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Alkaloids Indolizidine 235B', Quinolizidine 1-epi-207I, and the Tricyclic 205B are Potent and Selective Noncompetitive Inhibitors of Nicotinic Acetylcholine Receptors

Department of Clinical Pharmacology (H.T., Y.Y., S.Ka., S.Ko., T.S., I.K.) and Faculty of Pharmaceutical Sciences (N.T., H.N.), Toyama Medical and Pharmaceutical University, Toyama, Japan, and Division of Neuroscience, Baylor College of Medicine, Houston, Texas (J.A.D.)

Received for publication March 25, 2004.

Accepted for publication July 6, 2004.

	Abstract

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Nicotinic acetylcholine receptors are key molecules in cholinergic transmission in the nervous system. Because of their structural complexity, only a limited number of subtype-specific agonists and antagonists are available to study nicotinic receptor functions. To overcome this limitation, we used voltageclamp recordings to examine the effects of several frog skin alkaloids on acetylcholine-elicited currents in Xenopus laevis oocytes expressing major types of neuronal nicotinic receptors (42, 7, 32, 34, and 44). We found that the 5,8-disubstituted indolizidine (-)-235B' acted as a potent noncompetitive blocker of 42 nicotinic receptors (IC₅₀ = 74 nM). This effect was highly selective for 42 receptors compared with 32, 34, and 44 receptors. The inhibition of 42 currents by (-)-235B' was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 µM). Moreover, the blockade of 42 currents by (-)-235B' was voltage-dependent (more pronounced at hyperpolarized potentials) and use-dependent, indicating that (-)-235B' behaves as an open-channel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((-)-223A and (+)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((+)-207I) were less potent than (-)-235B', and none showed selectivity for 42 receptors. The quinolizidine (-)-1-epi-207I and the tricyclic (+)-205B had 8.7- and 5.4-fold higher sensitivity, respectively, for inhibition of the 7 nicotinic receptor than for inhibition of the 42 receptor. These results show that frog alkaloids alter the function of nicotinic receptors in a subtype-selective manner, suggesting that an analysis of these alkaloids may aid in the development of selective drugs to alter nicotinic cholinergic functions.

Evidence indicates that nAChRs are implicated in several neurological disorders. In particular, significant loss of 42 nAChRs has been observed in cortical autopsies from patients with Alzheimer's disease, and schizophrenia and bipolar disorder exhibit some linkage to the 7-subunit gene (Weiland et al., 2000). Mutations in the 4 or the 2 subunit also underlie autosomal-dominant nocturnal frontal lobe epilepsy in some families (Raggenbass and Bertrand, 2002). Nicotinic agonists and antagonists would therefore be very valuable and are being investigated for possible use in these diseases (Lloyd and Williams, 2000; Dani et al., 2004). However, selective ligands for the multiple subtypes of neuronal nicotinic receptors are still scarce.

Extracts from the skin of certain poison frogs provide a variety of pharmacologically active alkaloids, and more than 500 alkaloids have been isolated to date (Daly et al., 1999). Some of them are known to target nAChRs: a nicotinic agonist, epibatidine, and nicotinic antagonists, histrionicotoxins, pumiliotoxins, and indolizidines (Spivak et al., 1982; Warnick et al., 1982; Aronstam et al., 1986; Daly et al., 1991, 1999). However, the selectivity of most amphibian alkaloids for each subtype of nAChR remains to be characterized. In the present study, we investigated the selectivity of bicyclic alkaloids, indolizidines, and quinolizidines, and a tricyclic alkaloid, 8b-azaacenaphthylene, across several major types of nicotinic receptors (42, 7, 32, 34, and 44) expressed in Xenopus laevis oocytes.

	Materials and Methods

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Materials. All alkaloids and their analogs used in this study were synthesized and are shown in Fig. 1. The synthesis of indolizidine (-)-235B' was performed as reported previously (Momose and Toyooka, 1994; Toyooka et al., 1997; Toyooka and Nemoto, 2002). The structure of (-)-235B' is identical to that of the natural product. The synthetic indolizidines (5-n-propyl-8-n-butylindolizidine) I, II, and III were synthesized, and indicated that 223I was not a 5,8-disubstituted indolizdine. The synthesis of indolizidine I has been shown previously (Toyooka et al., 1997; Toyooka and Nemoto, 2002). The synthesis of 5,6,8-trisubstituted indolizidine (-)-223A along with revision of its structure has recently been reported (Toyooka and Nemoto, 2002; Toyooka et al., 2002) as well as the synthesis of a 6-epimer (223A proposed). This unnatural compound has been designated (+)-6-epi-223A in the present study. The 1,4-disubstituted quinolizidine (+)-207I, which is the opposite enantiomer of the natural product, was synthesized in recent studies (Toyooka and Nemoto, 2002, 2003). An unnatural 1-epimer of (+)-207I, named (-)-1-epi-207I in this study, was synthesized as described previously (Toyooka et al., 1997; Toyooka and Nemoto, 2002). Synthetic (+)-205B is the opposite enantiomer of natural (-)-205B, as reported by Toyooka et al. (2003).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Structures of frog skin alkaloids and their analogs. Absolute stereochemistry is as follows. (-)-235B', 5R, 8R, 9S; (-)-I, 5R, 8R, 9S; (+)-II: 5R, 8S, 9R; (-)-III, 5R, 8R, 9R; (+)-6-epi-223A, 5S, 6R, 8S, 9R; (-)-223A, 5R, 6R, 8R, 9S; (-)-1-epi-207I, 1R, 4S, 10S; (+)-207I, 1R, 4R, 10R; (+)-205B, 2aS, 5aS, 6R, 8R, 8aS

The other reagents were purchased from Sigma (St. Louis, MO), unless indicated otherwise. BAPTA-AM was purchased from Calbiochem-Novabiochem Corp. (San Diego, CA).

The cDNAs of the rat 3, 2, and 4 subunits in pcDNA1/Neo vectors were subcloned into HindIII and XhoI sites of pcDNA3.1 vectors (Invitrogen, Carlsbad, CA), and the cDNA of the rat 4 subunit in pcDNA1/Neo vectors was subcloned into HindIII and XbaI sites of pcDNA3.1 vectors (Invitrogen) to enhance the expression of 32, 34, and 44 nicotinic receptors in oocytes. To express 42 and 7 nAChRs, mouse cDNAs were used.

Expression in X. laevis Oocytes. All experiments were carried out in accordance with guidelines approved by the Toyama Medical and Pharmaceutical University Animal Research Committee. Oocytes were surgically removed from X. laevis frogs under anesthesia using Tricaine solution (2.4 g/l). The oocytes were rinsed in calcium-free OR2 buffer (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.6), then defolliculated in this buffer supplemented with 1.5 mg/ml collagenase A (Roche Diagnostics, Mannheim, Germany) for approximately 2 h at room temperature. Stage V-VI oocytes were selected and microinjected with 20 ng of cDNAs in the nucleus. The mixture of two subunit cDNAs, 3 or 4 in combination with 2 or 4, was injected in a ratio of 1:1, whereas 7 subunit was injected alone. Oocytes were incubated at 19°C in standard oocyte saline solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, 2.5 mM pyruvic acid, 1% BSA, and 25 µg/ml gentamycin, pH 7.5) for 3 to 8 days before recordings were made.

Electrophysiological Recording. An oocyte was placed in a 300-µl tube-like chamber in which Ringer's solution (82.5 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.4) containing 1 µM atropine to block endogenous muscarinic receptors was perfused by gravity (15 ml/min). Current responses were recorded under two-electrode voltage-clamp at a holding potential of -60 mV using a GeneClamp 500 amplifier and pClamp7 software (Axon Instruments, Union City, CA). The sampling rate was 20 Hz. Electrodes contained 3 M KCl and had resistances of <1 M.

To apply a pulse of acetylcholine (ACh) to the oocyte, the perfusion fluid was switched to one containing ACh for 5 s, using a three-way Teflon solenoid valve (Parker Hannifin Corp., General Valve Division, Fairfield, NJ) controlled by a PC computer with pClamp7 software. A 3-min wash between ACh applications produced reproducible control currents with no obvious desensitization. For responses in alkaloids (test responses), the perfusion fluid was stopped during alkaloid application for 3 min to conserve the compounds. Perfusion was started again 0.5 min before measuring responses to ACh in the presence of test alkaloid.

Control experiments in the Ca²⁺ chelator BAPTA were performed to minimize the activation of endogenous Ca²⁺-activated chloride channels in X. laevis oocytes. Those endogenous currents were prevented by BAPTA to be sure that they did not alter the pharmacological profiles of the tested alkaloids. Oocytes were loaded with BAPTA by incubating in a SOS solution containing BAPTA-AM (100 µM) for 4 h before recording, as described previously (Ibanez-Tallon et al., 2002). Otherwise, oocytes were incubated with a low-Ca²⁺ Ringer's solution (82.5 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl₂, 2 mM MgCl₂, and 5 mM HEPES, pH 7.4) to depress the endogenous chloride conductance while we investigated the mechanism of receptor blockade by the alkaloids.

Data Analysis. The average peak amplitudes of three control responses to ACh (1 µM for 42 and 44 receptors; 100 µM for 7, 32, and 34 receptors) just preceding exposure to alkaloids were used to normalize the amplitude of each test response. These ACh concentrations were determined to be approximately 30% of the maximum effective concentrations (EC₃₀), according to the ACh-concentration response curve (data not shown). Each data point of the concentration-response curve represents the average value ± S.E.M. of measurements from at least three oocytes.

Concentration-inhibition curves for alkaloids (antagonists) were fitted by a nonlinear regression to the equation: I = I_max - I_max/[1 + (IC₅₀/An)^nH], where I_max is the maximal normalized current response (in the absence of antagonist for inhibitory curves), An is the antagonist concentration, IC₅₀ is the antagonist concentration eliciting half-maximal current, and n_H is the Hill coefficient. Curve fittings were analyzed using Prism software (GraphPad Software, Inc., San Diego, CA). For antagonist efficacy curves, I_max was constrained to 1 and I_min was constrained to 0.

The ACh concentration-response curves for 42 nicotinic receptors were fitted to the following two-component (double sigmoid) equation using Prism software (GraphPad Software, Inc.): y = y_max x [a1/(1 + (EC_50,high/x)^nH,high) + (1 - a1)/(1 + (EC_50,low/x)^nH,low)], where y is the percentage amplitude, a1 is the percentage of receptors in the high-affinity component, high and low refer to relative ACh sensitivity, x is the acetylcholine concentration, and n_H is the Hill coefficient. Responses evoked by 1 mM ACh corresponded to 100% activation of the high- and low-affinity components. y_max is the maximal current amplitude (percentage), normalized by the amplitude of ACh (1 mM)-evoked currents in the absence of alkaloid. Moreover, fits to a single-component (sigmoid) equation y = y_max x [1/(1 + (EC₅₀/x)^nH] were performed, and relative improvements between the single-component fit and the two-component fit were assessed by using GraphPad Prism to calculate the F-statistic from the ratios of the minimum sums of squares of deviations.

Statistical Analysis. The significance of differences between two groups was assessed by Student's t test, and the significance of differences between multiple groups were assessed by one-way analysis of variance followed by the Dunnet's multiple range test. Values of P less than 0.05 were considered significant.

	Results

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The subtype selectivity of the frog alkaloids was investigated across five recombinant nAChRs (42, 7, 32, 34, and 44) expressed in X. laevis oocytes. The oocytes were voltage-clamped, and application of 0.1 to 1000 µM ACh elicited inward currents. When the currents were elicited by the EC₃₀ doses of ACh (see Data Analysis), 42 and 32 heteromeric receptors showed faster decay kinetics than 4-containing heteromeric receptors (34 and 44), and currents through 7 homomeric receptors decayed very rapidly during the ACh application (Fig. 2, A and B). These features were consistent with previous observations (Alkondon and Albuquerque, 1993; Chavez-Noriega et al., 1997; Fenster et al., 1997). We also confirmed that the ACh responses in oocytes expressing 42 nAChRs were inhibited by 5 µM dihydro--erythroidine (DHE, a competitive neuronal nAChR antagonist acting preferentially on non-7 receptor) and 7 nAChRs were inhibited by 10 nM methyllycaconitine (MLA, an antagonist of 7-containing nicotinic receptors) (Fig. 2A). The 42 nAChR-mediated currents gradually recovered during a 25-min washout of DHE, whereas 7 nAChR-mediated currents were completely recovered 15 min after removal of MLA.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Inhibitory effects of indolizidine (-)-235B' on ACh-induced currents in X. laevis oocytes expressing recombinant nicotinic receptors. Currents were recorded in voltage-clamp mode at -60 mV. A, typical traces showing inhibition by DHE (5 µM) on 42 currents elicited by ACh (1 µM) and inhibition by MLA (10 nM) on 7 currents elicited by ACh (100 µM). Horizontal bars indicate the period of perfusion with ACh for 5 s. Vertical scale bars represent 1 µA for 42 currents, and 0.5 µA for 7 currents. The ACh-elicited currents recovered after removals of these antagonists. B, representative traces showing the ACh-elicited currents in the absence and presence of (-)-235B' (0.3 µM). Concentrations near the 30% effective doses (EC30) of ACh were used to elicit the currents (i.e. 1 µM for 42 and 44 receptors and 100 µM for 7, 32, and 34 receptors). For test responses, oocytes were preincubated with (-)-235B' for 3 min and then exposed to ACh with (-)-235B'. Horizontal bars, 5 s. Vertical scale bars represent 1 µA for 42 currents, and 0.5 µA for 7, 32, 34, and 44 currents. C, concentration-response curves for (-)-235B' on recombinant nicotinic receptors. Current responses to ACh in the presence of (-)-235B' in each oocyte were normalized to the ACh responses (control responses) recorded in the same oocytes. Values represent the mean ± S.E.M. for five to nine separate experiments.

The responses mediated by recombinant nAChRs were blocked by most of the alkaloids used, as summarized in Table 1. When the oocytes expressing 42 nAChR were treated with indolizidine (-)-235B' (0.3 µM), the peak amplitude of the ACh-elicited currents was greatly decreased (Fig. 2B). This blocking effect was reversible within 10 min (data not shown). Compared with the efficacy on oocytes expressing the other receptor subtypes, (-)-235B' blocked the 42 receptor-mediated responses (IC₅₀ = 74 nM, n_H = 0.47 ± 0.04) with 6.0-fold higher sensitivity than blockade of 7 receptor-mediated responses (IC₅₀ = 442 nM, n_H = 0.48 ± 0.05), 40.5-fold higher than that of 32 receptor-mediated ones (IC₅₀ = 3.0 µM, n_H = 0.74 ± 0.05), 51.4-fold higher than that of 34 receptor-mediated ones (IC₅₀ = 3.8 µM, n_H = 0.49 ± 0.07), and 54.1-fold higher than that of 44 receptor-mediated ones (IC₅₀ = 4.0 µM, n_H = 0.91 ± 0.10) (Fig. 2, B and C).

To exclude the possibility that indolizidine (-)-235B' blocks nAChR signaling downstream, such as endogenous Ca²⁺-activated chloride channels that could amplify the nAChR responses in X. laevis oocytes, BAPTA control experiments were performed. Oocytes were incubated with the membrane-permeable Ca²⁺ chelator, BAPTA-AM, before recording. Similar blocking actions of indolizidine (-)-235B' were observed in the BAPTA-AM–treated oocytes expressing 42 nAChRs. Compared with untreated oocytes, the peak amplitude of the currents elicited by ACh (1 µM) in the absence of (-)-235B' was 459 ± 46 nA (3 oocytes), and in the presence of 0.1 and 0.3 µM (-)-235B', the amplitudes were decreased to 58.0 ± 3.2 and 35.7 ± 6.8%, respectively. These results indicate that indolizidine (-)-235B' directly blocks 42 nAChRs.

Analysis of the 42 peak current amplitude revealed that the ACh concentration-response curves were not adequately described by a single Hill equation (Fig. 3A, dashed line, EC₅₀ = 10.9 µM and n_H = 0.52). A significantly better fit was obtained with the sum of two Hill equations (Fig. 3A, continuous line), indicating the existence of a high- and low-affinity component (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Buisson and Bertrand, 2001; Nelson et al., 2003; Almeida et al., 2004; Khiroug et al., 2004). The high-affinity coefficients were EC_50,high = 1.3 µM and n_H,high = 0.56, whereas the low-affinity coefficients were EC_50,low = 119 µM and n_H,low = 1.5. The fractions of high- and low-affinity components were 58.3 and 41.7%, respectively. In the presence of indolizidine (-)-235B' (0.1 µM), the ACh concentration-response curve shifted downward, and the responses to ACh at the maximal concentration (1 mM) did not reach the levels observed in the absence of (-)-235B' (Fig. 3A). These results indicate that indolizidine (-)-235B' is not acting as a competitive antagonists of 42 receptors. The concentration-response relationship for ACh in the presence of (-)-235B' was well fit by a single Hill equation (Fig. 3A, dashed line, EC₅₀ = 8.8 µM and n_H = 0.44). When we applied the two-component model to the data, the fit obtained is shown as the continuous line in Fig. 3A, yielding the high-affinity coefficients of EC_50,high = 0.07 µM and n_H,high = 0.85 and the low-affinity coefficients of EC_50,low = 24.8 µM and n_H,low = 1.0. The fractions of high- and low-affinity components were 31.0 and 69.0%, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Blockade by indolizidine (-)-235B' of the ACh-induced currents in oocytes expressing 42-nicotinic receptors is noncompetitive in nature. Currents were recorded in voltage-clamp mode at -60 mV. A, concentration response curves for ACh in the absence and presence of indolizidine (-)-235B'. Oocytes were preincubated with (-)-235B' (0.1 µM) for 10 min and then exposed to ACh (0.1 µM–1 mM) in the presence of (-)-235B' in standard Ringer's solution. Each current response to ACh in the presence of (-)-235B' was normalized to the maximal current evoked by 1 mM ACh alone recorded in the same oocyte. Values represent the mean ± S.E.M. for five to eight separate experiments. The dashed lines indicate the fits by a single Hill equation, whereas the continuous lines indicate the fits by the sum of two Hill equations. B, the percentage inhibition by (-)-235B' (0.1 µM) of the 42 currents that were elicited by different concentrations of ACh (ranging from 10 nM to 1 mM) in a lower external Ca2+ concentration (0.5 mM). Values represent the mean ± S.E.M. for five to eight separate experiments. Inset shows 42 currents elicited by two different concentrations of ACh (0.1 and 100 µM) in the absence and presence of (-)-235B' (0.1 µM). Horizontal bars, 5 s. Vertical scale bars represent 150 nA for the ACh (0.1 µM)-elicited currents and 1000 nA for the ACh (100 µM)-elicited currents.

To know the mode of the receptor blockade by (-)-235B', 42 receptors were activated by increasing concentrations of ACh (ranging from 10 nM to 1 mM) in the presence of (-)-235B' (0.1 µM). The ACh-elicited currents were measured in a lower external Ca²⁺ concentration (0.5 mM) to avoid the Ca²⁺-activated chloride conductance. As shown in Fig. 3B, the relative blockade by (-)-235B' was larger at the higher concentrations of ACh. Thus, in the presence of the alkaloid (-)-235B' at a constant concentration (0.1 µM), the peak amplitudes of currents elicited by ACh at 0.1 and 100 µM were reduced by 32.8 ± 5.8 and 71.5 ± 3.4%, respectively; differences were statistically significant (P < 0.01, determined by unpaired Student's t test).

Moreover, the voltage dependence of the blocking effects of (-)-235B' on 42 currents was explored at different holding potentials (from -140 to +40 mV) in 20-mV steps. The currents were elicited by ACh (1 µM) in 0.5 mM Ca²⁺. As shown in Fig. 4A, the current-voltage relationship showed strong inward rectification for the ACh (1 µM)-elicited current at positive membrane potentials. Exposure to (-)-235B' (0.1 µM) induced a reduction of the peak amplitude of currents, and the magnitude of this reduction was more pronounced at hyperpolarized potentials. (-)-235B' blocked the 42 currents by 68 ± 6% at -140 mV and by only 38 ± 6% at -40 mV (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Voltage-dependent effect of (-)-235B' on ACh-induced currents in oocytes expressing 42-nicotinic receptors. Oocytes were voltage-clamped at various membrane potentials (from -140 to +40 mV) in 20-mV steps. The currents were elicited by ACh (1 µM, 5 s), 3 min apart, at different holding potentials. This protocol was repeated in the same oocyte but in the presence of (-)-235B' (0.1 µM). (-)-235B' was present 3 min before application of ACh and throughout the latter experiments, including the ACh pulse. A, a typical plot demonstrating the current-voltage relationship in one oocyte expressing 42 receptors in the absence and presence of (-)-235B' (0.1 µM). B, averaged values for the inhibition by (-)-235B' of the ACh-elicited currents through 42 receptors. Data were expressed as percentage of inhibition of the control current obtained without (-)-235B' at each potential; the control current was considered the peak amplitude of ACh-induced current before the addition of (-)-235B'. Values represent the mean ± S.E.M. for six to nine separate experiments. *, P < 0.05; **, P < 0.01, compared with the (-)-235B'-induced inhibition of 42 currents at -140 mV.

We also investigated whether (-)-235B' produces a use-dependent blockade of 42 receptors. When oocytes expressing 42 receptors were stimulated with short pulses of ACh (10 µM, 200 ms) every 8 s in 0.5 mM Ca²⁺, consistent, repeatable currents were obtained. Figure 5A shows a typical recording in one oocyte. In contrast, when (-)-235B' (0.1 µM) was superfused in the bath for 15 s preceding ACh application, then the responses to the repetitive pulses of ACh coapplied with (-)-235B' (0.1 µM) were progressively decreased in the same oocyte (Fig. 5A). Figure 5B summarizes the results obtained during the first 64 s in different oocytes. We further studied whether the blocking effect of (-)-235B' (0.1 µM) depends on the frequency of the ACh pulses. Using a lower frequency (16-s interval) rather than 8-s interval of stimulation, we found a significant difference in the extent of the blockade of 42 currents at the two frequencies (Fig. 5B). At any time point tested, (-)-235B' produced a stronger blockade of the currents elicited by ACh pulses at the higher frequency (8-s interval) than at the lower frequency (16-s interval).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Use-dependent blockade of 42 currents by (-)-235B'. Oocytes expressing 42 receptors were voltage-clamped at -60 mV, and the currents elicited every 8 or 16 s by short pulses of ACh (10 µM, 200 ms) were recorded in a lower external Ca2+ concentration (0.5 mM) until the currents became stable (IACh max). Thereafter, (-)-235B' (0.1 µM) was superfused in the bath for 15 s before ACh application, then the responses to the repetitive pulses of ACh coapplied with (-)-235B' (0.1 µM) were recorded in the same oocyte. A, top, a typical trace showing the stable currents elicited every 8 s by the ACh pulses (arrowheads) in the absence of (-)-235B'. Bottom, a typical trace showing the progressive inhibition of the ACh-elicited currents by (-)-235B'. Vertical scale bar, 100 nA; horizontal scale bar, 4 s. B, the ratios of the ACh-elicited currents in the presence of (-)-235B' (IACh) versus control IACh max at two different frequencies. Values represent the mean ± S.E.M. in 10 and 7 separate oocytes at a higher (8-s interval) and a lower (16-s interval) frequency of ACh stimulation, respectively. *, P < 0.05; **, P < 0.01, compared with the ratios of IACh/IACh max at the higher frequency (8 s) of ACh stimulation.

Next, we tested the effects of another type of the 5,8-disubstituted indolizidine analogs, 5-n-propyl-8-n-butylindolizidines I, II, and III, on the ACh-elicited currents in oocytes expressing recombinant nicotinic receptors. These three compounds are stereoisomers, and I has the same stereochemistry as (-)-235B' at the 5 and 9 positions (5,9-cis: 5,9Z), as shown in Fig. 1. The alkaloid I at 10 µM blocked the responses mediated by 42 receptors and 7 receptors similarly (Fig. 6A). In fact, these currents were blocked by this alkaloid in a concentration-dependent manner, with IC₅₀ values of 6.0 µM for 42 currents and 3.4 µM for 7 currents (Fig. 6B, Table 1). In contrast, the indolizidines II and III were more potent at blocking the responses mediated by 7 receptors than 42 receptors (Table 1). In addition, the indolizidine I had a negligible effect on the 34 receptor responses at a high concentration (10 µM), whereas the responses through this receptor were substantially blocked by the indolizidine II (IC₅₀ = 14.7 µM) and were potently blocked by the indolizidine III (IC₅₀ = 3.0 µM).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Inhibitory effects of indolizidine I on ACh-induced currents in X. laevis oocytes expressing recombinant nicotinic receptors. Currents were recorded in voltage-clamp mode at -60 mV. A, representative traces showing the ACh-elicited currents in the absence and presence of the indolizidine I (10 µM). Horizontal bars indicate the period of perfusion with ACh for 5 s. Concentrations of ACh used were 1 µM for 42 receptors and 100 µM for 7 and 34 receptors. For test responses, oocytes were preincubated with the indolizidine I for 3 min and then exposed to ACh with the indolizidine I. Vertical scale bars represent 1 µA for 42 currents and 0.5 µA for 7 and 34 currents. B, concentration-response curves for the indolizidine I on recombinant nicotinic receptors. Current responses to ACh in the presence of the indolizidine I in each oocyte were normalized to the ACh responses (control responses) recorded in the same oocytes. Values represent the mean ± S.E.M. for three to five separate experiments.

Stereoselective pharmacological properties were also observed with the 5,6,8-trisubstituted indolizidines (+)-6-epi-223A and (-)-223A. The alkaloid (+)-6-epi-223A (10 µM) had a negligible effect on the ACh-elicited currents in oocytes expressing 42 or 7 receptors (Table 1), whereas (-)-223A (10 µM) blocked both the 42 and 7 receptor-mediated currents to a similar extent (Fig. 7A). (-)-223A blocked those two receptors in a concentration-dependent manner with IC₅₀ values of about 4 µM (Fig. 7B, Table 1). The 34 receptor-mediated currents were also blocked by (-)-223A with an IC₅₀ value of 14 µM (Fig. 7, Table 1). This blocking effect was similar to that of (+)-6-epi-223A (IC₅₀ = 15 µM).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Inhibitory effects of indolizidine (-)-223A on ACh-induced currents in X. laevis oocytes expressing recombinant nicotinic receptors. Currents were recorded in voltage-clamp mode at -60 mV. A, typical traces showing the ACh-elicited currents in the absence and presence of (-)-223A (10 µM). Horizontal bars indicate the period of perfusion with ACh for 5 s. Concentrations of ACh used were 1 µM for 42 receptors and 100 µM for 7 and 34 receptors. For test responses, oocytes were preincubated with (-)-223A for 3 min and then exposed to ACh with (-)-223A. Vertical scale bars represent 1 µA for 42 currents, and 0.5 µA for 7 and 34 currents. B, concentration-response curves for (-)-223A on recombinant nicotinic receptors. Current responses to ACh in the presence of (-)-223A in each oocyte were normalized to the ACh responses (control responses) recorded in the same oocytes. Values represent the mean ± S.E.M. for five to seven separate experiments.

The 1,4-disubstituted quinolizidine (-)-1-epi-207I selectively blocked 7 receptor responses (IC₅₀ = 0.6 µM), and it did so with 8.7-fold higher sensitivity than blockade of 42 receptor responses (IC₅₀ = 5.2 µM) and 14.7-fold higher than that of 34 receptor responses (IC₅₀ = 8.8 µM) (Table 1). The alkaloid (+)-207I equally blocked the responses mediated by 42 receptors (IC₅₀ = 5.0 µM) and 7 receptors (IC₅₀ = 3.4 µM).

When the oocytes expressing 7 nAChRs were treated with 3 µM 8b-azaacenaphthylene (+)-205B, which is the unnatural enantiomer, the peak amplitude of the ACh-elicited currents was greatly decreased, whereas the responses via 42 and 34 nAChRs were not strongly affected (Fig. 8A). The blocking effect of (+)-205B (3 µM) on 7 nAChRs was reversible within 10 min (data not shown). When the concentration-response curves were compared across these nAChR sub-types, (+)-205B blocked the 7 receptor-mediated currents (IC₅₀ = 2.5 µM, n_H = 0.46 ± 0.08) with 5.4-fold higher sensitivity than blockade of the 42 receptor-mediated currents (IC₅₀ = 13.5 µM, n_H = 0.44 ± 0.05) and 4.5-fold higher than blockade of the 34 receptor-mediated currents (IC₅₀ = 11.3 µM, n_H = 0.65 ± 0.13) (Fig. 8B, Table 1). These results indicate that (+)-205B selectively blocked the responses mediated by 7 receptors, rather than 42 and 34 receptors. In the BAPTA-AM–treated oocytes expressing 7 nAChRs, the peak amplitudes of the ACh (100 µM)-elicited currents were decreased by (+)-205B at 3 and 10 µM to 45.7 ± 6.3% and 11.7 ± 0.3% (three oocytes), respectively. These results demonstrate that (+)-205B directly blocks 7 nAChRs.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Inhibitory effects of 8b-azaacenaphthylene (+)-205B on ACh-induced currents in X. laevis oocytes expressing recombinant nicotinic receptors. Currents were recorded in the voltage-clamp mode at -60 mV. Concentrations of ACh used were 1 µM for 42 receptors and 100 µM for 7 and 34 receptors. For test responses, oocytes were preincubated with (+)-205B for 3 min and then exposed to ACh with (+)-205B. A, representative traces showing the ACh-elicited currents in the absence and presence of (+)-205B (3 µM). Horizontal bars indicate the period of perfusion with ACh for 5 s. Vertical scale bars represent 1 µA on 42 receptor and 0.5 µA on 7 and 34 receptors. B, concentration-response curves for (+)-205B on recombinant nicotinic receptors. Current responses to ACh in the presence of (+)-205B in each oocyte were normalized to the ACh responses (control responses) recorded in the same oocytes. Values represent the mean ± S.E.M. for four to six separate experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The bicyclic "izidine" alkaloids (indolizidines, quinolizidines, pyrrolizidines, and azabicyclo[5.3.0]decanes) from amphibian skin have been examined. The alkaloids are mainly from neotropical dendrobatid frogs, Dendrobates spp., and most of the bicyclic alkaloids are known to be disubstituted, such as 5,8- or 3,5-disubstituted indolizidines and 1,4-disubstituted quinolizidines (Daly et al., 1999). Previously, it has been shown that particular 5,8-disubsutituted indolizidines, including (-)-235B', act as noncompetitive nicotinic antagonists and inhibit carbamylcholine-elicited ²²Na⁺-influx via nAChR channels in PC-12 cells (Daly et al., 1991). In this study, we examined the selectivity of indolizidines for multiple subtypes of recombinant nAChRs expressed in oocytes. In addition, we examined the effects of several synthetic compounds with structures of frog alkaloids. Among these compounds, we found that indolizidine (-)-235B' was a highly potent blocker of 42 nAChRs (IC₅₀ = 0.07 µM). (-)-235B' seems to block via direct interaction with 42 receptors, not through an indirect effect on the oocytes' endogenous Ca²⁺-activated chloride channels.

The concentration-response relationship for ACh on 42 nAChRs was best fit by the sum of two Hill equations. This finding is consistent with previous reports, indicating that there are at least two different populations of 42 nAChRs with different affinities for ACh when ectopically expressed in X. laevis oocytes (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Khiroug et al., 2004) and in HEK293 cell lines (Buisson and Bertrand, 2001; Nelson et al., 2003; Almeida et al., 2004). It has been proposed that these different properties are attributable to different subunit stoichiometries: the high-affinity nAChR is thought to be (4)₂(2)₃, whereas the low-affinity nAChR is (4)₃(2)₂ (Nelson et al., 2003).

The blockade of 42 currents by (-)-235B' was more pronounced as the ACh concentrations increased (from 10 nM to 100 µM). If the low-affinity population of 42 receptors were predominant compared with the high-affinity population in the present condition, selective blockade of the low-affinity receptors might explain the stronger effect of (-)-235B' on the responses to higher concentrations of ACh. In fact, the populations were almost equal between the high-affinity component (58.3%) and the low-affinity component (41.7%) based on the analysis of the ACh concentration-response curve (Fig. 3A). Furthermore, we estimate that activation of the high- and low-affinity receptors contributed to 58% and 42%, respectively, of the peak currents elicited by 1 µM ACh. The alkaloid (-)-235B' (0.1 nM-10 µM) blocked the ACh-elicited (1 µM) currents, and the concentration-response relationship for (-)-235B' was well described by a single sigmoid curve (Fig. 2C), without requiring a double sigmoid, indicating that (-)-235B' did not significantly differentiate between the high- and low-affinity receptors. Thus, the mode of action of (-)-235B' seems to be independent of these receptor populations. Because the probability of the 42 nAChR-channel opening increases as the ACh concentration increases, (-)-235B' blocks the receptor-channels that are more frequently open at higher ACh concentrations by entering and occluding the open-channel pores. In general, open channel blockers are known to exhibit both a voltage-dependent and a use-dependent inhibition of nAChRs (Buisson and Bertrand, 1998; Pintado et al., 2000). Consistent with that expectation, we observed that (-)-235B' exerted stronger inhibition of the 42 currents at hyperpolarized potentials. In addition, the 42 currents elicited by repetitive ACh pulses were progressively inhibited by (-)-235B' in a use-dependent manner. Therefore, the mode of action of (-)-235B' is likely to be open channel blockade.

The sensitivity of indolizidine (-)-235B' for 42 receptors was comparable with that of the best characterized antagonist of 42 nAChRs, DHE, observed in X. laevis oocytes expressing human 42 receptors (IC₅₀ = 0.11 µM) (Chavez-Noriega et al., 1997). Moreover, (-)-235B' selectively blocked 42 receptors more effectively than 32 receptors (40.5-fold) or 34 receptors (51.4-fold). These pharmacological properties are also comparable with DHE. However, the selectivity for 42 receptors over 7 or 44 receptors was different between (-)-235B' and DHE: the rank order of potency of (-)-235B' was 42>7>32>3444, whereas that of DHE is 44>42>32>347 (Chavez-Noriega et al., 1997). It may be possible to develop even more potent and selective ligands on the basis of the structure of indolizidine (-)-235B'.

The structure of natural 5,6,8-trisubstituted indolizidine (-)-223A has recently been revised from (+)-6-epi-223A (Toyooka et al., 2002). It is interesting that (-)-223A but not (+)-6-epi-223A exhibited blocking effects on 42 and 7 receptors. These results suggest that the alkaloid (-)-223A may bind to the nAChRs in a stereoselective manner.

We also examined the stereoselective pharmacological properties of three stereoisomers, 5,8-disubstituted indolizidines I, II, and III. The indolizidine I has a 5,9-cis (5,9Z) structure, whereas the other two compounds have 5,9-trans (5,9E) structures. Because the indolizidine I exhibited a greater sensitivity to 42 receptors than the indolizidines II and III, the 5,9-cis (5,9Z) structure may be important for binding to 42 receptors with high affinity. Indeed, indolizidine (-)-235B' that possesses the 5,9-cis (5,9Z) structure was a potent blocker of 42 receptors, as mentioned above. To test this hypothesis, it would be necessary to examine the effect of the (-)-235B' stereoisomer with a 5,9-trans (5,9E) structure on 42 receptors in a future study.

The alkaloid (+)-205B has a unique tricyclic structure of 8b-azaacenaphthylene (Daly et al., 1999). No other alkaloids from amphibian skin are known to belong to this class. The absolute stereochemistry of this alkaloid was determined by the total synthesis (Toyooka et al., 2003). The alkaloid (+)-205B produced a selective inhibition of 7 receptors over 42 or 34 receptors. We also found the high selectivity (8.7-fold) of 1,4-disubstituted quinolizidine (-)-1-epi-207I for 7 receptors over 42 receptors, which is greater than the 7 selectivity (5.4-fold) of (+)-205B. Therefore, we suggest that a novel class of nicotinic antagonists may be developed based on the structure of these compounds.

In conclusion, we found that indolizidine (-)-235B' is a potent noncompetitive blocker of 42 nAChRs, and its specificity is comparable with that of the competitive antagonist DHE. We also found that quinolizidine (-)-1-epi-207I and tricyclic (+)-205B are selective blockers of 7 nAChRs. It should also be noted that some of the alkaloids used in this study exhibited subtype-selective blockade of nAChRs in a stereoselective manner. These results suggest that it may be possible to obtain novel, potent, and subtype-selective blockers of nicotinic receptors by synthesizing stereoisomers of natural alkaloids. Thus, it is anticipated that the approach based on the frog alkaloids can provide lead compounds for the future design of drugs to treat cholinergic disorders in the central nervous system.

	Acknowledgements

 
We are grateful to Dr. Jerry A. Stitzel (University of Colorado) and Dr. Jim Patrick (Baylor College of Medicine) for providing us with plasmid DNA. We would also like to thank Dr. Hideki Sakai (Toyama Medical and Pharmaceutical University) for the continuous support on data acquisition.

	Footnotes

 
This work was supported in part by Smoking Research Foundation (Japan) (to I.K.) and by Grants-in-Aid 14771274 and 16590435 for Scientific Research from the Japanese Ministry of Science, Culture, Sports, and Technology (to H.T.). J.A.D. was supported by grants from the National Institute of Neurological Disorders and Stroke and National Institute on Drug Abuse of the National Institutes of Health (USA).

H.T. and Y.Y. contributed equally to this work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.000729.

ABBREVIATIONS: BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; ACh, acetylcholine; DHE, dihydro--erythroidine; MLA, methyllycaconitine; nAChR, nicotinic acetylcholine receptor.

Address correspondence to: Hiroshi Tsuneki, Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: htsuneki{at}ms.toyama-mpu.ac.jp

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