jun hu et al- iptakalim as a human nicotinic acetylcholine receptor antagonist

8/3/2019 Jun Hu et al- Iptakalim as a Human Nicotinic Acetylcholine Receptor Antagonist

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Iptakalim as a Human Nicotinic Acetylcholine ReceptorAntagonist

Jun Hu, Kari Lindenberger, Gang Hu, Hai Wang, Ronald J. Lukas, and Jie Wu

Divisions of Neurology (J.H., J.W.) and Neurobiology (K.L., R.J.L.), Barrow Neurological Institute, St. Josephs Hospital andMedical Center, Phoenix, Arizona; Department of Pharmacology, Nanjing Medical University, Nanjing, Peoples Republic ofChina (G.H., J.H., J.W.); and Department of Cardiovascular Pharmacology, Institute of Pharmacology and Toxicology, Beijing,Peoples Republic of China (H.W., J.W.)

Received August 30, 2005; accepted October 11, 2005

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) play many critical

roles in nervous system function and have been implicated in avariety of diseases. Drugs acting at nAChRs, perhaps in nAChRsubtype-selective manners, can be used to dissect receptorfunction and perhaps as medications. In the present study, weused patch-clamp whole-cell recording and pharmacologicalmanipulations to evaluate effects of iptakalim hydrochloride(Ipt), which is a drug reported to act as an ATP-sensitive po-tassium (K

ATP ) channel opener, on selected human nAChRs

heterologously expressed in the native nAChR-null SH-EP1human epithelial cell line. Ipt reduced both peak and steady-state whole-cell current amplitudes mediated by human 42-nAChRs in response to nicotinic agonists. It also accelerated

current decay, caused a decline in apparent efficacy of ago-

nists, and acted in voltage- and use-dependent manners at42-nAChRs. These findings and the inability of Ipt to blockradiolabeled epibatidine binding to 42-nAChRs suggest anoncompetitive mechanism of antagonism. Other studies dis-count effects of Ipt on nAChR internalization or involvement ofKATP channels in Ipt-induced inhibition of 42-nAChR func-tion. By comparison, 7-nAChRs were less sensitive than42-nAChRs to Ipt acting as an antagonist. Thus, 42-nAChRs are among the molecular targets of Ipt, which hasutility as a tool in functional characterization and pharmacolog-ical profiling of nAChRs.

nAChRs are prototypical members of the ligand-gated ionchannel superfamily of neurotransmitter receptors, and theyrepresent both classic and contemporary models for the es-tablishment of concepts pertaining to mechanisms of drugaction, synaptic transmission, and structure and function oftransmembrane signaling molecules (Lukas et al., 1999).Mammalian nAChRs exist as a diverse set of molecules com-posed of different combinations of multiple subunits encodedfrom a family of at least 16 genes (17, 910, 14, , ,and ). They play a variety of critical roles in nervous systemfunction and have been implicated in a number of neuropsy-

chiatric 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; Go-palakrishnan et al., 1996). 42-nAChRs bind nicotine withhigh affinity and respond to levels of nicotine found in theplasma of smokers (Fenster et al., 1997). 42-nAChRs alsohave been implicated in nicotine self-administration and indisorders such as Alzheimers disease, Parkinsons disease,and epilepsy (Cordero-Erausquin et al., 2000; Nakamura etal., 2001; ONeill et al., 2002; Quik, 2004). Knockout micelacking expression of 42-nAChRs fail to show nicotinicagonist-induced increases in striatal dopamine release ormidbrain 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 of4-containing nAChRs is sufficient fornicotine-induced reward, tolerance, and sensitization (Tap-per et al., 2004). Therefore, brain 42-nAChRs seem to playpivotal roles in mediation of nicotinic reinforcement and de-pendence. Furthermore, their loss, for example, in Alzhei-mers disease (Burghaus et al., 2000), may play a role indisease onset or progression.

However, the existence of nAChRs as a diverse family of

This work was supported by Barrow Neurological Institute Womens BoardFoundation (to J.W.), in part by endowment and capitalization funds from theWomens Boards of the Barrow Neurological Foundation (to R.J.L.), and bygrants from the Arizona Disease Control Research Commission (9615 and1-353, to R.J.L.) and the National Institutes of Health (NS40417 andDA015389, to R.J.L. and J.W.).

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.105.094987.

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.

0022-3565/06/3162-914925$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 2Copyright 2006 by The American Society for Pharmacology and Experimental Therapeutics 94987/3071780JPET 316:914925, 2006 Printed in U.S.A.

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subtypes has complicated their characterization. Differencesin ligand sensitivity of nAChR subtypes affords an opportu-nity to dissect roles of receptors in general and nAChR sub-types in particular, and, reciprocally, nAChR subtype phar-macological profiling provides a means for discriminatingroles of these subtypes in functions in health and disease.Drugs acting safely in mammals at nAChRs could be medi-cation candidates, and perhaps the effects of some com-

pounds known to be safe on brain or body function couldinclude interactions at nAChRs.

Iptakalim hydrochloride (Ipt) was initially designed andsynthesized as a novel antihypertensive drug (Wang, 1998).It is a small, water-soluble molecule that freely penetratesthe blood-brain barrier and has minimal toxic side effectsafter long-term systemic administration (Wang, 2003; Wanget al., 2004). Possible pharmacological mechanisms underly-ing its antihypertensive action include KATP channel activa-tion and endothelin antagonism (Wang, 2003). Tests in a variety of in vivo and in vitro ischemia and Parkinsonsdisease models indicate that Ipt has neuroprotective effects(Wang et al., 2004, 2005; Yang et al., 2004, 2005). Cardiovas-

cular and central effects of Ipt are thought to be mediated byopening of cytoplasmic and/or mitochondrial KATP channels(Wang, 2003; Wang et al., 2004). In contrast, neuroprotectiveeffects of Ipt also seem to be mediated by direct blockade ofpostsynaptic, ionotropic glutamate receptor function in cul-tured rat hippocampal neurons (Wang et al., 2004) and byenhancement of glutamate transporter function to increaseglutamate uptake (Wang et al., 2004; Yang et al., 2005). Bothmechanisms lead to a reduction of glutamate-induced neuro-toxicity. Furthermore, Ipt has potential in prevention of drugaddiction because it inhibits cocaine challenge-induced en-hancement of dopamine release in the rat nucleus accumbens(Liu et al., 2003).

In the present study, we have used patch-clamp recordingtechniques and pharmacological manipulations to evaluateeffects of Ipt on selected human nAChRs heterologously ex-pressed in the SH-EP1 cell-line. We found that Ipt inhibitshuman 42-nAChR function selectively relative to its ef-fects on 7-nAChR function through a noncompetitive mech-anism independent of effects on KATP channels.

Materials and Methods

Expression of Human Neuronal 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 intopcDNA3.1-zeocin or -hygromycin vectors, respectively, were intro-duced using established techniques (Puchacz et al., 1994; Peng et al.,1999) into native nAChR-null SH-EP1 cells (Lukas et al., 1993) tocreate the SH-EP1-h42 cell line. Cells were maintained as lowpassage number (126 from our frozen stocks) cultures in mediumaugmented with 0.5 mg/ml zeocin and 0.4 mg/ml hygromycin andpassaged once weekly by splitting just-confluent cultures 1/10 tomaintain cells in proliferative growth.

Epibatidine Binding Competition Studies. Membrane prepa-rations were processed and radioligand binding competition assayswere conducted as described previously (Eaton et al., 2003), takingspecial care to ensure that reaction mixtures were not ligand-de-pleted by conducting assays with no more than 25 fmol of bindingsites and 400 pM [3H]epibatidine (PerkinElmer Life and AnalyticalSciences, Boston, MA) in an 800-l volume containing competing

ligand at various concentrations. Data were plotted, analyzed, and

tested for statistical significance using Prism (GraphPad SoftwareInc., San Diego, CA).

Patch-Clamp Whole-Cell Current Recordings and Data Ac-

quisition. Conventional whole-cell current recording coupled withtechniques for fast application and removal of drugs (U-tube) wereapplied in this study as described previously (Zhao et al., 2003; Wuet al., 2004a,b). In brief, transfected cells plated on 35-mm culturedishes without poly(lysine) coating were placed on the stage of aninverted 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 andextracellular solutions) were used to form tight seals (1 G) on thecell surface until suction was applied to convert to conventionalwhole-cell recording and a period of 5 to 10 min lapsed to allow fullexchange between the pipette solution and the cytosol. Thereafter,recorded cells were lifted off the bottom of the culture plate, whichallows for improved kinetics of solution exchange and truer assess-ment of the kinetics of agonist-induced whole-cell currents. Beforecapacitance and series resistance compensation, access resistance(Ra) was measured and accepted for further experimentation if lessthan 20 M. Both pipette and whole-current capacitance were min-imized, 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 weremeasured (200B amplifier; Molecular Devices, Sunnyvale, CA). Cur-rent 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 com-puter hard drive. Data acquisition and analyses were done usingpClamp9.0 (Molecular Devices), and results were plotted using Ori-gin 5.0 (OriginLab Corp., North Hampton, MA). All experimentswere performed at room temperature (22 1C).

Solutions and Drug Application. The standard external solu-tion 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 mostexperiments, nicotine was used as the test agonist. In some experi-ments, acetylcholine (ACh) and RJR-2403 were applied. Atropinesulfate (1 M) was always added to ACh-containing standard exter-

nal solution to exclude any possible influences of muscarinic recep-tors. For conventional whole-cell current recordings, the pipette so-lution 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, pH7.3. To initiate whole-cell current responses, nicotinic agonists wererapidly delivered to the recorded cell by a computer-controlled U-tube system, allowing the applied drug to completely surround therecorded cell within 20 ms. The interval between drug applications (3min) was optimized specifically to ensure stability of nAChR respon-siveness (without functional rundown). Drugs used in the presentstudy were ()-nicotine, ACh, cytisine, choline, dihydro--erythroi-dine (DHE), mecamylamine, tolbutamide, and guanosine 5-O-(2-thio)diphosphate (GDPS) trilithium salt (Sigma Chemical, St.Louis, MO). RJR-2403, pinacidil, P1075, diazoxide, and gliben-clamide were purchased from Tocris Cookson Inc. (Ellisville, MO).

Ipt was kindly provided by Dr. H. Wang (Institute of Pharmacologyand Toxicology, Beijing, Peoples Republic of China). The chemicalstructure of iptakalim [N-(1-methylethyl)-1,1,2-trimethyl-pro-pylamine hydrochloride] is shown in Fig. 1. Because effects of Iptindicated enhanced functional inhibitory potency with prolonged orrepeated applications, data collection was more laborious than usualbecause many studies required recording from cells only through asingle exposure to the ligand.

Fig. 1. Chemical structure of iptakalim hydrochloride.

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Data Analysis and Statistics. nAChR whole-cell current re-

sponses 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) et/] Is. Data

usually were fit over the 10 to 90% period from inward current

peak until agonist exposure was terminated (4 s). The experimen-

tal data are presented as means standard errors. Statistical

analysis was done using paired t-tests comparing the data ob-

tained from a single cell or Students t test (unpaired values) or

one-way analysis of variance with Duncans 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 (Or-

igin software 5.0; OriginLab Corp.) using the logistic equation to

provide fits for maximal and minimal responses, the EC50 or IC50value, and Hill coefficients.

Results

Ipt Blocks 42-nAChR-Mediated Whole-Cell Cur-

rents. Relative to stable whole-cell current responses elicited

during challenge exposure to 3 M (EC50 concentration)nicotine alone, nicotine-induced inward currents mediated

via h42-nAChRs in the same SH-EP1-42 cell were re-

duced if assessed during coapplication with 3 M Ipt (Fig.

2A). In the presence of 3 M Ipt, the current peak, the ratioof 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 M) caused long-lasting inhi-

bition on 42-nAChR function (Fig. 2C). For example, thehalf-time for recovery of peak current response to nicotine

Fig. 2. Ipt inhibits 42-nAChR-medi-ated whole-cell currents. A, underwhole-cell voltage-clamp recording con-ditions, rapid application of 3 M nico-

tine through a U-tube to the recordedcell induced an inward current consist-ing of peak (Ip) and steady-state (Is)components (left trace). Coapplicationof 3 M Ipt and nicotine reduced bothpeak and steady-state current compo-nents and accelerated the decay fromthe peak to the steady-state current(middle trace). The whole-cell traces ofnicotinic responses with and withoutIpt are superimposed (right traces). Forthese and all subsequent traces, cur-rent amplitude and time calibrationbars are indicated, the VH was60 mVunless indicated otherwise, and the du-ration of ligand exposure is indicated bythe bars above the trace. B, statisticalanalysis shows that 3 M Ipt exposuresignificantly inhibits 42-nAChRfunction represented (ordinate, normal-ized values to those in the presence of 3M nicotine alone) as peak whole-cellcurrent (Ip, shaded bar), the ratio be-tween peak and steady-state currents(Is/Ip, solid bar), and the decay constant(, cross-hatched bar) for the decline ininward current from peak to steady-state levels. In this and all followingfigures, unless specifically mentioned,each column (or symbol) is the averagefrom four to sixcells tested, and verticalbars indicate S.E. A single asterisk() represents p 0.05, and double as-terisks () represent p 0.01 com-pared with the parameter for responses

to 3 M nicotine (horizontal dashedline). C, time dependence forrecoveryof42-nAChR function from Ipt inhibi-tion after a brief exposure (4 s) andwashout for the indicated period (min-utes) for responses to 3 M nicotine. D,normalized values ( S.E.; n 4 6cells) for the indicated parameters (Ip,f; Is/Ip, ; , F; ordinate) are plottedagainst the time of washout of 50 MIpt (abscissa, minutes) for responses to3 M nicotine. The horizontal dashedline at 100% represents the value forthe parameters for responses to 3 Mnicotine before exposure to Ipt. , p 0.01 compared with 50 M Ipt-inducedinhibition.

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challenge was 15 min, although effects on steady-stateinward current levels and on the time constant for currentdecay recovered more quickly (Fig. 2D). Inhibition by Ipt of42-nAChR responses to different nicotinic agonists alsowas evident. In the presence of EC50 concentrations of ace-tylcholine (10 M), RJR-2403 (10 M), or nicotine (3 M), 50M Ipt produced 66 1, 73 2, or 76 3% block of peakwhole-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. Initialtime dependence assays indicated that, compared with coap-plication, 3-min pretreatment with Ipt produced a more pro-found inhibition of the response to 3 M nicotine (Fig. 3A).Peak amplitudes of 3 M nicotine-induced currents werereduced to 62 6% of control values with 3-min pretreat-ment followed by continued coapplication with agonist plusIpt, to 79 3% without Ipt pretreatment but with coappli-cation with nicotine plus Ipt, and to 83 4% after 3-minpretreatment with Ipt ending before exposure to nicotinealone (Fig. 3B). Steady-state peak current amplitude ratios

and the time required for an e-fold loss from peak currentlevels during current decay rate () were lowest when Ipt wascoapplied with nicotine whether or not there was prior expo-sure to Ipt (Fig. 3B). Effects of Ipt applied at different con-

centrations during a 4-s nicotine exposure only or duringnicotine exposure and after 3-min pretreatment with Iptshow concentration dependence of functional block as well asgreater inhibitory efficiency after pretreatment (Fig. 3C).Concentration-response profiles for inhibition by Ipt of peakwhole-cell responses indicate IC50 values of 5.0 and 31.6 Mwith and without pretreatment, respectively (Fig. 3D). Uponcoapplication of nicotine and Ipt, the IC50 value for Ipt-

mediated inhibition of the whole-cell steady-state current is1.0 M and is lower than the coapplication IC50 value forinhibition of the peak component (Fig. 3E). The more pro-found inhibition after pretreatment suggests that some Iptbinding sites are accessible on nAChRs in the resting state.

Mechanism of Ipt Block of42-nAChR Function. Toexplore the nature of Ipt functional block, whole-cell currentresponses were obtained in the presence of nicotine alone orwith 10 or 50 M Ipt as shown in Fig. 4A. Assessment ofwhole-cell peak current amplitudes as a function of nicotineconcentration yielded apparent EC50 values of 5.1 M in thepresence of nicotine alone and 22 or 39 M in the presence ofnicotine plus 10 or 50 M Ipt, respectively (Fig. 4B). Al-

though nicotine up to 1 mM was unable to surmount func-tional block by 10 or 50 M Ipt of peak current responses, themagnitude of the inhibitory effect by Ipt decreased as nico-tine concentration was increased (Fig. 4B). However, the Hill

Fig. 3. Time- and dose-dependent effects of Ipt on 42-nAChR function. A, whole-cell current response traces forresponses to 3 M nicotine alone or in the presence of 3 MIpt for 3 min before and then continuing during 4-s agonistexposure (a), during agonist exposure only (b), or after Ipt

pretreatment terminated at the time of agonist exposure(c). B, bar graphs showing effects on 3 M nicotine-evoked,peak whole-cell currents (Ip), the ratio between steady-state and peak currents (Is/Ip), or the decay constant forrate e-fold decline from peak to steady-state current (), allnormalized to the parameter value for responses to 3 Mnicotine alone (ordinate), under the preplus coincubationcondition (solid bars), the coincubation condition (cross-hatched bars), or the pretreatment alone condition (openbars). , p 0.05 and , p 0.01 compared with 3 Mnicotine-induced current. Results are averages S.E. fromsix cells. C, effects on responses to 3 M nicotine alone(higher amplitude, lighter traces) or with exposure to Ipt atthe indicated concentrations (lower amplitude, darkertraces) are shown in representative traces for studies doneafter 3-min pretreatment with Ipt continuing during ago-nist application (a) or during coapplication of Ipt with

agonist (b). To prevent complications because of long-last-ing residual inhibition by Ipt, each pre- and post-Ipt expo-sure study was done using a different cell. D, curves show-ing effects of the indicated concentration of Ipt (abscissa,log micromolar scale) under the coincubation (no pretreat-ment; f) or 3-min pretreatment (F) conditions on peakcurrent responses to 3 M nicotine (ordinate, normalizedamplitude; mean S.E.; n 6 cells). E, concentration-inhibition relationships for effects of coincubation with Iptat the indicated concentrations (abscissa, log micromolarscale) on peak (f) and steady-state (F) components (ordi-nate, normalized amplitudes; mean S.E.; n 6 cells).



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slope for nicotine-elicited peak current responses becamemore shallow in the presence of higher concentrations of Ipt,inconsistent with a purely competitive mechanism of func-tional block (Fig. 4B). Moreover, the relative inhibition by Iptof steady state to peak current responses increased as nico-tine concentration increased (Fig. 4C).

To illuminate mechanisms involved in Ipt-induced func-tional inhibition, radioligand binding assays indicated that[3H]epibatidine binding was blocked by nicotine or DHEwith 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 radioligandbinding at concentrations up to 100 M (Fig. 5). Collectively,these results suggest that Ipt does not act on agonist bindingsites (i.e., via a purely competitive mechanism) to exert itsinhibition of42-nAChR function.

Voltage Dependence of Ipt Block of42-nAChR

Function. When ionized ligands act to block ion channels,their residence in the transmembrane region is affected bytransmembrane potential. Therefore, transmembrane volt-age dependence is one of the characteristic features of open-channel block by charged ligands. Whole-cell current re-sponses to nicotine alone and in the presence of 50 M Ipt

recorded at different holding potentials (VH; Fig. 6A) indicate

that Ipt exerts stronger inhibition of nicotinic responses atmore negative holding potentials (Fig. 6B). Fractional inhi-bition by Ipt of whole-cell peak current responses to nicotinewas 81 3, 76 3, and 69 4% at holding potentials of80,40, and 0 mV, respectively, and current decay constantswere 22 3, 30 1, and 44 8% of control, respectively,reaching significance for differences between VH 80 and0 mV (Fig. 6B; p 0.05; n 6). These results indicate thatthe inhibitory effects on the 42-nAChR function by Ipt arevoltage-dependent.

Use Dependence of Ipt Block of42-nAChR Func-tion. Under conditions where whole-cell current responses ofSH-EP1-h42 cells to repetitive applications of nicotine for4 s at 3-min intervals showed no significant response run-down (data not shown), six repetitive applications of nicotine(3 M) in the continuous presence of 3 M Ipt resulted in agradual reduction of nicotinic responses (Fig. 7Aa). However,after 15 min of Ipt pretreatment without repeated applica-tion of nicotine, there was less inhibition of a 3 M nicotine-induced response (Fig. 7Ab). That is, under conditions wherethe initial peak current response to nicotine in the presenceof coapplied Ipt was reduced relative to the response to nic-otine alone by the same amount (17 6 or 14 2%;p 0.05;

n 6), the response to nicotine challenge after 15 min of 3

Fig. 4. Investigating mechanism(s) of Ipt block of42-nAChR function. A, representative whole-cell current traces for responses to nicotine alone(higher amplitude, lighter traces) or during coapplication with 10 M Ipt (a) or 50 M Ipt (b) (lower amplitude, darker traces) are shown for studiesusing a different cell for each agonist-alone and agonist-Ipt pair. B, concentration-response curves for responses to nicotine at the indicatedconcentrations [abscissa, mean S.E. for peak currents normalized to that in the presence of 100 M nicotine alone (#); n 6 cells; log micromolar

scale] alone (), upon coapplication with 10 M Ipt (), or upon coapplication with 50 M Ipt (F). C, comparison of the extent of inhibitory effects ofIpt on steady-state relative to peak whole-cell currents (ordinate, percentage of control response to nicotine alone) as a function of nicotine challengeconcentration (abscissa, log molar scale) for coexposure to 10 M () or 50 M (F) Ipt.

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M Ipt exposure during repeated nicotine challenges wasonly 12 2% of control compared with 30 3% of control forthe response to nicotine challenge after 15 min of Ipt expo-

sure in the absence of intervening nicotine challenges (p 0.01; n 6; Fig. 7B). These results indicate that Ipt-mediatedblock of42-nAChR function is use-dependent.

Ipt Block of42-nAChRs Is Not Due to Opening of

KATP Channels. Ipt was initially designed as a novel KATPchannel opener, and until now its pharmacological effects onthe cardiovascular system and central nervous system func-tion have been thought to involve the opening of KATP chan-

nels (Wang, 2003; Wang et al., 2004, 2005). Because sucheffects could confound whole-cell current recording-based as-sessments of effects on 42-nAChR function, we testedwhether other KATP channel openers, such as pinacidil,P1750, and diazoxide shared the ability of Ipt to inhibit42-nAChR-mediated currents. At concentrations of 3 M,neither the relatively selective cytoplasmic membrane KATPchannel opener P1075 (Fig. 8Aa), the relatively selectivemitochondrial KATP channel opener diazoxide (Fig. 8Ab), northe cyanoguanidine KATP channel opener pinacidil (Fig.8Ac), inhibited nicotine-induced currents, although 3 M Ipthad a clear effect on the nicotine-evoked steady-state re-sponse and also significantly reduced the peak current re-

sponse (Fig. 8Ad

). These findings indicate that KATP channelopeners generally failed to mimic Ipt-induced inhibition of42-nAChR-mediated currents (Fig. 8B). We also assessedwhether classic KATP channel blockers were able to abolishIpt-induced inhibition of 42-nAChR function. Neither ofthe classic KATP channel blockers, glibenclamide (30 M) nortolbutamide (100 M), prevented Ipt-induced inhibition of

Fig. 5. Ligand competition profiles for blockade of specific [3H]epibatidinebinding to human 42-nAChR. Reaction mixtures containing SH-EP1-h42 cell membrane preparations (typically containing 1 to 5 g ofprotein), 400 pM [3H]epibatidine, and either nicotine (f), DHE (),mecamylamine (F), or Ipt (E) (coincubation, , 3-min preincubation) atthe indicated concentrations (abscissa, molar log scale) were used toassess the concentration dependence for competition toward specific[3H]epibatidine binding (ordinate, percentage of control). Results are theaverages of at least three separate experiments.

Fig. 6. Ipt inhibits nicotinic responses in a voltage-dependent manner. A, representative traces are su-perimposed for whole-cell current responses of SH-EP1-h42 cells to 3 M nicotine alone (largeramplitude, lighter traces) or during coapplicationwith 50 M Ipt (lower amplitude, darker traces) atthe indicated VH. B, voltage-dependent effects (ab-scissa) of coapplication of 50 M Ipt with 3 Mnicotine on peak currents (closed bars) and the cur-rent decay constant (; open bars) are complied (or-

dinate, normalized to the value for the parameter inthe presence of 3 M nicotine alone; mean S.E.;n 6 cells). , p 0.05 versus VH 80 mV.



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42-nAChR function (Fig. 8, C and D). We also found that30 M glibenclamide or 100 M tolbutamide alone inhibited42-nAChR function by 38 4 and 19 2%, respectively(data not shown). Collectively, whereas these findings indi-

cate that there may be some degree of interaction of agentsinitially 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 anyKATP channels that may be expressed in the SH-EP1 cell line.

Ipt Block of42-nAChRs Does Not Occur via Intra-

cellular Access. To assess whether Ipt exerts its action on42-nAChR via accessing intracellular sites, we directlyapplied it into the cell through the whole-cell recording pi-pette. In the absence of such application, repetitive extracel-lular applications of 3 M nicotine induced five stable re-sponses (2-s exposures at 15-s intervals; Fig. 9A). However,the response was gradually eliminated when nicotine and 3

M Ipt were coapplied extracellularly through the U-tube to

the same recorded cell (Fig. 9B). In the presence of 100 MIpt in the recording pipette, after conversion to the whole-cellrecording configuration for more than 20 min to allow fullloading of Ipt into the cell, there was no decline in responses

to five repetitive applications of nicotine (Fig. 9C). In thesame recorded cell, 3 M Ipt coapplied through the U-tubecaused an obvious nicotinic response reduction (Fig. 9D).When assessed collectively (Fig. 9E), these findings make itclear that access to intracellular sites is not involved inmediation of Ipt-induced block of42-nAChRs.

Effects of Internalization Block on Ipt-Mediated In-

hibition of 42-nAChR Function. Given evidence thatendocytosis or exocytosis of several kinds of transmembranereceptor occurs on the order of seconds to minutes throughprocesses modulated by agonists and antagonists, we ascer-tained effects of Ipt in cells preloaded with 600 M GDPSthrough the patch-clamp electrode for 20 min after convert-

ing to the whole-cell recording configuration. Treatment with

Fig. 7. Ipt inhibits nicotinic responsesin a use-dependent manner. A, SH-EP1 cells expressing 42-nAChRswere repetitively challenged with 3M nicotine (4-s exposure at 3-minintervals) in the continuous presenceof 3 M Ipt for 15 min (a) or nicotinewas applied for 4 s at the beginningand at the end of 15-min exposure to 3M Ipt (b) comparing sets of re-sponses recorded from different cells.B, bar graph compares effects of Iptduring repetitive exposure to nicotine(a; solid bars) or at the start and endof nicotine exposures (b, open bars) forwhole-cell peak current responses (or-dinate, normalized to responses in theabsence of Ipt) to nicotine at the onset(0 min) or at the end (15 min) of theIpt exposure period. , p 0.01 forthe difference in effects assessed un-der the two different nicotine chal-lenge protocols.

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GDPS has been reported to prevent -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor or GABAA receptorinternalization (Luscher et al., 1999; Blair et al., 2004). Pre-loading with GDPS for 20 min itself neither affected nico-tine-induced currents (Fig. 10A) nor affected Ipt-induced in-hibition of 42-nAChR function (Fig. 10, B and C). Theseresults argue against a role for 42-nAChR internalizationin the mechanism of Ipt functional antagonism.

Ipt More Selectively Blocks 4- Than 7-Containing

nAChRs. When effects of Ipt on function of heterologouslyexpressed human 42-, 44-, or 7-nAChRs in SH-EP1

cell lines were evaluated, 50 M Ipt exhibited similar inhi-bition of 42- or 44-nAChR-mediated currents butshowed little inhibition of7-nAChR-mediated current (Fig.11, A and B). However, Ipt-mediated functional block per-sisted for 42- but not 44-nAChR after 3 min of drugwashout (Fig. 11, A and B). Concentration-inhibition curvesfor Ipt effects on peak current amplitudes demonstrate selec-tive inhibition of 4-containing nAChR function (Fig. 11C).Ipt IC50 values for acute coapplication with agonist were 31and 23 M, respectively, for 42- and 44-nAChR func-tional inhibition but higher than 1 mM for 7-nAChR block-ade. Even at lower concentrations of choline closer to its EC50value for activation of7-nAChRs, and after brief pretreat-

ment of cells with 10 M Ipt, Ipt exerted more profound

inhibition of 42-nAChR-mediated currents (70 11%)than of7-nAChR-mediated currents (3.5 0.4%; Fig. 11, Dand E).

Discussion

Ipt Blocks Human 42-nAChR Function. The princi-pal finding of this study is that Ipt acts with some nAChRsubtype selectivity as an antagonist of human 42-nAChRfunction. Ipt inhibits both peak and steady-state whole-cellcurrent responses of heterologously expressed human 42-

nAChR to nicotinic agonists and accelerates whole-cell cur-rent decay in a concentration-dependent manner whilehaving long-lasting effects after washout. Its effects are en-hanced by exposure to nAChR before agonist challenge andare not because of GDPS-regulated nAChR internalizationor effects on KATP channels. Use and voltage dependence ofIpt effects at 42-nAChR as well as its inability to blockbinding of [3H]epibatidine argue against a competitive mech-anism of functional blockade and suggest noncompetitiveinteraction with 42-nAChR. Ipt is more potent as an an-tagonist of42- or 44-nAChR than of7-nAChR, and itseffects are longer lasting at 42- than at 44-nAChR.

Mechanism of Ipt Action. Means for deciphering mech-

anisms involved in ligand-gated ion channel antagonism, and

Fig. 8. Effects of KATP channel ligands on 42-nAChR-mediated currents and their sensitivity to Ipt. A, representative whole-cell current traces forresponses to 3 M nicotine alone (ad) or in the presence of 3 M coapplied KATP channel openers P1075 (a), diazoxide (b), pinacidil (c), or Ipt (d)and recorded from different cells. B, bar graph compares the effects of pinacidil, P1075, diazoxide, or Ipt on peak (filled bars) and steady-state (openbars) whole-cell current responses (ordinate, normalized to responses to nicotine alone; mean S.E.; n 5 cells) to 3 M nicotine. p 0.05; , p 0.01. C, representative whole-cell current responses to 3 M nicotine alone (left traces each row) or in the presence of 30 or 100 M coapplied classicKATP channel blockers glibenclamide (a) (Gli) or tolbutamide (b) (Tol) (right traces each row). D, bar graph compares the effects of KATP channelblockers at 30 M Gli and 100 M Tol on Ipt-induced inhibition of peak (filled bars) and steady-state (open bars) components of nicotinic responses., p 0.01 compared with response to nicotine (Nic) alone. #, no significant difference between indicated pairs.



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interpretations of the results obtained, continue to becomemore refined. As a positively charged ligand at pH 7.4, Ipt isexpected to more easily enter the nicotinic channel pore at80 mV than at 0 mV, reasonably explaining voltage depen-dence of its effects, although it is possible that the bindingsite is in the transmembrane field other than in the open

channel. Use dependence indicates that Ipt-mediated blockinvolves agonist-induced transition to a nAChR conformationhaving higher affinity for the antagonist and/or allowingfreer access of it to its binding site (Zhao et al., 2004), and itis reasonable to conclude that this transition is to the openchannel state. When Ipt was coapplied with nicotine, therising phase of the whole-cell current response was not al-tered, but the decay phase was dramatically accelerated,again suggesting an open channel block. However, pretreat-ment with Ipt induced more profound inhibition of the nico-tinic response, suggesting that Ipt binding sites are at leastpartially accessible in the resting, closed state. Perhaps thereare 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 occupancyat a given concentration less than complete, but once recep-tors are exposed to Ipt, it itself induces conversion to a statewhere binding site affinities for Ipt are more uniformlyhigher (Hill coefficients are 1 for the Ipt pretreatmentcondition), as is fractional occupancy.

Interestingly, although effects of Ipt on peak current am-plitudes were reduced in the presence of higher concentra-tions of nicotine, the ratio of steady-state/peak current re-sponses in the presence of Ipt was dramatically decreased. Areasonable interpretation is that channel activation rate isslow and channels activate asynchronously at low nicotineconcentrations, giving Ipt enough time to block the channel.This is consistent with the observed larger fractional block ofthe peak current and minimal effect on its steady-state com-ponent in the presence of low nicotine concentrations,whereas at very high concentrations of nicotine, the agonistassociation rate (binding on-rate) and channel activation rateshould be much faster than the association with Ipt (at a

fixed concentration), so more channels will be activated syn-

Fig. 9. Intracellular application of Ipt did not in-hibit 42-nAChR function. A to D, whole-cell cur-rent responses of SH-EP1-h42 cells to repetitiveapplication of 3 M nicotine (2-s exposure at 15-sintervals) were recorded in the absence of Ipt (A),during coapplication with 3 M Ipt (B), or after 100M Ipt was diffused into the recorded cell over 20min following establishment of a whole-cell patch inthe absence (C) or in the presence (D) of 3 M Iptcoapplied with nicotine extracellularly through theU-tube. E, plot summarizing effects of Ipt deliveredunder conditions specified in A (f), B (), C (), orD (F) above on peak current responses (ordinate,normalized to the control response to 3 M nicotinealone; mean S.E.; n 6 cells) as a function ofnicotine challenge number in the sequence (ab-scissa).

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chronously before block, which is evident by the observationof lower fractional inhibition of the peak current and greaterblock of steady-state current responses and acceleration of42-nAChR desensitization by Ipt at high nicotine concen-trations. Furthermore, other experiments failed to show anydetectable competition of Ipt for [3H]epibatidine binding, ar-guing against competitive block. Collectively, these findingssuggest that Ipt has access to the receptor in the resting statebut 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/ormitochondrial KATP channel opener that exerts various phar-macological effects in cardiovascular and central nervoussystems (Wang, 2003; Wang et al., 2004, 2005; Yang et al.,2004, 2005) through mechanisms that could indirectly mod-ulate nAChR function. However, other agents that open orblock KATP channels neither mimicked nor blocked effects ofIpt, suggesting that Ipt has its effects by direct action at4-containing nAChRs. We also found that 30 M gliben-clamide or 100 M tolbutamide alone inhibited 42-nAChRfunction, respectively. Interestingly, coapplication of gliben-

clamide or tolbutamide with Ipt did not further increase the

inhibition, implying that these KATP channel blockers mayact on the same site as Ipt, but detailed mechanisms need tobe further elucidated. Inhibition of42-nAChR by Ipt doesnot occur after direct perfusion of Ipt into a recorded cell,suggesting that intracellular sites are not involved in medi-ation of Ipt-induced block of42-nAChRs. Ipt does not seemto promote internalization of cell surface nAChR, at least viathe GDPS-sensitive mechanism previously shown to be in-volved 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 de-signed and synthesized as a KATP channel opener (Wang,2003) and has been shown to be an effective counteragent ina variety of animal models, including those for ischemia/hypoxia and Parkinsons disease (Wang, 2003; Wang et al.,2005; Yang et al., 2004, 2005). Ipt also has promise as anantiaddiction agent based on its ability to inhibit cocainechallenge-induced enhancement of dopamine levels in thenucleus 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

Fig. 10. Effects of intracellular GDPS on Ipt-mediatedinhibition of42-nAChR function. A and B, representa-tive whole-cell current responses to 3 M nicotine alone (A)or in the presence of coapplied 3 M Ipt (B) were recordedafter 600 M GDPS was loaded into the cell during 20min after conversion to the whole-cell recording configura-tion. C, plot summarizing effects of 3 M Ipt on responsesto 3 M nicotine (ordinate, peak currents normalized tothose elicited by 3 M nicotine alone; mean S.E.; n 6

cells) during repeated challenges (abscissa, trace/challengenumber) with nicotine and coapplied Ipt in control cells (E)or in GDPS-preloaded cells (f).



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et al., 2004), thus exhibiting features of a useful medicinal.Concentration-dependent opening of KATP channels has beenreported in vivo and in vitro animal experiments and indi-cates effects on KATP channels in vitro that are evident butnot maximal at 1 M, concentrations for protection fromglutamate toxicity with IC50 values of approximately 10 M,behavioral effects in hypertensive animals occur at approxi-mately 1 mg/kg, and a 0.3 mg/kg dose in mice produces a

brain concentration of 2.25 g/g or 10 M (Wang, 2003;Wang et al., 2004). Although findings remain incomplete,indications are that Ipt exerts its pharmacological effects andactions 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 concen-trations. Mechanisms involved in actions of Ipt in cardiacand central nervous systems are thought to be through theopening of cytoplasmic and/or mitochondrial KATP channels(Wang, 2003; Wang et al., 2004), but the relevant, directexperimental evidence to support this notion is still missing.In the present investigation, we demonstrated Ipt block ofhuman 42-nAChRs independent of regulation of KATP

channel function, thus tangibly clarifying our understanding

of pharmacological bases for Ipt action in various in vivo andin vitro studies. For example, it is possible that the inhibitionby Ipt of cocaine challenge-induced enhancement of dopa-mine levels in the nucleus accumbens in vivo (Liu et al.,2003) could reflect, at least in part, Ipt block of midbrainnAChRs. Indeed, systematic administration of the nicotinicantagonist, mecamylamine, reduces cocaine self-administra-tion in rats (Levin et al., 2000). Furthermore, Ipt-induced

neuroprotective effects may involve interaction withnAChRs. Nicotine and other nAChR agonists have neuropro-tective actions in vivo and in vitro by unknown mechanisms,but cytoprotection often requires exposure to nicotine for upto 24 h (Jonnala and Buccafusco, 2001). Chronic exposure ofneural cells to nAChR agonists or selected antagonists in-creases expression of nAChR-like radioligand binding sites(up-regulation) (Gentry and Lukas, 2002; Lopez-Hernandezet al., 2004), which may play a neuroprotective role.

Known effects of Ipt on cardiovascular function also mayinvolve interaction with nAChRs. Tobacco smoking is astrong risk factor for cardiovascular morbidity, including ac-celerated atherosclerosis and increased risk of heart attacks.

The nicotinic antagonist mecamylamine was initially devel-

Fig. 11. nAChR subtype selectivity of Ipt-mediated

inhibition. A, representative whole-cell currents re-sponses of 42-nAChR to 3 M nicotine, 44-nAChR to 1 M nicotine, or 7-nAChR to 10 mMcholine as indicated alone (left trace each row), in thepresence of coapplied 50 M Ipt (middle trace eachrow), or after 3 min of Ipt washout. B, bar graphindicating extent of Ipt-mediated inhibition of function(ordinate, normalized peak current responses to thechallenge agonist alone as in A; mean S.E.; n 6cells) for the indicated nAChR subtype (abscissa) dur-ing coapplication with agonist (50 M Ipt, solid bars)or after 3 min of drug washout (open bars). , p 0.01. C, Ipt concentration (abscissa, log micromolarscale) dependence of inhibition of function (ordinate,peak whole-cell current responses normalized to thosein the absence of Ipt; mean S.E.; n 6 cells) ofhuman 42- (f), 44- (E), or 7- () nAChR when

Ipt is coapplied with agonists as indicated in A. D,representative whole-cell current responses of 42-nAChR to 3 M nicotine (a) or of7-nAChR to 1 mMcholine (b) as indicated alone (left traces) or after 3 minof pretreatment followed by continued exposure withagonist to 10 M Ipt (right traces). E, bar graph indi-cating extent of Ipt-mediated inhibition of function(ordinate, normalized peak current responses to thechallenge agonist alone as in D; mean S.E.; n 6cells) for 42-nAChR (solid bar) responding to 3 Mnicotine or 7-nAChR (cross-hatched bar) respondingto 1 mM choline during 10 M Ipt pretreatment fol-lowed by coapplication with agonist. , p 0.01.

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oped as an effective antihypertensive drug in the 1950s(Young et al., 2001) and could have revisited dual utility incontrol of blood pressure variability and atherogenetic lipidprofiles 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, block-ade of autonomic ganglionic nAChR function plays an impor-tant role in regulation of cardiovascular function (Ayajiki et

al., 1998; Naguib and Magboul, 1998), so studies of the effectsof Ipt on 3-containing nAChR are warranted, because it orligands like it could be exploited as cardiovascular medicines.Systematic comparisons of effects of Ipt on an extended groupof nAChR subtypes are ongoing to clarify these issues. At aminimum, Ipt has utility as another agent for characteriza-tion of nAChR subtypes.

Acknowledgments

We thank Lori Buhlman for preparation of cells and Dr. Yongchang Chang for illuminating discussions.

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Address correspondence to: Dr. Jie Wu, Division of Neurology, BarrowNeurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013-4496. E-mail: [email protected]


jun hu et al- iptakalim as a human nicotinic acetylcholine receptor antagonist

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