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Genistein directly blocks glycine receptors of rat neurons freshly isolated from the ventral tegmental area
Neuropharmacology 45 (2003) 270–280 www.elsevier.com/locate/neuropharm
Genistein directly blocks glycine receptors of rat neurons freshly isolated from the ventral tegmental area L. Zhu a, Z.L. Jiang a, K. Krnjevic´ b, F.S. Wang a, J.H. Ye a,∗ a
New Jersey Medical School (UMDNJ), Rutgers-UMDNJ Integrative Neuroscience Program, Departments of Anesthesiology, Pharmacology and Physiology, 185, South Orange Avenue, Newark, NJ 07103-2714, USA b Physiology Department, McGill University, Montre´al, Canada Received 30 August 2002; received in revised form 28 March 2003; accepted 28 March 2003
Abstract The effects of tyrosine kinase inhibitors on the glycine-induced current (IGly) were studied in rat neurons freshly isolated from the ventral tegmental area (VTA). Genistein reversibly and concentration-dependently depressed IGly, with an IC50 of 13 µM. Preincubation with genistein had no effect on IGly, indicating that genistein is effective only when glycine is bound to the receptor and channels are most likely open. Genistein depressed maximum IGly without significantly changing the EC50 for glycine. Genisteininduced inhibition of IGly was sensitive to membrane voltage, being greater at positive membrane potentials. A kinetic analysis indicated that genistein lengthens the time constant of IGly activation, but has no effect on deactivation or desensitization. When genistein was rapidly washed out, a transient rebound current probably reflected a faster dissociation of genistein, with respect to glycine. Results of competition experiments suggest that genistein acts on the same region of the glycine receptor as picrotoxin. Daidzein, an analog of genistein that does not act on protein kinases, also inhibited IGly. Co-application of lavendustin A, a specific inhibitor of tyrosine kinase, had no effect on IGly. Our results extend to neurons isolated from the VTA, the previous finding that genistein directly inhibits glycine receptors of hypothalamic brain slices. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Whole-cell patch clamp; Rebound current; Open channel block; Daidzein; Tyrosine kinase antagonists; Picrotoxin
1. Introduction Phosphorylation of ligand-gated ion channels is recognized as a potentially important mechanism of short- and long-term modulation of ion-channel function. Following the discovery of numerous sites of phosphorylation on ligand-gated ion channel proteins, recent studies have demonstrated that activation of serine/threonine, tyrosine and other kinases can modulate various neurotransmitter-induced membrane currents (Swope et al., 1999; Davis et al., 2001). The strychnine-sensitive glycine receptor (GlyR) con-
Abbreviations: IGly, glycine-induced current; PKA, cyclic AMP dependent kinase; PKC, protein kinase C; PTK, tyrosine kinase; GABAA, g-aminobutyric acid A; td, time constant of decay; ton, activation time constant; toff, deactivation time constant; VTA, ventral tegmental area ∗ Corresponding author. Tel.: +1-973-972-4399; fax: +1-973-9724172. E-mail address: [email protected] (J.H. Ye).
sists of two main subunits, a (48 kDa) and b (58 kDa), forming a pentameric channel structure (Betz et al., 1994). The glycine receptor–chloride channel complex is phosphorylated by several protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and tyrosine kinase (PTK) (Song and Huang, 1990; Agopyan et al., 1993; Ruiz-Gomez et al., 1994; Schonrock and Bormann, 1995; Aguayo et al., 1996; Gu and Huang, 1998; Albarran et al., 2001; Caraiscos et al., 2002). The tyrosine residue in the intracellular loop of the glycine b subunit is a potential site for PTK-mediated phosphorylation (Wagner et al., 1991). Immunological and molecular cloning studies reveal that GlyRs are widely distributed not only in the spinal cord and brain stem, but also throughout the mammalian central nervous system, including the ventral tegmental area (VTA) (Betz et al., 1994; Ye et al., 1998). The VTA is an important mediator of the rewarding effects of drugs of abuse, including ethanol (Gatto et al., 1994; Wise, 1996). We are interested in the interactions
0028-3908/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00151-5
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of ethanol and GlyRs of VTA neurons, and the role of protein phosphorylation in these interactions. Recently, we have shown that more than 80% of VTA neurons respond to glycine, and that the GlyRs of VTA neurons are regulated by PKA and PKC (Ren et al., 1998; Tao and Ye, 2002; Jiang and Ye, 2003). In order to better understand the role of PTK phosphorylation in regulation of the GlyRs of VTA neurons, we studied the effects of several relevant agents, including genistein, daidzein and lavendustin A. As a PTK inhibitor, genistein has been used widely to assess PTK-mediated regulation of receptor/channel function (Akiyama et al., 1987; Akiyama and Ogawara, 1991). A recent study showed that PTK enhances GlyR function in spinal and hippocampal neurons (Caraiscos et al., 2002); but according to another report, genistein directly blocks hypothalamic GlyRs through a tyrosine kinase-independent mechanism that is not well understood (Huang and Dillon, 2000). The aim of the present study was to determine whether genistein also depresses GlyRs of dissociated VTA neurons. The combination of dissociated neurons and patch clamp technique is powerful, because it allows a fast exchange of external solutions. Thus, a more accurate concentration–response relation could be obtained, and the kinetics of IGly analyzed, which is important for a better understanding of the underlying mechanisms.
2. Methods 2.1. Isolation of neurons and electrophysiological recording The care and use of animals and the experimental protocol of this study were approved by the Institutional Animal Care and Use Committee of University of Medicine and Dentistry of New Jersey. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Sprague–Dawley rats (5–14-day-old) were decapitated as described earlier (Ye et al., 2001). The brain was quickly excised, placed into ice-cold saline saturated with 95% O2 and 5% CO2, glued to the chilled stage of a vibratome (Campden Instruments, UK) and sliced to a thickness of 400 µm. Slices were incubated at 31 °C, for 15 min in standard external solution containing 1 mg pronase/12 ml and saturated with O2, and for an additional 15 min in 1 mg thermolysin/12 ml. Micropunches of the VTA were isolated and transferred to a 35-mm culture dish. Mild trituration through heatpolished pipettes of progressively smaller tip diameters dissociated single neurons. Within 20 min of trituration, isolated neurons attached to the bottom of the culture dish and electrophysiological experiments were started. The saline in which the brain was dissected contained
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(in mM): NaCl 128, KCl 5, NaH2PO4 1.2, NaHCO3 26, MgCl2 9, CaCl2 0.3 and glucose 2.5. The standard external solution contained (mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10. The pH was adjusted to 7.4 with Tris base and the osmolarity to 320 mOsm with sucrose. The patch electrodes had a resistance of 3–5 M⍀ when filled with solution containing (mM): CsCl 120, TEA-Cl 21, MgCl2 4, EGTA 11, CaCl2 1, HEPES 10 and Mg-ATP 2; the pH 7.2 adjusted with Tris base, the osmolarity 280 mOsm with sucrose. After establishing the giga-seal in the cell-attached configuration by gentle suction, whole-cell recording was obtained by a further suction. Throughout all experiments the bath was continually perfused with the standard external solution. All glycine-induced responses were elicited in this solution at an ambient temperature of 20–23 °C. Currents were recorded under voltage-clamp with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) interfaced to a data acquisition A/D converter Digidata 1320A (Axon Instruments) and directly digitized with pCLAMP 8 software for further off-line analysis. The junction potential between the patch pipette and the bath solutions was nulled just before forming the giga-seal. The liquid junction potential between the bath and the electrode was 3.3 mV, as calculated from the generalized Henderson equation using the Axoscope junction potential calculator (Barry, 1996). This value was corrected off-line when estimating the reversal potential of glycine-activated currents. In most experiments, the series resistance before compensation was 15–25 M⍀. Routinely, 80% of the series resistance was compensated; hence, there was a 3–5 mV error for 1 nA of current. 2.2. Chemical applications Most of the chemicals including glycine, picrotoxin and strychnine were obtained from Sigma Chemical Company (St. Louis, MO, USA), and genistein, daidzein and lavendustin A were obtained from CalbioChem (La Jolla, CA). Their solutions were prepared on the day of experimentation. Genistein, daidzein and lavendustin were predissolved in dimethyl sulphoxide (DMSO) and diluted to its final concentration in standard external solution. At the maximum concentration applied (0.05% (v/v)), DMSO neither induced nor blocked membrane currents and had no effect on the glycine responses. Solutions were applied to a dissociated neuron via a multibarreled superfusion system, consisting of five needles aligned and held together with heat-shrinkable plastic tubing and exiting into a common outflow tube (as described previously, Ye and McArdle, 1997). Each barrel of the pipette was connected to a separate reservoir containing solutions of various drugs. Solutions were exchanged by simultaneously closing and opening
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valves controlling flow from the reservoirs. These maneuvers were performed manually and the flow rate was regulated hydrostatically. The tip of the superfusion pipette was usually placed 50–100 µm away from a cell, a position that allowed rapid as well as uniform drug application while preserving the neuron’s mechanical stability. By keeping the dead volume small and the flow rate high, solutions could be exchanged within 20 ms. The speed of solution exchange was measured by reducing the external Na+ concentration from 140 to 10 mM (plus 130 mM N-methyl-D-glucamine, NMDG), while recording a stable kainate current: the rate of decrease of the kainate current reflected the rate of solution change. The time constant of such change was ⬍20 ms. 2.3. Data analysis Whole-cell current decays were fitted by a Chebychev algorithm (pCLAMP). Concentration–response data were analyzed with a nonlinear curve-fitting program (Sigma Plot; Jandel Scientific, San Rafael, CA). Data were statistically compared using Student’s t-test at a significance level of P ⬍ 0.05. Throughout, means are given ±SEM, with the number of experiments in brackets.
3. Results 3.1. Genistein depresses IGly of VTA neurons In agreement with our earlier report, glycine elicited a current (IGly) in approximately 82% of VTA neurons from young rats (Ye et al., 1998; Ye, 2000). As expected, IGly was antagonized by 0.1 µM strychnine (Ye et al., 1998; Ye, 2000). Genistein (1–300 µM) alone had no detectable effect on membrane current; but when co-applied with the agonist, it decreased IGly in all neurons tested (n = 95), in a concentration-dependent manner. Fig. 1A shows examples of current activated by 30 µM glycine alone (A-a) and in the presence of 3, 10 and 30 µM genistein (A-b, -c, and -d), respectively. After a transient rebound of inward current at the end of the genistein application, IGly rapidly decayed to the base-line. The depressant effect of genistein was fully reversible after washout of genistein (A-e). In similar tests on five neurons, 3, 10, and 30 µM genistein decreased peak IGly to 66 ± 8% (n = 4), 52 ± 2% (n = 5), and 36 ± 2% (n = 5), respectively. The concentration–response analysis of Fig. 1B reveals that genistein suppressed IGly in response to 30 µM glycine with an apparent IC50 of 13 µM and a Hill coefficient of 0.6. Genistein (10 µM) effectively suppressed IGly evoked by subsaturating and saturating concentrations of glycine (data not shown). This is further demonstrated by the
Fig. 1. Genistein depresses IGly. (A) Records of IGly induced by 30 µM glycine in absence (A-a and e) and presence of 3 (A-b), 10 (Ac), and 30 (A-d) µM genistein. Sequence of current traces (from left to right) was obtained from single neuron. The bar above each trace indicates duration of drug application. At washout of higher genistein concentrations (ⱖ10 µM), there was a transient increase of inward rebound of current (A-c and d). (B) Concentration–response relation for genistein-induced depression of IGly. Data from between four and five neurons are means (±SEM) of normalized peak IGly elicited by 30 µM glycine in the presence of several concentrations of genistein. Peak IGly was normalized to peak response to 30 µM glycine in the absence of genistein. Continuous line is least square fit of the following form of the Hill equation, I / IGly = 1 / [1 + (C /IC50)n] to the data; where I is peak current during co-application of genistein, IGly is control peak current and C concentration of genistein, IC50, the concentration at which genistein produced 50% of inhibition and n, Hill coefficient. For this and other figures, all currents were recorded at a holding potential of –50 mV, unless otherwise indicated. (C) Glycine concentration–response curves show mean data (±SEM, from 6 to 10 neurons) for glycine alone (䊊) or glycine plus 10 µM genistein (쎲). Before pooling, peak currents were normalized to the peak current evoked by 300 µM glycine alone. For individual neurons, three-point glycine concentration–response curves were determined first; and then in the presence of genistein. Continuous lines are least square fits of the following form of the Hill equation to the experimental data: IGly / IGlymax = 1 / [1 + (EC50 / C)n], where IGlymax is maximal IGly, C is glycine concentration, EC50 the concentration at which IGly is 50% of maximum and n the Hill coefficient.
concentration–response plots obtained in the absence and presence of genistein (Fig. 1C). On average, 10 µM genistein reduced the currents induced by 15, 30, 100 and 300 µM glycine to 53 ± 8 (n = 6), 67 ± 3 (n = 10), 64 ± 6 (n = 6), and 68 ± 7% (n = 7) of original values, respectively. Genistein did not significantly change
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either the EC50 of IGly or the Hill coefficient, their respective values being 27.9 µM and 3.2 in the absence, and 25.7 µM and 3.1 in the presence of 10 µM genistein. 3.2. Genistein-induced depression of IGly is sensitive to membrane voltage, but is not use-dependent In view of recent evidence that the gating of GlyRchannels is voltage-dependent (Legendre, 1999) we examined the effect of membrane voltage on the depressant action of genistein on IGly. As illustrated by the current–voltage ramps in Fig. 2A–C, genistein decreased IGly to a significantly greater extent at positive membrane potentials. For example, 30 µM genistein depressed IGly to 31 ± 4% of control at +50 mV, but to 74 ± 10% of control value at –50 mV (P ⬍ 0.05, n = 5, Fig. 2D). However, the reversal potential of IGly remained close to the calculated Nernst potential for Cl⫺ (⫺5 mV in our experimental conditions), in agreement with a previous report (Huang and Dillon, 2000). The voltage sensitivity of genistein’s effect suggests that it may act at least partly as an open channel blocker. To examine this possibility, we performed several additional experiments. First, we compared genistein’s effect on IGly when it was preapplied to neurons for 30 s before co-application with glycine (++ protocol, Fig. 3A-b) to its effect without this prepulse (⫺+ protocol, Fig. 3A-c). The effects were similar with and without the prepulse, as also shown by the data from three other neurons (Fig. 3B). Similar results were obtained when preincubation in genistein was extended to 120 s (data no shown). Thus, genistein has a depressant effect only when the GlyR channels are open. This notion is further supported by the experiments illustrated in Fig. 3A-e and A-g. In these tests, genistein was applied alone for 30 s before the application of glycine alone (+⫺ protocol). These typical traces show that genistein had no effect on IGly. Similar results were obtained from three other neurons (Fig. 3B). Furthermore, repeated pulses of 30 µM genistein during a longer pulse of glycine (30 µM) depressed IGly to a similar extent (Fig. 3C). When compared with the initial effect of genistein, the depression of IGly was 101 ± 2% and 102 ± 4% at the second, and third pulse (P ⬎ 0.05, n = 5; Fig. 3C-b). The effect of genistein shows no use-dependence.
Fig. 2. Genistein depression of IGly is sensitive to membrane voltage. (A) Genistein’s effect on current–voltage relationship of IGly was studied with a pair of voltage ramps (from +50 to ⫺60 mV, at rate of 1 mV/10 ms), first in the absence and then the presence of drugs, as indicated. The initial small current at left in A-a and A-b is the leakage current, which was subtracted from the much larger currents recorded in the presence of 30 µM glycine (alone or plus 30 µM genistein). (B) Current–voltage relations obtained from the ramp protocol in the presence of glycine alone (䊊) or glycine plus genistein (쎲). (C) Normalized current–voltage relations from the same experiment of B show a greater effect of genistein at positive membrane potentials (all currents were normalized to values at ⫺60 mV). (D) Histograms are means (±SEM) of normalized peak IGly obtained in the presence of 30 µM genistein from neurons held at +50 and –50 mV; IGly was normalized to peak IGly in the absence of genistein. Genistein caused significantly greater depression at +50 mV than at –50 mV (P ⬍ 0.01, n = 5).
3.3. Kinetic studies The on-rate time constant of genistein’s action was measured as follows. A VTA neuron was exposed to 30 µM glycine. Once a stable current was attained, the solution was rapidly switched to one containing glycine 30 µM + 10 µM genistein; when the current change became stable, the solution was switched back to glycine alone (Fig. 4A). As shown in Fig. 4B, both the onset
and the offset of genistein’s effect could be fitted by single exponentials. The genistein-induced block therefore behaves as a simple, reversible bimolecular reaction. kon
R ⫹ D,RD koff
(1)
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Fig. 3. Preincubation in genistein does not increase the inhibition of IGly produced by co-applications of glycine and genistein. (A) A-a, Ad and A-f are currents evoked by glycine alone (open bars). A-b, 30 s prepulse of 10 µM genistein was applied (hatched bars) before the co-application of genistein and glycine. A-c, co-application of genistein and glycine depressed IGly to a similar extent. A-e and A-g, 30 s prepulse of 10 µM genistein did not affect IGly induced by subsequent pulse of glycine alone. Traces a–c, d–e, and f–g were from three different neurons. (B) Histograms show means (±SEM) of normalized peak IGly in presence of 10 µM genistein, applied as indicated; IGly was normalized to peak value in absence of genistein. (C) C-a, repeated pulses of 30 µM genistein during continuous application of 30 µM glycine depressed IGly to a similar extent. C-b, means (±SEM, n = 4) of normalized IGly during second and third pulse of similar sequences of pulses of genistein (30 µM); IGly was normalized to peak IGly during the first pulse of genistein.
where R is the GlyRs, D is genistein, and RD is genistein-bound GlyRs. The apparent antagonist dissociation constant (KD) was calculated from the measured on- (ton) and off- (toff) rate constants by first deriving the estimated forward (kon) and reverse (koff) binding rate constants; koff is the reciprocal of the measured toff and kon was obtained from: k on = (1 / t on–k off) / [D], and
Fig. 4. The inhibition of IGly by a short pulse of genistein (10 µM) is very rapid. (A, B) The onset and offset of genistein’s effect could be fitted by single exponentials (solid lines). (C) Kinetic analysis of genistein-provoked block of steady-state IGly induced by 30 µM glycine. In plots of on- and off-rate constants vs. genistein concentration, points are means (±SEM) and lines were fitted by linear regression. The apparent association rate increased with genistein concentration, whereas the apparent dissociation rate was independent of antagonist concentration (n = 10 from five cells).
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K D = k off / k on. For example, in Fig. 4B the time constants of onset and offset were 310 and 350 ms for the block produced by a brief pulse of 10 µM genistein. From these values, we calculated K D = 77 µM which is sixfold greater than the IC50 of 13 µM given by the genistein dose–response curves. Thus, the simple model (Eq. (1)) is not appropriate; as also indicated by the low forward rate constant kon ((1.76 ± 0.03) × 104 M⫺1s⫺1, n = 4), which is five orders of magnitude slower than the rate of diffusion to a fully exposed site (109 M⫺1s⫺1, Burgen, 1966; Chang and Weiss, 1999). In a similar manner, we estimated the on- and off-rate constants of the block of steady-state IGly (induced by 30 µM glycine) by 3, 10 and 30 µM genistein. A plot of 1/ton vs. genistein concentration was linear over this range (Fig. 4C, open circles). From the slope of this plot the apparent association rate constant was calculated to be near 0.7 × 105 M⫺1 s⫺1 and from the zero y-intercept the apparent dissociation rate constant was 2.5 s⫺1,which is in good agreement with that obtained from the plot of 1/toff (2.6 s⫺1). The value of 1/toff (closed circles) did not alter with changes in genistein concentration. From the apparent association and dissociation rate constants, a KD of 40 µM was calculated for genistein in 30 µM glycine, which is in broad agreement with the KD of 77 µM already obtained with a single concentration of genistein (10 µM). The overall rate of block not only reflects a possibly limited access to the binding site, but also suggests bound genistein closes the channel. We then studied the effects of genistein on the kinetics of IGly. To allow accurate measurement of time constants within the limits of the fast perfusion system, glycine was applied at concentrations less than 30 µM. In agreement with previous observations (Ye et al., 2001), after a brief latent period, the onset of the inward current following glycine concentration jumps could be fitted by a single exponential function (Fig. 5A). Genistein significantly increased the activation time of IGly. Fig. 5A shows that in the presence of 0, 3 and 30 µM genistein, the activation time constant was 241, 487 and 711 ms, respectively. The plot in Fig. 5B indicates that the relationship between 1/ton and the concentration of glycine is linear. The slopes of these curves—which give estimates of kon for glycine—were significantly reduced by genistein: 1.0 × 105 M⫺1 s⫺1, 7.3 × 104 M⫺1 s⫺1, and 2.9 × 104 M⫺1 s⫺1 in 0, 3 and 30 µM genistein, respectively (ANOVA, P ⬍ 0.05; n = 3–6). We also measured the deactivation time constant (toff) for glycine-activated channels from the time course of responses when glycine was rapidly washed from the external medium. The values of toff obtained by fitting single exponentials to the current decay after glycine concentration jumps did not change significantly, either with glycine or with genistein concentration (Fig. 5A and C, ANOVA, P ⬎ 0.05; n = 3–6). Furthermore, the intercept of these plots was used to estimate the apparent
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Fig. 5. Effects of genistein on kinetics of IGly. (A) Whole-cell recordings of currents activated by 30 µM glycine in the absence and presence of 3 and 30 µM genistein. Both activation and deactivation were well fitted by single exponentials (solid lines). To eliminate possible complicating effect of slower onset and offset of genistein’s action, genistein application started 10 s before and ended after that of glycine. Note that both 3 and 30 µM genistein greatly increased the activation time-constant (ton), but had little effect on the deactivation time-constant (toff). (B) Graphs of mean values of 1/ton (±SEM) as function of glycine concentration in absence (䊊) and presence of genistein (3 (쎲) and 30 µM (䊏)). Note approximately linear relation between 1/ton and glycine concentration. The slopes of these curves give estimates of the forward rate of glycine binding. (C) Graph of mean values of 1/toff vs. glycine concentration in the absence (䊊) and presence of genistein (3 (쎲) and 30 µM (䊏)). The y-intercepts of the lines yield estimates of the reverse rate of unbinding (koff). Mean 1/toff for IGly was not sensitive to the concentration of either genistein (by ANOVA, P ⬎ 0.05; n = 3–6) or glycine (by ANOVA, P ⬎ 0.5; n = 3–6).
dissociation rate (k off = 2.2 s⫺1), which did not change significantly in the presence of genistein and was in good agreement with that obtained from the plot of 1/toff (2.3 s⫺1). These koff values and the data in Fig. 1C indicate that genistein does not affect the apparent affinity of the receptor for glycine.
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3.4. Genistein does not increase receptor desensitization The depression of glycine-activated currents by genistein could result from increased receptor desensitization. To test this hypothesis, we studied the desensitization of IGly in the absence and presence of genistein. As shown in Fig. 6, the simple exponential rate of decay of currents activated by 30 µM glycine did not significantly change. The ratio of the decay time constants (tgenistein/tcontrol) was 1.07 in Fig. 6, and 1.09 ± 0.2 (P ⬎ 0.05) for a total of four cells. 3.5. Genistein-induced rebound of IGly Fig. 7A illustrates in more detail the transient increase in inward current seen immediately after the rapid withdrawal of genistein (see also Fig. 1A-c, d; Fig. 2A-b, Fig. 3C-a). When various concentrations of genistein (3– 30 µM) were co-applied with 30 µM glycine, the rebound current increased with the concentration of
Fig. 7. Rebound current induced by genistein washout may reflect genistein-induced potentiation of IGly. (A) Rebound current at the end of glycine and genistein co-application. (B) The magnitude of the rebound current increased with the concentration of genistein. (C) Currents in response to 30 µM glycine alone (a) and with superimposed brief applications of 10 or 30 µM genistein (hatched bars in b, c). Note rebound IGly at end of genistein pulse can exceed initial peak IGly (IT, in C-c). (D) Concentration–response relation of genistein-induced rebound of IGly. Data are ratios of IT to IGly immediately preceding genistein application (means ± SEM from between four and five neurons). Fig. 6. Genistein does not increase desensitization of IGly. (A) Superimposed traces illustrate decay of currents elicited by long application of 30 µM glycine, in absence and presence of 10 µM genistein. (B) Same currents normalized with reference to peak control current. As suggested by the indicated time-constants of decay (td), average values of td obtained in absence and presence of 10 µM genistein did not differ significantly (6.2 ± 0.7 and 6.8 ± 1.1 s, respectively; P ⬎ 0.05, n = 4).
genistein (Fig. 7B–D). After a 10-µM pulse of genistein, the rebound IGly was greater than would be expected if GlyRs had remained in the desensitized state (Fig. 7Cb). When 30 µM genistein and 30 µM glycine were coapplied (Fig. 7C-c), the rebound current was so large that it greatly exceeded the peak of control IGly. Such an increase seems to indicate some genistein-induced
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Fig. 8. Interaction of genistein and picrotoxin. The suppressive effect of genistein in indicated concentrations on IGly (50 µM) is diminished by 20 µM picrotoxin (PTX). The effects of co-applied genistein with picrotoxin on IGly were much smaller than the sum of the suppressive effects when they were applied individually with glycine. Bars are the mean ± SEM of 4 to 11 neurons.
potentiation of IGly. This phenomenon was also observed in six other neurons. The amplitudes of the rebound IGly, normalized to that of control IGly, are summarized in Fig. 7D as a function of genistein concentration. Genistein increases IGly rebound in a concentration-dependent manner. 3.6. Genistein may act at the same region of the glycine receptor as picrotoxin To further investigate how genistein suppresses IGly, we performed competition experiments with picrotoxin, an open channel blocker of homomeric GlyR channels. To simplify the comparison between the suppressive effects of picrotoxin, genistein and of their mixture on IGly, we assumed that if genistein and picrotoxin act by completely different mechanisms, the suppressive effects produced by the mixture of genistein and picrotoxin should be equal or close to the sum of their individual Fig. 9. Further evidence that genistein-induced rapid block of IGly is not mediated by tyrosine kinase. (A) IGly traces from same VTA neuron show typical IGly depression by co-application of 50 µM daidzein (close genistein derivative that does not block tyrosine kinases) and lack of effect of 5 µM lavendustin A (specific blocker of tyrosine kinases). (B) B-a demonstrates rapid onset and offset of IGly depression by brief pulse of daidzein (50 µM) during continued application of glycine. B-b, both onset and offset of daidzein’s effect could be fitted by single exponentials (solid lines). (C) Bar graphs are means (±SEM) of normalized peak IGly in the presence of 50 µM daidzein or 5 µM lavendustin A; peak IGly was normalized to peak response to glycine alone. (D) Glycine current–voltage relations from voltage ramps in absence (a) and presence (b) of co-applied daidzein. Corresponding difference currents show no voltage-sensitivity of daidzein effect, neither in original plots (E), nor in plots normalized to control current at –80 mV (F).
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effects. In agreement with our previous reports (Ye et al., 1998; Ye, 2000), VTA GlyRs of neonatal rats are sensitive to picrotoxin. Furthermore, although the sensitivity to picrotoxin of GlyRs in VTA neurons changed with development (Ye, 2000), this change is not significant among the neurons of 5–14-day-old rats (data not shown). For this series of neurons, 20 µM picrotoxin and 30 µM genistein suppressed IGly in response to 50 µM glycine by 41 ± 6% (n = 11), and by 34 ± 5% (n = 7) of control current, respectively. When 20 µM picrotoxin and 30 µM genistein were coapplied with 50 µM glycine, IGly was suppressed by 51 ± 16% of control (n = 4), which is significantly smaller than 75%, the sum of their individual effects (P ⬍ 0.05, n = 4). Similar results were observed with other concentrations of genistein. While 3 and 100 µM genistein alone suppressed IGly by 28 ± 7% (n = 7) and by 65 ± 7% (n = 6) of control, respectively, when coapplied with 20 µM picrotoxin, they suppressed IGly by 42 ± 11% (n = 7), and 69 ± 10% (n = 4) of control, respectively. These values are much smaller then the sum of 75 and 96%, respectively (Fig. 8). The mutual occlusion between their effects suggests that genistein and picrotoxin share a common binding site on the GlyR or act on sites that are functionally related. 3.7. Effects of other tyrosine antagonists on glycine response To further assess the nature of genistein’s actions, we examined the effect of daidzein, an inactive structural analogue of genistein (Akiyama and Ogawara, 1991). As in hypothalamic neurons (Huang and Dillon, 2000), daidzein reversibly suppressed IGly of VTA neurons: 50 µM daidzein reduced the peak current elicited by 30 µM glycine to 76.6 ± 3.6% of control (P ⬍ 0.05, n = 5; Fig. 9). In some neurons, a rebound current was seen after daidzein (data no shown). Both the onset and offset of daidzein action could be fitted by a single exponential, which suggests that the daidzein-induced block also behaved as a simple bimolecular reaction. In Fig. 9B, in the presence of 50 µM daidzein, the time constants of onset and offset are 410 and 507 ms, the mean values being 400 ± 40 and 500 ± 30 ms, respectively (n = 14–16 measurements from six neurons). These values were used to calculate K D = 200 ± 15 µM. Because the forward rate constant kon (1 × 104 M⫺1 s⫺1, n = 4) is much slower than free diffusion in solution, the binding site for daidzein is not freely accessible. To examine the voltage dependence, a voltage ramp protocol was used (Fig. 9D). As illustrated in Fig. 9E–F, the current–voltage curves were linear and daidzein decreased IGly to a similar extent at all membrane voltages between ⫺80 and +30 mV. Thus, in contrast to genistein, daidzein acts in a membrane voltage-insensitive manner. In addition, we examined the effects of lavendustin
A, a highly selective inhibitor of protein tyrosine kinase. As in hypothalamic neurons (Huang and Dillon, 2000), co-applications of 5 µM lavendustin A with 30 µM glycine did not affect IGly of VTA neurons, the peak current was 96.9 ± 2.7% (n = 18, P ⬎ 0.05, t-test) of control (Fig. 9). These results further support the notion that genistein inhibits IGly independently of tyrosine kinase. 4. Discussion Confirming and extending previous observations on hypothalamic cells in slices (Huang and Dillon, 2000), we have found that 1–300 µM genistein depressed IGly in neurons freshly isolated from the VTA of young rats. This is the first report of such an effect of genistein on GlyRs of isolated VTA neurons. The fact that the inhibition of IGly was rapid and reversible suggested that genistein acts directly on the GlyRs (Huang and Dillon, 2000). Our experiments offer further evidence in support of this idea. On the following basis, we propose that genistein most likely inhibits GlyRs by an open channel block. First, genistein inhibited IGly only when it was co-applied with the agonist, and to the same extent without and with preincubation; when applied alone before an application of glycine alone, genistein did not inhibit IGly. Therefore genistein can bind to and inhibit the channel only after the binding of glycine, presumably when GlyR channels are open. Second, genistein’s effect was sensitive to membrane voltage, being greater at positive potentials: the binding site is therefore likely to be within the GlyR channel, at a site part of the way across the electric field of the membrane. Furthermore, the competition between the suppressive effects of genistein and picrotoxin further support the idea that genistein acts as an open channel blocker. The rebound of inward current seen at the end of a co-application could be simply explained by if genistein dissociated faster from GlyRs than does glycine, in keeping with the observed dissociation constants (koff) of 2.5 s⫺1 for genistein and 2.2 s⫺1 for glycine. Note that the koff of daidzein is only 2.0 s⫺1 and less rebound current was seen after application of daidzein, in keeping with this explanation. The block of an increasing number of channels when the concentration of genistein is raised at least partly accounts for the rebound’s marked concentration-dependence. Small rebound currents have also been observed after block of GlyR channels by 2,3-butanedione monoxime (Ye and McArdle, 1996), by Waglerin (Ye et al., 1999) and GABAAR channels by picrotoxin (Ye et al., 1998) and genistein (Huang et al., 1999). Picrotoxin is known to bind to a site inside the channel lumen (Edwards and Lees, 1997). However, the greater than expected rebound current shown in Fig. 7C and D suggests an additional effect of genistein. One possible
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explanation is that genistein reverses glycine-induced desensitization. However, as shown in Fig. 7C, IGly evoked by 30 µM glycine showed only minor desensitization. Reduced desensitization therefore cannot be the only mechanism. Another possibility is that such rebound currents reflect the transition from a ‘channelblocked’ to a ‘reopened’ state, a mechanism underlying the rebound currents induced by the rapid withdrawal of isoflurane (Hapfelmeier et al., 2001). Our data also support their notion that an open-channel block at ligandgated receptors can prolong postsynaptic currents (Hapfelmeier et al., 2001). The other possibility is that, genistein has a biphasic action: a fast suppression and a delayed potentiation of GlyRs, which is similar to the biphasic action of penicillin on IGly (Tokutomi et al., 1992). The mechanism underlying the biphasic effect of these agents requires further study. In the current study, the use-dependence often associated with open channel block was not evident, peak and steady state IGly being inhibited to the same extent, and repeated pulses of genistein equally effective during a longer pulse of glycine. The degree of use-dependence is a function of the microscopic dissociation rate constant. While ‘slow’ blockers show extreme use-dependence, use-dependence of ‘fast’ blockers may not be so clear (Gibb, 2002). As genistein has a dissociation rate of 3.1 s⫺1, the mean lengths of the blockage gaps is ⬍300 ms. It is useful to compare the suppressing effect of genistein on IGly with that of picrotoxin. As with picrotoxin, genistein suppresses IGly only when the channels are open. This observation and the occlusion between the effects of genistein and picrotoxin on IGly suggest a similar mechanism and site of action. Therefore, we suggest that both genistein and picrotoxin compete for the picrotoxin binding site of the GlyR. When picrotoxin occupies this site, genistein can no longer bring about suppression of IGly. The sensitivity of IGly to picrotoxin also indicates the absence of the b-subunit of the GlyRs, which support the argument ruling out a possible action through PTKmediated phosphorylation, since the potential site for PTK-mediated phosphorylation is in the b-subunit. The effect of genistein was mimicked by daidzein, a close analogue of genistein that does not block activation of PTK. Interestingly, the forward rate (kon) for daidzein (1 × 10⫺4 M⫺1 s⫺1) was 17 times slower than that for genistein (1.7 × 10 - 5 M⫺1 s⫺1); this may reflect a lower rate of access. A somewhat different site of action is suggested by the lack of voltage-dependence of daidzein’s effect on IGly. Both features are perhaps ascribable to the additional OH group on the daidzein molecule. The comparable block of IGly by daidzein gives further support for the idea that genistein acts independently of PTK-mediated phosphorylation. In the previous study on hypothalamic neurons
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(Huang and Dillon, 2000) 100 µM genistein reduced IGly by only 30%, whereas our VTA data yielded an IC50 of 13 µM. Several technical reasons may account for this difference: notably our recording from a quite different brain area, as well as the use of isolated VTA neurons instead of brain slices: a fast and accurate change of external solutions is important, because of rapid desensitization of glycine receptors. As a tyrosine kinase inhibitor, genistein has frequently been used to detect an involvement of tyrosine phosphorylation in the modulation of various receptors— most often with daidzein as an inactive control. However, evidence has been accumulating that, independently of tyrosine kinase, genistein can also directly interact with many receptor/ion channels: including those for GABAA (Dunne et al., 1998; Huang et al., 1999) and glycine (Huang and Dillon, 2000), as well as voltage-sensitive Na+ channels (Paillart et al., 1997), voltage-gated K+ channels (Ogata et al., 1997; Zhang and Wang, 2000), L-type Ca2+ channels (Yokoshiki et al., 1996; Katsube et al., 1998), ATP-sensitive K+ channels (Ogata et al., 1997) and cystic fibrosis transmembrane conductance regulator (CFTR) Cl⫺ channels (French et al., 1997; Weinreich et al., 1997; Al-Nakkash et al., 2001). But we did not eliminate the possibility that longer applications of genistein (and lavendustin A) also affect PTK-mediated phosphorylation of glycine receptors. In summary, genistein appears to be an open channel blocker producing a rapid direct block of GlyRs in VTA, but a possible role of tyrosine kinase in longer-term modulation of this receptor has not been ruled out. Acknowledgements This study is supported by National Institute of Alcohol Abuse and Alcoholism, National Institute of Health Grant AA-11989 (to JHY). The authors are grateful to Dr. Yongchang Chang for his critical reading and his comments on this manuscript. References Agopyan, N., Tokutomi, N., Akaike, N., 1993. Protein kinase Amediated phosphorylation reduced only the fast desensitizing glycine current in acutely dissociated ventromedial hypothalamic neurons. Neuroscience 56, 605–615. Aguayo, L.G., Tapia, J.C., Pancetti, F.C., 1996. Potentiation of the glycine-activated Cl⫺ current by ethanol in cultured mouse spinal neurons. Journal of Pharmacology and Experimental Therapeutics 279, 1116–1122. Akiyama, T., Ogawara, H., 1991. Use and specificity of genistein as inhibitor of protein-tyrosine kinase. Methods in Enzymology 201, 362–370. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., Fukami, Y., 1987. Genistein, a specific inhibitor of protein-tyrosine kinases. Journal of Biological Chemistry 262, 5592–5595.
280
L. Zhu et al. / Neuropharmacology 45 (2003) 270–280
Albarran, F.A., Roa, J.P., Navarrete, R., Castillo, R., Nualart, F., Aguayo, L.G., 2001. Effect of protein kinase C activation on the glycine evoked Cl(⫺) current in spinal cord neurons. Brain Research 902, 1–10. Al-Nakkash, L., Hu, S., Li, M., Hwang, T.C., 2001. A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs. Journal of Pharmacology and Experimental Therapeutics 296, 464–472. Barry, P., 1996. Axoscope junction potential calculator. Axobits 18, 3–4. Betz, H., Kuhse, J., Fischer, M., Schmieden, V., Laure, B., Kuryatov, A., Langosch, D., Meyer, G., Bormann, J., Rundstrom, N., Matzenbach, B., Kirsch, J., Ramming, M., 1994. Structure, diversity and synaptic localization of inhibitory glycine receptors. Journal of Physiology (Lond) 88, 243–248. Burgen, A.S., 1966. The drug–receptor complex. Journal of Pharmacy and Pharmacology 18, 137–149. Caraiscos, V.B., Mihic, S.J., MacDonald, J.F., Orser, B.A., 2002. Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human alpha(1)beta glycine receptors. Journal of Physiology (Lond) 539, 495–502. Chang, Y., Weiss, D.S., 1999. Channel opening locks agonist onto the GABAC receptor. Nature Neuroscience 2, 219–225. Davis, M.J., Wu, X., Nurkiewicz, T.R., Kawasaki, J., Gui, P., Hill, M.A., Wilson, E., 2001. Regulation of ion channels by protein tyrosine phosphorylation. American Journal of Physiology, Heart and Circulation Physiology 281, H1835–H1862. Dunne, E.L., Moss, S.J., Smart, T.G., 1998. Inhibition of GABAA receptor function by tyrosine kinase inhibitors and their inactive analogues. Molecular and Cellular Neuroscience 12, 300–310. Edwards, M.D., Lees, G., 1997. Modulation of a recombinant invertebrate gamma-aminobutyric acid receptor-chloride channel complex by isoflurane: effects of a point mutation in the M2 domain. British Journal of Pharmacology 122, 726–732. French, P.J., Bijman, J., Bot, A.G., Boomaars, W.E.M., Scholte, B.J., Jonge, H.R.D., 1997. Genistein activates CFTR Cl⫺ channels via a tyrosine kinase- and protein phosphatase-independent mechanism. American Journal of Physiology 273, C747–C753. Gatto, G.J., Mcbride, W.J., Murphy, J.M., Lumeng, L., Li, T.K., 1994. Ethanol self-infusion into the ventral area by alcohol-preferring rats. Alcohol 11, 557–564. Gibb, A.J., 2002. Receptors linked to ion channels: mechanisms of activation and block. In: Foreman, J.C., Johansen, T. (Eds.), Textbook of receptor pharmacology, second ed. CRC Press Inc, Florida, USA. Gu, Y., Huang, L.-Y.M., 1998. Cross-modulation of glycine-activated Cl⫺ channels by protein kinase C and cAMP-dependent protein kinase in the rat. Journal of Physiology (Lond) 284, 826–831. Hapfelmeier, G., Haseneder, R., Kochs, E., Beyerle, M., Zieglgansberger, W., 2001. Coadministered nitrous oxide enhances the effect of isoflurane on GABAergic transmission by an increase in open-channel block. Journal of Pharmacology and Experimental Therapeutics 298, 201–208. Huang, R.Q., Dillon, G.H., 2000. Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor. Neuropharmacology 39, 2195–2204. Huang, R.Q., Fang, M.J., Dillon, G.H., 1999. The tyrosine kinase inhibitor genistein directly inhibits GABAA receptors. Molecular Brain Research 67, 177–183. Jiang, Z.L., Ye, J.H., 2003. Protein kinase C⑀ is involved in ethanol potentiation of glycine-gated Cl⫺ current in rat neurons of ventral tegmental area. Neuropharmacology 44, 493–502. Katsube, Y., Yokoshiki, H., Nguyen, L., Yamamoto, M., Sperelakis, N., 1998. Inhibition of Ca2+ current in neonatal and adult rat ventricular myocytes by the tyrosine kinase inhibitor, genistein. European Journal of Pharmacology 345, 309–314. Legendre, P., 1999. Voltage dependence of the glycine receptor-chan-
nel kinetics in the zebrafish hindbrain. Journal of Neurophysiology 82, 2120–2129. Ogata, R., Kitamura, K., Ito, Y., Nakano, H., 1997. Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle. British Journal of Pharmacology 122, 1395–1404. Paillart, C., Carlier, E., Guedin, D., Dargent, B., Couraud, F., 1997. Direct block of voltage-sensitive sodium channels by genistein, a tyrosine kinase inhibitor. Journal of Pharmacology and Experimental Therapeutics 280, 521–526. Ren, J., Ye, J.H., McArdle, J.J., 1998. cAMP-dependent protein kinases modulation of glycine-activated chloride current in neurons freshly isolated from rat ventral tegmental area. Brain Research 811, 71–78. Ruiz-Gomez, A., Vallo, M.L., Valdivieso, F., Mayor, F.J., 1994. Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C. Journal of Biological Chemistry 266, 559–566. Schonrock, B., Bormann, J., 1995. Modulation of hippocampal glycine receptor channels by protein kinases C. Neuroreport 6, 301–304. Song, Y., Huang, L.-Y.M., 1990. Modulation of glycine receptor chloride channel by cyclic AMP-dependent protein kinases in spinal trigeminal neurons. Nature 348, 242–245. Swope, S.L., Moss, S.I., Raymond, L.A., Huganir, R.L., 1999. Regulation of ligand-gated ion channels by protein phosphorylation. Advances in Second Messenger and Phosphoprotein Research 33, 49–78. Tao, L., Ye, J.H., 2002. Protein kinase C modulates ethanol inhibition of glycine-activated current in dissociated neurons of rat ventral tegmental area. Journal of Pharmacology and Experimental Therapeutics 300, 967–975. Tokutomi, N., Agopyan, N., Akaike, N., 1992. Penicillin-induced potentiation of glycine receptor-operated chloride current in rat ventro-medial hypothalamic neurones. British Journal of Pharmacology 106, 73–78. Wagner, K., Edson, K., Heginbotham, L., Post, M., Huganir, R.L., Czernik, A.J., 1991. Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor. Journal of Biological Chemistry 266, 23784–23789. Weinreich, F., Wood, P.G., Riordan, J.R., Nagel, G., 1997. Direct action of genistein on CFTR. Pflu¨ gers Archiv 434, 484–491. Wise, R.A., 1996. Additive drugs and brain stimulation reward. Annual Review of Neurosciences 19, 319–340. Ye, J.H., 2000. Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons. Brain Research 862, 74–82. Ye, J.H., McArdle, J.J., 1996. 2,3-Butanedione monoxime modifies the glycine-gated chloride current of acutely isolated murine hypothalamic neurons. Brain Research 735, 20–29. Ye, J.H., McArdle, J.J., 1997. Waglerin-1 modulates gamma-aminobutyric acid activated current of murine hypothalamic neurons. Journal of Pharmacology and Experimental Therapeutics 282, 74–80. Ye, J.H., Ren, J., Liu, P.L., McArdle, J.J., 1998. Glycine-activated chloride currents of neurons freshly isolated from the ventral tegmental area of rats. Brain Research 796, 53–62. Ye, J.H., Ren, J., McArdle, J.J., 1999. Waglerin-1 inhibits GABA(A) current of neurons in the nucleus accumbens of neonatal rats. Brain Research 837, 29–37. Ye, J.H., Tao, L., Zhu, L., Krnjevic´ , K., McArdle, J.J., 2001. Ethanol inhibition of glycine-activated responses in neurons of ventral tegmental area of neonatal rats. Journal of Neurophysiology 86, 2426–2434. Yokoshiki, H., Sumii, K., Sperelakis, N., 1996. Inhibition of L-type calcium current in rat ventricular cells by the tyrosine kinase inhibitor, genistein and its inactive analog, daidzein. Journal of Molecular Cell Cardiology 28, 807–814. Zhang, Z.H., Wang, Q., 2000. Modulation of a cloned human A-type voltage-gated potassium channel (hKv1.4) by the protein kinase. Pflu¨ gers Archiv 440, 784–792.
Genistein directly blocks glycine receptors of rat neurons freshly isolated from the ventral tegmental area L. Zhu a, Z.L. Jiang a, K. Krnjevic´ b, F.S. Wang a, J.H. Ye a,∗ a
New Jersey Medical School (UMDNJ), Rutgers-UMDNJ Integrative Neuroscience Program, Departments of Anesthesiology, Pharmacology and Physiology, 185, South Orange Avenue, Newark, NJ 07103-2714, USA b Physiology Department, McGill University, Montre´al, Canada Received 30 August 2002; received in revised form 28 March 2003; accepted 28 March 2003
Abstract The effects of tyrosine kinase inhibitors on the glycine-induced current (IGly) were studied in rat neurons freshly isolated from the ventral tegmental area (VTA). Genistein reversibly and concentration-dependently depressed IGly, with an IC50 of 13 µM. Preincubation with genistein had no effect on IGly, indicating that genistein is effective only when glycine is bound to the receptor and channels are most likely open. Genistein depressed maximum IGly without significantly changing the EC50 for glycine. Genisteininduced inhibition of IGly was sensitive to membrane voltage, being greater at positive membrane potentials. A kinetic analysis indicated that genistein lengthens the time constant of IGly activation, but has no effect on deactivation or desensitization. When genistein was rapidly washed out, a transient rebound current probably reflected a faster dissociation of genistein, with respect to glycine. Results of competition experiments suggest that genistein acts on the same region of the glycine receptor as picrotoxin. Daidzein, an analog of genistein that does not act on protein kinases, also inhibited IGly. Co-application of lavendustin A, a specific inhibitor of tyrosine kinase, had no effect on IGly. Our results extend to neurons isolated from the VTA, the previous finding that genistein directly inhibits glycine receptors of hypothalamic brain slices. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Whole-cell patch clamp; Rebound current; Open channel block; Daidzein; Tyrosine kinase antagonists; Picrotoxin
1. Introduction Phosphorylation of ligand-gated ion channels is recognized as a potentially important mechanism of short- and long-term modulation of ion-channel function. Following the discovery of numerous sites of phosphorylation on ligand-gated ion channel proteins, recent studies have demonstrated that activation of serine/threonine, tyrosine and other kinases can modulate various neurotransmitter-induced membrane currents (Swope et al., 1999; Davis et al., 2001). The strychnine-sensitive glycine receptor (GlyR) con-
Abbreviations: IGly, glycine-induced current; PKA, cyclic AMP dependent kinase; PKC, protein kinase C; PTK, tyrosine kinase; GABAA, g-aminobutyric acid A; td, time constant of decay; ton, activation time constant; toff, deactivation time constant; VTA, ventral tegmental area ∗ Corresponding author. Tel.: +1-973-972-4399; fax: +1-973-9724172. E-mail address: [email protected] (J.H. Ye).
sists of two main subunits, a (48 kDa) and b (58 kDa), forming a pentameric channel structure (Betz et al., 1994). The glycine receptor–chloride channel complex is phosphorylated by several protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and tyrosine kinase (PTK) (Song and Huang, 1990; Agopyan et al., 1993; Ruiz-Gomez et al., 1994; Schonrock and Bormann, 1995; Aguayo et al., 1996; Gu and Huang, 1998; Albarran et al., 2001; Caraiscos et al., 2002). The tyrosine residue in the intracellular loop of the glycine b subunit is a potential site for PTK-mediated phosphorylation (Wagner et al., 1991). Immunological and molecular cloning studies reveal that GlyRs are widely distributed not only in the spinal cord and brain stem, but also throughout the mammalian central nervous system, including the ventral tegmental area (VTA) (Betz et al., 1994; Ye et al., 1998). The VTA is an important mediator of the rewarding effects of drugs of abuse, including ethanol (Gatto et al., 1994; Wise, 1996). We are interested in the interactions
0028-3908/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00151-5
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of ethanol and GlyRs of VTA neurons, and the role of protein phosphorylation in these interactions. Recently, we have shown that more than 80% of VTA neurons respond to glycine, and that the GlyRs of VTA neurons are regulated by PKA and PKC (Ren et al., 1998; Tao and Ye, 2002; Jiang and Ye, 2003). In order to better understand the role of PTK phosphorylation in regulation of the GlyRs of VTA neurons, we studied the effects of several relevant agents, including genistein, daidzein and lavendustin A. As a PTK inhibitor, genistein has been used widely to assess PTK-mediated regulation of receptor/channel function (Akiyama et al., 1987; Akiyama and Ogawara, 1991). A recent study showed that PTK enhances GlyR function in spinal and hippocampal neurons (Caraiscos et al., 2002); but according to another report, genistein directly blocks hypothalamic GlyRs through a tyrosine kinase-independent mechanism that is not well understood (Huang and Dillon, 2000). The aim of the present study was to determine whether genistein also depresses GlyRs of dissociated VTA neurons. The combination of dissociated neurons and patch clamp technique is powerful, because it allows a fast exchange of external solutions. Thus, a more accurate concentration–response relation could be obtained, and the kinetics of IGly analyzed, which is important for a better understanding of the underlying mechanisms.
2. Methods 2.1. Isolation of neurons and electrophysiological recording The care and use of animals and the experimental protocol of this study were approved by the Institutional Animal Care and Use Committee of University of Medicine and Dentistry of New Jersey. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Sprague–Dawley rats (5–14-day-old) were decapitated as described earlier (Ye et al., 2001). The brain was quickly excised, placed into ice-cold saline saturated with 95% O2 and 5% CO2, glued to the chilled stage of a vibratome (Campden Instruments, UK) and sliced to a thickness of 400 µm. Slices were incubated at 31 °C, for 15 min in standard external solution containing 1 mg pronase/12 ml and saturated with O2, and for an additional 15 min in 1 mg thermolysin/12 ml. Micropunches of the VTA were isolated and transferred to a 35-mm culture dish. Mild trituration through heatpolished pipettes of progressively smaller tip diameters dissociated single neurons. Within 20 min of trituration, isolated neurons attached to the bottom of the culture dish and electrophysiological experiments were started. The saline in which the brain was dissected contained
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(in mM): NaCl 128, KCl 5, NaH2PO4 1.2, NaHCO3 26, MgCl2 9, CaCl2 0.3 and glucose 2.5. The standard external solution contained (mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10. The pH was adjusted to 7.4 with Tris base and the osmolarity to 320 mOsm with sucrose. The patch electrodes had a resistance of 3–5 M⍀ when filled with solution containing (mM): CsCl 120, TEA-Cl 21, MgCl2 4, EGTA 11, CaCl2 1, HEPES 10 and Mg-ATP 2; the pH 7.2 adjusted with Tris base, the osmolarity 280 mOsm with sucrose. After establishing the giga-seal in the cell-attached configuration by gentle suction, whole-cell recording was obtained by a further suction. Throughout all experiments the bath was continually perfused with the standard external solution. All glycine-induced responses were elicited in this solution at an ambient temperature of 20–23 °C. Currents were recorded under voltage-clamp with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) interfaced to a data acquisition A/D converter Digidata 1320A (Axon Instruments) and directly digitized with pCLAMP 8 software for further off-line analysis. The junction potential between the patch pipette and the bath solutions was nulled just before forming the giga-seal. The liquid junction potential between the bath and the electrode was 3.3 mV, as calculated from the generalized Henderson equation using the Axoscope junction potential calculator (Barry, 1996). This value was corrected off-line when estimating the reversal potential of glycine-activated currents. In most experiments, the series resistance before compensation was 15–25 M⍀. Routinely, 80% of the series resistance was compensated; hence, there was a 3–5 mV error for 1 nA of current. 2.2. Chemical applications Most of the chemicals including glycine, picrotoxin and strychnine were obtained from Sigma Chemical Company (St. Louis, MO, USA), and genistein, daidzein and lavendustin A were obtained from CalbioChem (La Jolla, CA). Their solutions were prepared on the day of experimentation. Genistein, daidzein and lavendustin were predissolved in dimethyl sulphoxide (DMSO) and diluted to its final concentration in standard external solution. At the maximum concentration applied (0.05% (v/v)), DMSO neither induced nor blocked membrane currents and had no effect on the glycine responses. Solutions were applied to a dissociated neuron via a multibarreled superfusion system, consisting of five needles aligned and held together with heat-shrinkable plastic tubing and exiting into a common outflow tube (as described previously, Ye and McArdle, 1997). Each barrel of the pipette was connected to a separate reservoir containing solutions of various drugs. Solutions were exchanged by simultaneously closing and opening
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valves controlling flow from the reservoirs. These maneuvers were performed manually and the flow rate was regulated hydrostatically. The tip of the superfusion pipette was usually placed 50–100 µm away from a cell, a position that allowed rapid as well as uniform drug application while preserving the neuron’s mechanical stability. By keeping the dead volume small and the flow rate high, solutions could be exchanged within 20 ms. The speed of solution exchange was measured by reducing the external Na+ concentration from 140 to 10 mM (plus 130 mM N-methyl-D-glucamine, NMDG), while recording a stable kainate current: the rate of decrease of the kainate current reflected the rate of solution change. The time constant of such change was ⬍20 ms. 2.3. Data analysis Whole-cell current decays were fitted by a Chebychev algorithm (pCLAMP). Concentration–response data were analyzed with a nonlinear curve-fitting program (Sigma Plot; Jandel Scientific, San Rafael, CA). Data were statistically compared using Student’s t-test at a significance level of P ⬍ 0.05. Throughout, means are given ±SEM, with the number of experiments in brackets.
3. Results 3.1. Genistein depresses IGly of VTA neurons In agreement with our earlier report, glycine elicited a current (IGly) in approximately 82% of VTA neurons from young rats (Ye et al., 1998; Ye, 2000). As expected, IGly was antagonized by 0.1 µM strychnine (Ye et al., 1998; Ye, 2000). Genistein (1–300 µM) alone had no detectable effect on membrane current; but when co-applied with the agonist, it decreased IGly in all neurons tested (n = 95), in a concentration-dependent manner. Fig. 1A shows examples of current activated by 30 µM glycine alone (A-a) and in the presence of 3, 10 and 30 µM genistein (A-b, -c, and -d), respectively. After a transient rebound of inward current at the end of the genistein application, IGly rapidly decayed to the base-line. The depressant effect of genistein was fully reversible after washout of genistein (A-e). In similar tests on five neurons, 3, 10, and 30 µM genistein decreased peak IGly to 66 ± 8% (n = 4), 52 ± 2% (n = 5), and 36 ± 2% (n = 5), respectively. The concentration–response analysis of Fig. 1B reveals that genistein suppressed IGly in response to 30 µM glycine with an apparent IC50 of 13 µM and a Hill coefficient of 0.6. Genistein (10 µM) effectively suppressed IGly evoked by subsaturating and saturating concentrations of glycine (data not shown). This is further demonstrated by the
Fig. 1. Genistein depresses IGly. (A) Records of IGly induced by 30 µM glycine in absence (A-a and e) and presence of 3 (A-b), 10 (Ac), and 30 (A-d) µM genistein. Sequence of current traces (from left to right) was obtained from single neuron. The bar above each trace indicates duration of drug application. At washout of higher genistein concentrations (ⱖ10 µM), there was a transient increase of inward rebound of current (A-c and d). (B) Concentration–response relation for genistein-induced depression of IGly. Data from between four and five neurons are means (±SEM) of normalized peak IGly elicited by 30 µM glycine in the presence of several concentrations of genistein. Peak IGly was normalized to peak response to 30 µM glycine in the absence of genistein. Continuous line is least square fit of the following form of the Hill equation, I / IGly = 1 / [1 + (C /IC50)n] to the data; where I is peak current during co-application of genistein, IGly is control peak current and C concentration of genistein, IC50, the concentration at which genistein produced 50% of inhibition and n, Hill coefficient. For this and other figures, all currents were recorded at a holding potential of –50 mV, unless otherwise indicated. (C) Glycine concentration–response curves show mean data (±SEM, from 6 to 10 neurons) for glycine alone (䊊) or glycine plus 10 µM genistein (쎲). Before pooling, peak currents were normalized to the peak current evoked by 300 µM glycine alone. For individual neurons, three-point glycine concentration–response curves were determined first; and then in the presence of genistein. Continuous lines are least square fits of the following form of the Hill equation to the experimental data: IGly / IGlymax = 1 / [1 + (EC50 / C)n], where IGlymax is maximal IGly, C is glycine concentration, EC50 the concentration at which IGly is 50% of maximum and n the Hill coefficient.
concentration–response plots obtained in the absence and presence of genistein (Fig. 1C). On average, 10 µM genistein reduced the currents induced by 15, 30, 100 and 300 µM glycine to 53 ± 8 (n = 6), 67 ± 3 (n = 10), 64 ± 6 (n = 6), and 68 ± 7% (n = 7) of original values, respectively. Genistein did not significantly change
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either the EC50 of IGly or the Hill coefficient, their respective values being 27.9 µM and 3.2 in the absence, and 25.7 µM and 3.1 in the presence of 10 µM genistein. 3.2. Genistein-induced depression of IGly is sensitive to membrane voltage, but is not use-dependent In view of recent evidence that the gating of GlyRchannels is voltage-dependent (Legendre, 1999) we examined the effect of membrane voltage on the depressant action of genistein on IGly. As illustrated by the current–voltage ramps in Fig. 2A–C, genistein decreased IGly to a significantly greater extent at positive membrane potentials. For example, 30 µM genistein depressed IGly to 31 ± 4% of control at +50 mV, but to 74 ± 10% of control value at –50 mV (P ⬍ 0.05, n = 5, Fig. 2D). However, the reversal potential of IGly remained close to the calculated Nernst potential for Cl⫺ (⫺5 mV in our experimental conditions), in agreement with a previous report (Huang and Dillon, 2000). The voltage sensitivity of genistein’s effect suggests that it may act at least partly as an open channel blocker. To examine this possibility, we performed several additional experiments. First, we compared genistein’s effect on IGly when it was preapplied to neurons for 30 s before co-application with glycine (++ protocol, Fig. 3A-b) to its effect without this prepulse (⫺+ protocol, Fig. 3A-c). The effects were similar with and without the prepulse, as also shown by the data from three other neurons (Fig. 3B). Similar results were obtained when preincubation in genistein was extended to 120 s (data no shown). Thus, genistein has a depressant effect only when the GlyR channels are open. This notion is further supported by the experiments illustrated in Fig. 3A-e and A-g. In these tests, genistein was applied alone for 30 s before the application of glycine alone (+⫺ protocol). These typical traces show that genistein had no effect on IGly. Similar results were obtained from three other neurons (Fig. 3B). Furthermore, repeated pulses of 30 µM genistein during a longer pulse of glycine (30 µM) depressed IGly to a similar extent (Fig. 3C). When compared with the initial effect of genistein, the depression of IGly was 101 ± 2% and 102 ± 4% at the second, and third pulse (P ⬎ 0.05, n = 5; Fig. 3C-b). The effect of genistein shows no use-dependence.
Fig. 2. Genistein depression of IGly is sensitive to membrane voltage. (A) Genistein’s effect on current–voltage relationship of IGly was studied with a pair of voltage ramps (from +50 to ⫺60 mV, at rate of 1 mV/10 ms), first in the absence and then the presence of drugs, as indicated. The initial small current at left in A-a and A-b is the leakage current, which was subtracted from the much larger currents recorded in the presence of 30 µM glycine (alone or plus 30 µM genistein). (B) Current–voltage relations obtained from the ramp protocol in the presence of glycine alone (䊊) or glycine plus genistein (쎲). (C) Normalized current–voltage relations from the same experiment of B show a greater effect of genistein at positive membrane potentials (all currents were normalized to values at ⫺60 mV). (D) Histograms are means (±SEM) of normalized peak IGly obtained in the presence of 30 µM genistein from neurons held at +50 and –50 mV; IGly was normalized to peak IGly in the absence of genistein. Genistein caused significantly greater depression at +50 mV than at –50 mV (P ⬍ 0.01, n = 5).
3.3. Kinetic studies The on-rate time constant of genistein’s action was measured as follows. A VTA neuron was exposed to 30 µM glycine. Once a stable current was attained, the solution was rapidly switched to one containing glycine 30 µM + 10 µM genistein; when the current change became stable, the solution was switched back to glycine alone (Fig. 4A). As shown in Fig. 4B, both the onset
and the offset of genistein’s effect could be fitted by single exponentials. The genistein-induced block therefore behaves as a simple, reversible bimolecular reaction. kon
R ⫹ D,RD koff
(1)
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Fig. 3. Preincubation in genistein does not increase the inhibition of IGly produced by co-applications of glycine and genistein. (A) A-a, Ad and A-f are currents evoked by glycine alone (open bars). A-b, 30 s prepulse of 10 µM genistein was applied (hatched bars) before the co-application of genistein and glycine. A-c, co-application of genistein and glycine depressed IGly to a similar extent. A-e and A-g, 30 s prepulse of 10 µM genistein did not affect IGly induced by subsequent pulse of glycine alone. Traces a–c, d–e, and f–g were from three different neurons. (B) Histograms show means (±SEM) of normalized peak IGly in presence of 10 µM genistein, applied as indicated; IGly was normalized to peak value in absence of genistein. (C) C-a, repeated pulses of 30 µM genistein during continuous application of 30 µM glycine depressed IGly to a similar extent. C-b, means (±SEM, n = 4) of normalized IGly during second and third pulse of similar sequences of pulses of genistein (30 µM); IGly was normalized to peak IGly during the first pulse of genistein.
where R is the GlyRs, D is genistein, and RD is genistein-bound GlyRs. The apparent antagonist dissociation constant (KD) was calculated from the measured on- (ton) and off- (toff) rate constants by first deriving the estimated forward (kon) and reverse (koff) binding rate constants; koff is the reciprocal of the measured toff and kon was obtained from: k on = (1 / t on–k off) / [D], and
Fig. 4. The inhibition of IGly by a short pulse of genistein (10 µM) is very rapid. (A, B) The onset and offset of genistein’s effect could be fitted by single exponentials (solid lines). (C) Kinetic analysis of genistein-provoked block of steady-state IGly induced by 30 µM glycine. In plots of on- and off-rate constants vs. genistein concentration, points are means (±SEM) and lines were fitted by linear regression. The apparent association rate increased with genistein concentration, whereas the apparent dissociation rate was independent of antagonist concentration (n = 10 from five cells).
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K D = k off / k on. For example, in Fig. 4B the time constants of onset and offset were 310 and 350 ms for the block produced by a brief pulse of 10 µM genistein. From these values, we calculated K D = 77 µM which is sixfold greater than the IC50 of 13 µM given by the genistein dose–response curves. Thus, the simple model (Eq. (1)) is not appropriate; as also indicated by the low forward rate constant kon ((1.76 ± 0.03) × 104 M⫺1s⫺1, n = 4), which is five orders of magnitude slower than the rate of diffusion to a fully exposed site (109 M⫺1s⫺1, Burgen, 1966; Chang and Weiss, 1999). In a similar manner, we estimated the on- and off-rate constants of the block of steady-state IGly (induced by 30 µM glycine) by 3, 10 and 30 µM genistein. A plot of 1/ton vs. genistein concentration was linear over this range (Fig. 4C, open circles). From the slope of this plot the apparent association rate constant was calculated to be near 0.7 × 105 M⫺1 s⫺1 and from the zero y-intercept the apparent dissociation rate constant was 2.5 s⫺1,which is in good agreement with that obtained from the plot of 1/toff (2.6 s⫺1). The value of 1/toff (closed circles) did not alter with changes in genistein concentration. From the apparent association and dissociation rate constants, a KD of 40 µM was calculated for genistein in 30 µM glycine, which is in broad agreement with the KD of 77 µM already obtained with a single concentration of genistein (10 µM). The overall rate of block not only reflects a possibly limited access to the binding site, but also suggests bound genistein closes the channel. We then studied the effects of genistein on the kinetics of IGly. To allow accurate measurement of time constants within the limits of the fast perfusion system, glycine was applied at concentrations less than 30 µM. In agreement with previous observations (Ye et al., 2001), after a brief latent period, the onset of the inward current following glycine concentration jumps could be fitted by a single exponential function (Fig. 5A). Genistein significantly increased the activation time of IGly. Fig. 5A shows that in the presence of 0, 3 and 30 µM genistein, the activation time constant was 241, 487 and 711 ms, respectively. The plot in Fig. 5B indicates that the relationship between 1/ton and the concentration of glycine is linear. The slopes of these curves—which give estimates of kon for glycine—were significantly reduced by genistein: 1.0 × 105 M⫺1 s⫺1, 7.3 × 104 M⫺1 s⫺1, and 2.9 × 104 M⫺1 s⫺1 in 0, 3 and 30 µM genistein, respectively (ANOVA, P ⬍ 0.05; n = 3–6). We also measured the deactivation time constant (toff) for glycine-activated channels from the time course of responses when glycine was rapidly washed from the external medium. The values of toff obtained by fitting single exponentials to the current decay after glycine concentration jumps did not change significantly, either with glycine or with genistein concentration (Fig. 5A and C, ANOVA, P ⬎ 0.05; n = 3–6). Furthermore, the intercept of these plots was used to estimate the apparent
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Fig. 5. Effects of genistein on kinetics of IGly. (A) Whole-cell recordings of currents activated by 30 µM glycine in the absence and presence of 3 and 30 µM genistein. Both activation and deactivation were well fitted by single exponentials (solid lines). To eliminate possible complicating effect of slower onset and offset of genistein’s action, genistein application started 10 s before and ended after that of glycine. Note that both 3 and 30 µM genistein greatly increased the activation time-constant (ton), but had little effect on the deactivation time-constant (toff). (B) Graphs of mean values of 1/ton (±SEM) as function of glycine concentration in absence (䊊) and presence of genistein (3 (쎲) and 30 µM (䊏)). Note approximately linear relation between 1/ton and glycine concentration. The slopes of these curves give estimates of the forward rate of glycine binding. (C) Graph of mean values of 1/toff vs. glycine concentration in the absence (䊊) and presence of genistein (3 (쎲) and 30 µM (䊏)). The y-intercepts of the lines yield estimates of the reverse rate of unbinding (koff). Mean 1/toff for IGly was not sensitive to the concentration of either genistein (by ANOVA, P ⬎ 0.05; n = 3–6) or glycine (by ANOVA, P ⬎ 0.5; n = 3–6).
dissociation rate (k off = 2.2 s⫺1), which did not change significantly in the presence of genistein and was in good agreement with that obtained from the plot of 1/toff (2.3 s⫺1). These koff values and the data in Fig. 1C indicate that genistein does not affect the apparent affinity of the receptor for glycine.
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3.4. Genistein does not increase receptor desensitization The depression of glycine-activated currents by genistein could result from increased receptor desensitization. To test this hypothesis, we studied the desensitization of IGly in the absence and presence of genistein. As shown in Fig. 6, the simple exponential rate of decay of currents activated by 30 µM glycine did not significantly change. The ratio of the decay time constants (tgenistein/tcontrol) was 1.07 in Fig. 6, and 1.09 ± 0.2 (P ⬎ 0.05) for a total of four cells. 3.5. Genistein-induced rebound of IGly Fig. 7A illustrates in more detail the transient increase in inward current seen immediately after the rapid withdrawal of genistein (see also Fig. 1A-c, d; Fig. 2A-b, Fig. 3C-a). When various concentrations of genistein (3– 30 µM) were co-applied with 30 µM glycine, the rebound current increased with the concentration of
Fig. 7. Rebound current induced by genistein washout may reflect genistein-induced potentiation of IGly. (A) Rebound current at the end of glycine and genistein co-application. (B) The magnitude of the rebound current increased with the concentration of genistein. (C) Currents in response to 30 µM glycine alone (a) and with superimposed brief applications of 10 or 30 µM genistein (hatched bars in b, c). Note rebound IGly at end of genistein pulse can exceed initial peak IGly (IT, in C-c). (D) Concentration–response relation of genistein-induced rebound of IGly. Data are ratios of IT to IGly immediately preceding genistein application (means ± SEM from between four and five neurons). Fig. 6. Genistein does not increase desensitization of IGly. (A) Superimposed traces illustrate decay of currents elicited by long application of 30 µM glycine, in absence and presence of 10 µM genistein. (B) Same currents normalized with reference to peak control current. As suggested by the indicated time-constants of decay (td), average values of td obtained in absence and presence of 10 µM genistein did not differ significantly (6.2 ± 0.7 and 6.8 ± 1.1 s, respectively; P ⬎ 0.05, n = 4).
genistein (Fig. 7B–D). After a 10-µM pulse of genistein, the rebound IGly was greater than would be expected if GlyRs had remained in the desensitized state (Fig. 7Cb). When 30 µM genistein and 30 µM glycine were coapplied (Fig. 7C-c), the rebound current was so large that it greatly exceeded the peak of control IGly. Such an increase seems to indicate some genistein-induced
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Fig. 8. Interaction of genistein and picrotoxin. The suppressive effect of genistein in indicated concentrations on IGly (50 µM) is diminished by 20 µM picrotoxin (PTX). The effects of co-applied genistein with picrotoxin on IGly were much smaller than the sum of the suppressive effects when they were applied individually with glycine. Bars are the mean ± SEM of 4 to 11 neurons.
potentiation of IGly. This phenomenon was also observed in six other neurons. The amplitudes of the rebound IGly, normalized to that of control IGly, are summarized in Fig. 7D as a function of genistein concentration. Genistein increases IGly rebound in a concentration-dependent manner. 3.6. Genistein may act at the same region of the glycine receptor as picrotoxin To further investigate how genistein suppresses IGly, we performed competition experiments with picrotoxin, an open channel blocker of homomeric GlyR channels. To simplify the comparison between the suppressive effects of picrotoxin, genistein and of their mixture on IGly, we assumed that if genistein and picrotoxin act by completely different mechanisms, the suppressive effects produced by the mixture of genistein and picrotoxin should be equal or close to the sum of their individual Fig. 9. Further evidence that genistein-induced rapid block of IGly is not mediated by tyrosine kinase. (A) IGly traces from same VTA neuron show typical IGly depression by co-application of 50 µM daidzein (close genistein derivative that does not block tyrosine kinases) and lack of effect of 5 µM lavendustin A (specific blocker of tyrosine kinases). (B) B-a demonstrates rapid onset and offset of IGly depression by brief pulse of daidzein (50 µM) during continued application of glycine. B-b, both onset and offset of daidzein’s effect could be fitted by single exponentials (solid lines). (C) Bar graphs are means (±SEM) of normalized peak IGly in the presence of 50 µM daidzein or 5 µM lavendustin A; peak IGly was normalized to peak response to glycine alone. (D) Glycine current–voltage relations from voltage ramps in absence (a) and presence (b) of co-applied daidzein. Corresponding difference currents show no voltage-sensitivity of daidzein effect, neither in original plots (E), nor in plots normalized to control current at –80 mV (F).
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effects. In agreement with our previous reports (Ye et al., 1998; Ye, 2000), VTA GlyRs of neonatal rats are sensitive to picrotoxin. Furthermore, although the sensitivity to picrotoxin of GlyRs in VTA neurons changed with development (Ye, 2000), this change is not significant among the neurons of 5–14-day-old rats (data not shown). For this series of neurons, 20 µM picrotoxin and 30 µM genistein suppressed IGly in response to 50 µM glycine by 41 ± 6% (n = 11), and by 34 ± 5% (n = 7) of control current, respectively. When 20 µM picrotoxin and 30 µM genistein were coapplied with 50 µM glycine, IGly was suppressed by 51 ± 16% of control (n = 4), which is significantly smaller than 75%, the sum of their individual effects (P ⬍ 0.05, n = 4). Similar results were observed with other concentrations of genistein. While 3 and 100 µM genistein alone suppressed IGly by 28 ± 7% (n = 7) and by 65 ± 7% (n = 6) of control, respectively, when coapplied with 20 µM picrotoxin, they suppressed IGly by 42 ± 11% (n = 7), and 69 ± 10% (n = 4) of control, respectively. These values are much smaller then the sum of 75 and 96%, respectively (Fig. 8). The mutual occlusion between their effects suggests that genistein and picrotoxin share a common binding site on the GlyR or act on sites that are functionally related. 3.7. Effects of other tyrosine antagonists on glycine response To further assess the nature of genistein’s actions, we examined the effect of daidzein, an inactive structural analogue of genistein (Akiyama and Ogawara, 1991). As in hypothalamic neurons (Huang and Dillon, 2000), daidzein reversibly suppressed IGly of VTA neurons: 50 µM daidzein reduced the peak current elicited by 30 µM glycine to 76.6 ± 3.6% of control (P ⬍ 0.05, n = 5; Fig. 9). In some neurons, a rebound current was seen after daidzein (data no shown). Both the onset and offset of daidzein action could be fitted by a single exponential, which suggests that the daidzein-induced block also behaved as a simple bimolecular reaction. In Fig. 9B, in the presence of 50 µM daidzein, the time constants of onset and offset are 410 and 507 ms, the mean values being 400 ± 40 and 500 ± 30 ms, respectively (n = 14–16 measurements from six neurons). These values were used to calculate K D = 200 ± 15 µM. Because the forward rate constant kon (1 × 104 M⫺1 s⫺1, n = 4) is much slower than free diffusion in solution, the binding site for daidzein is not freely accessible. To examine the voltage dependence, a voltage ramp protocol was used (Fig. 9D). As illustrated in Fig. 9E–F, the current–voltage curves were linear and daidzein decreased IGly to a similar extent at all membrane voltages between ⫺80 and +30 mV. Thus, in contrast to genistein, daidzein acts in a membrane voltage-insensitive manner. In addition, we examined the effects of lavendustin
A, a highly selective inhibitor of protein tyrosine kinase. As in hypothalamic neurons (Huang and Dillon, 2000), co-applications of 5 µM lavendustin A with 30 µM glycine did not affect IGly of VTA neurons, the peak current was 96.9 ± 2.7% (n = 18, P ⬎ 0.05, t-test) of control (Fig. 9). These results further support the notion that genistein inhibits IGly independently of tyrosine kinase. 4. Discussion Confirming and extending previous observations on hypothalamic cells in slices (Huang and Dillon, 2000), we have found that 1–300 µM genistein depressed IGly in neurons freshly isolated from the VTA of young rats. This is the first report of such an effect of genistein on GlyRs of isolated VTA neurons. The fact that the inhibition of IGly was rapid and reversible suggested that genistein acts directly on the GlyRs (Huang and Dillon, 2000). Our experiments offer further evidence in support of this idea. On the following basis, we propose that genistein most likely inhibits GlyRs by an open channel block. First, genistein inhibited IGly only when it was co-applied with the agonist, and to the same extent without and with preincubation; when applied alone before an application of glycine alone, genistein did not inhibit IGly. Therefore genistein can bind to and inhibit the channel only after the binding of glycine, presumably when GlyR channels are open. Second, genistein’s effect was sensitive to membrane voltage, being greater at positive potentials: the binding site is therefore likely to be within the GlyR channel, at a site part of the way across the electric field of the membrane. Furthermore, the competition between the suppressive effects of genistein and picrotoxin further support the idea that genistein acts as an open channel blocker. The rebound of inward current seen at the end of a co-application could be simply explained by if genistein dissociated faster from GlyRs than does glycine, in keeping with the observed dissociation constants (koff) of 2.5 s⫺1 for genistein and 2.2 s⫺1 for glycine. Note that the koff of daidzein is only 2.0 s⫺1 and less rebound current was seen after application of daidzein, in keeping with this explanation. The block of an increasing number of channels when the concentration of genistein is raised at least partly accounts for the rebound’s marked concentration-dependence. Small rebound currents have also been observed after block of GlyR channels by 2,3-butanedione monoxime (Ye and McArdle, 1996), by Waglerin (Ye et al., 1999) and GABAAR channels by picrotoxin (Ye et al., 1998) and genistein (Huang et al., 1999). Picrotoxin is known to bind to a site inside the channel lumen (Edwards and Lees, 1997). However, the greater than expected rebound current shown in Fig. 7C and D suggests an additional effect of genistein. One possible
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explanation is that genistein reverses glycine-induced desensitization. However, as shown in Fig. 7C, IGly evoked by 30 µM glycine showed only minor desensitization. Reduced desensitization therefore cannot be the only mechanism. Another possibility is that such rebound currents reflect the transition from a ‘channelblocked’ to a ‘reopened’ state, a mechanism underlying the rebound currents induced by the rapid withdrawal of isoflurane (Hapfelmeier et al., 2001). Our data also support their notion that an open-channel block at ligandgated receptors can prolong postsynaptic currents (Hapfelmeier et al., 2001). The other possibility is that, genistein has a biphasic action: a fast suppression and a delayed potentiation of GlyRs, which is similar to the biphasic action of penicillin on IGly (Tokutomi et al., 1992). The mechanism underlying the biphasic effect of these agents requires further study. In the current study, the use-dependence often associated with open channel block was not evident, peak and steady state IGly being inhibited to the same extent, and repeated pulses of genistein equally effective during a longer pulse of glycine. The degree of use-dependence is a function of the microscopic dissociation rate constant. While ‘slow’ blockers show extreme use-dependence, use-dependence of ‘fast’ blockers may not be so clear (Gibb, 2002). As genistein has a dissociation rate of 3.1 s⫺1, the mean lengths of the blockage gaps is ⬍300 ms. It is useful to compare the suppressing effect of genistein on IGly with that of picrotoxin. As with picrotoxin, genistein suppresses IGly only when the channels are open. This observation and the occlusion between the effects of genistein and picrotoxin on IGly suggest a similar mechanism and site of action. Therefore, we suggest that both genistein and picrotoxin compete for the picrotoxin binding site of the GlyR. When picrotoxin occupies this site, genistein can no longer bring about suppression of IGly. The sensitivity of IGly to picrotoxin also indicates the absence of the b-subunit of the GlyRs, which support the argument ruling out a possible action through PTKmediated phosphorylation, since the potential site for PTK-mediated phosphorylation is in the b-subunit. The effect of genistein was mimicked by daidzein, a close analogue of genistein that does not block activation of PTK. Interestingly, the forward rate (kon) for daidzein (1 × 10⫺4 M⫺1 s⫺1) was 17 times slower than that for genistein (1.7 × 10 - 5 M⫺1 s⫺1); this may reflect a lower rate of access. A somewhat different site of action is suggested by the lack of voltage-dependence of daidzein’s effect on IGly. Both features are perhaps ascribable to the additional OH group on the daidzein molecule. The comparable block of IGly by daidzein gives further support for the idea that genistein acts independently of PTK-mediated phosphorylation. In the previous study on hypothalamic neurons
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(Huang and Dillon, 2000) 100 µM genistein reduced IGly by only 30%, whereas our VTA data yielded an IC50 of 13 µM. Several technical reasons may account for this difference: notably our recording from a quite different brain area, as well as the use of isolated VTA neurons instead of brain slices: a fast and accurate change of external solutions is important, because of rapid desensitization of glycine receptors. As a tyrosine kinase inhibitor, genistein has frequently been used to detect an involvement of tyrosine phosphorylation in the modulation of various receptors— most often with daidzein as an inactive control. However, evidence has been accumulating that, independently of tyrosine kinase, genistein can also directly interact with many receptor/ion channels: including those for GABAA (Dunne et al., 1998; Huang et al., 1999) and glycine (Huang and Dillon, 2000), as well as voltage-sensitive Na+ channels (Paillart et al., 1997), voltage-gated K+ channels (Ogata et al., 1997; Zhang and Wang, 2000), L-type Ca2+ channels (Yokoshiki et al., 1996; Katsube et al., 1998), ATP-sensitive K+ channels (Ogata et al., 1997) and cystic fibrosis transmembrane conductance regulator (CFTR) Cl⫺ channels (French et al., 1997; Weinreich et al., 1997; Al-Nakkash et al., 2001). But we did not eliminate the possibility that longer applications of genistein (and lavendustin A) also affect PTK-mediated phosphorylation of glycine receptors. In summary, genistein appears to be an open channel blocker producing a rapid direct block of GlyRs in VTA, but a possible role of tyrosine kinase in longer-term modulation of this receptor has not been ruled out. Acknowledgements This study is supported by National Institute of Alcohol Abuse and Alcoholism, National Institute of Health Grant AA-11989 (to JHY). The authors are grateful to Dr. Yongchang Chang for his critical reading and his comments on this manuscript. References Agopyan, N., Tokutomi, N., Akaike, N., 1993. Protein kinase Amediated phosphorylation reduced only the fast desensitizing glycine current in acutely dissociated ventromedial hypothalamic neurons. Neuroscience 56, 605–615. Aguayo, L.G., Tapia, J.C., Pancetti, F.C., 1996. Potentiation of the glycine-activated Cl⫺ current by ethanol in cultured mouse spinal neurons. Journal of Pharmacology and Experimental Therapeutics 279, 1116–1122. Akiyama, T., Ogawara, H., 1991. Use and specificity of genistein as inhibitor of protein-tyrosine kinase. Methods in Enzymology 201, 362–370. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., Fukami, Y., 1987. Genistein, a specific inhibitor of protein-tyrosine kinases. Journal of Biological Chemistry 262, 5592–5595.
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L. Zhu et al. / Neuropharmacology 45 (2003) 270–280
Albarran, F.A., Roa, J.P., Navarrete, R., Castillo, R., Nualart, F., Aguayo, L.G., 2001. Effect of protein kinase C activation on the glycine evoked Cl(⫺) current in spinal cord neurons. Brain Research 902, 1–10. Al-Nakkash, L., Hu, S., Li, M., Hwang, T.C., 2001. A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs. Journal of Pharmacology and Experimental Therapeutics 296, 464–472. Barry, P., 1996. Axoscope junction potential calculator. Axobits 18, 3–4. Betz, H., Kuhse, J., Fischer, M., Schmieden, V., Laure, B., Kuryatov, A., Langosch, D., Meyer, G., Bormann, J., Rundstrom, N., Matzenbach, B., Kirsch, J., Ramming, M., 1994. Structure, diversity and synaptic localization of inhibitory glycine receptors. Journal of Physiology (Lond) 88, 243–248. Burgen, A.S., 1966. The drug–receptor complex. Journal of Pharmacy and Pharmacology 18, 137–149. Caraiscos, V.B., Mihic, S.J., MacDonald, J.F., Orser, B.A., 2002. Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human alpha(1)beta glycine receptors. Journal of Physiology (Lond) 539, 495–502. Chang, Y., Weiss, D.S., 1999. Channel opening locks agonist onto the GABAC receptor. Nature Neuroscience 2, 219–225. Davis, M.J., Wu, X., Nurkiewicz, T.R., Kawasaki, J., Gui, P., Hill, M.A., Wilson, E., 2001. Regulation of ion channels by protein tyrosine phosphorylation. American Journal of Physiology, Heart and Circulation Physiology 281, H1835–H1862. Dunne, E.L., Moss, S.J., Smart, T.G., 1998. Inhibition of GABAA receptor function by tyrosine kinase inhibitors and their inactive analogues. Molecular and Cellular Neuroscience 12, 300–310. Edwards, M.D., Lees, G., 1997. Modulation of a recombinant invertebrate gamma-aminobutyric acid receptor-chloride channel complex by isoflurane: effects of a point mutation in the M2 domain. British Journal of Pharmacology 122, 726–732. French, P.J., Bijman, J., Bot, A.G., Boomaars, W.E.M., Scholte, B.J., Jonge, H.R.D., 1997. Genistein activates CFTR Cl⫺ channels via a tyrosine kinase- and protein phosphatase-independent mechanism. American Journal of Physiology 273, C747–C753. Gatto, G.J., Mcbride, W.J., Murphy, J.M., Lumeng, L., Li, T.K., 1994. Ethanol self-infusion into the ventral area by alcohol-preferring rats. Alcohol 11, 557–564. Gibb, A.J., 2002. Receptors linked to ion channels: mechanisms of activation and block. In: Foreman, J.C., Johansen, T. (Eds.), Textbook of receptor pharmacology, second ed. CRC Press Inc, Florida, USA. Gu, Y., Huang, L.-Y.M., 1998. Cross-modulation of glycine-activated Cl⫺ channels by protein kinase C and cAMP-dependent protein kinase in the rat. Journal of Physiology (Lond) 284, 826–831. Hapfelmeier, G., Haseneder, R., Kochs, E., Beyerle, M., Zieglgansberger, W., 2001. Coadministered nitrous oxide enhances the effect of isoflurane on GABAergic transmission by an increase in open-channel block. Journal of Pharmacology and Experimental Therapeutics 298, 201–208. Huang, R.Q., Dillon, G.H., 2000. Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor. Neuropharmacology 39, 2195–2204. Huang, R.Q., Fang, M.J., Dillon, G.H., 1999. The tyrosine kinase inhibitor genistein directly inhibits GABAA receptors. Molecular Brain Research 67, 177–183. Jiang, Z.L., Ye, J.H., 2003. Protein kinase C⑀ is involved in ethanol potentiation of glycine-gated Cl⫺ current in rat neurons of ventral tegmental area. Neuropharmacology 44, 493–502. Katsube, Y., Yokoshiki, H., Nguyen, L., Yamamoto, M., Sperelakis, N., 1998. Inhibition of Ca2+ current in neonatal and adult rat ventricular myocytes by the tyrosine kinase inhibitor, genistein. European Journal of Pharmacology 345, 309–314. Legendre, P., 1999. Voltage dependence of the glycine receptor-chan-
nel kinetics in the zebrafish hindbrain. Journal of Neurophysiology 82, 2120–2129. Ogata, R., Kitamura, K., Ito, Y., Nakano, H., 1997. Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle. British Journal of Pharmacology 122, 1395–1404. Paillart, C., Carlier, E., Guedin, D., Dargent, B., Couraud, F., 1997. Direct block of voltage-sensitive sodium channels by genistein, a tyrosine kinase inhibitor. Journal of Pharmacology and Experimental Therapeutics 280, 521–526. Ren, J., Ye, J.H., McArdle, J.J., 1998. cAMP-dependent protein kinases modulation of glycine-activated chloride current in neurons freshly isolated from rat ventral tegmental area. Brain Research 811, 71–78. Ruiz-Gomez, A., Vallo, M.L., Valdivieso, F., Mayor, F.J., 1994. Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C. Journal of Biological Chemistry 266, 559–566. Schonrock, B., Bormann, J., 1995. Modulation of hippocampal glycine receptor channels by protein kinases C. Neuroreport 6, 301–304. Song, Y., Huang, L.-Y.M., 1990. Modulation of glycine receptor chloride channel by cyclic AMP-dependent protein kinases in spinal trigeminal neurons. Nature 348, 242–245. Swope, S.L., Moss, S.I., Raymond, L.A., Huganir, R.L., 1999. Regulation of ligand-gated ion channels by protein phosphorylation. Advances in Second Messenger and Phosphoprotein Research 33, 49–78. Tao, L., Ye, J.H., 2002. Protein kinase C modulates ethanol inhibition of glycine-activated current in dissociated neurons of rat ventral tegmental area. Journal of Pharmacology and Experimental Therapeutics 300, 967–975. Tokutomi, N., Agopyan, N., Akaike, N., 1992. Penicillin-induced potentiation of glycine receptor-operated chloride current in rat ventro-medial hypothalamic neurones. British Journal of Pharmacology 106, 73–78. Wagner, K., Edson, K., Heginbotham, L., Post, M., Huganir, R.L., Czernik, A.J., 1991. Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor. Journal of Biological Chemistry 266, 23784–23789. Weinreich, F., Wood, P.G., Riordan, J.R., Nagel, G., 1997. Direct action of genistein on CFTR. Pflu¨ gers Archiv 434, 484–491. Wise, R.A., 1996. Additive drugs and brain stimulation reward. Annual Review of Neurosciences 19, 319–340. Ye, J.H., 2000. Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons. Brain Research 862, 74–82. Ye, J.H., McArdle, J.J., 1996. 2,3-Butanedione monoxime modifies the glycine-gated chloride current of acutely isolated murine hypothalamic neurons. Brain Research 735, 20–29. Ye, J.H., McArdle, J.J., 1997. Waglerin-1 modulates gamma-aminobutyric acid activated current of murine hypothalamic neurons. Journal of Pharmacology and Experimental Therapeutics 282, 74–80. Ye, J.H., Ren, J., Liu, P.L., McArdle, J.J., 1998. Glycine-activated chloride currents of neurons freshly isolated from the ventral tegmental area of rats. Brain Research 796, 53–62. Ye, J.H., Ren, J., McArdle, J.J., 1999. Waglerin-1 inhibits GABA(A) current of neurons in the nucleus accumbens of neonatal rats. Brain Research 837, 29–37. Ye, J.H., Tao, L., Zhu, L., Krnjevic´ , K., McArdle, J.J., 2001. Ethanol inhibition of glycine-activated responses in neurons of ventral tegmental area of neonatal rats. Journal of Neurophysiology 86, 2426–2434. Yokoshiki, H., Sumii, K., Sperelakis, N., 1996. Inhibition of L-type calcium current in rat ventricular cells by the tyrosine kinase inhibitor, genistein and its inactive analog, daidzein. Journal of Molecular Cell Cardiology 28, 807–814. Zhang, Z.H., Wang, Q., 2000. Modulation of a cloned human A-type voltage-gated potassium channel (hKv1.4) by the protein kinase. Pflu¨ gers Archiv 440, 784–792.