250-300 g) remained laterally extended for at least 10 sec. To detect prostration each rat was observed for at least 5 min after injection. All rats that were not prostrated after injection were killed after an additional i.c.v. injection of Evan's blue dye to ascertain that the cannula was operational; the result was not used if the dye was absent from the lateral ventricular space.

	Results

Abstract Introduction Materials & Methods Results Discussion References

Activation of frog neuromuscular nicotinic receptors by anabaseine. Anabaseine acted as a potent nicotinic agonist on frog rectus abdominis muscle. A wide variety of natural toxins and synthetic compounds have previously been tested on this preparation, which facilitated quantitative comparison of anabaseine with these other substances (table 1). When the median effective concentrations of the active, monocationic forms of each compound were compared, anabaseine was only 14- and 3.7-fold less potent, respectively, than epibatidine and anatoxin-a, which are the most potent nicotinic agonists thus far reported. Nicotine was 6.7-fold and cytisine 23-fold less potent than anabaseine.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Ability of muscle nicotinic receptor antagonists to affect the contracting action of anabaseine. (A) The reversible antagonist d-tubocurarine (10 µM) shifts the anabaseine concentration-response curve to the right. n = 6 muscles per point, except the lowest two concentrations of anabaseine alone, where n = 4. The EC50 for anabaseine in the presence of TC was calculated assuming that the maximum contractile response was the same as when anabaseine was applied alone. (B) BTX (30 minutes initial exposure) irreversibly reduces the response at all anabaseine concentrations. n = 12 muscles per point for anabaseine alone and n = 6 for points with BTX.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Relative actions of anabaseine, anabasine and nicotine upon frog rectus abdominis muscle at pH 7.2 For anabaseine, n = 8 muscles per point; for nicotine, n = 6 muscles per point except the highest concentration where n = 4; for anabasine, n = 6 except for the two highest concentrations where n = 4.

Interaction of nicotinoid compounds with electric fish muscle nicotinic receptors. The relative abilities of the three nicotinoid compounds to inhibit ¹²⁵I-BTX binding to Torpedo electric organ membranes are shown in figure 4. Anabaseine displayed the highest affinity of the three compounds although anabasine displayed the lowest affinity (table 2). The relative K_d for the electric fish muscle were very similar with the frog rectus muscle potency (EC₅₀) estimates shown in table 3.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Nicotinoid displacement of 125I-BTX binding to Torpedo electric organ membranes. Membranes containing 10 µg protein by Lowry assay were preincubated 15 min with anabaseine in buffer (50 mM Tris-HCl, pH 7.4) containing 2 mg/ml bovine serum albumin and then incubated for 1 hr with 125I-BTX in a final volume of .25 ml. Nonspecific binding was measured in the presence of 1 mM nicotine. After incubation samples were diluted with 1.2 ml of ice cold buffer and bound radioligand was separated from free ligand by filtration under vacuum through glass fiber filters (Whatman GF/C) at 4°C. The filters were presoaked for 15 min in 0.5% (v/v) polyethyleneimine containing 0.25 mg/ml BSA and washed with 2.5 ml buffer before filtration. The membranes were washed twice on the filters with 4 ml of ice cold buffer and then counted with a Biogamma counter. Each point is the average of triplicate measurements. Data were fitted with EBDA software (Ligand).

Activation of mammalian neuromuscular nicotinic receptors by anabaseine. The ability of anabaseine to activate mouse embryonic neuromuscular type nicotinic receptors was examined using cell-attached single channel recordings from the clonal BC3H-1 cell line. The single channel conductance in the presence of anabaseine was indistinguishable from that obtained using ACh. As reported for other nicotinic agonists (Colquhoun and Sakmann, 1985; Sine and Steinbach, 1986; Papke et al., 1988), a low anabaseine concentration (40 nM) caused two types of open channel activity: short duration (<500 µsec) bursts resulting primarily from single brief openings and long duration (>3 msec) bursts that were often interrupted by brief closures (results not shown). Histograms of burst durations revealed two components. The average duration of the longer component was somewhat shorter than for bursts activated by ACh, although the voltage-dependence of the burst durations was similar for both agonists. In some cases three components better described the distribution of burst durations, a characteristic also noted for bursts activated by ACh (Colquhoun and Sakmann, 1985).

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Nicotinic agonist activity of anabaseine and ACh upon BC3H-1 cells. Data were recorded using the cell-attached voltage clamp method. Groupings of openings activated by either ACh (A) or anabaseine (B) are shown. Groups for analysis were selected as defined in "Materials and Methods." Qualitatively, closed intervals within groups of openings become shorter with increases in agonist concentration. With anabaseine, open intervals become shorter with increases in agonist concentration as a result of channel block by anabaseine, and an increase in frequency of a short duration gap is apparent.

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View larger version (26K): [in this window] [in a new window]   Fig. 6.   Rapid channel block of BC3H-1 receptor channels by anabaseine. In A, the frequency of occurrence (events per msec open time) of a closed interval component of duration of about 100 msec is plotted as a function of anabaseine concentration. Patches with calculated membrane potential between -75 and -100 were included in this analysis. Error bars are S.E. for 6, 4, 7, 7, 9, 8 and 5 files for 0.04, 5, 10, 20, 50, 100 and 200 µM, respectively. The fitted line has a slope of 1.2 × 107 M1 sec1, with intercept of 102 ± 88/s. In B, the blocking gap duration (open circles) and longer open time component (filled circles) are plotted as a function of anabaseine concentration. Error bars are S.E.s. Values were corrected for missed events. The open durations were fit with t([Anabaseine])= 1/(a * f[Anabaseine]), where a is the channel closing rate at low [Anabaseine]. The fitted values were a = 210 ± 10 sec1, with f = 1.8 (± .3) × 107 M1 sec1. In C, the duration of fast gaps corrected for missed events is plotted as a function of membrane potential. Only values obtained with 100 µM anabaseine were included in both C and D. The fitted line is described by tc(V) = tc(0)* e(A*V) with tc(0) = 0.02 ± .01 msec and A = -0.017 ± 0.003/mV. In D, the frequency of fast gaps scaled by the anabaseine concentration (100 µM) is plotted as a function of membrane potential. The 0-voltage frequency was 7.13 ± 2.56 sec1 mM1, with a voltage-dependence of -0.007 ± 0.003/mV.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Dependence of BC3H-1 nicotinic receptor channel cluster open probability on anabaseine. Channel open probability was calculated from the interval distributions from selected groups as described in "Materials and Methods." Closures attributed to channel block or short-lived desensitized states were excluded by this method. Each point is the mean of 4, 5, 8, 7, 6, and 5 values for 5, 10, 20, 50, 100 and 200 µM anabaseine, respectively, with error bars showing the S.E. The points were fit to po([Anabaseine])= po(max)/(1+(EC50/[Anabaseine])n) with po(max) = 0.75 ± 0.08, EC50 = 9.6 ± 2.3, and n = 1.6 ± .5.

Rat brain neuronal nicotinic receptors: Xenopus oocyte experiments. At alpha7 receptors anabaseine and anabasine displayed very similar efficacies, although nicotine was significantly less efficacious (fig. 8A). Anabaseine and anabasine displayed the highest potencies for homomeric alpha7 receptor. The concentration dependence of recovery in responsiveness to ACh to each compound displayed the same concentration dependence as its agonist activity (fig. 8B).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Full agonist actions of anabaseine and anabasine upon rat brain alpha7 homomeric receptors expressed in the Xenopus oocyte. Agonist responses were normalized to the individual oocyte's response to a control ACh (500 µM) application made 5 min before the experimental applications. Each point represents the average response (±S.E.) of at least four oocytes to that agonist concentration.(A) concentration-response curves for activation of the receptor. (B) Concentration-response curves showing residual inhibitory activity 5 min after washing away the nicotinic agonist. The ACh data, previously reported in Papke et al. (1994), are included for comparison with the three alkaloids.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Weak partial agonist actions of anabaseine and anabasine upon alpha4-beta2 receptors expressed in the Xenopus oocyte. Agonist responses were normalized to the individual oocyte's response to a control ACh (500 µM) application made 5 min before the experimental applications. Each point represents the average response (±S.E.) of at least four oocytes to that agonist concentration. The ACh data, previously reported in Papke et al. (1994), are included for comparison with the three alkaloids.

Rat brain neuronal nicotinic receptors: radioligand binding experiments. Next we examined the ability of anabaseine, anabasine and nicotine to bind to rat brain neuronal receptors using two radioligand binding assays, involving displacement of ¹²⁵I-BTX and ³H-MCC binding. Displacement of the first radioligand predominantly measures interaction with alpha7-containing receptors although displacement of the second one measures alpha4-beta2 receptor binding (Flores et al., 1992).

View larger version (26K): [in this window] [in a new window]   Fig. 10.   Nicotinic agonist displacement of specific [125I]-alpha-bungarotoxin binding to rat brain membranes. The BTX concentration was 1 nM. Membranes were equilibrated with the radioligand for 1 hr at 37 C before washing and filtration with ice-cold saline. Nonspecific binding was determined in the presence of 1 mM nicotine. Each point represents the mean value for triplicate samples. The IC50s of the three alkaloids, determined by EBDA software, are shown in the box.

View larger version (25K): [in this window] [in a new window]   Fig. 11.   Scatchard plot showing anabaseine effect upon binding of [3H]-methylcarbamylcholine to rat brain high nicotine affinity receptors. Each point represents the mean value for triplicate samples. The Kd of [3H]-MCC was calculated to be 11 nM.

View larger version (30K): [in this window] [in a new window]   Fig. 12.   Nicotinic agonist displacement of specific [3H]-MCC binding to rat cerebral cortex membranes. The tritiated MCC concentration was 5 nM and the saline pH was 7.4. Nonspecific binding was determined in the presence of 10 µM carbamylcholine. Each point represents a mean value obtained from triplicate estimates. The IC50s shown in the box were determined with EBDA software.

Anabaseine stimulation of autonomic nicotinic receptors. All three nicotinic agonists acted as high efficacy nicotinic receptor agonists on PC12 cells (table 3). The major ganglionic nicotinic receptor (Rogers et al., 1992) in this transformed cell line consists of alpha3 and beta4 subunits, and was previously shown to possess relatively low affinity for nicotine and ACh in functional assays (Lukas and Cullen, 1988; Lukas, 1989; Wong et al., 1995). Because maximum nicotinic stimulation of the cells by the three alkaloids and carbamylcholine caused the release of only a small (usually less than 10%) fraction of the internal rubidium, our efflux measurements with this ion should reflect the average extent of receptor activation over the time (1 min) of the measurement. All three compounds acted as full agonists relative to carbachol (concentration-response curves not shown). When concentration was expressed in terms of the active cationic form of each compound, the potencies of the three nicotinoid compounds were very similar (table 3).

View larger version (22K): [in this window] [in a new window]   Fig. 13.   Relaxing action of anabaseine and nicotine upon rat colon longitudinal muscle. The relaxing ability of a particular concentration of agonist was determined by adding it to the bath as soon as the peak contraction caused by 320 nM oxotremorine had been reached The amplitude of relaxation was expressed as a percentage of the oxotremorine-stimulated contractile force. TTX inhibited the relaxing effect of anabaseine in a noncompetitive fashion. n = 6 for both anabaseine and nicotine curves, although n = 4 for the anabaseine plus TTX curve.

Intracerebroventricular administration of anabaseine: rat prostration experiments. On a mole basis, anabaseine was approximately 2.7-fold less potent than nicotine in causing prostration when administered into the lateral ventricle of the unanesthetized rat (fig. 14). If only the monocationic forms of the two compounds are active, this equipotent mole ratio would become nearly one. Nicotine was less potent in our experiments than was previously reported by Abood et al. (1981). This may have been due to our use of a slightly more stringent behavioral endpoint for assessing prostration, as described in the Methods section. Mecamylamine and DHBE both inhibited the prostrating action of anabaseine. A large dose (80 µg) of DMAB-anabaseine failed to prostrate rats but did partially inhibit the prostrating action of nicotine.

View larger version (40K): [in this window] [in a new window]   Fig. 14.   Anabaseine causes immediate prostration in the rat. Also shown is the inhibition of 80 µg anabaseine prostration by pretreatment either with 40 µM mecamylamine or dihydro-B-erythroidine, and the inhibition of 20 µg nicotine prostration by 80 µg DMAB-anabaseine. The number of animals receiving injections (i.c.v.) at each dose is indicated within parentheses. Doses (µg) of anabaseine were based on its dihydrochloride form, whereas those of nicotine were based on its free base form.

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

Anabaseine selectively stimulates nicotinic receptors. Although anabaseine stimulated all of the nicotinic receptor preparations that we investigated, its high potency upon neuromuscular and alpha7 nicotinic receptors is particularly noteworthy. On the skeletal muscle membrane, anabaseine appears to work entirely on nicotinic receptors, because its action could be completely blocked by the insurmountable antagonist BTX. Nerve action potentials were unaffected by this compound, even at millimolar concentrations (Kem, 1971). However, an effect at nerve terminals cannot yet be ruled out, as some mammalian motoneuron terminals seem to possess nicotinic receptors. Anabaseine affected neither rat brain muscarinic receptors nor plasma cholinesterase, except at very high concentrations (>100 µM) where nonspecific membrane effects often occur (Kem et al., 1994c). Stimulation of the rat brain 5-HT₃ receptor, which possesses a subunit sequence homologous with nicotinic receptor subunits, was only inhibited by 24% in the presence of 100 µM anabaseine (Machu et al., 1996). Thus, anabaseine is expected to selectively act on nicotinic receptors at concentrations of less than 100 µM.

Anabaseine actions upon single neuromuscular channels. The results show that the characteristics of openings and groups of openings activated by anabaseine at both low and high concentrations share many similarities to properties of openings activated by ACh. Based on the total concentration (~10 µM) of anabaseine at which a half maximal channel open probability is achieved, anabaseine activates the mouse embryonic neuromuscular nicotinic receptor with an apparent affinity slightly less than that of ACh (2-10 µM; Sine and Steinbach, 1987; C. Lingle, unpublished results). However, because the cyclic minimum concentration of anabaseine is only 29% of its total concentration at pH 7.4, this active form of anabaseine is probably slightly more potent than ACh on this mammalian receptor, as at the amphibian neuromuscular receptor (table 1). In our experiments the intrinsic activity or true efficacy (after correcting for its channel-blocking action) of anabaseine displayed a limiting open probability of less than 0.8 compared with values in excess of 0.9 for ACh (Sine and Steinbach, 1987; Zhang et al., 1995). A higher limiting p_o value for the compound might have been achieved if higher anabaseine concentrations had been tested.

Electrophysiological comparison of the three alkaloids on oocyte-expressed neuronal nicotinic receptors. Anabaseine (Papke et al., 1994) and anabasine (table 3) both displayed low efficacies on the Xenopus oocyte expressed alpha4-beta2 receptor. A submaximal efficacy on this receptor has previously been observed with other potent nicotinic agonists, including cytisine, anatoxin-a, epibatidine, nicotine and the synthetic nicotinic agonist ABT-418 (Papke and Heinemann, 1994; Alkondon and Albuquerque, 1995; Papke et al., in press). Apparently the ligand molecular requirements for activating this receptor subtype are even more stringent than those for high affinity binding. Because most of the agonists that display high affinity are larger, less flexible molecules, high efficacy may be related to an ability to bind within a relatively restricted space on this receptor. An alternative interpretation would be that the alpha4-beta2 receptor channel is more readily blocked by receptor agonists, which would be reflected in a smaller maximum response or efficacy (Papke et al., 1997b). Patch-clamp analyses of the actions of these agonists are clearly needed to determine the basis for the reduced apparent efficacy of these compounds on alpha4-beta2 and alpha7 nicotinic receptors.

Anabaseine interaction with rat brain membrane nicotinic receptors. Our binding data with the naturally expressed BTX-binding nicotinic receptor is in agreement with our functional data on the oocyte-expressed homomeric alpha7 receptor. Quik et al. (1996) have reported an excellent correspondence between the ligand binding properties of the rat brain alpha7-containing receptor and those of the homomeric alpha7 receptor expressed in a transfected cell line, which is consistent with the notion that the alpha7 receptor in the rat brain may also be homomeric. However, Anand et al. (1993) have observed some pharmacological differences between the artificially expressed homomeric chick alpha7 receptor and brain receptors containing the alpha7 subunit, so at least in the chick brain the receptors are not the same.

Anabaseine actions on PC12 cells and parasympathetic neurons. On PC12 cell receptors anabaseine displayed a potency similar to nicotine and anabasine when the extent of ionization was taken into consideration, and the maximal responses (data not shown) were nearly identical with that of carbachol. The uncorrected EC₅₀ value of 29 µM for nicotine stimulation of ⁸⁶Rb efflux is in excellent agreement with other reported nicotine EC₅₀ values for these cells (29 µM, Kemp and Edge, 1987; 20 µM, Lukas, 1989). However, our observation that the maximal effect of nicotine on PC12 cells is comparable with the 1 mM carbamylcholine response differs from some previously reported data that indicated that nicotine's maximal effect was significantly less than the effect of 1 mM carbamylcholine (Lukas and Cullen, 1988; Lukas, 1989). Several factors, such as differences in the composition or degree of expression of nicotinic receptors between different PC12 cultures, could possibly contribute to such a difference. PC12 cells express other nicotinic receptor subunits besides alpha4 and beta3 (Rogers et al., 1992). PC12 alpha7 receptors bind BTX, but probably have at most, only a small contribution to the rubidium fluxes we measured over a 1-min interval (Kemp and Edge, 1987; Rogers et al., 1991).

Whole animal actions of anabaseine. At an initial stage of this investigation the rat prostration response to lateral ventricular injection of nicotinic agonists was selected as an in vivo bioassay for demonstrating neuronal nicotinic agonist activity of anabaseine. The in vivo agonistic and antagonistic activities of a variety of nicotinic compounds, neurotransmitters and toxins had previously been demonstrated using this assay (Abood et al., 1981). The prostration response displayed pronounced stereo-specificity for the (S)-form of nicotine, as had been observed in radioligand binding experiments with the brain high nicotine affinity binding site. Because displacement of BTX binding to low affinity receptors shows little stereospecificity (Wonnacott, 1986), the existing data suggest that alpha7 type receptors do not play a major role in causing this prostration response. Abood et al. (1981) reported that i.c.v. injection of hexamethonium or mecamylamine immediately preceding nicotine administration partially inhibited its prostrating action. They also reported that preadministration of BTX or TC failed to inhibit the action of prostrating action of nicotine, although TC injected alone caused seizures.

Preferential actions of the three alkaloids upon particular nicotinic receptors. To compare nicotinic agonists in molecular terms, it is necessary to quantitatively express potencies in terms of the concentration of the active form of each compound. This can be estimated with knowledge of the bulk pH of the saline and the pKa of the ionizable group. Fixed negative charges might alter the local pH at the ACh recognition site, so that it may differ from that of the bulk pH (Stauffer and Karlin, 1994). For instance, a slightly lower local pH at the ACh recognition site would enhance agonist ionization and increase the estimated potencies of secondary and tertiary amine compounds relative to a quaternary ammonium salt like carbamylcholine. Correction for the local pH effect would probably not greatly affect the potency comparisons of the non-quaternary compounds in table 1.

Structural comparison of anabaseine with the two tobacco alkaloids. Both nicotine and anabasine possess a tetrahedral chiral carbon at position 2 of the saturated ring, whereas the same carbon atom in anabaseine is part of a trigonal imine bond whose pi electrons are conjugated with those of the pyridyl ring. Conformational analyses predict that the saturated ring is twisted approximately 90 degrees out of the plane of the pyridyl ring in nicotine and anabasine (Whidby and Seeman, 1976; Seeman, 1984), although the two rings of anabaseine are coplanar (Prokai et al., in preparation).

Comparison of anabaseine with 3-substituted anabaseines. Our study now provides a foundation for understanding the pharmacological properties of the benzylidene and cinnamylidene derivatives of anabaseines, including DMAB-, DMXB- and DMAC-anabaseines, which preferentially stimulate neuronal nicotinic receptors containing alpha7 subunits (Kem et al., 1994c; Meyer et al., 1994; Papke et al., 1994; de Fiebre et al., 1995) and enhance cognitive behavior (Woodruff-Pak et al., 1994; Meyer et al., 1994; Arendash et al., 1995b; Bjugstad et al., in press). As with anabaseine, these compounds display high affinity and efficacy on alpha7 receptors but low affinity and efficacy with alpha4-beta2 receptors. Thus, the 3-substitution of anabaseine merely increases further an alpha7 vs. alpha4-beta2 preferential activity already present in anabaseine. 3-Substituted anabaseines also lack significant agonist activity on peripheral nicotinic receptors of the autonomic and neuromuscular types (Kem et al., 1994c). It is extremely interesting that the addition of a 3-substituent to anabaseine seems to diminish its peripheral nervous system and alpha4-beta2 stimulation without reducing central alpha7 stimulation. This is probably the major pharmacodynamic advantage of the 3-substituted anabaseines over the parent toxin. One of these derivatives, DMXB-anabaseine (also known as GTS-21; Kem et al., 1996), is currently in clinical trials for possible treatment of Alzheimer's dementia. Further studies are needed to fully understand the nature of the molecular differences between the 3-substituted derivatives of anabaseine and the natural toxin.