Introduction

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A growing body of experimental evidence accumulated over the last several years indicates that the activity of L-type Ca²⁺ channels can be directly regulated by protein tyrosine kinase (PTK)-dependent phosphorylation. Initially, this idea came from the finding that genistein, a specific PTK inhibitor, can inhibit basal L-type Ca²⁺ current in a number of preparations (Davis et al., 2001). Such observations are consistent with the conclusion that basal PTK activity produces a stimulatory effect on L-type Ca²⁺ channels. This conclusion has been substantiated by numerous studies involving smooth muscle and neuronal Ca_v1.2 channel isoforms. In vascular smooth muscle preparations, inhibitors of protein tyrosine phosphatases (PTPs) can stimulate the L-type Ca²⁺ channel current (Wijetunge et al., 1998; Wu et al., 2001). Furthermore, it has been reported that the activation of tyrosine kinases by platelet-derived growth factor (PDGF) or Src kinase-activating peptide as well as intracellular application of constitutively active Src (c-Src) kinase result in an augmentation of L-type Ca²⁺ channel current in smooth muscle cells (Wijetunge and Hughes, 1995a,b, 1996; Hu et al., 1998). In addition, the ₁ subunit of smooth muscle Ca_v1.2 channels was shown to coimmunoprecipitate with c-Src (Hu et al., 1998). This is consistent with the recent report by Bence-Hanulec et al. (2000) that insulin-like growth factor-1 (IGF-1) potentiates the L-type Ca²⁺ current in cultured cerebellar granule neurons through Src-mediated phosphorylation of a specific tyrosine residue (Tyr²¹²²) near the C terminus of the ₁ subunit of the neuronal Ca_v1.2 channel.

However, it remains unclear whether or not cardiac L-type Ca²⁺ channels can be directly regulated by PTK activity. On the one hand, in the study of Bence-Hanulec et al. (2000), IGF-1 did not potentiate activity of the cardiac L-type Ca²⁺ channel ₁ subunit. Furthermore, IGF-1 failed to produce an effect on the L-type Ca²⁺ current in ventricular myocytes (Sims et al., 2000). On the other hand, the ability of genistein to inhibit the basal L-type Ca²⁺ current in a variety of cardiac myocytes has been used as an argument to support the idea that these channels may be actually regulated by basal tyrosine kinase activity (Yokoshiki et al., 1996; Hool et al., 1998; Katsube et al., 1998; Wang and Lipsius, 1998; Ogura et al., 1999). Although genistein inhibits PTK activity with little or no effect on serine/threonine protein kinases, it can also produce effects that are unrelated to its ability to inhibit PTKs. For example, genistein has been reported to directly block ligand-gated (Huang and Dillon, 2000) and voltage-gated ion channels (Smirnov and Aaronson, 1995; Paillart et al., 1997; Washizuka et al., 1997). Therefore, the main objective of the present study was to investigate the contribution of PTK regulation to the inhibitory effect that genistein has on the L-type Ca²⁺ current in guinea pig ventricular myocytes.

	Materials and Methods

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Cell Isolation. Single ventricular myocytes were isolated from adult Hartley guinea pigs using a modification of a method described previously (Hool et al., 1998). The methods used in this study were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University. Briefly, animals were anesthetized by intraperitoneal injection of pentobarbital (150 mg kg¹). Hearts were then quickly excised and the coronary arteries were perfused via the aorta with a solution containing 140 mM NaCl, 5.4 mM KCl, 2.5 mM MgCl₂, 1.5 mM CaCl₂, 11 mM glucose, and 5.5 mM HEPES, pH 7.4. The heart was perfused with this solution for 5 min, nominally Ca²⁺-free solution for 5 min, and then nominally Ca²⁺-free solution containing ~0.2 mg/ml collagenase (class B; Roche Diagnostics, Indianapolis, IN) for about 30 min. The ventricles were then removed and minced in a modified Kraft-Brühe solution containing 110 mM potassium glutamate, 10 mM KH₂PO₄, 25 mM KCl, 2 mM MgSO₄, 20 mM taurine, 5 mM creatine, 0.5 mM EGTA, 20 mM glucose, and 5 mM HEPES, pH 7.4. Single cells were obtained by filtering through nylon mesh. After settling, cells were resuspended in Ca²⁺-containing solution and used on the day of isolation only.

Data Acquisition and Analysis. The L-type Ca²⁺ current was studied using the conventional whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Patch pipettes (1 to 2 M) were filled with an intracellular solution containing 130 mM CsCl, 20 mM tetraethylammonium chloride (TEA-Cl), 5 mM MgATP, 5 mM EGTA, 0.1 mM Tris-GTP, and 5 mM HEPES, pH 7.2. In experiments employing ATPS the following pipette solution was used: 120 mM CsCl, 20 mM TEA-Cl, 5 mM EGTA, 5 mM Li₄ATPS, 5 mM MgCl₂, 0.1 mM Tris-GTP, and 5 mM HEPES, pH 7.2. Cells were bathed in a K⁺-free control extracellular solution containing 140 mM NaCl, 5.4 mM CsCl, 2.5 mM CaCl₂, 0.5 mM MgCl₂, 11 mM glucose, and 5.5 mM HEPES, pH 7.4. The voltage dependence and kinetics of L-type Ca²⁺ current inactivation were studied using the following extracellular solution: 100 mM TEA-Cl, 45.4 mM CsCl, 2.5 mM CaCl₂, 0.5 mM MgCl₂, 11 mM glucose, and 5.5 mM HEPES, pH 7.4. Isolated myocytes were placed in a 0.5-ml chamber on the stage of an inverted microscope, where they were superfused with either control or drug containing extracellular solution at a rate of 1 to 2 ml/min. In some experiments, cells were exposed to different experimental solutions using a fast flow system. The system consisted of a cFlow 8 channel flow controller, cF-8VS valve assembly unit, and MPRE8 miniature manifold (Cell MicroControls, Norfolk, VA). This method allowed rapid (<1 s) changes in extracellular solutions bathing myocytes being voltage clamped. A 3 M KCl-agar bridge was used to ground the bath. All experiments were performed at room temperature.

Drugs and Reagents. Genistein (Alexis Corp., San Diego, CA) and lavendustin A (Calbiochem, San Diego, CA) were prepared as stock solutions in dimethyl sulfoxide. To achieve the final concentrations used, these stock solutions were then diluted in external solution and sonicated prior to use. The final concentration of dimethyl sulfoxide in extracellular solutions was never more than 0.1%. It is important to note that in solutions containing 100 and 300 µM genistein, aggregates of crystals were clearly visible when viewed through the microscope. This suggests that under our experimental conditions, genistein at concentrations higher than 50 µM is not completely soluble in aqueous solution. PVN was prepared as previously described (Hool et al., 1998) by combining 10 mM H₂O₂ and 10 mM Na₃VO₄ in an aqueous solution containing 50 mM HEPES, pH 7.4. This mixture was allowed to stand at room temperature for 15 min, after which excess H₂O₂ was eliminated by adding catalase. The resulting stock solution contained a mixture of vanadate and peroxovanadate complexes (Posner et al., 1994). The final concentration of PVN used in our experiments is based on the concentration of Na₃VO₄ used in preparing the stock solution. All solutions containing genistein, PVN, and isoproterenol (Iso) were stored in light-resistant containers. All drugs were obtained from Sigma/RBI (Natick, MA), except where noted.

	Results

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Effect of Extracellular Genistein on the L-Type Ca²⁺ Current. Bath application of 50 µM genistein inhibited the amplitude of basal L-type Ca²⁺ current in guinea pig ventricular myocytes by 49 ± 1.9% (n = 34, Fig. 1). We used a fast flow system to rapidly exchange solutions bathing the cell and measure the onset of the inhibitory effect of genistein. The peak Ca²⁺ current measured within 5 s of exposure to genistein was already at a level equal to about 50% of the steady-state effect (Fig. 1A). The inhibitory effect of 50 µM genistein developed monoexponentially, with an average time constant of 11 ± 1.0 s (n = 11), and reached steady-state within 40 s. Upon washout of genistein, the amplitude of the L-type Ca²⁺ current returned to 88 ± 2.6% (n = 18) of its initial level with a time constant of 20 ± 0.4 s (n = 11). The apparent incomplete reversibility of the genistein effect can be explained by basal current rundown observed in some cells. It should be noted that 74% of the cells exposed to 50 µM genistein exhibited this type of inhibitory response. The remaining 26% exhibited an inhibitory response followed by a more slowly developing stimulatory reaction. This is consistent with previous reports that genistein can have both inhibitory and stimulatory effects (Hool et al., 1998; Wang and Lipsius, 1998). Cells displaying a biphasic response were not included in our analysis of the inhibitory effect of genistein.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Inhibitory effect of genistein on L-type Ca2+ current. A, time course of changes in amplitude of L-type Ca2+ current under control conditions (a) and during exposure to 50 µM genistein (b). B, examples of L-type Ca2+ current recorded under conditions indicated in panel A. The dotted line above current traces represents the zero current level. C, current-voltage relationships of peak L-type Ca2+ current density in the absence () and presence () of 50 µM genistein. Currents were evoked by 100 ms depolarizing steps to test potentials from 30 to 60 mV in 5 mV increments. D, dose-response relationship for the inhibitory effect (I) of genistein on L-type Ca2+ current. The dashed line represents the best fit of the data points to the equation: (100  I) = Imax/(1 + {[genistein]/IC50}k) + (100  Imax). The IC50, apparent Hill coefficient (k), and maximal effect (Imax) were 20 ± 3.5 µM, 1.5 ± 0.34 and 68 ± 4.6%, respectively. The number of cells tested at each concentration of genistein is indicated in brackets.

Effect of Intracellular Genistein on the L-Type Ca²⁺ Current. If the genistein-induced reduction of the Ca²⁺ current is really due to inhibition of PTK activity, then one might expect that intracellular application of genistein would produce the same inhibitory response, and preempt any inhibitory effect produced by subsequent extracellular application of this compound. However, this was not the case (Fig. 2). The peak current density measured after 7 min of dialysis with a control pipette solution was 6.2 ± 0.50 pA/pF (n = 8). The peak current density measured after 7 min of dialysis with a pipette solution containing 50 µM genistein was 5.3 ± 0.44 pA/pF (n = 14). Although the L-type Ca²⁺ current density was 14% smaller in cells dialyzed with a pipette solution containing genistein, this difference was not statistically significant (P > 0.2, unpaired t test). Furthermore, cell dialysis with a pipette solution containing 50 µM genistein did not change the sensitivity of the L-type Ca²⁺ current to subsequent extracellular application of this compound (Fig. 2). In cells dialyzed with 50 µM genistein, exposure to 50 µM extracellular genistein resulted in inhibition of the Ca²⁺ current by 45 ± 1.9%. This is not significantly different from the magnitude of the response to the same concentration of genistein in cells dialyzed with a control pipette solution (P > 0.2, unpaired t test).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Intracellular application of genistein does not prevent the inhibitory effect produced by extracellular application of genistein but increases the sensitivity of the Ca2+ current to -adrenergic stimulation. A, time course of changes in amplitude of L-type Ca2+ current in cells dialyzed with a pipette solution containing 50 µM genistein before (a) and during (b) exposure to extracellular genistein (50 µM). Cells were dialyzed with a pipette solution containing 50 µM genistein for at least 7 min before extracellular application of 50 µM genistein. B, examples of L-type Ca2+ current recorded under conditions indicated in panel A. The dotted line above current traces represents the zero current level. C, inhibitory effect produced by extracellular application of genistein (50 µM) was not significantly different (P > 0.2, unpaired t test) in cells dialyzed with control pipette solution () and with a pipette solution containing 50 µM genistein (). However, stimulatory effect of 0.5 nM Iso was significantly increased in cells dialyzed with pipette solution containing 50 µM genistein (, P < 0.05, unpaired t test).

Effect of PVN on Genistein-Induced Inhibition of L-Type Ca²⁺ Current. Assuming that inhibition of PTK activity was responsible for inhibition of the Ca²⁺ current by extracellular genistein, then an increase in PTK-dependent phosphorylation might be expected to produce a stimulatory effect on this current. However, exposure to 100 µM PVN, a potent phosphotyrosine phosphatase (PTP) inhibitor (Posner et al., 1994), did not result in an increase in the amplitude of the L-type Ca²⁺ current. In fact, the current actually decreased by 15 ± 4.0% (n = 5, Fig. 3A). However, this small decrease was most likely due to current rundown and not a true inhibition of the current. The fact that the Ca²⁺ current did not respond to PVN suggests that either the L-type Ca²⁺ channels in guinea pig ventricular myocytes are not regulated by PTKs, or they have already been maximally stimulated by basal PTK-dependent phosphorylation. If the latter were true, then inhibition of PTP activity would be expected to attenuate the inhibitory effect of genistein. However, application of genistein to cells pretreated with PVN was still able to inhibit the L-type Ca²⁺ current (Fig. 3, A and B). In the presence of 100 µM PVN, 50 µM genistein reduced the peak Ca²⁺ current by 56 ± 1.8% (n = 5), which is not significantly different from 49% inhibition observed under control conditions (P > 0.1, unpaired t test). Conversely, when cells were first exposed to genistein, subsequent addition of 100 µM PVN did not affect genistein-induced inhibition of the Ca²⁺ current (Fig. 3, C and D). The inhibitory effect of 50 µM genistein measured at the end of a 3- to 4-min application of 100 µM PVN was 45 ± 3.9% (n = 9), which was not significantly different from the 45 ± 2.0% reduction in L-type Ca²⁺ current amplitude measured just before PVN addition in the same cells (P > 0.5, paired t test).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   The protein tyrosine phosphatase inhibitor PVN affects neither basal L-type Ca2+ current nor genistein-mediated inhibition of L-type Ca2+ current. A, time course of changes in amplitude of L-type Ca2+ current under control conditions (a), during exposure to 100 µM PVN alone (b), and 100 µM PVN plus 50 µM genistein (c). Cells were exposed to 100 µM PVN for at least 4 min before application of genistein. B, time course of changes in amplitude of L-type Ca2+ current under control conditions (a), during exposure to 50 µM of genistein alone (b), and 50 µM genistein plus 100 µM PVN (c). C and D, examples of L-type Ca2+ current recorded under conditions indicated in panels A and B, respectively. The dotted lines above current traces represent the zero current level.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Intracellular application of PVN does not prevent the inhibitory effect of genistein, but it significantly reduces -adrenergic stimulation of the Ca2+ current. A, time course of changes in amplitude of L-type Ca2+ current in ventricular myocytes dialyzed with a pipette solution containing 100 µM PVN before (a) and during (b) exposure to 50 µM genistein. B, examples of L-type Ca2+ current recorded under conditions indicated in panel A. The dotted line above current traces represents the zero current level. C, stimulatory effects of 10 nM and 10 µM Iso recorded in myocytes dialyzed with a pipette solution containing 100 µM PVN () were significantly reduced (, P < 0.001) when compared to those recorded under control conditions ().

Effect of ATPS on Genistein-Induced Inhibition of L-Type Ca²⁺ Current. Another piece of evidence arguing against the contribution of PTKs in genistein-mediated inhibition of cardiac L-type Ca²⁺ current was obtained from cells dialyzed with ATPS, a non-hydrolyzable analog of ATP. ATPS can substitute for ATP in kinase reactions. The product is thiophosphorylated proteins that are known to be poor substrates for both serine/threonine phosphatases (Gratecos and Fischer, 1974) and PTPs (Hiriyanna et al., 1994). Again, if the inhibition of basal PTK activity were the mechanism responsible for genistein's inhibitory effect, then dialysis of cells with a pipette solution containing ATPS would be expected to produce irreversible, tyrosine thiophosphorylation of the Ca²⁺ channel protein (or other auxiliary proteins) and blunt the genistein-induced inhibition. However, in the presence of ATPS, 50 µM genistein inhibited the amplitude of the L-type Ca²⁺ current by 53 ± 3.4% (n = 8, Fig. 5), which is not significantly different from the magnitude of the inhibitory effect observed in cells dialyzed with the control pipette solution (P > 0.3, unpaired t test). It should be noted that ATPS alone produced a slowly developing stimulatory effect on the Ca²⁺ current. In myocytes dialyzed with a pipette solution containing ATPS, the amplitude of the basal L-type Ca²⁺ current increased by 59 ± 22% (n = 8). The fact that the inhibitory response to genistein was not altered suggests that this increase in basal current was not due to PTK activity. As illustrated in Fig. 5, subsequent exposure to Iso produced further irreversible stimulation of the current, suggesting that ATPS was effective in attenuating dephosphorylation associated with the activation of protein kinase A. 

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Intracellular ATPS has no effect on genistein-induced inhibition of the L-type Ca2+ current. A, time course of changes in amplitude of L-type Ca2+ current in ventricular myocytes dialyzed with a pipette solution containing 5 mM ATPS at the beginning of cell dialysis (a), after 14 minutes of cell dialysis (b), following exposure to 50 µM genistein (c), following washout of genistein (d), following exposure to 1 µM Iso (e), and after washout of Iso (f). B, examples of L-type Ca2+ current recorded under conditions indicated in panel A. The dotted lines above current traces represent the zero current level.

Effect of Lavendustin A on L-Type Ca²⁺ Current. A common approach used to determine whether an effect produced by genistein is due to inhibition of PTK activity is to determine whether or not structural analogs such as daidzein and/or genistin, which do not significantly inhibit PTK activity, produce the same type of response. However, neither daidzein nor genistin were sufficiently soluble in our extracellular solutions to allow us to attempt this type of experiment. As an alternative approach, we determined whether lavendustin A, a broad range PTK inhibitor that is structurally unrelated to genistein (Onoda et al., 1989), produces the same type of effect. As shown in Fig. 6, treatment with 5 µM lavendutsin A for 4 min did not significantly affect the basal L-type Ca²⁺ current. The magnitude of the Ca²⁺ current measured in the presence of lavendustin A (5 µM) was 96 ± 4.1% (n = 7) of that measured before exposure to lavendustin (P > 0.4, paired t test). We have previously demonstrated that this same concentration of lavendustin A can antagonize PTK-dependent inhibition of -adrenergic responses associated with PVN treatment in guinea pig ventricular myocytes (Belevych et al., 2001). Therefore, the inability of lavendustin A to inhibit the basal Ca²⁺ current further supports the idea that basal PTK activity does not significantly contribute to the regulation of the basal L-type Ca²⁺ current in guinea pig ventricular myocytes.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   The tyrosine kinase inhibitor lavendustin A has no effect on the basal L-type Ca2+ current. A, time course of changes in amplitude of L-type Ca2+ current under control conditions (a) and in the presence of 5 µM lavendustin A (b). B, examples of L-type Ca2+ current recorded under conditions indicated in panel A. The dotted line above current traces represents the zero current level.

Effect of Genistein on Kinetic and Voltage-Dependent Properties of Ca²⁺ Channels. It has previously been reported that PTK activity selectively enhances neuronal L-type Ca²⁺ channel activity at hyperpolarized membrane potentials, which is consistent with it causing a shift in the voltage dependence of channel activation. In addition, PTK activity increased the rate of current activation but only at more hyperpolarized test potentials, and it had no significant effect on the rate of current inactivation (Blair & Marshall, 1997; Bence-Hanulec et al., 2000). To determine whether genistein might affect cardiac L-type Ca²⁺ channel activity by inhibiting the same type of PTK-dependent regulatory responses, we studied the effect that genistein has on the kinetic and voltage-dependent properties of the current in guinea pig ventricular myocytes.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Genistein slows the rate of L-type Ca2+ current activation but does not affect the voltage dependence of peak current activation. A, the time to peak current following depolarization to indicated membrane potentials under control conditions () and in the presence of 50 µM genistein () (, P < 0.05, paired t test, n = 8). Inset, examples of Ca2+ current recorded at 0 mV under control conditions and in the presence of 50 µM genistein. The traces were normalized to peak current amplitude. B, voltage dependence of L-type Ca2+ current activation observed under control conditions () and in the presence of 50 µM genistein (). Activation curves were derived from I-V relationships (Fig. 1) by calculating the conductance at each test potential. Lines through data points represent a least-squares fit to a Boltzmann equation. The membrane potential at which activation was half-maximal was 1.6 and 1.2 mV, and the slope factor was 7.3 and 7.2 mV in the absence and presence of genistein, respectively (P > 0.3).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Genistein significantly alters the voltage dependence of L-type Ca2+ current inactivation. A, L-type Ca2+ currents evoked by 200-ms test pulses following 5-s conditioning steps from 90 to 30 mV in 20 mV increments under control conditions and in the presence of 50 µM genistein. B, voltage dependence of L-type Ca2+ current inactivation before (), during (), and after () exposure to 50 µM genistein. Lines through the data points represent least-squares fit to a Boltzmann equation. The membrane potential at which inactivation was half-maximal (V0.5) was 23, 36, and 28 mV, and the slope factor was 5.1, 10, and 6.1 mV before, during, and after cell exposure to genistein, respectively. There is a statistically significant difference in V0.5 and slope factor obtained in the absence and presence of genistein (P < 0.001). Inset, the voltage-clamp protocol used to define the voltage dependence of inactivation. Each conditioning pulse was preceded by a 200-ms pretest pulse to 0 mV and followed by a 10-ms step to 80 mV before depolarizing to the test pulse at 0 mV. The magnitude of the current recorded during the pretest pulse to 0 mV was used to normalize responses and correct for changes in current magnitude due to rundown.

	Discussion

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In the present study, we demonstrated that genistein is able to inhibit the L-type Ca²⁺ current in native cardiac myocytes. This is consistent with what has been previously reported (Chiang et al., 1996; Yokoshiki et al., 1996; Hool et al., 1998; Katsube et al., 1998; Wang and Lipsius, 1998; Ogura et al., 1999). The basal current was inhibited by 68% in the presence of maximally effective concentrations of genistein, and the apparent IC₅₀ was 20 µM. Although the accuracy of these values is most likely limited by the insolubility of genistein at higher concentrations, our results are in line with maximal inhibitory effects of 40 to 79% and IC₅₀ values of 11 to 47 µM, which have been reported by others (Yokoshiki et al., 1996; Ogura et al., 1999).

Although genistein's ability to inhibit L-type Ca²⁺ channel activity has been a consistent observation, the conclusion as to whether or not this is due to changes in PTK activity has not. Several approaches have been used to investigate the underlying mechanism. One that has been employed by most groups involves the use of daidzein, a structural analog of genistein that does not inhibit PTK activity. The ability of daidzein to at least partially mimic the inhibitory effect of genistein has been reported by some, but not all, investigators (Chiang et al., 1996; Yokoshiki et al., 1996; Wang and Lipsius, 1998; Ogura et al., 1999). In the present study, we did not examine the effects of daidzein, because it was only partially soluble in our experimental solutions. However, we previously reported that daidzein could inhibit the L-type Ca²⁺ current in guinea pig ventricular myocytes but that the magnitude of its inhibitory effect was only about 50% of that produced by genistein (Hool et al., 1998). It is conceivable that the lower potency of daidzein might be explained at least in part by its lower solubility reducing the effective amount of this compound that is actually in solution.

Another approach that has been used to address the potential role of changes in tyrosine phosphorylation in mediating genistein-induced inhibition of the cardiac L-type Ca²⁺ current has been to determine whether PTP inhibitors can block or reverse such responses. Wang and Lipsius (1998) reported that 1 mM Na₃VO₄ was able to completely block the inhibitory effect of genistein. This is at least partially consistent with the work of Ogura et al. (1999) who reported that this same concentration of Na₃VO₄ antagonized the ability of low, but not high, concentrations of genistein to inhibit the Ca²⁺ current in guinea pig ventricular myocytes. On the contrary, in the present study, we found that the PTP inhibitor PVN had no such effect on the inhibitory response to genistein. The explanation for this apparent discrepancy is unclear. The negative response to PVN is unlikely to be explained by the inability of this compound to actually inhibit PTP activity in cardiac myocytes. Even though we used a concentration of only 100 µM, PVN contains a mixture of peroxovanadate derivatives that are significantly more potent inhibitors of PTP activity than Na₃VO₄ (Posner et al., 1994). The lack of response to externally applied PVN is also unlikely to be due to the inability of these compounds to cross the plasma membrane and reach an effective concentration inside the cell, because dialysis with a PVN-containing pipette solution did not alter the response to genistein, even though it did inhibit the response to -adrenergic receptor stimulation. Furthermore, we have previously demonstrated that exposure of isolated guinea pig ventricular myocytes to bathing solutions containing this concentration of PVN produces a profound increase in the level of protein tyrosine phosphorylation (Belevych et al., 2001).

The ability of other tyrosine kinase inhibitors to mimic the inhibitory response to genistein has also been used to support the idea that tyrosine kinase activity actually does regulate cardiac L-type Ca²⁺ channel activity. Ogura et al. (1999) demonstrated that the inhibitory effect of genistein could be mimicked by tyrphostins A23 and A25, which are also broad range PTK inhibitors. However, tyrphostin A1, a structural analog with no PTK inhibitory activity, has also been reported to block L-type Ca²⁺ channel activity in vascular smooth muscle cells, as well as cardiac myocytes (Wijetunge et al., 1992; Ogura et al., 1999). For that reason, we did not use any of the tyrphostins. Instead, we tested the response to lavendustin A, another broad range inhibitor of PTK activity that is significantly more potent than genistein (Onoda et al., 1989). Because of its greater potency, we could use lower concentrations, potentially avoiding any nonspecific effects. However, at a concentration that we previously found to antagonize other PTK-dependent responses in these cells (Belevych et al., 2001), lavendustin A had no effect on the basal L-type Ca²⁺ current. This observation supports the idea that the inhibitory effect of genistein has nothing to do with inhibition of PTK activity. However, this interpretation is based on the assumption that both genistein and lavendustin A are able to inhibit the same contingent of PTKs.

In the present study, we attempted to shed new light on the mechanism responsible for the inhibitory effect that genistein has on cardiac L-type Ca²⁺ channel activity by determining whether there is any sidedness to its effect. If genistein does produce its inhibitory effect by inhibiting PTK activity, then it must be acting at a site inside the cell. However, we found that the inhibitory effect of genistein was only observed when the compound was applied from the outside. Introduction of genistein intracellularly, through cell dialysis, did not produce obvious inhibition of the basal current. Furthermore, it did not alter the magnitude of the response to subsequent external application of genistein (Fig. 2). The lack of an inhibitory response to genistein added to the pipette solution is unlikely to be explained by ineffective dialysis of genistein into these cells since, as expected, the response to -adrenergic stimulation was enhanced (Hool et al., 1998). This supports the idea that genistein produces its inhibitory effect by acting at an external site, which is not consistent with a mechanism involving regulation of PTK activity.

Another new approach employed in the present study addressed the mechanism responsible for the inhibitory effect of genistein by using ATPS. It is well established that ATPS can substitute for ATP in protein kinase-dependent phosphorylation reactions. The result is a thiophosphorylated protein that is resistant to dephosphorylation by both serine/threonine and tyrosine phosphatases (Gratecos and Fischer, 1974; Hiriyanna et al., 1994; Sorota, 1995). However, dialyzing cells with a pipette solution containing ATPS did not alter the inhibitory response to genistein. Again, the absence of an effect on the genistein response cannot be attributed to inadequate dialysis with ATPS, since exposure of these cells clearly produced irreversible PKA-dependent enhancement of the L-type Ca²⁺ current following exposure to Iso. Therefore, these results argue against the possibility that the response to genistein is due to PTK inhibition, which then allows basal PTP activity to dephosphorylate a site responsible for maintaining basal channel activity.

In the final set of experiments, we demonstrated that genistein shifts the voltage dependence of Ca²⁺ channel inactivation. This confirms the effect reported by Yokoshiki et al. (1996). It is also consistent with the ability of genistein to shift the voltage dependence of L-type Ca²⁺ channel inactivation in smooth muscle cells (Wijetunge et al., 2000). However, PTK-dependent regulation of this current in smooth muscle cells is not necessarily associated with a change in the voltage dependence of channel inactivation (Wijetunge and Hughes, 1996; Wijetunge et al., 1998). This suggests that the ability of genistein to affect the voltage dependence of channel gating may be independent of its ability to inhibit PTK activity. Consistent with this idea, PTK-dependent regulation of the neuronal and smooth muscle isoforms of the L-type Ca²⁺ channel are believed to involve phosphorylation of the tyrosine residue located at position 2122 of the ₁ subunit (Bence-Hanulec et al., 2000; Davis et al., 2001). However, the cardiac isoform of the L-type Ca²⁺ channel lacks this tyrosine residue. This could then explain why genistein inhibition of the L-type Ca²⁺ current in cardiac myocytes is associated with a change in voltage dependence but not inhibition of PTK activity.

The most likely explanation for the ability of genistein to inhibit the cardiac L-type Ca²⁺ current is that the drug directly blocks the channel. It should be noted that such an effect is not restricted to L-type Ca²⁺ channels. It has been reported that genistein exerts a direct, non-catalytic blocking effect on glycine receptors (Huang and Dillon, 2000), voltage-gated Na⁺ channels, (Paillart et al., 1997), and voltage-gated K⁺ channels (Smirnov and Aaronson, 1995; Washizuka et al., 1997; Zhang and Wang, 2000). Thus our study provides further support for the conclusion that genistein is actually a promiscuous ion channel blocker, and its use as a probe for assessing the role of PTK activity in regulating ion channel activity should be avoided.