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NEUROPHARMACOLOGY
Divisions of Neurology (J.H., J.W.) and Neurobiology (K.L., R.J.L.), Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona; Department of Pharmacology, Nanjing Medical University, Nanjing, People's Republic of China (G.H., J.H., J.W.); and Department of Cardiovascular Pharmacology, Institute of Pharmacology and Toxicology, Beijing, People's Republic of China (H.W., J.W.)
Received August 30, 2005; accepted October 11, 2005.
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4
2-nAChRs in response to nicotinic agonists. It also accelerated current decay, caused a decline in apparent efficacy of agonists, and acted in voltage- and use-dependent manners at 42-nAChRs. These findings and the inability of Ipt to block radiolabeled epibatidine binding to 42-nAChRs suggest a noncompetitive mechanism of antagonism. Other studies discount effects of Ipt on nAChR internalization or involvement of KATP channels in Ipt-induced inhibition of 42-nAChR function. By comparison, 7-nAChRs were less sensitive than 42-nAChRs to Ipt acting as an antagonist. Thus, 42-nAChRs are among the molecular targets of Ipt, which has utility as a tool in functional characterization and pharmacological profiling of nAChRs.
). Mammalian nAChRs exist as a diverse set of molecules composed of different combinations of multiple subunits encoded from a family of at least 16 genes (1-7, 9-10, 1-4, 
, 
, and 
). They play a variety of critical roles in nervous system function and have been implicated in a number of neuropsychiatric conditions as well as in nicotine dependence.
For example, the most abundant form of nAChR in the brain contains 4 and 2 subunits (42-nAChR; Gopalakrishnan et al., 1996). 42-nAChRs bind nicotine with high affinity and respond to levels of nicotine found in the plasma of smokers (Fenster et al., 1997). 42-nAChRs also have been implicated in nicotine self-administration and in disorders such as Alzheimer's disease, Parkinson's disease, and epilepsy (Cordero-Erausquin et al., 2000; Nakamura et al., 2001; O'Neill et al., 2002; Quik, 2004). Knockout mice lacking expression of 42-nAChRs fail to show nicotinic agonist-induced increases in striatal dopamine release or midbrain dopaminergic neuronal discharge frequency and rapidly cease nicotine, but not cocaine, self-administration (Picciotto et al., 1998; Marubio et al., 2003). By contrast, nicotine activation of 4-containing nAChRs is sufficient for nicotine-induced reward, tolerance, and sensitization (Tapper et al., 2004). Therefore, brain 42-nAChRs seem to play pivotal roles in mediation of nicotinic reinforcement and dependence. Furthermore, their loss, for example, in Alzheimer's disease (Burghaus et al., 2000), may play a role in disease onset or progression.
However, the existence of nAChRs as a diverse family of subtypes has complicated their characterization. Differences in ligand sensitivity of nAChR subtypes affords an opportunity to dissect roles of receptors in general and nAChR subtypes in particular, and, reciprocally, nAChR subtype pharmacological profiling provides a means for discriminating roles of these subtypes in functions in health and disease. Drugs acting safely in mammals at nAChRs could be medication candidates, and perhaps the effects of some compounds known to be safe on brain or body function could include interactions at nAChRs.
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). It is a small, water-soluble molecule that freely penetrates the blood-brain barrier and has minimal toxic side effects after long-term systemic administration (Wang, 2003; Wang et al., 2004). Possible pharmacological mechanisms underlying its antihypertensive action include KATP channel activation and endothelin antagonism (Wang, 2003). Tests in a variety of in vivo and in vitro ischemia and Parkinson's disease models indicate that Ipt has neuroprotective effects (Wang et al., 2004, 2005; Yang et al., 2004, 2005). Cardiovascular and central effects of Ipt are thought to be mediated by opening of cytoplasmic and/or mitochondrial KATP channels (Wang, 2003; Wang et al., 2004). In contrast, neuroprotective effects of Ipt also seem to be mediated by direct blockade of postsynaptic, ionotropic glutamate receptor function in cultured rat hippocampal neurons (Wang et al., 2004) and by enhancement of glutamate transporter function to increase glutamate uptake (Wang et al., 2004; Yang et al., 2005). Both mechanisms lead to a reduction of glutamate-induced neurotoxicity. Furthermore, Ipt has potential in prevention of drug addiction because it inhibits cocaine challenge-induced enhancement of dopamine release in the rat nucleus accumbens (Liu et al., 2003).
In the present study, we have used patch-clamp recording techniques and pharmacological manipulations to evaluate effects of Ipt on selected human nAChRs heterologously expressed in the SH-EP1 cell-line. We found that Ipt inhibits human 42-nAChR function selectively relative to its effects on 7-nAChR function through a noncompetitive mechanism independent of effects on KATP channels.
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Materials and Methods |
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42-nAChR in SH-EP1 Human Epithelial Cells. Heterologous expression of human 42-nAChRs has been described in detail previously (Eaton et al., 2003; Wu et al., 2004b). In brief, human 4 and 2 subunits subcloned into pcDNA3.1-zeocin or -hygromycin vectors, respectively, were introduced using established techniques (Puchacz et al., 1994; Peng et al., 1999) into native nAChR-null SH-EP1 cells (Lukas et al., 1993) to create the SH-EP1-h42 cell line. Cells were maintained as low passage number (1-26 from our frozen stocks) cultures in medium augmented with 0.5 mg/ml zeocin and 0.4 mg/ml hygromycin and passaged once weekly by splitting just-confluent cultures 1/10 to maintain cells in proliferative growth.
Epibatidine Binding Competition Studies. Membrane preparations were processed and radioligand binding competition assays were conducted as described previously (Eaton et al., 2003), taking special care to ensure that reaction mixtures were not ligand-depleted by conducting assays with no more than 
25 fmol of binding sites and 400 pM [3H]epibatidine (PerkinElmer Life and Analytical Sciences, Boston, MA) in an 800-µl volume containing competing ligand at various concentrations. Data were plotted, analyzed, and tested for statistical significance using Prism (GraphPad Software Inc., San Diego, CA).
Patch-Clamp Whole-Cell Current Recordings and Data Acquisition. Conventional whole-cell current recording coupled with techniques for fast application and removal of drugs (U-tube) were applied in this study as described previously (Zhao et al., 2003; Wu et al., 2004a,b). In brief, transfected cells plated on 35-mm culture dishes without poly(lysine) coating were placed on the stage of an inverted microscope (Olympus IX7; Olympus, Lake Success, NY) and continuously superfused with standard external solution (2 ml/min). Glass microelectrodes (3- to 5-M
resistance between pipette and extracellular solutions) were used to form tight seals (>1G)onthe cell surface until suction was applied to convert to conventional whole-cell recording and a period of 5 to 10 min lapsed to allow full exchange between the pipette solution and the cytosol. Thereafter, recorded cells were lifted off the bottom of the culture plate, which allows for improved kinetics of solution exchange and truer assessment of the kinetics of agonist-induced whole-cell currents. Before capacitance and series resistance compensation, access resistance (Ra) was measured and accepted for further experimentation if less than 20 M. Both pipette and whole-current capacitance were minimized, and series resistance was routinely compensated to 80%. Cells were then voltage-clamped at holding potentials of -60 mV, and ion currents in response to application of nicotinic ligands were measured (200B amplifier; Molecular Devices, Sunnyvale, CA). Current signals were typically filtered at 2 kHz, acquired at 5 kHz, displayed and digitized on-line (Digidata 1322 series A/D board; Molecular Devices, Sunnyvale, CA), and subsequently stored to computer hard drive. Data acquisition and analyses were done using pClamp9.0 (Molecular Devices), and results were plotted using Origin 5.0 (OriginLab Corp., North Hampton, MA). All experiments were performed at room temperature (22 ± 1°C).
Solutions and Drug Application. The standard external solution contained 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM D-glucose, and 10 mM HEPES, pH 7.4 (Tris-base). In most experiments, nicotine was used as the test agonist. In some experiments, acetylcholine (ACh) and RJR-2403 were applied. Atropine sulfate (1 µM) was always added to ACh-containing standard external solution to exclude any possible influences of muscarinic receptors. For conventional whole-cell current recordings, the pipette solution contained 110 mM Tris-phosphate dibasic, 28 mM Tris-base, 11 mM EGTA, 2 mM MgCl2, 0.5 mM CaCl2, and 4 mM Na-ATP, pH 7.3. To initiate whole-cell current responses, nicotinic agonists were rapidly delivered to the recorded cell by a computer-controlled U-tube system, allowing the applied drug to completely surround the recorded cell within 20 ms. The interval between drug applications (3 min) was optimized specifically to ensure stability of nAChR responsiveness (without functional rundown). Drugs used in the present study were (-)-nicotine, ACh, cytisine, choline, dihydro--erythroidine (DHE), mecamylamine, tolbutamide, and guanosine 5'-O-(2-thio)diphosphate (GDPS) trilithium salt (Sigma Chemical, St. Louis, MO). RJR-2403, pinacidil, P1075, diazoxide, and glibenclamide were purchased from Tocris Cookson Inc. (Ellisville, MO). Ipt was kindly provided by Dr. H. Wang (Institute of Pharmacology and Toxicology, Beijing, People's Republic of China). The chemical structure of iptakalim [N-(1-methylethyl)-1,1,2-trimethyl-propylamine hydrochloride] is shown in Fig. 1. Because effects of Ipt indicated enhanced functional inhibitory potency with prolonged or repeated applications, data collection was more laborious than usual because many studies required recording from cells only through a single exposure to the ligand.
Data Analysis and Statistics. nAChR whole-cell current responses were analyzed to fit for decay time constant (tau; 
), peak current (Ip), and steady-state current (Is) using fits to the mono- or double-exponential expression I = [(Ip - Is) e-t/] + Is. Data usually were fit over the 10 to 90% period from inward current peak until agonist exposure was terminated (4 s). The experimental data are presented as means ± standard errors. Statistical analysis was done using paired t-tests comparing the data obtained from a single cell or Student's t test (unpaired values) or one-way analysis of variance with Duncan's multiple comparison comparing the data obtained from different cells. Values of p less than 0.05 were considered significant. Curve fitting for agonist and antagonist concentration-response data were performed (Origin software 5.0; OriginLab Corp.) using the logistic equation to provide fits for maximal and minimal responses, the EC50 or IC50 value, and Hill coefficients.
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42-nAChR-Mediated Whole-Cell Currents. Relative to stable whole-cell current responses elicited during challenge exposure to 3 µM (EC50 concentration) nicotine alone, nicotine-induced inward currents mediated via h42-nAChRs in the same SH-EP1-42 cell were reduced if assessed during coapplication with 3 µM Ipt (Fig. 2A). In the presence of 3 µM Ipt, the current peak, the ratio of steady-state to peak current, and the rate of decay from peak to steady-state current were reduced by 21 ± 3, 69 ± 2, and 21 ± 3%, respectively (Fig. 2, A and B). Surprisingly, a brief (4-s) exposure of Ipt (50 µM) caused long-lasting inhibition on 42-nAChR function (Fig. 2C). For example, the half-time for recovery of peak current response to nicotine challenge was 15 min, although effects on steady-state inward current levels and on the time constant for current decay recovered more quickly (Fig. 2D). Inhibition by Ipt of 42-nAChR responses to different nicotinic agonists also was evident. In the presence of EC50 concentrations of acetylcholine (10 µM), RJR-2403 (10 µM), or nicotine (3 µM), 50 µM Ipt produced 66 ± 1, 73 ± 2, or 76 ± 3% block of peak whole-cell currents, respectively. These results indicate that Ipt blocks h42-nAChR-mediated currents.
Ipt Blocks a42-nAChR-Mediated Currents in a Time- and Concentration-Dependent Manner. Initial time dependence assays indicated that, compared with coapplication, 3-min pretreatment with Ipt produced a more profound inhibition of the response to 3 µM nicotine (Fig. 3A). Peak amplitudes of 3 µM nicotine-induced currents were reduced to 62 ± 6% of control values with 3-min pretreatment followed by continued coapplication with agonist plus Ipt, to 79 ± 3% without Ipt pretreatment but with coapplication with nicotine plus Ipt, and to 83 ± 4% after 3-min pretreatment with Ipt ending before exposure to nicotine alone (Fig. 3B). Steady-state peak current amplitude ratios and the time required for an e-fold loss from peak current levels during current decay rate () were lowest when Ipt was coapplied with nicotine whether or not there was prior exposure to Ipt (Fig. 3B). Effects of Ipt applied at different concentrations during a 4-s nicotine exposure only or during nicotine exposure and after 3-min pretreatment with Ipt show concentration dependence of functional block as well as greater inhibitory efficiency after pretreatment (Fig. 3C). Concentration-response profiles for inhibition by Ipt of peak whole-cell responses indicate IC50 values of 5.0 and 31.6 µM with and without pretreatment, respectively (Fig. 3D). Upon coapplication of nicotine and Ipt, the IC50 value for Ipt-mediated inhibition of the whole-cell steady-state current is 1.0 µM and is lower than the coapplication IC50 value for inhibition of the peak component (Fig. 3E). The more profound inhibition after pretreatment suggests that some Ipt binding sites are accessible on nAChRs in the resting state.
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Mechanism of Ipt Block of 42-nAChR Function. To explore the nature of Ipt functional block, whole-cell current responses were obtained in the presence of nicotine alone or with 10 or 50 µM Ipt as shown in Fig. 4A. Assessment of whole-cell peak current amplitudes as a function of nicotine concentration yielded apparent EC50 values of 5.1 µM in the presence of nicotine alone and 22 or 39 µM in the presence of nicotine plus 10 or 50 µM Ipt, respectively (Fig. 4B). Although nicotine up to 1 mM was unable to surmount functional block by 10 or 50 µM Ipt of peak current responses, the magnitude of the inhibitory effect by Ipt decreased as nicotine concentration was increased (Fig. 4B). However, the Hill slope for nicotine-elicited peak current responses became more shallow in the presence of higher concentrations of Ipt, inconsistent with a purely competitive mechanism of functional block (Fig. 4B). Moreover, the relative inhibition by Ipt of steady state to peak current responses increased as nicotine concentration increased (Fig. 4C).
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To illuminate mechanisms involved in Ipt-induced functional inhibition, radioligand binding assays indicated that [3H]epibatidine binding was blocked by nicotine or DHE with previously reported IC50 values (Eaton et al., 2003) but that mecamylamine, a noncompetitive inhibitor of 42-nAChR function, as well as Ipt, failed to block radioligand binding at concentrations up to 100 µM (Fig. 5). Collectively, these results suggest that Ipt does not act on agonist binding sites (i.e., via a purely competitive mechanism) to exert its inhibition of 42-nAChR function.
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42-nAChR Function. When ionized ligands act to block ion channels, their residence in the transmembrane region is affected by transmembrane potential. Therefore, transmembrane voltage dependence is one of the characteristic features of open-channel block by charged ligands. Whole-cell current responses to nicotine alone and in the presence of 50 µM Ipt recorded at different holding potentials (VH; Fig. 6A) indicate that Ipt exerts stronger inhibition of nicotinic responses at more negative holding potentials (Fig. 6B). Fractional inhibition by Ipt of whole-cell peak current responses to nicotine was 81 ± 3, 76 ± 3, and 69 ± 4% at holding potentials of -80, -40, and 0 mV, respectively, and current decay constants were 22 ± 3, 30 ± 1, and 44 ± 8% of control, respectively, reaching significance for differences between VH = -80 and 0 mV (Fig. 6B; p < 0.05; n = 6). These results indicate that the inhibitory effects on the 42-nAChR function by Ipt are voltage-dependent.
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42-nAChR Function. Under conditions where whole-cell current responses of SH-EP1-h42 cells to repetitive applications of nicotine for 4 s at 3-min intervals showed no significant response rundown (data not shown), six repetitive applications of nicotine (3 µM) in the continuous presence of 3 µM Ipt resulted in a gradual reduction of nicotinic responses (Fig. 7Aa). However, after 15 min of Ipt pretreatment without repeated application of nicotine, there was less inhibition of a 3 µM nicotine-induced response (Fig. 7Ab). That is, under conditions where the initial peak current response to nicotine in the presence of coapplied Ipt was reduced relative to the response to nicotine alone by the same amount (17 ± 6or14 ± 2%; p > 0.05; n = 6), the response to nicotine challenge after 15 min of 3 µM Ipt exposure during repeated nicotine challenges was only 12 ± 2% of control compared with 30 ± 3% of control for the response to nicotine challenge after 15 min of Ipt exposure in the absence of intervening nicotine challenges (p < 0.01; n = 6; Fig. 7B). These results indicate that Ipt-mediated block of 42-nAChR function is use-dependent.
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Ipt Block of 42-nAChRs Is Not Due to Opening of KATP Channels. Ipt was initially designed as a novel KATP channel opener, and until now its pharmacological effects on the cardiovascular system and central nervous system function have been thought to involve the opening of KATP channels (Wang, 2003; Wang et al., 2004, 2005). Because such effects could confound whole-cell current recording-based assessments of effects on 42-nAChR function, we tested whether other KATP channel openers, such as pinacidil, P1750, and diazoxide shared the ability of Ipt to inhibit 42-nAChR-mediated currents. At concentrations of 3 µM, neither the relatively selective cytoplasmic membrane KATP channel opener P1075 (Fig. 8Aa'), the relatively selective mitochondrial KATP channel opener diazoxide (Fig. 8Ab'), nor the cyanoguanidine KATP channel opener pinacidil (Fig. 8Ac'), inhibited nicotine-induced currents, although 3 µM Ipt had a clear effect on the nicotine-evoked steady-state response and also significantly reduced the peak current response (Fig. 8Ad'). These findings indicate that KATP channel openers generally failed to mimic Ipt-induced inhibition of 42-nAChR-mediated currents (Fig. 8B). We also assessed whether classic KATP channel blockers were able to abolish Ipt-induced inhibition of 42-nAChR function. Neither of the classic KATP channel blockers, glibenclamide (30 µM) nor tolbutamide (100 µM), prevented Ipt-induced inhibition of 42-nAChR function (Fig. 8, C and D). We also found that 30 µM glibenclamide or 100 µM tolbutamide alone inhibited 42-nAChR function by 38 ± 4 and 19 ± 2%, respectively (data not shown). Collectively, whereas these findings indicate that there may be some degree of interaction of agents initially identified as KATP channel ligands with 42-nAChR, they also indicate that inhibition of human 42-nAChR function by Ipt is not mediated by opening of any KATP channels that may be expressed in the SH-EP1 cell line.
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Ipt Block of 42-nAChRs Does Not Occur via Intracellular Access. To assess whether Ipt exerts its action on 42-nAChR via accessing intracellular sites, we directly applied it into the cell through the whole-cell recording pipette. In the absence of such application, repetitive extracellular applications of 3 µM nicotine induced five stable responses (2-s exposures at 15-s intervals; Fig. 9A). However, the response was gradually eliminated when nicotine and 3 µM Ipt were coapplied extracellularly through the U-tube to the same recorded cell (Fig. 9B). In the presence of 100 µM Ipt in the recording pipette, after conversion to the whole-cell recording configuration for more than 20 min to allow full loading of Ipt into the cell, there was no decline in responses to five repetitive applications of nicotine (Fig. 9C). In the same recorded cell, 3 µM Ipt coapplied through the U-tube caused an obvious nicotinic response reduction (Fig. 9D). When assessed collectively (Fig. 9E), these findings make it clear that access to intracellular sites is not involved in mediation of Ipt-induced block of 42-nAChRs.
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42-nAChR Function. Given evidence that endocytosis or exocytosis of several kinds of transmembrane receptor occurs on the order of seconds to minutes through processes modulated by agonists and antagonists, we ascertained effects of Ipt in cells preloaded with 600 µM GDPS through the patch-clamp electrode for 20 min after converting to the whole-cell recording configuration. Treatment with GDPS has been reported to prevent -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor or GABAA receptor internalization (Luscher et al., 1999; Blair et al., 2004). Preloading with GDPS for 20 min itself neither affected nicotine-induced currents (Fig. 10A) nor affected Ipt-induced inhibition of 42-nAChR function (Fig. 10, B and C). These results argue against a role for 42-nAChR internalization in the mechanism of Ipt functional antagonism.
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Ipt More Selectively Blocks 4-Than 7-Containing nAChRs. When effects of Ipt on function of heterologously expressed human 42-, 44-, or 7-nAChRs in SH-EP1 cell lines were evaluated, 50 µM Ipt exhibited similar inhibition of 42- or 44-nAChR-mediated currents but showed little inhibition of 7-nAChR-mediated current (Fig. 11, A and B). However, Ipt-mediated functional block persisted for 42- but not 44-nAChR after 3 min of drug washout (Fig. 11, A and B). Concentration-inhibition curves for Ipt effects on peak current amplitudes demonstrate selective inhibition of 4-containing nAChR function (Fig. 11C). Ipt IC50 values for acute coapplication with agonist were 31 and 23 µM, respectively, for 42- and 44-nAChR functional inhibition but higher than 1 mM for 7-nAChR blockade. Even at lower concentrations of choline closer to its EC50 value for activation of 7-nAChRs, and after brief pretreatment of cells with 10 µM Ipt, Ipt exerted more profound inhibition of 42-nAChR-mediated currents (70 ± 11%) than of 7-nAChR-mediated currents (3.5 ± 0.4%; Fig. 11, D and E).
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42-nAChR Function. The principal finding of this study is that Ipt acts with some nAChR subtype selectivity as an antagonist of human 42-nAChR function. Ipt inhibits both peak and steady-state whole-cell current responses of heterologously expressed human 42-nAChR to nicotinic agonists and accelerates whole-cell current decay in a concentration-dependent manner while having long-lasting effects after washout. Its effects are enhanced by exposure to nAChR before agonist challenge and are not because of GDPS-regulated nAChR internalization or effects on KATP channels. Use and voltage dependence of Ipt effects at 42-nAChR as well as its inability to block binding of [3H]epibatidine argue against a competitive mechanism of functional blockade and suggest noncompetitive interaction with 42-nAChR. Ipt is more potent as an antagonist of 42- or 44-nAChR than of 7-nAChR, and its effects are longer lasting at 42- than at 44-nAChR.
Mechanism of Ipt Action. Means for deciphering mechanisms involved in ligand-gated ion channel antagonism, and interpretations of the results obtained, continue to become more refined. As a positively charged ligand at pH 7.4, Ipt is expected to more easily enter the nicotinic channel pore at -80 mV than at 0 mV, reasonably explaining voltage dependence of its effects, although it is possible that the binding site is in the transmembrane field other than in the open channel. Use dependence indicates that Ipt-mediated block involves agonist-induced transition to a nAChR conformation having higher affinity for the antagonist and/or allowing freer access of it to its binding site (Zhao et al., 2004), and it is reasonable to conclude that this transition is to the open channel state. When Ipt was coapplied with nicotine, the rising phase of the whole-cell current response was not altered, but the decay phase was dramatically accelerated, again suggesting an open channel block. However, pretreatment with Ipt induced more profound inhibition of the nicotinic response, suggesting that Ipt binding sites are at least partially accessible in the resting, closed state. Perhaps there are several classes of binding sites with different affinities for Ipt on the nAChR at rest (Hill coefficients for block if Ipt is coapplied with agonist are >1), making fractional occupancy at a given concentration less than complete, but once receptors are exposed to Ipt, it itself induces conversion to a state where binding site affinities for Ipt are more uniformly higher (Hill coefficients are 1 for the Ipt pretreatment condition), as is fractional occupancy.
Interestingly, although effects of Ipt on peak current amplitudes were reduced in the presence of higher concentrations of nicotine, the ratio of steady-state/peak current responses in the presence of Ipt was dramatically decreased. A reasonable interpretation is that channel activation rate is slow and channels activate asynchronously at low nicotine concentrations, giving Ipt enough time to block the channel. This is consistent with the observed larger fractional block of the peak current and minimal effect on its steady-state component in the presence of low nicotine concentrations, whereas at very high concentrations of nicotine, the agonist association rate (binding on-rate) and channel activation rate should be much faster than the association with Ipt (at a fixed concentration), so more channels will be activated synchronously before block, which is evident by the observation of lower fractional inhibition of the peak current and greater block of steady-state current responses and acceleration of 42-nAChR desensitization by Ipt at high nicotine concentrations. Furthermore, other experiments failed to show any detectable competition of Ipt for [3H]epibatidine binding, arguing against competitive block. Collectively, these findings suggest that Ipt has access to the receptor in the resting state but principally acts as a noncompetitive antagonist with its presumed binding site in the 42-nAChR pore.
Possible Roles of Other Ipt Targets in Effects on Human 42-nAChRs. Ipt is a novel cytoplasmic and/or mitochondrial KATP channel opener that exerts various pharmacological effects in cardiovascular and central nervous systems (Wang, 2003; Wang et al., 2004, 2005; Yang et al., 2004, 2005) through mechanisms that could indirectly modulate nAChR function. However, other agents that open or block KATP channels neither mimicked nor blocked effects of Ipt, suggesting that Ipt has its effects by direct action at 4-containing nAChRs. We also found that 30 µM glibenclamide or 100 µM tolbutamide alone inhibited 42-nAChR function, respectively. Interestingly, coapplication of glibenclamide or tolbutamide with Ipt did not further increase the inhibition, implying that these KATP channel blockers may act on the same site as Ipt, but detailed mechanisms need to be further elucidated. Inhibition of 42-nAChR by Ipt does not occur after direct perfusion of Ipt into a recorded cell, suggesting that intracellular sites are not involved in mediation of Ipt-induced block of 42-nAChRs. Ipt does not seem to promote internalization of cell surface nAChR, at least via the GDPS-sensitive mechanism previously shown to be involved in ligand-induced endocytosis of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (Luscher et al., 1999) or GABAA receptors (Blair et al., 2004).
Pharmacological and Potential Clinical Significance of Ipt Block of nAChR Function. Ipt was initially designed and synthesized as a KATP channel opener (Wang, 2003) and has been shown to be an effective counteragent in a variety of animal models, including those for ischemia/hypoxia and Parkinson's disease (Wang, 2003; Wang et al., 2005; Yang et al., 2004, 2005). Ipt also has promise as an antiaddiction agent based on its ability to inhibit cocaine challenge-induced enhancement of dopamine levels in the nucleus accumbens (Liu et al., 2003). It is water-soluble, penetrates the blood-brain barrier, and has a low side effect profile when administered systemically (Wang, 2003; Wang et al., 2004), thus exhibiting features of a useful medicinal. Concentration-dependent opening of KATP channels has been reported in vivo and in vitro animal experiments and indicates effects on KATP channels in vitro that are evident but not maximal at 1 µM, concentrations for protection from glutamate toxicity with IC50 values of approximately 10 µM, behavioral effects in hypertensive animals occur at approximately 1 mg/kg, and a 0.3 mg/kg dose in mice produces a brain concentration of 2.25 µg/g or 10 µM (Wang, 2003; Wang et al., 2004). Although findings remain incomplete, indications are that Ipt exerts its pharmacological effects and actions on KATP channels in the low micromolar range (Wang, 2003; Wang et al., 2004, 2005; Yang et al., 2005). Thus far, no data are available about human tissue concentrations. Mechanisms involved in actions of Ipt in cardiac and central nervous systems are thought to be through the opening of cytoplasmic and/or mitochondrial KATP channels (Wang, 2003; Wang et al., 2004), but the relevant, direct experimental evidence to support this notion is still missing. In the present investigation, we demonstrated Ipt block of human 42-nAChRs independent of regulation of KATP channel function, thus tangibly clarifying our understanding of pharmacological bases for Ipt action in various in vivo and in vitro studies. For example, it is possible that the inhibition by Ipt of cocaine challenge-induced enhancement of dopamine levels in the nucleus accumbens in vivo (Liu et al., 2003) could reflect, at least in part, Ipt block of midbrain nAChRs. Indeed, systematic administration of the nicotinic antagonist, mecamylamine, reduces cocaine self-administration in rats (Levin et al., 2000). Furthermore, Ipt-induced neuroprotective effects may involve interaction with nAChRs. Nicotine and other nAChR agonists have neuroprotective actions in vivo and in vitro by unknown mechanisms, but cytoprotection often requires exposure to nicotine for up to 24 h (Jonnala and Buccafusco, 2001). Chronic exposure of neural cells to nAChR agonists or selected antagonists increases expression of nAChR-like radioligand binding sites (up-regulation) (Gentry and Lukas, 2002; Lopez-Hernandez et al., 2004), which may play a neuroprotective role.
Known effects of Ipt on cardiovascular function also may involve interaction with nAChRs. Tobacco smoking is a strong risk factor for cardiovascular morbidity, including accelerated atherosclerosis and increased risk of heart attacks. The nicotinic antagonist mecamylamine was initially developed as an effective antihypertensive drug in the 1950s (Young et al., 2001) and could have revisited dual utility in control of blood pressure variability and atherogenetic lipid profiles and as an aid to cessation in smokers with mild-to-moderate hypertension (Shytle et al., 2002). Although 3*- and 7-, but not 4*-nAChR subtypes, are found there, blockade of autonomic ganglionic nAChR function plays an important role in regulation of cardiovascular function (Ayajiki et al., 1998; Naguib and Magboul, 1998), so studies of the effects of Ipt on 3-containing nAChR are warranted, because it or ligands like it could be exploited as cardiovascular medicines. Systematic comparisons of effects of Ipt on an extended group of nAChR subtypes are ongoing to clarify these issues. At a minimum, Ipt has utility as another agent for characterization of nAChR subtypes.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; Ipt, iptakalim hydrochloride; ACh, acetylcholine; RJR-2403, (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine; DHE, dihydro--erythroidine; P1075, N-cyano-N'-(1,1-dimethylpropyl)-N'-3-pyridylguanidine; GDPS, guanosine 5'-O-(2-thio)diphosphate.
Address correspondence to: Dr. Jie Wu, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013-4496. E-mail: jwu2{at}chw.edu
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; Is/Ip, 
; 
; ordinate) are plotted against the time of washout of 50 µM Ipt (abscissa, minutes) for responses to 3 µM nicotine. The horizontal dashed line at 100% represents the value for the parameters for responses to 3 µM nicotine before exposure to Ipt. **, p < 0.01 compared with 50 µM Ipt-induced inhibition.
), or upon coapplication with 50 µM Ipt (
) or 50 µM (
), mecamylamine (
) (coincubation, 
), C (
