Neuroscience 135 (2006) 1075–1086
17-ESTRADIOL INHIBITS OUTWARD POTASSIUM CURRENTS RECORDED IN RAT PARABRACHIAL NUCLEUS CELLS IN VITRO M. FATEHI,a,b S. B. KOMBIANc AND T. M. SALEHa,b*
Key words: autonomic, patch-clamp electrophysiology, postsynaptic currents, neurosteroids.
a
Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3
The parabrachial nucleus (PBN) is an important autonomic regulatory nucleus that serves to integrate and relay visceral information projecting to the thalamus and cortex (Chamberlin and Saper, 1992; Huang et al., 2003; Kang et al., 2004; Saleh and Cechetto, 1994). Immunocytochemical evidence exists for the presence of both estrogen receptors (ER␣ and ER) on cell bodies, axons and terminals of autonomic regulatory nuclei throughout the neuraxis including the PBN (Turcotte and Blaustein, 1993). In addition to the receptors, endogenous estrogens are also present in these nuclei (Saleh et al., 2003). The presence of estrogen and its receptors on cells in these autonomic nuclei suggest that estrogen may exert rapid regulatory and modulatory effects on integrated responses and information to and from these nuclei. It is believed that 17-estradiol influences neuronal communication and subsequently cardiovascular function through different mechanisms, ranging from trans-synaptic modulation of neurotransmitter synthesis and release, to development and remodeling of synaptic circuitry, and via the response of the target postsynaptic cell. Despite the presence of estrogen receptors in these cardiovascular regulatory nuclei, their role in the function of these nuclei is not well known. The modulatory role of 17-estradiol in the PBN has been the subject of recent investigations in our laboratory (Saleh and Connell, 2003a,b; Saleh et al., 2005). Bilateral microinjection of 17estradiol into the PBN was shown to rapidly (within 30 min) decrease sympathetic nerve activity and increase the sensitivity of the cardiac baroreceptor reflex (Saleh et al., 2000, 2003; Saleh and Connell, 2003a). At the cellular level, the effects of estrogens on neuronal excitability and synaptic transmission are also not well understood. Microinjection of estrogen into the PBN of male rats while recording extracellular neuronal activity in the thalamus in vivo, rapidly enhanced GABAergic neurotransmission within the PBN and attenuated visceral afferent activity to the thalamus (Saleh and Saleh, 2001). Longterm, genomic effects of estradiol have also been observed with responses occurring hours or days following pretreatment. For example, it has also been reported that estrogen enhanced sensorimotor performance following 2 week implantation of estradiol containing pellets into the neostriatum (Becker et al., 1987). Also, one week pretreatment of neostriatal neurons with estrogen, caused an enhancement of the firing rate in response to dopamine in these cells (Arnauld et al., 1981). Much of what is known about the rapid (within 5 min of application), cellular effects
b
Prince Edward Island Health Research Institute, University of Prince Edward Island, Charlottetown, P.E.I., Canada c
Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait University, Safat, Kuwait
Abstract—Evidence is increasingly accumulating in support of a role for the steroid hormone 17-estradiol to modify neuronal functions in the mammalian CNS, especially in autonomic centers. In addition to its well known slowly developing and long lasting actions (genomic), estrogen can also rapidly modulate cell signaling events by affecting membrane excitability (non-genomic). Little, however, is known regarding the mechanism(s) by which 17-estradiol produces its rapid effects on neuronal membrane excitability. As potassium channels play a crucial role in cell excitability, we hypothesized that 17-estradiol caused excitability by modulating potassium flux through the neuronal cell membrane. We tested this hypothesis by examining the effects of 17-estradiol on outward potassium currents recorded in cells from the parabrachial nucleus of rats, in vitro. Bath application of 17-estradiol (10 –100 M) reversibly reduced voltage-activated outward potassium currents in a concentration-dependent manner. This effect was mimicked by BSA-17-estradiol but not mimicked by 17␣-estradiol and was significantly reduced by ICI 182,780, a selective estrogen receptor antagonist. The inhibitory effect of 17-estradiol was dependent on extracellular potassium concentration, with more profound effects observed at lower concentrations. The 17-estradiolinduced inhibition of the outward current was blocked by pretreatment with the potassium channel blockers tetraethylammonium and 4-aminopyridine. The time constants of deactivation of tail currents were decreased by 17-estradiol over a range of test potentials (ⴚ140 to ⴚ80 mV). Finally, the inhibitory effect of 17-estradiol on the outward potassium currents was blocked following pre-incubation of slices in lavendustin A, a tyrosine kinase inhibitor. Taken together, these results suggest that 17-estradiol acts rapidly at an extracellular membrane receptor to reduce tetraethylammonium- and 4-aminopyridine-sensitive outward potassium currents by accelerating the closure of potassium channels. This may be the ionic basis of 17-estradiol-induced enhancement of neuronal excitability. © 2005 Published by Elsevier Ltd on behalf of IBRO. *Correspondence to: T. M. Saleh, Prince Edward Island Health Research Institute, Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3. Tel: ⫹1-902-566-0819; fax: ⫹1-902-566-0832. E-mail address:
[email protected] (T. M. Saleh). Abbreviations: aCSF, artificial cerebrospinal fluid; DMSO, dimethyl sulfoxide; Erev, reversal potential; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; I–V, current-voltage; PBN, parabrachial nucleus; TEA, tetraethylammonium; TTX, tetrodotoxin; Vh, holding potential; 4-AP, 4-aminopyridine. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.07.024
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of estrogen within the CNS comes from studies in hypothalamic and hippocampal neurons. It has been shown that 17-estradiol decreased the spontaneous firing of medial preoptic area neurons (Kelly et al., 1977), enhanced the firing rate of pituitary cells (Dufy et al., 1979) and potentiated excitatory postsynaptic potentials in hippocampus (Wong and Moss, 1992). Thus, although the effects of 17-estradiol on neuronal function in several regions of the brain are widespread, the underlying mechanism(s) of action remain uncertain. Generally, the most reported effects of estrogen on the electrophysiological properties of CNS neurons are those of increased membrane excitability. To begin to understand the ionic basis of this excitability, we hypothesized that estrogens may modulate potassium conductances. The present study therefore examined the effects of 17-estradiol, the endogenously bioactive estrogen, on voltage-activated potassium currents recorded from parabrachial neurons contained in pontine slice preparations in vitro.
EXPERIMENTAL PROCEDURES Slice preparation All efforts were made to minimize the number of animals used in this study. The handling and maintenance of animals met the guidelines of the Canadian Council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee (protocol # 04 – 032). Male Sprague–Dawley rats (Charles River, Montreal, PQ, Canada) weighing 150 –200 g were deeply anesthetized with isoflurane (Abbott Laboratories, SaintLaurent, PQ, Canada) vapor in a closed environment and then decapitated. After quick removal, the brain was immersed in icecold (2–3 °C) artificial cerebrospinal fluid (aCSF) with the following composition (in mM): 126 NaCl, 2.5 KCl, 11 D-glucose, 18 NaHCO3, 1.2 NaH2PO4, 1.3 MgCl2, 2.5 CaCl2 (pH 7.4), which was continuously bubbled with 95% O2–5% CO2. Coronal slices (300 or 400 m thick) containing the PBN were prepared from a tissue block of the brain maintained in ice cold carbogenated aCSF, using a vibratome (Model 1000 plus, Ted Pella, Inc., Redding, CA, USA). Slices were incubated in aCSF at room temperature (22– 25 °C) for at least 45 min prior to recording. A single slice was then transferred to a 500 l recording chamber and submerged in continuously flowing extracellular solution (2–3 ml/min) gassed with 95% O2–5% CO2.
In vitro electrophysiological recordings “Blind” whole-cell patch-clamp recordings (Blanton et al., 1989) were performed on cells from the PBN using an EPC-10 amplifier (Heka Electronics Inc., Mahone Bay, NS, Canada) controlled by the Pulse (data acquisition and analysis) software program (Heka Electronics Inc.). All experiments were carried out at temperatures of 30⫾1 °C. A tight giga ohm seal on each cell was obtained using micropipettes (6 – 8 M⍀) pulled from thin-walled (outer diameter, 1.5 mm) borosilicate glass capillaries (World Precision Instruments) by a vertical puller (Model PIP5; Heka Electronics Inc., Germany). The composition of the intracellular (pipette-filling) solution was (in mM): 130 K-gluconate, 6 NaCl, 10 HEPES, 2.5 Na-ATP, 0.1 Na-guanosine 5-triphosphate (pH adjusted to 7.2 with KOH). The extracellular solution had the same composition as that used for the dissection. Sodium and calcium conductances were respectively blocked by adding tetrodotoxin (TTX) (10 nM) and CdCl2 (0.1 mM) to the external solution. The fast electrode capacitance was first compensated. After achieving whole-cell configuration, capacitance transients were cancelled by using an
automatic compensation function of the EPC-10 (about 70 – 80%) and were monitored periodically. Access resistance (series resistance) was also carefully monitored throughout the experiment and only those cells that showed less than 10% change in access resistance (ranged 20 –25 M⍀) over the period of experiments were included in the analysis of the data. Following adequate access to the cell, the amplifier was switched to voltage clamp mode. Data acquisition and analysis were performed using Pulse and Pulsefit software (Heka Electronics Inc.), respectively. Current–voltage relationships (I–V curves) were generated as described below. Currents were filtered at a frequency of 1 kHz. Membrane currents were subjected to leak subtraction to remove the passive component of the membrane conductance. This was performed on-line using the p/4 method (Bezanilla and Armstrong, 1977), and applied by activating this protocol in the Pulse software. Tail currents were fitted by a single exponential function to measure their time constant of decay using a Pulsefit program (Tristani-Firouzi and Sanguinetti, 1998; Wigmore and Lacey, 2000). All cells were voltage clamped at a holding potential (Vh) of ⫺65 mV near their resting potential. Voltagedependent outward potassium currents were elicited by applying depolarizing voltage pulses. Acquired cells were pulsed up to ⫹60 mV (50 ms duration) in voltage steps of 10 mV from the Vh unless otherwise stated. A family of outward currents was recorded under control conditions and then at 5 min intervals following drug exposure. The shift in the I–V relationship was measured as the voltage difference between the curve under control conditions and 5 min after exposure to 17-estradiol (0.1–100 M) and taken as the estrogen effect. All drugs were applied to the cells by bath perfusion of the slices with aCSF containing the final concentration of the drug.
Statistics Data are expressed as mean percentage change from control values⫾standard error of the mean (S.E.M.). Each individual cell served as its own internal control. For comparison of various groups, means were analyzed by one-way analysis of variance (ANOVA) followed by a Tukey-Kramer multiple comparison test. Results expressed as percentages of control were considered to be nonparametric data and analyzed by employing the MannWhitney U test. Statistical significance was determined at P⬍0.05. Graphing was performed using the SigmaPlot®, GraphPad® and CorelDraw software®.
Drugs Isoflo (isoflurane) was purchased from Abbott Laboratories. Cadmium chloride and ICI 182,780 were obtained from Fisher Scientific (Fair Lawn, NJ, USA) and Tocris (Ellisville, MO, USA), respectively. 17␣-Estradiol, 17-estradiol, BSA-17-estradiol, lavendustin A, TTX, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and all the salts in the aCSF were purchased from Sigma (St. Louis, MO, USA). When 4-AP and TEA were included in the extracellular solution, the pH was adjusted to 7.4 by using HCl. Appropriate stock solutions were made and diluted with aCSF just before application.
RESULTS Bath application of low concentrations of 17-estradiol (⬍50 M) at the Vh of ⫺65 mV did not induce any measurable change in the resting (holding) current. This suggests that 17-estradiol does not alter a resting or leak conductance to excite PBN neurons. However, 17-estradiol at a high concentration (50 M) caused a slight, but significant cell membrane depolarization (8⫾3 mV; 12.8⫾2.2% of control, P⬍0.05; n⫽5). We therefore studied depolarization-induced potassium currents.
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Effects of 17-estradiol on depolarization-induced outward potassium currents Prior to exposure of each brain slice to various concentrations of 17-estradiol, a family of outward currents were recorded using depolarizing voltage steps from a Vh of ⫺65 mV. These recordings were used as internal control against which the effects of 17-estradiol (1–100 M) were compared. Representative recordings shown in Fig. 1A, B and C, illustrate the inhibitory effect of 50 M 17-estradiol on depolarizationinduced outward potassium currents. The inhibitory effect of
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17-estradiol appeared to be more profound on the slow inactivating currents. Fig. 1B clearly demonstrates that the fast inactivating component of the outward potassium currents is affected to a lesser extent by 17-estradiol. Washout of the drug resulted in partial recovery of the outward potassium currents (Fig. 1C). As shown in Fig. 1D, 17-estradiol caused a rightward shift in the I–V curve. For these cells, the input resistance estimated between ⫺20 mV and 20 mV was 330⫾100 MS in control and 610⫾115 M⍀ in the presence of 50 M 17-estradiol (P⬍0.05; n⫽4; paired t-test). The in-
Fig. 1. 17-Estradiol inhibits voltage-activated, outward potassium currents recorded in PBN cells. Representative current traces recorded in a neuron (Vh⫽⫺65 mV) in control (A), and in the presence of 50 M 17-estradiol (B). (B) Shows the inhibitory effect of 17-estradiol on these currents at all tested potentials (⫺30 to ⫹70 mV). Above each family of traces is the voltage step protocol used to elicit these currents (50 ms duration in 10 mV increments). This effect was partially reversible following washout of 17-estradiol as illustrated by the superimposed current traces (C) taken at the maximal voltage step. Also shown in (C) is the control trace (a), the trace in the presence of 50 M 17-estradiol (b) and the recovery trace following a 15 min washout (c). (D) A I–V relationship (I–V curve) showing the voltage dependence of the potassium current in control (●) and in the presence of 50 M 17-estradiol (o). Note the reduction of current amplitude at all test potentials. (E) Bar graph summarizing the concentration-dependent effect of 17-estradiol on the potassium currents. This graph was generated by calculating the effect of 17-estradiol at the maximum test potential of ⫹70 mV. Note the maximum blockade of the outward currents induced by 50 M 17-estradiol (* P⬍0.05, ** P⬍0.01 vs control, Mann-Whitney U test, n⫽3–11). (F) The above data in E were then re-plotted as a concentration-response curve that yielded an EC50 value of 26.8 M. (G) A normalized and averaged time-effect plot of the effect of 50 M 17-estradiol on the outward potassium currents (n⫽5).
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Fig. 2. Effect of 17-estradiol is dependent on extracellular potassium ion concentration ([K⫹]o). (A) Averaged I–V curves showing the dependence of the currents on [K⫹]o (0 mM: Œ; 2.5 mM: ●; 5 mM: ’ and 10 mM: L) (n⫽4 – 6 cells). For these experiments, currents were recorded over a voltage range of ⫺120 to ⫹30 mV and each step current was averaged across all cells. (B) Nernst plot of Erevs recorded in the PBN at various [K⫹]o (). Also plotted on this graph for comparison is the theoretical (dotted) line assuming an intracellular [K⫹] of 130 mM for both plots. Note the depolarizing shift in the Erev from ⫺92 mV to ⫺56 mV induced by elevating [K⫹]o from 2.5–10 mM. (C) In the upper panel are representative current traces in the absence (left panels) or presence (right panels) of 20 M 17-estradiol recorded in 0 mM [K⫹]o. In the lower panel are the I–V curves (●: in control and Œ: in the presence of 17-estradiol (20 M)) obtained from the above traces. The currents were evoked with depolarizing voltage steps from ⫺40 to ⫹50 mV, in 10 mV increments (Vh⫽⫺65 mV; 50 ms duration). (D) These are similar to (C) except that recordings were performed in 10 mM [K⫹]o. (E) Summary of the effect of varying external [K⫹]o on inhibition of the outward currents induced by 17-estradiol (20 M). These data were calculated by using the current amplitudes at maximum depolarization and normalizing all responses (open bars) to the control values (solid bars) (* P⬍0.01 vs control, ## P⬍0.01 vs #, one-way analysis of variance followed by a Tukey-Kramer multiple comparison test, n⫽3– 4 cells).
crease in input resistance suggests that 17-estradiol produced this inhibition by closing potassium channels. This effect of 17-estradiol was concentration-dependent. At 100 nM, 17-estradiol had no significant effect on the currents. At 1 M, 17-estradiol appeared to slightly increase the amplitude of these currents. However, at 10, 20, 50 and 100 M concentrations, 17-estradiol reduced the maximum amplitude of the outward currents by 17⫾2% (n⫽6), 27⫾2% (n⫽11), 50⫾3% (n⫽4) and 52⫾4% (n⫽3) of control, respectively (Fig. 1E). The estimated EC50 of this effect was
26.8 M (Fig. 1F). The inhibitory effect of 17-estradiol appeared within 5 min following bath application. Fig. 1G shows the time course of the effect and partial recovery of the outward currents after washout of 17-estradiol (50 M). Characterization of 17-estradiol-sensitive currents To further determine if potassium was the ion responsible for the outward currents sensitive to 17-estradiol, three different sets of experiments were performed. In the first set
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of experiments, inhibition of the outward currents induced by 17-estradiol (20 M) was measured when the extracellular concentration of potassium ions was varied from 0 to 10 mM. In the second set of experiments, TEA and 4-AP were added to the external solution containing 17-estradiol (50 M). Finally, in the third set of experiments, prior to exposure to 17-estradiol, the tissue was perfused with an external solution containing TEA (5 mM) and 4-AP (1 mM). A family of outward currents was then recorded prior to and 5 min after continuous exposure to each drug. Effects of extracellular potassium ions on the inhibitory action of 17-estradiol To determine if the effects of 17-estradiol were dependent on potassium conductance, we varied the extracellular potassium concentration ([K⫹]o) prior to exposure of brain slices to 17-estradiol. Under increasing [K⫹]o, the reversal potential (Erev) shifted to more depolarized potentials (Fig. 2A) showing that the Erev for these estrogen-sensitive currents was dependent on [K⫹]o. Further, this result suggests that potassium is the likely ion carrying the current under study. The Erev of these currents estimated from the I–V curve was about ⫺92 mV in the external solution containing 2.5 mM K⫹ (Fig. 2A). This value was close to the theoretical potassium ion equilibrium potential of ⫺99.8 mV calculated from the Nernst equation, when the K⫹ concentration in the external solution is 2.5 mM and in the pipette is 130 mM. As the extracellular concentration of K⫹ was increased to 10 mM, the Erev shifted to about ⫺56 mV, which is reasonably close to the calculated K⫹ equilibrium potential of ⫺64.8 mV. The slope of the Erev values for a four-fold increase in extracellular K⫹ concentration is 36 mV which is almost identical to the predicted value (35 mV) of the slope (Fig. 2B). We further tested if the effect of 17-estradiol (20 M) was dependent on [K⫹]o by testing the current inhibition at the various [K⫹]o. When extracellular concentration of potassium was reduced to zero (0 mM KCl in external solution), the inhibition of the outward currents induced by 17-estradiol (20 M) was increased significantly (47⫾4% of control; n⫽4; Fig. 2C and 2E) compared with that detected when these currents were recorded under control conditions with the extracellular solution containing 2.5 mM KCl (27⫾2% (n⫽11); P⬍0.05; Fig. 2E). In contrast, when high extracellular concentration of potassium (10 mM) was applied, the inhibitory effect of 20 M 17-estradiol was no longer significantly different when compared with control (6⫾3%, n⫽4; P⬎0.05; Fig. 2D and 2E). Therefore, the inhibitory effect of 17-estradiol on these currents was dependent on the concentration of extracellular potassium, diminishing in magnitude as the concentration increased from 0 to 10 mM (Fig. 2E). Blockade of 17-estradiol-sensitive outward currents by TEA and 4-AP To further characterize the effect of 17-estradiol on the recorded outward currents, we used two potassium channel blockers, TEA and 4-AP. Application of TEA and 4-AP
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after exposure of the brain slice to 17-estradiol caused a further depression of the outward currents (Fig. 3A). The currents recovered following washout of all the drugs. In a second set of experiments, TEA and 4-AP were first applied and they markedly, but not completely, inhibited the current (Fig. 3B). The remaining current (TEA and 4-AP resistant current) was not sensitive to 17-estradiol inhibition (2.0⫾1%; P⬎0.05; n⫽4; Fig. 3B and C). Thus, the TEA- and 4-AP-insensitive residual current was also insensitive to 17-estradiol suggesting that estrogen inhibits the TEA- and 4-AP-sensitive potassium channels. Effects of 17-estradiol on potassium tail currents To further investigate the mechanism by which 17-estradiol inhibits potassium currents, we examined its effect on tail currents using a prepulse protocol. This involved applying a prepulse from Vh (⫺65 mV) to ⫹40 mV (30 ms duration), followed by hyperpolarizing pulses to voltages between ⫺140 mV and ⫺30 mV (30 ms duration), in increments of 10 mV (Fig. 4A). The decay of these tail currents was best described by a single exponential. The time constant for deactivation progressively increased as a function of the test potential to a maximum ⫺80 mV (Fig. 4D). Representative tail currents evoked as described above, before and after 17-estradiol (50 M) application are shown in Fig. 4A and B. 17-Estradiol profoundly reduced the amplitude of tail currents recorded at depolarized and hyperpolarized potentials (⫺30 mV and ⫺140 mV; Fig. 4C; n⫽4). 17-Estradiol (50 M) also accelerated the deactivation time course of potassium channels. The time course of the tail currents was approximated by a single exponential and the averaged time constant values are shown as a function of voltage in Fig. 4D. Acceleration of the tail current time course induced by 17-estradiol appeared greater at test potentials of ⫺100 to ⫺80 mV compare with more hyperpolarized potentials (Fig. 4D). This suggests that 17-estradiol produces inhibition of potassium channels by also accelerating their deactivation. Involvement of estrogen receptors in 17-estradiolinduced inhibition of potassium currents To determine if the inhibitory effect of 17-estradiol on potassium currents was specific through estrogen receptors, slices were pretreated with the potent and selective estrogen receptor antagonist, ICI 182,780 (1 mM dissolved in 0.1% v/v dimethyl sulfoxide (DMSO)). Exposure of the brain slices to DMSO alone (up to 0.5% v/v), or ICI 182,780 (1 mM dissolved in 0.1% v/v DMSO) had no significant effect on these outward currents (Fig. 5A, n⫽3). This observation would indicate that any effect of ICI 182,780 would not be due to the solvent, DMSO. Following pretreatment with ICI 182,780, the inhibitory effect of 17estradiol on the step depolarization-activated currents was significantly attenuated (Fig. 5). This attenuation occurred at all concentrations of 17-estradiol tested in the presence of ICI 182,780 (n⫽4; P⬍0.05 compared with control inhibition; Fig. 5D). Estrogens have been reported to produce effects by activating receptors located on both the extracellular membrane and intracellularly located recep-
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we performed experiments using a membrane impermeant analog of 17-estradiol, BSA-17-estradiol. Similar to the actions of 17-estradiol, bath application of the estrogen conjugate at two different concentrations resulted in a concentration-dependent inhibition of the outward potassium current. For example, 20 M BSA-17-estradiol produced an inhibition of 24⫾2% (n⫽5, Fig. 6) an effect that was similar in magnitude that was produced by 17-estradiol (27⫾2%; n⫽11; illustrated in Fig. 1E, P⬎0.05). Stereospecificity of the 17-estradiol-mediated inhibitory effect To verify if the 17-estradiol-induced inhibition of the outward potassium currents was stereospecific, we tested the effects of 17␣-estradiol (a biologically inactive enantiomer) on these currents. Fig. 7 shows that 17␣-estradiol (20 M) had no significant effect on the outward recorded currents (n⫽4) indicating that the effect of estrogen observed in the present study is stereospecific and limited to the biologically-active form of estrogen. The slight enhancement of the outward current observed following 17␣-estradiol bath application was likely due to the effect of the vehicle, as ethanol application also resulted in a similar enhancement of this current (Fig. 7; n⫽4). Involvement of tyrosine kinase in the potassium current inhibiting effect of 17-estradiol To explore the second messenger system that may couple the estrogen receptors to the potassium current inhibition, we performed additional experiments to investigate the involvement of tyrosine kinase in mediating this effect of 17-estradiol. This was done by using the selective tyrosine kinase inhibitor lavendustin A (O’Dell et al., 1991). When brain slices were incubated with lavendustin A (10 M) for 2 h, application of 17-estradiol (20 and 50 M) did not significantly alter the recorded outward currents (Fig. 8, n⫽4 –5). Lavendustin A by itself had no direct effect on these cells as assessed by the resting membrane potential (data not shown).
DISCUSSION Fig. 3. TEA and 4-AP block the inhibitory effect of 17-estradiol. (A) Superimposed traces of a representative recording of outward potassium currents in PBN cells (n⫽3). Currents were elicited by 50-ms pulses applied from ⫺65 to ⫹30 mV, in control conditions (a), in the presence of 50 M 17-estradiol (b), in the additional presence of a combination of 5 mM TEA and 1 mM 4-AP (c) and after washout of all the drugs (d). Note that a fraction of the current remaining after exposure to 17-estradiol (50 M) was further reduced by TEA⫹4-AP application. (B) Superimposed traces of currents elicited by 50 ms pulses applied from ⫺65 to ⫹30 mV (n⫽6) in control (a), in the presence of 5 mM TEA alone (b), in the additional presence of 1 mM 4-AP (c) and finally in the presence of a cocktail containing 5 mM TEA, 1 mM 4-AP and 50 M 17-estradiol (d). Note the lack of further inhibition of the remaining current by 17-estradiol in the presence TEA and 4-AP. (C) A bar chart summarizing the above effects (** P⬍0.01 vs control, one-way analysis of variance followed by a Tukey-Kramer multiple comparison test).
tors (Gu and Moss, 1998). To determine if the effects of 17-estradiol were limited to the extracellular receptors,
Our data show that 17-estradiol, the biologically active estrogen, depresses TEA- and 4-AP-sensitive potassium channels in a rapid, partially reversible and in a concentration dependent manner. This effect was stereospecific and dependent on the activation of cell membrane estrogen receptors as it was blocked by an estrogen receptor antagonist, mimicked by BSA-17-estradiol but not by 17␣-estradiol, the inactive analog. This inhibition of potassium currents by 17-estradiol may underlie the reported neuroexcitatory effects of estrogens. The observation that the estradiol effect did not typically fully recover following washout is consistent with observations made with other steroids and peptides following bath application (Gu and Moss, 1998; Wooley, 1999; Wooley et al., 1997). It is believed that the hydrophobic nature of these drugs does not allow for full recovery in addition to the fact that these drugs could be activating
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Fig. 4. 17-Estradiol affects potassium channel deactivation. Tail currents recorded during 30 ms test pulses to potentials between ⫺140 and ⫺30 mV, at 10 mV increments in control (A) and in the presence of 50 M 17-estradiol (B). Each test pulse was preceded by a pre-pulse (voltage step to ⫹40 mV for 30 ms from a Vh of ⫺65 mV). Note the attenuation of the pre-pulse current amplitude as well as the increased rate of deactivation (*) of the tail currents in the presence of 17-estradiol (50 M). (C) I–V curves plotted with data from four experiments (⫽control and Œ⫽17-estradiol (50 M)). (D) Graph showing the voltage dependence of the deactivation time course. Deactivation time constants (tau, J) were calculated by fitting a single exponential curve to the tail currents recorded from four different cells in control conditions () and in the presence of 50 M 17-estradiol (Œ).
long-term changes in the cell. In general, 17-estradiol acts via at least two broad-spectrum mechanisms, genomic and non-genomic, to regulate morphology, metabolism and electrical properties of various cells. The genomic effects of 17-estradiol exposure are not immediate and, in fact may take several hours or days to be observed. The non-genomic effects of 17-estradiol are quite rapid, in that they occur within a few minutes of exposure and are usually reversible after hormone removal. The results of this study are focused on nongenomic effects of 17-estradiol and specifically on outward potassium currents recorded from cells in the PBN. The present data clearly demonstrate that exposure of PBN cells to 17-estradiol (in vitro) modifies an outward potassium conductance. To date, we are not aware of any studies on the effects of 17-estradiol on potassium cur-
rents in cardiovascular regulatory nuclei such as the PBN. With the protocol used to evoke currents in the present study, there is no evidence to suggest that any voltageoperated conductances other than potassium conductances contributed to the outward currents recorded here. Under increasing [K⫹]o, the Erev shifted to more depolarized potentials in a Nernstian fashion indicating that the Erev was dependent on [K⫹]o and suggesting that K⫹ was the likely ion carrying the current under study. That 17estradiol depressed a potassium current is supported by the fact that the magnitude of the 17-estradiol-induced depression was accompanied by an increase in input resistance of the recorded cells and was dependent on the [K⫹]o. In other words, we observed that the inhibitory effect of 17-estradiol was more pronounced at the more physiological extracellular potassium conditions. Raising extra-
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Fig. 5. 17-Estradiol effect on outward currents is blocked by an estrogen receptor antagonist, ICI 182,780. (A) Superimposed current traces illustrating the lack of effect of DMSO at 0.1 and 0.5% v/v and ICI 182,780 recorded in PBN cells. (B) Superimposed current traces illustrate attenuation of the inhibitory effect of 17-estradiol (20 M; b) on the outward currents in the presence of ICI 182,780 (1 mM; c). (C) A representative I–V curve in control (●) and after exposure to 17-estradiol (20 M) in the presence of ICI 182,780 (Œ). (D) Concentration-response bar graph for 17-estradiol in the absence () and in the presence (□) of ICI 182,780 (1 mM).
cellular potassium ions to high concentrations reduces the driving force, and hence an estradiol effect becomes difficult to detect. Furthermore, the 17-estradiol effect was blocked by the well-established potassium channel blockers TEA and 4-AP. Thus, our evidence strongly favors the possibility that estrogens may inhibit potassium channels to cause cellular excitation. Although the exact nature of the potassium channel involved in the effect of 17-estradiol is not known, it can be assumed that the recorded outward currents are mainly due to the activation of delayed rectifier potassium channels, as they were very sensitive to TEA and 4-AP. Considering the significant inhibition of outward potassium channel activity observed in the present investigation, it is possible that 17-estradiol at high concentrations (e.g. 10 –50 M, physiological levels as measured in plasma)
may increase cell excitability by this mechanism alone although under control conditions, other ionic currents such as chloride, sodium or calcium may contribute to the effects of estrogens. This is consistent with other observations on the effect of estradiol in CNS nuclei. From our experiments performed in the presence of TEA and 4-AP, we can conclude that the portion of potassium channels which are not blocked by TEA and 4-AP, are not affected by 17-estradiol. A significant contribution of a calcium-dependent potassium current to these outward currents is unlikely because of the presence of cadmium in our external solution which blocked the influx of calcium ions from the extracellular environment. However, we cannot completely exclude the role of calcium-activated potassium channels as we did not block intracellular sources of calcium (i.e. endoplasmic reticulum or mito-
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Fig. 6. Inhibitory effect of 17-estradiol on outward potassium currents is mimicked by a membrane impermeant analog. (A) Superimposed are current traces illustrating the inhibitory effect of BSA-17estradiol, a membrane impermeant analog of 17-estradiol on the outward potassium currents in control (a), 5 min after exposure to 20 M BSA-17-estradiol (b), 5 min after exposure to 50 M BSA17-estradiol (c), and 3 min after exposure to 2 mM 4-AP and 10 mM TEA (d) all activated at the maximum test potential. (B) A normalized and averaged time-effect plot obtained from six cells showing the effect of 50 M BSA-17-estradiol. (C) A bar graph summarizing the inhibitory effect of two concentrations of BSA-17-estradiol (BSA-E2).
chondria) or used selective blockers of these currents. Nonetheless, our evidence indicates that not all potassium currents or channels are modulated by estrogens. It has been reported that estradiol pretreatment enhanced the response to synaptic stimulation (Wooley et al., 1997). Also, estradiol treatment enhanced ion conductances, such as voltage-dependent, high- and low-threshold calcium currents in CA1 pyramidal cells (Pozzo-Miller et al., 1999). Furthermore, in these neurons, estradiol caused potentiation of kainate-induced currents (Gu and
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Moss, 1998). For this effect, estradiol was required on both sides of the plasma membrane. It was suggested that extracellular estrogen activated a G-protein-coupled receptor, whereas the intracellular action of estrogen appeared to modulate the balance between phosphorylation and dephosphorylation. Using slices from ER␣-knockout mice, these rapid actions of estradiol have been reproduced in hippocampal neurons lacking the intracellular estrogen receptor. In both wild-type and knockouts, the potentiating effect of 17-estradiol on kainate-induced currents was completely abolished using an inhibitor of protein kinase A (Gu and Moss, 1998). The results obtained from our experiments using the membrane-impermeable BSA-17-estradiol support the hypothesis that the inhibitory effect of this steroid on the outward potassium currents is a cell membrane-mediated action. Since BSA-17estradiol mimicked the inhibitory effect of 17-estradiol, it may be that the hormone-binding site of the estrogen receptor is accessible from the extracellular surface on the plasma membrane. This is consistent with recent findings by other investigators that BSA-17-estradiol inhibits Gprotein-coupled inwardly rectifying potassium channels in hypothalamic neurones (Qiu et al., 2003). In addition to these postsynaptic effects of estrogens, estradiol can also produce rapid effects on neurotransmitter release (Becker, 1990), an effect which lasts as long as the steroid is present and is not blocked by transcription or protein synthesis inhibitors (Moss et al., 1997). Further to enhancing or potentiating excitatory postsynaptic currents, estrogen has also been shown to have inhibitory effects within some CNS nuclei (Weiland and Orchinick, 1995). Our results show that 17-estradiol decreases the decay time constant of tail currents evoked for examining the deactivation kinetics of potassium channels. The analysis of tail currents suggest that 17-estradiol may also depress the outward currents by decreasing the duration of the opened state of the channels. Similar observations have been made with other potassium channel modifiers (e.g. indoles) on the outward and tail potassium currents (Avdonin and Hoshi, 2001). In both control conditions and after exposure to 17-estradiol, the decay time of the tail currents was strongly dependent on the preceding membrane potential. Deactivation time of the channels was much faster at greater hyperpolarizations and decreased at more depolarized potentials. Other studies have reported a similar pattern of deactivation for KvLQT1 channels which produce a potassium current upon their activation (Tristani-Firouzi and Sanguinetti, 1998). Although our data suggest that 17-estradiol may affect the activation and inactivation of sensitive potassium channels to produce the observed effects, the precise mechanism of this effect was not addressed in this study. Two types of potassium channel inactivation mechanisms have been reported, C-type or N-type mechanisms. C-type inactivation has been suggested to require conformational alterations very close to the external mouth of the pore which causes channel closure (Lopez-Barneo et al., 1993). N-type inactivation on the other hand has been shown to be mediated by movement of the NH2-terminus to physically occlude
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Fig. 7. 17␣-Estradiol has no effect on outward potassium currents recorded in PBN cells. (A) Superimposed sample current traces (step from ⫺65 to ⫹50 mV) taken from a representative cell exposed to 17␣-estradiol (20 M) where (a) is under control conditions, (b) and (c) are in the presence of ethanol (1%), and 17␣-estradiol dissolved in 1% ethanol, respectively. (B) A bar graph summarizing the lack of effect of 17␣-estradiol (20 M; n⫽5) and ethanol (1%, n⫽4), the solvent in which 17␣-estradiol was dissolved.
the internal mouth of the open state of the channel (Hoshi et al., 1990). Thus, 17-estradiol may modulate one or both of these mechanisms to cause the effects observed in this study. The effects of 17-estradiol reported here are quite specific, involving estrogenic receptors and limited to only the biologically active analog. Although we showed that the effect was blocked by a non-selective estrogen receptor antagonist, we could not examine the specific estrogen receptor involved as, to our knowledge there are currently no selective ␣ or  receptor antagonists commercially available. The stereospecific actions of 17-estradiol as demonstrated by the lack of effect by 17␣-estradiol, as well as the receptor dependence of the effect are consistent with in vivo reports of estrogenic effects observed in autonomic regulatory nuclei (Saleh et al., 2005) and on membrane excitability in other CNS nuclei (Gu and Moss, 1998; Wooley, 1999). It is well established that phosphorylation of tyrosine residues can modulate the activity of voltage-gated potassium channels. This modulation could happen either via direct protein–protein interaction with the tyrosine kinase
or through complex cell signaling events involving activation of a protein tyrosine kinase (Berninger and Poo, 1996; Bolton et al., 2000). There are several lines of evidence supporting the hypothesis that tyrosine kinases modulate outward voltage-dependent potassium channel activity in neurons (Tucker and Fadool, 2002; Yu et al., 1999). Our finding in this study that an inhibitor of protein tyrosine kinase, lavendustin A blocked the ability of 17-estradiol to suppress potassium currents suggests that alterations in the outward potassium current amplitude induced by 17estradiol may have resulted from an action involving tyrosine kinase.
CONCLUSION In conclusion, our results obtained with 17-estradiol indicate that estrogens may act to increase neuronal or cellular excitability by modulating TEA and 4-AP sensitive potassium channels. The pathophysiological consequences of such an action are currently not clear. However, we speculate that by acting to increase neuronal excitability in the PBN, estrogens may influence the function of this
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cise mechanism of the modulation of potassium channels by 17-estradiol and whether these effects may have clinical implications. Acknowledgments—This work was supported by a grant from the Heart and Stroke Foundation of PEI and the Canadian Institutes for Health Research (CIHR; MOP 50095). Authors are grateful to Dr. J. A. Zidichouski for his critical reading of the manuscript.
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Fig. 8. The inhibitory effect of 17-estradiol on the outward potassium currents is abolished by lavendustin A (a tyrosine kinase inhibitor) pre-treatment. (A) Superimposed are current traces illustrating the lack of effect of 17-estradiol on the outward potassium currents after brain slices have been incubated with lavendustin A (10 M) for 2 h. Shown are the currents recorded in cells pretreated with lavendustin A (a), 5 min after exposure of these lavendustin A pretreated cells to 17estradiol (50 M) (b), and 3 min after exposure to 1 mM 4-AP and 10 mM TEA (c). (B) A normalized and averaged time-effect plot showing the lack of effect of 17-estradiol in lavendustin A pretreated cells (n⫽5). (C) A bar graph summarizing the results of these experiments.
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(Accepted 19 July 2005) (Available online 13 September 2005)
