Dexmedetomidine, an alpha2-adrenergic agonist inhibits neuronal delayed-rectifier potassium current

NEUROSCIENCES AND NEUROANAESTHESIADexmedetomidine,an a 2-adrenergic agonist,inhibits neuronaldelayed-rectifier potassium current and sodium currentB.-S.Chen 13,H.Peng 2and S.-N.Wu 12*1Institute of Basic Medical Sciences and 2Department of Physiology,National Cheng Kung University Medical College,No.1,University Road,Tainan 70101,Taiwan,Republic of China.3Department ofAnesthesiology,Buddhist Dalin Tzu Chi General Hospital,Chiayi County,Taiwan,Republic of China*Corresponding author.E-mail:snwu@.twBackground.Dexmedetomidine (DEX),a selective agonist of a 2-adrenergic receptors,isrecognized to facilitate analgesia and anaesthesia in humans.Despite the potential for wideuse,its effects on ion currents and membrane potential in neurones remain largely unclear.Methods.W e investigated the effects of DEX on ion channels in NG108-15neuronal cells dif-ferentiated with dibutyryl cyclic AMP and in cultured cerebellar neurones.Results.DEX suppressed the amplitude of delayed rectifier K +current [I K(DR)]in a concen-tration-dependent manner with an IC 50value of 4.6m M in NG108-15cells.No change in thesteady-state inactivation of I K(DR)was evident in the presence of DEX.A minimal bindingscheme was also used to evaluate DEX-induced block of I K(DR).Inhibition of I K(DR)by DEXwas still observed in cells preincubated with yohimbine (10m M)or efaroxan (10m M).DEXdepressed the peak amplitude of Na +current (INa ),whereas it had minimal effect on L-typeCa 2+current.Under current-clamp configuration,DEX increased the duration of action poten-tials (APs).I K(DR)and I Na in response to AP waveforms were more sensitive to block by DEXthan those elicited during rectangular pulses.In isolated cerebellar granule cells,DEX alsoeffectively suppressed I K(DR).Conclusions.The effects of DEX are not limited to its interactions with a 2-adrenergic recep-tors.Inhibitory effects on I K(DR)and I Na constitute one of the underlying mechanisms throughwhich DEX and its structurally related compounds might affect neuronal activity in vivo .Br J Anaesth 2009;103:244–54Keywords :ions,ion channels,pharmacology;ions,ion channels,voltage-gated;model,neuroblastoma cells;pharmacology,dexmedetomidineAccepted for publication:April 7,2009Dexmedetomidine (DEX)is a potent and highly selective agonist of the a 2-adrenergic receptors that is approved by the US Food and Drug Administration for the provision of short-term sedation of adult patients in the intensive care unit.It exerts a variety of actions on the human brain,including sedation,anaesthetic-sparing effects,and analge-sia.12Because this agent has minimal effects on venti-lation,it is relatively safe in the setting of functional neurosurgery.3Its sedative action is thought to involve inhibition of the locus coeruleus and subsequent disinhibi-tion of the ventrolateral preoptic nucleus.4DEX has been reported to decrease the convulsive potency of bupivacaine and levobupivacaine in awake rats.5A recent study showed that as an anaesthetic adjunct,DEX at a high concentration can affect transcranial motor evoked responses.6However,there are limited reports regarding its ionic mechanism of action on neurones.Recent evidence has emerged that the effects on ion channels may be an important mechanism underlying DEX-induced actions in neurones.For example,previous studies demonstrated the ability of DEX to modify L-type Ca 2+current (I Ca,L )in pheochromocytoma cells 7and to inhibit M 3muscarinic receptors in dorsal root ganglion cells.8In paraventricular nucleus neurones or substantia gelatinosa neurones,DEX increases G-protein-coupled inwardly recti-fying K +currentand inhibits hyperpolarization-elicited inward current.910Voltage-gated K +(K V )channels play an essential role in determining the excitability of neurones.#The Author [2009].Published by Oxford University Press on behalf of The Board of Directors of the British Journal of Anaesthesia.All rights reserved.British Journal of Anaesthesia 103(2):244–54(2009)doi:10.1093/bja/aep107Advance Access publication June 20,2009Among them,delayed rectifier K +(K DR )channels are ubi-quitous in neurones.In fast-spiking neurones,a causal relationship between K V 3,especially K V 3.1,and the delayed rectifier K +current [I K(DR)]has been well estab-lished.The K V 3recombinant channel has properties suit-able for the generation of high-frequency firing.High activation threshold (about 220mV)and fast deactivationkinetics of the channels make them ideal for narrowingspike width and inter-spike interval.11The I K(DR)containingK V 3subunits contributes to changes in membrane potential in differentiated NG108-15cells.12–14Previous studies in our laboratory have shown that tra-madol suppresses the amplitude of I K(DR)in NG108-15cells.14If K V 3-encoded K +currents are located at nerve terminals,modulation of current amplitude might be expected indirectly to alter Ca 2+influx and to affect neuro-transmitter release in the absence of overt changes in firing patterns.1115However,whether DEX can interact with I K(DR)or other types of ion currents to modify mem-brane potential in neurones remains largely unclear,despite its clinical use in a variety of neurosurgical pro-cedures.2–4In this study,we investigated the effects of DEX on ion currents (e.g.delayed rectifier K +current [I K(DR)],Na +current [I Na ],and L-type Ca 2+current [I Ca,L ])and mem-brane potential in NG108-15neuronal cells differentiated with dibutyryl cyclic AMP and in cultured cerebellar neur-ones.DEX was able to suppress both I K(DR)and I Na .The major action of DEX on I K(DR)is thought to be through anopen-channel mechanism.These inhibitory effects are not mediated by a 2-adrenergic receptors and might contributeto its pharmacological actions in vivo .Methods Drugs and solutions DEX (dexmedetomidine HCl;(S )-4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole hydrochloride;Precedex w )was obtained from Abbott Laboratories (Abbott Park,IL,USA).Collagenase,dibutyryl cyclic AMP,DNase I,efaroxan,tetraethylammonium chloride,and tetrodotoxin were purchased from Sigma Chemicals (St Louis,MO,USA),and yohimbine was from Tocris (Bristol,UK).Tissue culture media,L -glutamine penicillin–streptomycin,fungi-zone,and trypsin were obtained from Life Technologies (Carlsbad,CA,USA).All other chemicals,including CsCl 2and CdCl 2,were commercially available and of reagent grade.The composition of normal Tyrode’s solution was 136.5mM NaCl,5.4mM KCl,1.8mM CaCl 2,0.53mM MgCl 2,5.5mM glucose,and 5.5mM HEPES–NaOH buffer,pH 7.4.To record I K(DR)or membrane potential,the patch pipette was filled with a solution consisting of 140mM KCl,1mM MgCl 2,3mM Na 2ATP,0.1mM Na 2GTP,0.1mM EGTA,and 5mM HEPES–KOH buffer,pH 7.2.To measure I Na or I Ca,L ,K +ions inside the pipette solution were replaced with equimolar Cs +ions,and the pH was adjusted to 7.2with CsOH.In some experiments,the pipette was filled with the solution which contained 10m M DEX.Cell preparation and differentiationThe clonal strain NG108-15cell line,formed by Sendaivirus-induced fusion of the mouse neuroblastoma cloneN18TG-2and the rat glioma clone C6BV-1,was obtained from the European Collection of Cell Cultures(ECACC-88112302;Wiltshire,UK).Cellswere kept in monolayer cultures at a density of 106ml 21in plastic disks containing Dulbecco’s modified Eagle’s medium supplemented with 100m M hypoxanthine,1m M amino-pterin,16m M thymidine,and 5%fetal bovine serum asthe culture medium,in a humidified incubator equilibrated with 90%air/10%CO 2at 378C.14Media were replenished every 2–3days with fresh media.To induce neuronal differentiation,culture medium was replaced with medium containing 1mM dibutyryl cyclic AMP and cells were cultured in the incubator for 1–7days.16In a separate set of experiments,differentiated NG108-15cells were prein-cubated with yohimbine (10m M)or efaroxan (10m M)for 5h before electrophysiological experiments were performed.Isolation of cerebellar granule cellsCerebellar granule cells were isolated from Wistar rat pupsof post-natal days 9–10.Cerebella of 5–7animals wereexcised after decapitation,and minced and digested withcollagenase (50mg ml 21)for 10min.Mechanical tritura-tion with a Pasteur pipette was made in a DNase I/soybeantrypsin inhibitor solution.Dissociated cells were centri-fuged and suspended in normal Tyrode’s solution.Cells were used for experiments within 2–3h after isolation.Animal experiments were conducted according to the pro-tocols that follow the NIH standards and the guidelines for the Care and Use of Experimental Animals.The procedure was approved by the Animal Care and Use Committee of the National Cheng Kung University,Tainan,Taiwan,Republic of China.Electrophysiological measurementsImmediately before each experiment,cells were disso-ciated and an aliquot of cell suspension was transferred to a recording chamber mounted on the stage of an inverted DM-IL microscope (Leica,Wetzlar,Germany).Cells were bathed at room temperature (20–258C)in normal Tyrode’ssolution containing 1.8mM CaCl 2.Patch pipettes werepulled from Kimax-51glass capillaries (Kimble Glass,Vineland,NJ,USA)on a PP-830microelectrode puller (Narishige,Tokyo,Japan),and their tips were fire-polishedDexmedetomidine inhibits neuronal ion currentsby on August 7, 2010 Downloaded fromwith an MF-83microforge (Narishige).The resistances of the patch pipettes when filled with the internal solutions were between 3and 5M V .Ion currents were measured with glass pipettes in a whole-cell configuration of the patch-clamp technique,using an RK-400patch-clamp amplifier (Biologic,Claix,France).1317Data recordings and analyses Signals were displayed on an HM-507oscilloscope (Hameg Inc.,East Meadow,NY,USA).Data were stored online in a Slimnote VX 3computer (Lemel,Taipei,Taiwan)at 10kHz equipped with a Digidata-1322A inter-face (Molecular Devices,Sunnyvale,CA,USA).Series resistance,in the range of 5–15M V ,was electronically compensated.Data were analysed by the use of pCLAMP9.0software (Molecular Devices),Origin 7.5software(Microcal Software,Northampton,MA,USA),or custom-made macros built in Excel 2007spreadsheet running onWindows Vista (Microsoft,Redmond,WA,USA).ThepCLAMP-generated voltage-step protocols were used to measure the current–voltage (I–V )relations for ion cur-rents.Action potential duration (APD)was measured at 90%of repolarization (APD 90).To evaluate the percentage inhibition of DEX on I K(DR),NG108-15cells were bathed in normal Ca 2þ-free Tyrode’s solution and each cell was depolarized from 250to +50mV.The amplitude of I K(DR)at the end of depolarizing pulse during exposure to different concentrations (0.1–30m M)of DEX was measured.Concentration–response data for inhibition of I K(DR)were fitted with a modified form of the Hill equation:y ¼ð1Àa Þ½DEX Àn H½DEX Àn H þIC Àn H 50þa ;where y is the normalized amplitude of I K(DR);[DEX]rep-resents the DEX concentration;and IC 50and n H are the concentration required for 50%inhibition and Hill coeffi-cient,respectively.Maximal inhibition (i.e.12a )was also estimated.By the use of a least squares method,the smooth curve shown in Figure 1B was allowed to converge on the experimental data when the maximal block was left undefined.Time constants of current activation (t act )in the absence or presence of DEX were determined by fittingthe trajectory of each current trace with single exponentialcurves.To determine the effect of DEX on the steady-state inactivation of I K(DR),the relationship between the con-ditioning potentials and the normalized amplitude of I K(DR)obtained with or without addition of DEX (3m M)was fitted by the Boltzmann function:I ¼I max 1þexp ðV Àa =b Þwhere I max represents the maximal activated I K(DR),V is the membrane potential in mV,a the membrane potential for a half-maximal inactivation,and b the slope factor of the inactivation curve of I K(DR).Values are provided as means (SEM )with sample sizes (n )indicating the number of cells from which the data were taken.The paired or unpaired Student’s t -test andone-way analysis of variance (ANOV A )with the least signifi-cant difference method for multiple comparisons were used for the statistical evaluation of differences among means.Non-parametric Kruskal–Wallis test was then used,if the assumption of normality underlying ANOVA wasviolated.Statistical analyses were performed using SPSS 14.0(SPSS Inc.,Chicago,IL,USA).Statistical signifi-cance was determined at a P -value of ,0.05.ResultsEffect of DEX on delayed rectifier K +current [I K(DR)]in differentiated NG108-15cellsIn an initial set of experiments,a whole-cell configuration of the patch-clamp technique was used to evaluate the effects of DEX on ion currents in NG108-15cells differ-entiated with dibutyryl cyclic AMP (1mM).To record I K(DR),cells were bathed in Ca 2+-free Tyrode’s solution which contained tetrodotoxin (1m M)and CdCl 2(0.5mM),and the recording pipette was filled with a K +-containing solution.Figure 1shows that DEX can depress the amplitude of I K(DR)in a concentration-dependent manner.In these experiments,each cell wasdepolarized from 250to +50mV with a duration of 300ms,and current amplitudes of I K(DR)were measured at theend of depolarizing pulses.DEX (0.1–30m M)suppressedthe amplitude of I K(DR)in a concentration-dependentmanner.The relationship between the DEX concentration and the percentage inhibition of I K(DR)was constructed and plotted in Figure 1B .The IC 50value for DEX-induced inhibition of I K(DR)was 4.6m M.Figure 1C illustrates aver-aged I–V relationships of I K(DR)obtained in the control and during cell exposure to 3m M DEX.The results demonstrate that DEX has a significant depressant action on I K(DR)functionally expressed in differentiated NG108-15cells.Kinetic studies of DEX-induced block of I K(DR)Because the activation time course of I K(DR)during exposure to DEX tends to be progressively reduced,it is important to determine the kinetics of DEX-induced block in these cells.As shown in Figure 1D ,when cells weredepolarized from 250to +50mV,exposure to DEX wasnoted to produce a reduction of t act in a concentration-dependent fashion.Therefore,the results suggest that theinhibitory effects of DEX on I K(DR)can be primarilyexplained by state-dependent blocker where it can bind toChen et al.by on August 7, 2010 Downloaded fromthe open state of the channel according to the minimal kinetic scheme:18C O O · Dwhere a and b are kinetic constants for the opening andclosing of the channel,k 1and k 21are those for forward and reverse rate constants of DEX binding,and D is theDEX concentration.C,O,and O .D represent the closed,open,and open-bound states,respectively.Forward and reverse rate constants,k 1and k 21,were estimated from the values of tact for DEX-induced change in the trajectory ofI K(DR)(Fig.1E ).The values were calculated using therelation:t À1¼k 1½D þk À1In particular,k 1and k 21,respectively,result from the slope and from the y -axis intercept at [D ]=0of the linear p A 1000020003000–50050m V [DEX] (μM)D A C u rr e n t a m p li t ude (pA)ms 002000e d c b a pA 2040Time (ms)Fig 1Inhibitory effect of DEX on I K(DR)in NG108-15cells differentiated with dibutyryl cyclic AMP.Cells were bathed in Ca 2+-free Tyrode’s solution that contained tetrodotoxin (1m M)and CdCl 2(0.5mM).(A )Superimposed current traces obtained in the absence and presence of DEX.The upper part indicates the voltage protocol used.Inset shown in the right side of (A )indicates expanded time scale of current traces (dashed box).a,control;b,0.1m M DEX;c,0.3m M DEX;d,1m M DEX;and e,3m M DEX.(B )Concentration–response relationship for DEX-induced inhibition of I K(DR).Each cell was depolarized from 250to 50mV.Each point represents the mean (SEM )value (n =6–9).The smooth line represents the best fit to the Hill equation as described in Methods.The IC 50value was derived from the non-linear curve fit.The IC 50value,maximally inhibited percentage of I K(DR),and Hill coefficient (n H )were 4.6m M,90%(i.e.a =0.1),and 1.1,respectively.(C )Averaged I–V relationships of I K(DR)obtained in the absence and presence of 3m M DEX.Mean (SEM )(n =7–12).In (D ),the time courses of current activation in the absence and presence of DEX were well fitted by a single exponential.a,control;b,0.1m M DEX;c,0.3m M DEX;d,1m M DEX;and e,3m M DEX.The inset indicates the voltage protocol used.In (E ),the reciprocal of the activation time constant (t act ),obtained by a single-exponential fit of the increasing phase of I K(DR),was plotted against the DEX concentration.Data points were fitted by a linear regression,suggesting that the interaction occurs with a stoichiometry of 1.Forward (k 1)and reverse (k 21)rate constants,given by the slope and the y -axis intercept of the interpolated line,were 0.056ms 21m M 21and 0.34ms 21,respectively.Each point represents the mean (SEM )value (n =8–11).Dexmedetomidine inhibits neuronal ion currentsby on August 7, 2010 Downloaded fromregression interpolating the reciprocal of time constants (i.e.t 21)vs different DEX concentrations (i.e.[D ]).In agreement with this kinetic scheme,the relationship between k 1and [D ]was found to be linear with a corre-lation coefficient of 0.95(Fig.1E ).The forward and reverse rate constants obtained from 6to 8different cells were calculated to be 0.056ms 21m M 21and 0.34ms 21,respectively.On the basis of these rate constants,a value of 6.1m M for the dissociation constant (K D =k 21Âk 121)ofthis drug was derived.Notably,this value was found to agree with its IC 50value determined from the concen-tration–response curve (Fig.1B ).However,the backward rate constant (i.e.k 21)showed little dependence on theDEX concentration:k 21was 0.34ms 21(n =6)at 1m Mand 0.34ms 21(n =6)at 3m ck of effect of DEX on steady-state inactivation curve of I K(DR)To characterize the inhibitory effect of DEX on I K(DR),wenext investigated whether this drug affects the steady-state inactivation curve of I K(DR)in differentiated NG108-15cells.Figure 2illustrates the steady-state inactivation curve of I K(DR)obtained in the absence and presence of DEX(3m M).In these experiments,a 10s conditioning pulse to different membrane potentials preceded the test pulse to +50mV from a holding potential of 250mV.Therelationships between the conditioning potentials and thenormalized amplitudes of I K(DR)with or without appli-cation of DEX (3m M)were plotted and fitted by the Boltzmann equation as described in Methods.In control,a =212.3(2.2)mV,b =6.6(0.8)mV (n =6),whereas in the presence of DEX (3m M),a =212.1(2.1)mV,b =6.3(0.7)mV (n =6).The values for a and b were not significantly modified in the presence of DEX (3m M),although it reduced the maximal conductance of I K(DR)by about 50%.Effect of DEX on averaged I–V relations of I K(DR)in NG108-15cells treated with yohimbineDEX was reported to be a selective agonist ofa 2-adrenergic receptors.1Previous studies showed the ability of yohimbine to reverse both increased K +outwardcurrents and anticonvulsant effect caused by DEX.5910We next evaluated whether inhibition of a -adrenergic receptors can influence DEX-induced block of I K(DR)inNG108-15cells.In these experiments,differentiated NG108-15cells preincubated with yohimbine (10m M)for5h.Yohimbine is an a 2-adrenoceptor antagonist,whereas efaroxan is an antagonist of imidazoline-I 1and a 2-adrenergic receptors.In yohimbine-treated cells,theinhibitory effect of DEX on the I–V relationship of I K(DR)remained unaltered (Fig.3).No significant difference inthe magnitude of DEX-induced inhibition of I K(DR)between control cells and cells treated with DEX can be seen.Moreover,subsequent application of yohimbine (10m M)or efaroxan (10m M)did not reverse the inhibition ofI K(DR)caused by DEX (3m M)(data not shown).Similarly,in cells treated with efaroxan (10m M)for 5h,the magnitude of DEX-induced inhibition of I K(DR)remained unchanged.These results are thus interpreted to mean that the inhibitory effect of DEX on I K(DR)in these cells is unrelated to its binding to a 2-adrenergic receptors.Effect of DEX on voltage-gated Na +current (I Na )in differentiated NG108-15cellsWe next investigated whether DEX has any effects on I Na present in these cells.Cells were bathed in Ca 2+-free Tyrode’s solution containing 0.5mM CdCl 2and the pipette was filled with a Cs +-containing solution.The effect of DEX on I Na was examined at different potentials and an I–V relationship of I Na was constructed.As shownI/Im axConditioning potential (mV)BC u r re n ta m pli tud e(pA )010 000Time (ms)500020 00015 0000100020003000A ms 0–50050mV 10000Fig 2Steady-state inactivation curve of I K(DR)in the absence or presence of DEX in differentiated NG108-15cells.Conditioning voltage pulses with a duration of 10s to potentials between 260and +60mV were applied from a holding potential of 250mV.After each conditioning pulse,a test pulse to +50mV was applied to evoke I K(DR).An example of current traces obtained by two-pulse protocol in the control is illustrated in (A ).The inset shown in (A )indicates the voltage protocol used.(B )Steady-state inactivation curve of I K(DR)obtained with or without application of DEX (3m M).The normalized amplitude of I K(DR)(I /I max )was constructed against the conditioning potentials,and the smooth curves were fitted by the Boltzmann equation (see text for details).Mean (SEM )(n =5–9).Note that the steady-state inactivation curves of I K(DR)in the absence and presence of DEX are virtually superimposable.Chen et al.by on August 7, 2010 Downloaded fromin Figure 4,DEX at a concentration of 10m M could significantly decrease the peak amplitude of I Na ,whencells were depolarizing from 280mV to different poten-tials ranging from 280to +40mV.For example,when the cell was depolarized from 280to 240mV,application of DEX (10m M)significantly decreased I Na from 0.79(0.07)to 0.62(0.07)nA (n =7).After washout of the drug,I Nawas returned to 0.76(0.06)nA (n =6).However,the overall I–V configuration of I Na did not differ between theabsence and the presence of DEX.A subsequent appli-cation of yohimbine (10m M)did not reverse the inhibition of I Na caused by DEX (data not shown).Lack of effect of DEX on L-type Ca 2+current (I Ca,L )in differentiated NG108-15cellsPrevious reports showed that DEX is able to modify Ca 2+current in phaeochromocytoma cells.7We also determined the ability of this agent to alter the amplitude of I Ca,L inthese cells.The experimental results are illustrated inFigure 5.When the cell was depolarized from 240to different potentials ranging from 240to +40mV,thepeak amplitude of I Ca,L was not found to differ between the absence and the presence of 10m M DEX.Moreover,when cells were exposed to this agent,no modification ofthe I–V relationship of I Ca,L can be demonstrated.Therefore,unlike I Na ,I Ca,L is relatively insensitive to inhi-bition by DEX in these cells.Effect of DEX on action potentials in differentiated NG108-15cellsIn another set of experiments,we determined the ability ofDEX to alter membrane potential.In these experiments,cells were bathed in normal Tyrode’s solution containing 1.8mM CaCl 2,and current-clamp recordings were madein a K +-containing pipette solution.The resting membranepotential and APD measured at 90%repolarization(APD 90)in differentiated NG108-15cells were 270(8)mV and 119(20)ms,respectively (n =21).A representativeexample of DEX-induced effects on action potentials(APs)in differentiated NG108-15cells is illustrated inFigure 6.DEX at a concentration of 10m M significantly decreased the amplitude of APs from 86(3)to 75(3)mV and prolonged the duration of APs from 76(9)to 143(21)ms (n =7).However,no discernible change in resting mem-brane potential can be demonstrated in the presence of C u rre n ta mp l itud e(n A)m V Control DEXTime (ms)050100150Fig 4Inhibitory effect of DEX on I Na in differentiated NG108-15neuronal cells.Cells were bathed in Ca 2+-free Tyrode’s solution containing 10mM tetraethylammonium chloride and 0.5mM CdCl 2.The recording pipette was filled with a Cs +-containing solution.(A )Superimposed current traces when the cell was depolarized from 280mV to different potentials.Upper panel is the control,and lower panel was obtained 2min after application of 10m M DEX.The uppermost part indicates the voltage protocol used.(B )Averaged I–V relationships of peakI Na obtained in the absence or presence of 10m M DEX.Mean (SEM )(n =6–11).DEX reduced the peak amplitude of I Na with no change in the overall I–V configuration.C ur re n tam pli tu d e(p A )Membrane potential (mV)Fig 3Effect of DEX on averaged I–V relationships of I K(DR)inNG108-15cells treated with yohimbine.Before each experiment,cells were preincubated with yohimbine (10m M)for 5h.In each cell examined,I K(DR)was elicited from 250mV to different potentials ranging from 250to +50mV with 10mV increments.The I–V relationships of I K(DR)obtained in the absence or presence of 3m M DEX are illustrated.Mean (SEM )(n =5–10).Dexmedetomidine inhibits neuronal ion currentsby on August 7, 2010 Downloaded fromDEX.The widening of APs caused by DEX could be pri-marily explained by its block of I K(DR)and I Na ,but not by the activation of I Ca,L .Inhibitory effects of DEX on ion currents in response to AP waveforms in differentiated NG108-15cells The size and time course of ion currents [e.g.I Na andI K(DR)]in response to change in AP waveforms tend to bedifferent from those elicited by rectangular voltage-clamp pulses.The effects of DEX on ion currents during APs generated from NG108-15cells were also investigated.In these experiments,cells were bathed in normal Tyrode’s solution,and the waveforms of APs were digitally gener-ated as voltage templates and replayed to evoke ion cur-rents.19As shown in Figure 7,the amplitudes of I Na andI K(DR)in response to AP waveforms were reduced in thepresence of DEX.For example,after application of 1m MDEX,the peak amplitude of I Na elicited by the waveforms of APs was decreased by about 10%.Moreover,I K(DR)in response to AP waveforms was suppressed by about 30%.The results led us to suggest that I Na and I K(DR)evoked by AP waveforms are more sensitive to block by this agent than those during rectangular pulses.Effect of DEX on I K(DR)in isolated rat cerebellargranule cells Previous studies have demonstrated the presence of I K(DR)(i.e.K V 3.3-encoded current)in cerebellar granule cells.20DEX was shown to influence the cyclic GMP effector system in the mouse cerebellum.21Therefore,to verify whether the DEX-induced inhibition of I K(DR)could also be observed in another type of cells,we also investigatedthe effect of DEX on I K(DR)in isolated cerebellar granularcells.In isolated cerebellar granule cells,the I K(DR),which was clearly identified as a slowly inactivating I K(DR),could be elicited.As shown in Figure 8,DEX was effec-tive in suppressing I K(DR)found in these cells.When depolarizing pulses from –50to +50mV were applied,DEX (30m M)significantly decreased current amplitude at the end of the voltage pulses from 2018(152)to 1196(81)pA (n =7).Consistent with the observations made in differentiated NG108-14cells,these data indicate that DEX is capable of inducing the block of I K(DR)andincreasing current activation in cerebellar granule cells.DiscussionThe present results demonstrate that in differentiated NG108-15neuronal cells,DEX produces a depressant action on I K(DR)in a concentration-and state-dependent fashion.DEX also blocks the peak amplitude of I Na ,whereas it had little or no effect on I Ca,L .This studysuggests that the exposure to this agent can produce inhibi-tory effects on I K(DR)and I Na .In addition to activation ofa 2-adrenergic receptors,DEX-induced block of these cur-rents could thus be potential mechanisms through which itmay depress neuronal excitability.A notable feature of the block of I K(DR)by DEX in NG108-15cells is that the activating phase of the current C u rr en tam p l it u d e (p A)m VControl DEX Time (ms)Fig 5Lack of effect of DEX on I Ca,L in differentiated NG108-15cells.Cells,bathed in normal Tyrode’s solution containing 1.8mM CaCl 2and 1m M tetrodotoxin,were held at 240mV and voltage pulses ranging from 240to +50mV in 10mV increments with a duration of 300ms were applied.In (A ),superimposed current traces shown in the upper part are control,and those in the lower part were recorded 2min after application of 10m M DEX.Voltage protocol is shown in the uppermost part.(B )Averaged I–V relationships of peak I Ca,L obtained under control or during exposure to 10m M DEX.Mean (SEM )(n =6–8).The I–V relationships of I Ca,L in the absence and presence of DEX are virtually superimposable.Chen et al.by on August 7, 2010 Downloaded from。