Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Nicotine and nicotinic agonists elicit many diverse responses in vivo (Brioni et al., 1997). This physiological and behavioral diversity arises in part from differences in composition of, anatomical localization of, and physiological processes stimulated by nicotinic receptors. Nicotinic receptors are distributed on skeletal muscle, in the autonomic nervous system, on secretory tissue, and in the brain. In addition, nicotinic receptors themselves are complex pentameric assemblies of homologous subunits. Receptor subtypes composed of different homomeric or heteromeric combinations of subunits display markedly diverse physiological and pharmacological properties (Sargent, 1993). Responses secondary to the activation or inhibition of nicotinic receptors (such as neurotransmitter or hormonal release) also influence the response to nicotinic agonists (Wonnacott, 1997). Thus, the responses observed after acute or chronic exposure to nicotine or other nicotinic drugs are determined by a complex array of factors.

One approach to investigate the diversity of nicotinic responses in the brain is to use biochemical assays for receptor function. For example, nicotinic receptors have been shown to modulate the release of many neurotransmitters such as dopamine, norepinephrine, acetylcholine (ACh), and GABA (Wonnacott, 1997). Because nicotinic receptors are ligand-gated ion channels, measurement of the agonist-stimulated uptake or efflux of isotopically labeled Na⁺, K⁺ (or Rb), and Ca²⁺, has been used to measure receptor function. Theoretically, ion flux assays have an inherent advantage of universal applicability to receptors located either presynaptically or postsynaptically and can be used for tissue prepared from any source. Receptor-mediated stimulation of ⁸⁶Rb⁺ efflux has been successfully applied to the measurement of receptor function in cell lines (Lukas, 1989), in cells transfected with defined nicotinic receptor subunits (Gopalakrishnan et al., 1996), and in synaptosomes prepared from rodent brain (Marks et al., 1993). One limitation of these measurements has been the relatively long sampling times (10 s to 5 min) used. Inasmuch as nicotinic receptors desensitize with prolonged stimulation and that some receptor subtypes desensitize very rapidly, the sampling times may be inadequate to measure rapidly desensitizing subtypes such as those containing 7 subunits (Couturier et al., 1990; Seguela et al., 1993; Alkondon and Albuquerque, 1993). The results presented in this paper describe studies of nicotinic agonist-stimulated ⁸⁶Rb⁺ efflux from mouse brain synaptosomes measured with an on-line continuous flow radiation monitor. This methodology reduces sampling time to 3 s. Two distinct components of nicotinic agonist-mediated efflux were revealed, one that was relatively sensitive to inhibition by dihydro--erythroidine (DHE) and one that was less sensitive to inhibition. Nicotinic agonist activation and antagonist inhibition differed for the DHE-sensitive and DHE-resistant components. Failure of either [¹²⁵I]-bungarotoxin (-Bgt) or low concentrations of methyllycaconitine (MLA) to inhibit either component indicates that the 7 subunit was not involved. Both components were widely distributed throughout the brain and experiments with 2-null mutants demonstrated that each required the 2 subunit. The DHE-sensitive component appears to be identical with a process described previously, whereas the DHE-resistant component appears to be a unique, as yet undescribed, response that may have a major functional role in mouse brain.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. The following were purchased from Sigma Chemical Co. (St. Louis, MO): ()-nicotine tartrate, (+)-nicotine-(+)-di-p-toluoyltartrate, ACh, carbachol iodide, (±)-epibatidine hydrochloride, cytisine, (±)-nornicotine, (±)-anabasine, tetramethylammonium chloride (TMA), d-tubocurarine chloride (dTC), hexamethonium chloride, decamethonium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, atropine sulfate, tetrodotoxin, diisopropylflourophosphate (DFP), bovine serum albumin, and polyethylenimine. The following were purchased from Research Biochemicals International (Natick, MA): (+)-epibatidine hydrochloride, ()-epibatidine hydrochloride, (+)-anatoxin-a, DHE, MLA, epiboxidine and, 3-(2-(S)-azetidinylmethoxy)pyridine dihydrochloride (A-85380). Sucrose and HEPES were purchased from Boehinger-Mannheim (Indianapolis, IN). Dimethylphenylpiperazinium iodide (DMPP) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Cesium chloride and Budget Solve Scintillation fluid were purchased from Research Products International (Mt. Prospect, IL). (S)-3-methyl-5-(1-methyl-2-pyrrodinyl)isoxazole (ABT-418) was a gift from Abbott Laboratories (Abbott Park, IL). [³H]Nicotine (81.5 Ci/mmol), [³H]epibatidine (33.8 Ci/mmol), and carrier-free ⁸⁶RbCl were purchased from DuPont-NEN (Boston, MA). -Bungarotoxin (initial specific activity 210 Ci/mmol) was purchased from Amersham, Inc. (Arlington Heights, IL).

Mice. Female C57BL/6J and 2 nicotinic acetylcholine receptor (nAChR)-null mutant mice (Picciotto et al., 1995) of either sex were bred at the Institute for Behavioral Genetics (University of Colorado, Boulder, CO). C57BL/6 mice were housed five per cage and 2 mutant mice were housed with like sex littermates (2-5 per cage). The mice were housed in a vivarium maintained at 22°C with a 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM). The mice were allowed free access to food (Harlan Teklad Rodent Diet) and water. Animals were 60- to 90-days old when used. Animal care and experimental procedures were performed in accordance with the guidelines and with the approval of the Animal Care and Utilization Committee of the University of Colorado, Boulder.

Synaptosome Preparation. Crude synaptosomes were prepared from mouse forebrain or dissected brain regions. Samples were homogenized by hand (20 strokes with a Teflon-glass tissue grinder) in 10 volumes ice-cold 0.32 M sucrose buffered to pH 7.5 with 5 mM HEPES. The homogenate was centrifuged at 500g for 10 min. The resulting supernatant was subsequently centrifuged at 12,000g for 20 min to yield the crude synaptosomal pellet (P2).

⁸⁶Rb⁺ Uptake. The uptake of ⁸⁶Rb⁺ into the synaptosomal fractions was achieved by incubating the resuspended P2 for 30 min in uptake buffer (NaCl, 140 mM; KCl, 1.5 mM; CaCl₂, 2 mM; MgSO₄, 1 mM; HEPES hemisodium, 25 mM; glucose, 20 mM; pH, 7.5) containing 4 µCi of carrier-free ⁸⁶RbCl. The final incubation volume was 35 µl. Uptake was terminated by filtration of the sample onto a 6-mm diameter glass fiber filter (Type AE; Gelman, Ann Arbor, MI) under gentle vacuum (0.2 atmospheres) followed by two washes with 0.5 ml of uptake buffer. Samples to be tested for the effects of ACh were incubated with 10 µM diisopropyl fluorophosphate during the final 10 min of uptake to inhibit acetylcholinesterase.

Sample Perfusion. After filtration and wash, the glass fiber filter containing the loaded synaptosomes was transferred to a polypropylene platform. Perfusion buffer (NaCl, 135 mM; CsCl, 5 mM; KCl, 1.5 mM; CaCl₂, 2 mM; MgSO₄, 1 mM; HEPES hemisodium, 25 mM; glucose, 20 mM; tetrodotoxin, 50 nM; atropine, 1 µM; bovine serum albumin (fraction V), 0.1%; pH, 7.5) was delivered to the filter at a rate of 2.5 ml/min using a Gilson Minipuls 3 peristaltic pump (Gilson, Middleton, WI). Atropine and tetrodotoxin were included in the buffer to prevent activation of muscarinic receptors and sodium channels, respectively. The 1 µM concentration of atropine is comparable to that used in studies of heterologously expressed receptors (Leutje and Patrick, 1991) and of nAChR in cell culture (Alkondon and Albuquerque, 1993, 1995). Preliminary experiments indicated that this concentration of atropine had no effect on nicotine-stimulated ⁸⁶Rb⁺ efflux. TTX (50 nM) inhibits a fraction of nicotine-stimulated ⁸⁶Rb⁺ efflux apparently resulting from secondary activation of Na⁺ channels (Marks et al., 1995) and was included to block this secondary response. Buffer was actively removed using a second Gilson peristaltic pump set for a flow rate of 3.2 ml/min. The use of two pumps prevented the accumulation of perfusion buffer on the filter holding the sample. Sample effluent was pumped through a 200 µl volume flow-through Cherenkov cell in a -RAM Radioactivity HPLC Detector (IN/US Systems, Inc., Tampa, FL) to achieve continuous monitoring of ⁸⁶Rb efflux from the sample. Stimulation of the synaptosomes was accomplished by diverting perfusion buffer flow through a 200 µl loop containing the test solution by means of a 4-way rotary Teflon injection valve (Alltech Associates, Inc., Deerfield, IL). Thus, stimulation time was 5 s. The ⁸⁶Rb⁺ efflux was monitored for 4 min and timing was adjusted so that any efflux resulting from stimulation was located in the middle of the sampling period. This timing permitted the definition of basal efflux rate measured before and after agonist application.

Agonist Application. The stimulation of samples by agonists was achieved by filling the 200 µl sample loop with a solution of defined concentration of the agonist being evaluated. Each sample was stimulated once.

Antagonist Effects. The effect of DHE on ⁸⁶Rb⁺ efflux stimulated by ACh, nicotine, or epibatidine was evaluated at two concentrations of each agonist (30 and 1000 µM, 10 and 300 µM, and 0.3 and 10 µM, respectively).

Agonist-Stimulated ⁸⁶Rb⁺ Efflux in Several Brain Regions. The following fifteen brain regions were dissected and P2 fractions prepared: olfactory bulbs (OB), olfactory tubercles (OT), cerebral cortex (Cx), septum (Se), hippocampus (Hp), striatum (St), habenula (Hab), thalamus (Th), hypothalamus (HT), interpeduncular nucleus (IPN), midbrain (MB), superior colliculus (SC), inferior colliculus (IC), hindbrain (HB), and cerebellum (Cb). The P2 fractions were loaded with ⁸⁶Rb⁺ and evaluated for the efflux stimulated by 10 µM nicotine and 10 µM epibatidine plus 2 µM DHE. Nicotine was used to stimulate in the absence of DHE to provide data directly comparable to those obtained previously, whereas epibatidine was used in the presence of DHE because of the large response obtained with this agonist. To obtain adequate samples from the small brain areas (OT, Se, Hab, HT, IPN, SC, IC), tissue from several mice was pooled before homogenization.

Ligand Binding. Particulate fractions were prepared from P2 after lysis of the crude synaptosomes by three cycles of suspension in hypotonic buffer (NaCl, 14 mM; KCl, 0.15 mM; CaCl₂, 0.2 mM; MgSO₄, 0.1 mM; HEPES, hemisodium salt, 2.5 mM; pH = 7.5) followed by centrifugation at 20,000g for 15 min.

Protein. Protein was measured using the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Data Calculation. The magnitude of agonist stimulated ⁸⁶Rb⁺ efflux was determined as the counts exceeding basal efflux during time of exposure to agonist. Samples were normalized to the counts remaining in the sample at the end of the stimulation period such that the signal size represented the percent of total tissue ⁸⁶Rb⁺ that was stimulated by agonist exposure.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

ACh-Stimulated ⁸⁶Rb⁺ Efflux. The ⁸⁶Rb⁺ efflux stimulated by a 5-s exposure to several concentrations of ACh is illustrated in the left panel of Fig. 1. Each point represents datum collected for a 3-s time period using a continuous flow detector. The amount of ⁸⁶Rb⁺ efflux increased with increasing concentrations of ACh between 1 and 1000 µM. The peak observed after stimulation with 1000 µM ACh was approximately 3-fold greater than the basal efflux. The concentration-response curve for ACh-stimulated ⁸⁶Rb⁺ deviated markedly from simple Michaelis-Menten kinetics (Fig. 1, right). The Hill coefficient for this curve was 0.44 ± 0.08. The results were consistent with those of a two-component process displaying EC₅₀ values of 0.80 ± 0.57 µM and 82.6 ± 40.2 µM with maximal efflux representing 0.079 ± 0.019% and 0.160 ± 0.018% of total tissue ⁸⁶Rb⁺ during the 5-s stimulation, respectively. These results suggest that more than one process mediates ACh-stimulated ⁸⁶Rb⁺ efflux from mouse brain synaptosomes.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Stimulation of 86Rb+ efflux by ACh. Crude whole brain synaptosomes stimulated for 5 s with ACh. The traces (left) are actual data obtained at the indicated concentrations of ACh. Each point represents the counts measured during the 3-s sampling period. Basal efflux for each condition was approximately 50 counts per fraction. The ACh concentration-effect curve is shown in the right panel. Each point represents the mean ± S.E.M. of six determinations from three separate experiments. The curve is that obtained for a two-site fit of the data.

Effects of DHE and -Bgt. The effects of the nicotinic antagonists DHE and -Bgt on ⁸⁶Rb⁺ efflux stimulated by either 30 or 1000 µM ACh were evaluated in an attempt to pharmacologically differentiate the putative multiple processes underlying ACh-stimulated ⁸⁶Rb⁺ efflux from mouse brain synaptosomes (Fig. 2). Samples were treated with DHE, an antagonist that is somewhat selective for 4/2 nicotinic receptors, for 8 min or with -Bgt, an antagonist that is selective for neuronal 7 nicotinic receptors, for 70 min before stimulation with ACh. The main panel on the left side of Fig. 2 presents data from representative perfusion profiles obtained with a 5-s stimulation with 30 µM ACh without antagonist treatment, and after treatment with 2 µM DHE, 100 nM -Bgt, or both antagonists. DHE treatment inhibited ACh-stimulated ⁸⁶Rb⁺ efflux approximately 75%, whereas -Bgt treatment had no significant effect. -Bgt also had no additional effect in the presence of DHE (Fig. 2, left, inset). The main panel on the right side of Fig. 2 presents data from representative perfusion profiles obtained with a 5-s stimulation with 1000 µM ACh. The response was inhibited approximately 40% by 2 µM DHE, but was unaffected by 100 nM -Bgt alone or in combination with DHE. These results indicate that a significant fraction of ACh-stimulated ⁸⁶Rb⁺ efflux is sensitive to inhibition by DHE, but not by -Bgt. Furthermore, the DHE-sensitive component was the same (0.8% of tissue content) whether the samples were stimulated with 30 or 1000 µM ACh.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effect of 2 µM DHE and 100 nM -Bgt on 86Rb+ efflux stimulated by 30 or 1000 µM ACh. The main panel of each figure illustrates primary data collected from samples of crude whole-brain synaptosomes stimulated with 30 µM ACh (left) or 1000 µM ACh (right) in the presence of 2 µM DHE, 100 nM -Bgt, or both agents, as indicated. Samples were stimulated with ACh for 5 s. Samples were treated with DHE for 8 min before stimulation and with -Bgt for 70 min before stimulation. DHE and -Bgt were present before, during, and after exposure to ACh. The insets summarize the responses (mean ± S.E.M., n = 6) obtained under each experimental condition: C, control; D, 2 µM DHE; B, 100 nM -Bgt; D+B, 2 µM DHE plus 100 nM -Bgt.

DHE Inhibition. The partial inhibition of ACh-stimulated ⁸⁶Rb⁺ efflux by 2 µM DHE and the difference in the magnitude of this effect at different ACh concentrations suggests that this antagonist may differentially inhibit subsets of responses. Thus, the inhibitory effects of DHE were examined by constructing concentration-response curves for responses stimulated by 30 or 1000 µM ACh (Fig. 3). Samples were exposed to DHE for 8 min before stimulation and DHE was present during and after the 5-s stimulation with ACh. DHE produced a concentration-dependent decrease in ⁸⁶Rb⁺ efflux at both concentrations of ACh. An apparently monophasic inhibition was observed for samples stimulated with 30 µM ACh with an estimated IC₅₀ value of 0.17 ± 0.02 µM. Higher concentrations of DHE were required to inhibit ⁸⁶Rb⁺ efflux stimulated by 1000 µM ACh. Inasmuch as DHE is a competitive antagonist, the shift in the concentration-effect curve could have arisen merely because of the increased ACh concentration. Inhibition by DHE was reversible (k = 0.021/s, T_1/2 = 33 s), however with this dissociation rate, reversal of inhibition was approximately 5% during the 5-s stimulation.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Inhibition of ACh-stimulated 86Rb+ efflux by DHE. Crude whole-brain synaptosomes were stimulated with 30 µM ACh () or 1000 µM ACh () for 5 s after an 8-min exposure to the indicated concentrations of DHE. The inset displays the difference in response at 1000 µM and 30 µM ACh. Each data point represents the mean ± S.E.M. of eight determinations from four separate experiments.

Stimulation of DHE-Sensitive and DHE-Resistant ⁸⁶Rb⁺ Efflux by Nicotinic Agonists. The existence of ACh-stimulated responses that are differentially sensitive to inhibition by DHE was used to evaluate the concentration dependence of nicotinic agonist-stimulated ⁸⁶Rb⁺ efflux. Concentration-effect curves for ACh activation of ⁸⁶Rb⁺ efflux from mouse forebrain were constructed with either 0 or 2 µM DHE present in the perfusion buffer and are shown in Fig. 4. Similar to the results presented in Fig. 1, ⁸⁶Rb⁺ efflux stimulated by ACh displayed a biphasic concentration-effect curve with estimated EC₅₀ values of 5.4 ± 4.7 and 244 ± 328 µM and efflux of 0.12 ± 0.05 and 0.16 ± 0.05%, respectively. However, at concentrations higher than 1 mM the responses decreased. In the presence of 2 µM DHE, a monophasic concentration-effect curve with an EC₅₀ value of 540 ± 60 µM and maximal efflux of 0.16 ± 0.01% was obtained. These parameters are comparable to the low-affinity component of the full concentration-effect curve in the absence of DHE. The difference in response between total ACh-stimulated efflux and that observed in the presence of 2 µM DHE is shown in the inset to Fig. 4. At ACh concentrations at or below 1000 µM, the ACh-stimulated ⁸⁶Rb⁺ efflux appeared to be a monophasic process with an EC₅₀ of 7.5 ± 2.6 µM and a maximal efflux of 0.15 ± 0.01%. These parameters are comparable to the high-affinity component of the full dose-response curve in the absence of DHE. Also note that a substantial decrease in efflux occurred at concentrations above 1 mM. These results indicate that differential inhibition by DHE is a useful tool to study agonist stimulation of ⁸⁶Rb⁺ efflux.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves for ACh in the presence of 0 or 2 µM DHE. Crude whole-brain synaptosomes were treated with 0 µM DHE () or 2 µM DHE () for 8 min before a 5-s exposure to the indicated concentrations of ACh. Each point represents the mean ± S.E.M. for six determinations from three separate experiments. The inset displays the difference between the results in the absence or presence of DHE. Curves shown for the response in the presence of DHE and the inset are theoretical fits of the data to the Michaelis-Menten equation. The curve for the response in the absence of DHE is a two-site fit of these data.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Agonist simulation of DHE-sensitive and DHE-resistant 86Rb+ efflux. Crude whole brain synaptosomes were stimulated with the indicated concentrations of each agonist in the presence or absence of 2 µM DHE. Data points represent results obtained in the presence of 2 µM DHE (; DHE-resistant) or the difference between the response measured in the absence or presence of 2 µM DHE (; DHE-sensitive). Each point represents the mean ± S.E.M. of six to eight determinations from three to four separate experiments. Curves are theoretical fits of the data to the Michaelis-Menten equation.

View this table: [in this window] [in a new window]   TABLE 1 EC50 values and maximal efflux rates for DHE-sensitive and DHE-resistant 86Rb+ efflux

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Correlations between EC50 values and maximal efflux for DHE-sensitive and DHE-resistant 86Rb+ efflux. EC50 values (left) and maximal efflux (right) determined for the agonist curves of Fig. 5 for the DHE-sensitive response are compared to the corresponding values for the DHE-resistant response.

Effects of Nicotinic Antagonists. Inhibition of DHE-sensitive (measured with 10 µM nicotine) and DHE-resistant (measured with 10 µM epibatidine plus 2 µM DHE) nicotinic responses by six antagonists was studied. Concentration-effect curves for these antagonists are shown in Fig. 7 and IC₅₀ values are summarized in Table 2. In contrast to the agonist effects, where every agonist was more potent in stimulating DHE-sensitive ⁸⁶Rb⁺ efflux, the antagonists were not uniformly more potent inhibitors of DHE-sensitive response. Results obtained for DHE under these conditions confirm the differential effect of DHE. An IC₅₀ value of 0.15 ± 0.05 µM was calculated for DHE-sensitive efflux and an IC₅₀ value of 8.3 ± 1.7 µM or 13.7 ± 3.3 was calculated for the response stimulated by 10 µM epibatidine in the presence of 2 µM DHE or 2 µM MLA, respectively. MLA is also a more potent inhibitor of DHE-sensitive efflux (IC₅₀ = 0.20 ± 0.09 µM) than it is of the DHE-resistant response (IC₅₀ = 4.4 ± 1.6 µM). Decamethonium inhibited both responses with equal potency (IC₅₀ = 4.1 µM). Mecamylamine and dTC were slightly more potent inhibitors of the DHE-resistant efflux (IC₅₀ = .16 ± 0.03 µM and 1.1 ± 0.2 µM, respectively) than of the DHE-resistant efflux (IC₅₀ = .59 ± 0.09 and 2.8 ± 0.7 µM, respectively). Hexamethonium was a significantly more potent inhibitor of the DHE-resistant response (IC₅₀ = .89 ± 0.17 µM) than the DHE-sensitive (IC₅₀ = 17.6 ± 6.1 µM). Thus, all six nicotinic antagonists inhibited both DHE-sensitive and DHE-resistant ⁸⁶Rb⁺ efflux, but the relative potency of the compounds as inhibitors of the two processes varied markedly.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Inhibition of DHE-sensitive and DHE-resistant 86Rb+ efflux by nicotinic antagonists. Crude whole-brain synaptosomes were exposed to the indicated concentration of antagonists for 8 min before a 5-s exposure to 10 µM L-nicotine () or 10 µM (±)-epibatidine plus 2 µM DHE (). Those samples to be stimulated with (±)-epibatidine were treated with 2 µM DHE as well as the test antagonist for 8 min before stimulation. In a separate set of of experiments, samples for which DHE inhibition of the DHE-resistant response were measured were treated with 2 µM MLA () instead of DHE to allow analysis of DHE inhibition in the absence of this antagonist. Each point represents the mean ± S.E.M. of six to eight individual measurements from three to four separate experiments. The curves are theoretical one-site fits using the equation: AI = 100/(1+(I/IC50)), where AI is the percentage of control activity at antagonist concentration, I, and IC50 is the antagonist concentration that gives 50% inhibition.

View this table: [in this window] [in a new window]   TABLE 2 IC50 values for inhibition of DHE-sensitive and DHE-resistant 86Rb+ efflux

Responses in 2 Mutant Mice. Analysis of responses of mice expressing mutant nicotinic receptors provides one means by which the molecular basis of nicotinic responses can be investigated. The effect of null mutation of the 2 nAChR subunit on DHE-sensitive and DHE-resistant ⁸⁶Rb⁺ efflux was determined. Perfusion profiles for ⁸⁶Rb⁺ efflux stimulated by a 5-s exposure to 10 µM nicotine for 2 homozygote wild type, heterozygote, and homozygote mutant mice are shown in Fig. 8 (top left). Although exposure to nicotine produced substantial increases in ⁸⁶Rb⁺ efflux from whole brain synaptosomes of wild type and heterozygote mice, little response was observed in the homozygote mutants. The effect of 2 genotype is summarized in Fig. 8 (bottom left). A small decrease (12%) in response was observed for the heterozygotes, but ⁸⁶Rb⁺ efflux stimulated by 10 µM nicotine decreased 96% in the 2 null mutants. The efflux remaining in the homozygote mutants was not significantly different from zero.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of 2 genotype on DHE-sensitive and DHE-resistant 86Rb+ efflux. Crude synaptosomes were prepared from whole brains of mice with the following genotype for the 2 nAChR subunit: +/+, homozygote wild type; ±, heterozygote; /, homozygote mutant. The top panels illustrate primary data for mice of each genotype receiving a 5-s stimulation with 10 µM L-nicotine (left) or 10 µM (±)-epibatidine plus 2 µM DHE (right). DHE was present before, during, and after the stimulation with epibatidine. The bottom panels summarize data (mean ± S.E.M.) obtained for four wild type, seven heterozygote, and five mutant mice.

Distribution of DHE-Sensitive and DHE-Resistant ⁸⁶Rb⁺ Efflux in Mouse Brain. The experiments described above were performed using crude synaptosomes prepared from whole mouse forebrain. To determine the distribution of DHE-sensitive and DHE-resistant nicotinic responses throughout the brain, ⁸⁶Rb⁺ efflux stimulated by 10 µM nicotine or 10 µM epibatidine in the presence of 2 µM DHE was measured in crude synaptosomes prepared from 15 brain areas. The results of these experiments are shown in Fig. 9.

View larger version (31K): [in this window] [in a new window]   Fig. 9.   DHE-sensitive and DHE-resistant 86Rb+ efflux in 15 brain regions. Crude synaptosomes were prepared from the following brain regions: OB, OT, Cx, Se, Hp, St, Hab, Th, HT, IPN, MB, SC, IC, HB, Cb. 86Rb+ efflux was stimulated by a 5-s exposure to 10 µM nicotine (top left) or 10 µM epibatidine plus 2 µM DHE (bottom left). DHE was present before, during, and after stimulation with DHE. Values represent mean ± S.E.M. of four separate experiments. The magnitude of the DHE-resistant response is compared to the magnitude of the DHE-sensitive response in the right panel.

Comparison of nAChR-Mediated ⁸⁶Rb⁺ Efflux to Nicotinic Binding Sites. Because the pharmacological properties, and to a lesser extent the regional distribution, of the DHE-sensitive response differ from those of the DHE-resistant response, a comparison of the distribution of the functional responses to that of nicotinic binding sites may indicate a relationship between binding and function. Several nicotinic binding sites can be measured using radioligand binding assays. High-affinity [³H]nicotine binding and -Bgt binding have been used to measure two distinct binding sites (Marks et al., 1986). Recently the binding of [³H]epibatidine has been characterized (Houghtling et al., 1995). This ligand measures several nicotinic binding sites: a high-affinity site sensitive to inhibition by cytisine (identical with the high-affinity agonist site), a high-affinity site relatively resistant to inhibition by cytisine and that may include more than one site, and a low-affinity site (Houghtling et al., 1995; Marks et al., 1998; Zoli et al., 1998).

View larger version (26K): [in this window] [in a new window]   Fig. 10.   [3H]Epibatidine binding and inhibition by cytisine and dTC. The main top panel illustrates a representative saturation curve for the binding of [3H]epibatidine to whole mouse brain particulate fraction. Actual data are represented by the points (). The solid curve is the least-squares fit of the data to a two-site model and the dotted curve is the theoretical binding to the high-affinity site. The inset to this panel is the Scatchard plot of these data. The main panel at the bottom left displays the inhibition of [3H]epibatidine binding by cytisine and the main panel at the bottom right displays the inhibition of binding by dTC at 0.44 nM () or 12.5 nM () [3H]epibatidine. The solid curve at 0.44 nM [3H]epibatidine is the least-squares two-site fit of the data, whereas the dotted curve presents the theoretical low-affinity binding site. The dotted curve at 12.5 nM is the least-squares two-site fit of these data. The inset to this panel is the calculated inhibition profile for the low-affinity binding site calculated as described in Experimental Procedures. The curve is the theoretical one-site fit of these data.

View larger version (37K): [in this window] [in a new window]   Fig. 11.   Comparison of regional distribution of DHE-sensitive or DHE-resistant 86Rb+ efflux to the regional distribution of nicotinic binding sites. 86Rb+ efflux stimulated by 10 µM nicotine (top) or by 10 µM epibatidine plus 2 µM DHE (bottom) in 15 brain regions is compared to the density of nicotinic binding sites measured with 17 nM [3H]nicotine (Nicotine Binding), 0.36 nM [3H]epibatidine binding in the presence of 50 nM cytisine (Cytisine-Resistant High-Affinity Epibatidine Binding), as the difference between binding of 10 nM [3H]epibatidine in the absence or presence of 300 µM dTC (Low-Affinity Epibatidine Binding), or with 2 nM -Bgt (-Bungarotoxin Binding). Each point represents the mean ± S.E.M. of the efflux or binding in each of 15 brain regions. Correlation coefficients are displayed in each panel.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Two pharmacologically distinct components of nicotinic agonist-stimulated ⁸⁶Rb⁺ efflux from mouse brain synaptosomes were analyzed using an online continuous flow radioactivity monitor. Differential sensitivity to inhibition by the nicotinic antagonist DHE was exploited to study the pharmacology and regional distribution of these responses. The efflux inhibited by low concentrations of DHE displayed a higher affinity for nicotinic agonists than did the response that was less sensitive to DHE inhibition. Both the DHE-sensitive and DHE-resistant components were reduced more than 95% in 2 null mutant mice, revealing an absolute requirement for this subunit. The DHE-sensitive and DHE-resistant responses were widely distributed throughout the brain with similar, but not identical, regional distribution. Thus, a novel DHE-resistant functional nicotinic response is widely distributed in mouse brain. This response is robust and pharmacologically distinct from those characterized previously. Whether this response is mediated by a single receptor subtype remains to be determined.

Differential inhibition by DHE was used to measure the two pharmacologically distinct nicotinic responses. Because DHE is a competitive antagonist, the DHE-resistant response may have occurred because inhibition was overcome at high agonist concentrations. To reduce agonist-dependent changes in IC₅₀ values and subsequent changes in the amount of inhibition, samples were treated with DHE before stimulation. Therefore, reversal of blockade requires dissociation of bound DHE. Although dissociation is relatively rapid (k = 0.02 sec^-1), little reversal (about 5%) of inhibition would occur during the 5-s stimulation. Thus, under the conditions used in these experiments, differential inhibition by DHE appears to be a valid experimental approach to resolve different nicotinic responses.

Differential sensitivity of nicotinic agonist-stimulated responses to inhibition by DHE is not a unique observation. Pharmacologically distinct nicotinic responses in hippocampal cell cultures (Alkondon and Albuquerque, 1993) and in neurons isolated from the IPN compared to neurons isolated from medial habenula (Mulle et al., 1991) displayed differential responses to DHE inhibition. Indeed, differential DHE sensitivity was used to investigate nAChR subtypes in hippocampal cultures (Alkondon and Albuquerque, 1995) in a manner analogous to that used in the current study. Biochemical assays of nicotinic receptor function also exhibit differential sensitivity to inhibition by DHE. For example, nicotine-stimulated dopamine release is relatively more sensitive to inhibition by DHE (Grady et al., 1992; Sacaan et al., 1995; Clarke and Reuben, 1996), than is nicotine-stimulated norepinephrine release (Sacaan et al., 1995; Clarke and Reuben, 1996). Defined nicotinic receptor subtypes expressed in Xenopus oocytes are differentially affected by DHE (Harvey et al., 1996; Chavez-Noriega et al., 1997). Structural determinants for differential DHE sensitivity are present in both  (Harvey et al., 1996) and  subunits (Harvey and Leutje, 1996).

In addition to DHE, two antagonists, MLA and hexamethonium, differed markedly in their inhibitory potency. The selectivity demonstrated by MLA was comparable to that of DHE. In contrast, hexamethonium was a more potent inhibitor of the DHE-resistant response. Three other antagonists displayed similar inhibition of the DHE-sensitive and DHE-resistant components. Potency and efficacy for both DHE-sensitive and DHE-resistant ⁸⁶Rb⁺ efflux differed among nicotinic agonists, but every agonist tested was more potent for the DHE-sensitive component. Differential effects of antagonists and agonists on DHE-sensitive and DHE-resistant ⁸⁶Rb⁺ efflux indicate the existence of at least two receptors.

Determining the molecular compositions of the nAChR subtypes that mediate the DHE-sensitive and DHE-resistant responses is important. Both DHE-sensitive and DHE-resistant ⁸⁶Rb⁺ efflux were reduced more than 95% in 2-null mutant mice (Picciotto et al., 1995). Thus, both responses are nicotinic and the 2 subunit is an essential component of the nAChR mediating these responses. The identity of the  subunits remains to be unequivocally established.

In the absence of definitive assignment of subunit composition beyond the absolute requirement for 2, pharmacological and physiological data provide information about the potential subunit identity. Differences in sensitivity to agonist stimulation have been observed for other nicotinic receptors and these differences have been used to investigate potential molecular composition of the receptors. Four classical nicotinic agonists (ACh, ()-nicotine, cytisine, and DMPP) show distinct differences in the activation of heterologously expressed receptors of defined composition (Leutje and Patrick, 1991; Chavez-Noriega et al., 1997). DHE-sensitive ⁸⁶Rb⁺ efflux stimulated by these agonists most closely resembles the responses observed for the 42 nAChR. Several other agonists that were evaluated in the current study have also been measured in systems of defined receptor composition. The relative potency and efficacy of epibatidine (Gerzanich et al., 1995; Gopalakrishnan et al., 1996), ABT-418 (Gopalakrishnan et al., 1996), and A-85380 (Sullivan et al., 1996) measured for 42 nAChR are comparable to those determined for DHE-sensitive ⁸⁶Rb⁺ efflux. Furthermore, the potencies and relative efficacies of ACh, anatoxin-a, cytisine, (+)-epibatidine, ()-epibatidine, ()-nicotine, and DMPP, for which both the Type II responses of hippocampal cells (Alkondon and Albuquerque, 1995; Zoli et al., 1998) and DHE-sensitive ⁸⁶Rb⁺ efflux were determined, are comparable. The Type II response most probably is mediated by 42 the nAChR (Alkondon et al., 1994). Finally, the close correlation between the regional distribution of high-affinity nicotine binding and DHE-sensitive ⁸⁶Rb⁺ efflux reported here and previously (Marks et al., 1993, 1996) suggests that this component is an 42 nAChR because high-affinity agonist binding measures primarily, if not exclusively, this subtype (Whiting and Lindstrom, 1988; Flores et al., 1992). Thus, the receptor mediating DHE-sensitive ⁸⁶Rb⁺ efflux in the majority of brain regions is likely to be the 42 subtype, perhaps with other subunits contributing in certain brain regions.

The possible molecular structure(s) of the DHE-resistant component is less clear. Results with null mutant mice demonstrate that the 2 subunit is required. The broad distribution of the DHE-resistant response makes it unlikely to be mediated by receptors containing either 2 or 3 nAChR subunits because the mRNA encoding these subunits has restricted expression in rat and mouse brain (Wada et al., 1989; Marks et al., 1992). Furthermore, the poor correlation between DHE-resistant ⁸⁶Rb⁺ efflux and cytisine-resistant high-affinity epibatidine binding, which may measure 3 nAChR (Marks et al., 1998; Zoli et al., 1998), suggests no relationship between the functional response and this binding component. The failure of -Bgt or low concentrations of MLA to measurably inhibit either the DHE-sensitive or DHE-resistant responses indicates that 7-containing nAChRs are not involved. Considering the rapid desensitization and rundown that is displayed by 7 nAChRs, the experimental conditions used in these studies may not be well suited to measure these receptors.

The DHE-resistant response may be mediated by a receptor with an as yet unidentified  subunit. Because subunit composition profoundly affects nAChR pharmacology and physiology (Luetje and Patrick, 1991; Chavez-Noriega et al., 1997), expression of an nAChR with an  subunit other than 4 could produce the DHE-resistant response. If such a subunit exists, it must be widely distributed, inasmuch as the DHE-resistant response is found throughout the brain. Receptors assembled from more than two different subunits also can display distinct pharmacology. For example, when the 5 subunit coassembles with other nAChR subunits, properties of the resulting receptor are altered (Ramirez-Latorre et al., 1996; Wang et al., 1996; Conroy and Berg, 1998). Although receptors containing 5 subunits are known, this subunit may be too sparsely expressed in rat and mouse brain to account for all of the DHE-resistant response (Wada et al., 1990; Marks et al., 1992). The DHE-resistant response may also be mediated by an 42 receptor with a subunit stoichiometry other than (4)₂(2)₃, a receptor that exists in an alternative conformational state, or a receptor that has undergone differential post-translational processing. The response might also be mediated by receptor subunits containing alternatively spliced subunits. Splice variants at the C terminus of rat 4 subunit are known (Goldman et al., 1987; Yu et al., 1996), but the properties of the subsequent nAChR formed from these variants do not differ. However, changes in the amino acids in the ligand binding domain can substantially change agonist (Corringer et al., 1998) or antagonist (Harvey et al., 1996) affinity. Splice variants with changes at or near the ligand binding sites could conceivably alter both agonist and antagonist affinity to produce the DHE-resistant receptors.

Whatever the molecular identity of the DHE-resistant response, it is likely to serve an important functional role. This response is expressed throughout the brain, and, if the in vitro measurements are any indication, would produce rapid, large responses. Although higher concentrations of ACh or nicotine are required to activate the DHE-resistant response than the DHE-sensitive response, the potency of these agonists for the DHE-resistant response is comparable to that observed for many nAChR subtypes (Couturier et al., 1990; Gross et al., 1991; Seguela et al., 1993; Alkondon and Albuquerque, 1993; Gerzanich et al., 1995; Chavez-Noriega et al., 1997).

In summary, the experiments presented in this paper describe two components of nicotinic receptor-mediated ⁸⁶Rb⁺ efflux that are of similar magnitude but are differentially sensitive to inhibition to DHE, and are predominantly, if not exclusively, composed of 2 containing nAChR. The DHE-sensitive component appears to be identical with one described previously. The DHE-resistant component represents a previously undescribed, but ubiquitous and robust nAChR-mediated response that may be of significant functional consequence.