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Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Guinea pig ventricular myocytes in whole cell configuration were treated with tyrosine kinase (TK) inhibitors [genistein (Gst), tyrphostin A23 (T23), and tyrphostin A25 (T25)] and with inactive analogs [daidzein, genistin, and tyrphostin A1 (T1)] to measure effects on L-type Ca2+ current (ICa,L). Gst inhibited ICa,L (IC50 = 47 µM) without affecting its time course or shifting the ICa,L-voltage relationship. At the highest concentration of isoflavone tested (200 µM), ICa,L was inhibited by 66 ± 7% (Gst), 22 ± 2% (daidzein), and 1 ± 3% (genistin). Inhibition of ICa,L by the active tyrphostins was significantly larger than inhibition by T1; at 200 µM the inhibitions were 72 ± 6% (T23), 71 ± 6% (T25), and 27 ± 6% (T1). The phosphotyrosine phosphatase inhibitor orthovanadate (1 mM) had a small stimulatory effect (6 ± 2%) on basal ICa,L and blocked the inhibition of ICa,L by TK inhibitors. The data suggest a role for the TK-phosphotyrosine phosphatase system in the regulation of cardiac Ca2+ channels.
genistein; daidzein; tyrphostins; orthovanadate; protein tyrosine kinase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE ISOFLAVONE GENISTEIN (Gst) and a number of tyrphostin compounds inhibit tyrosine kinase (TK) on extracellular application (2, 7, 12, 13) and are therefore widely used to evaluate the roles of tyrosine phosphorylation in cell function (1, 3, 11), including acute regulation of ion channel activity (5, 13, 15, 24, 30). Recent studies on L-type Ca2+ current (ICa,L) in smooth muscle cells indicate that Gst and tyrphostin inhibit the current, whereas TK-inactive analogs do not (19, 20), and phosphotyrosine phosphatase (PTP) inhibitors have stimulatory effects (33). These results led to the conclusion that tonic tyrosine phosphorylation of the channels may be required for channel opening in the myocytes, and a similar conclusion has been reached in regard to Ca2+ channels in neuroblastoma-glioma hybrid cells (22) and retinal pigment epithelial cells (26).
Studies on ICa,L in neonatal and adult rat ventricular myocytes treated with Gst and the TK-inactive analog daidzein (17, 34) have not supported a role for TK, because daidzein inhibited ICa,L to the same extent as Gst. On the other hand, we found that orthovanadate, a PTP inhibitor (14, 27), reversed the inhibition (35 ± 6%, n = 9) of rat ventricular ICa,L induced by 100 µM Gst and that 50-100 µM Gst also inhibited ICa,L in guinea pig ventricular myocytes (48 ± 4%, n = 4) (25). Furthermore, Wang and Lipsius (29) recently reported that 50 µM daidzein has no significant effect on ICa,L in feline atrial myocytes and that orthovanadate antagonized the inhibition caused by Gst.
In the present study on guinea pig ventricular myocytes, we have 1) compared the effects of Gst with those of daidzein and genistin, two compounds that closely resemble Gst but have far smaller inhibitory effects on TK (2), 2) compared the effects of two TK-inhibitory tyrphostins [tyrphostin A23 (T23) and tyrphostin A25 (T25)] with those of the inactive analog tyrphostin A1 (T1) (7), and 3) determined whether orthovanadate antagonizes the effects of TK inhibitors.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell Preparation
Guinea pigs (250-350 g) were killed by cervical dislocation. Hearts were quickly excised, and single ventricular myocytes were enzymatically isolated as described previously (23, 24). Briefly, the excised hearts were mounted on a Langendorff column and retrogradely perfused (37°C) through the aorta with Ca2+-free Tyrode solution containing collagenase (0.08-0.12 mg/ml; Yakult Pharmaceutical, Tokyo, Japan) for 10-15 min. The cells were dispersed and maintained at room temperature in a high-K+, nutrient-supplemented storage solution.Solutions
The following superfusates were used: 1) normal Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (pH 7.4 with NaOH); 2) K+-free Tyrode solution (KCl omitted, 10 mM CsCl added); and 3) solution 2 with 0.2 mM CdCl2 added.All myocytes were dialyzed with
Cs+ pipette-filling solution.
Standard Cs+ solution contained
(in mM) 40 CsCl, 106 CsOH, 106 aspartic acid, 5 MgATP, 5 EGTA, and 5 HEPES (pH 7.2 with CsOH). In some experiments, dialysate
Cl
concentration was
elevated to 130 mM (replacement of aspartate).
Storage solution contained (in mM) 80 KOH, 30 KCl, 30 KH2PO4, 3 MgSO4, 50 glutamic acid, 20 taurine, 20 glucose, 0.5 EGTA, and 10 HEPES (pH 7.4 with KOH).
Electrophysiology
A few drops of the cell suspension were placed in a 0.3-ml perfusion chamber mounted on an inverted microscope stage. After the cells had settled to the bottom, the chamber was perfused (~2 ml/min) with Tyrode solution at 36°C. Whole cell membrane currents were recorded using an EPC-7 amplifier (List Electronic, Darmstadt, Germany). Recording pipettes were fabricated from thick-walled borosilicate glass capillaries (H15/10/137, Jencons Scientific, Bedfordshire, UK) and had resistances of 1.5-2.5 M
when filled with pipette solution. Liquid
junction potentials between external and pipette-filling solutions were
offset before the pipette touched the cell. The series resistance was
3-7 M and was compensated by 60-80%. Leakage compensation was
not used. The current signals were low-pass filtered at 3 kHz
and digitized with an analog-to-digital converter (Digidata 1200A, Axon
Instruments, Foster City, CA) and pCLAMP software (Axon Instruments) at
a sampling rate of 8 kHz before analysis.
All experiments were performed on the day of cell isolation with
superfusate heated to 36°C. K+
currents were suppressed by using
Cs+-rich dialysate and
K+-free
Cs+-containing superfusate. The
cell membrane was held at 
40 mV to inactivate
Na+ current and any T-type
Ca2+ current, and 200-ms test
steps were applied at 0.2 Hz.
Drugs
Gst (Sigma Chemical, St. Louis, MO), daidzein (Calbiochem, San Diego, CA), and genistin (Sigma Chemical) and T1, T23, and T25 (Calbiochem) were prepared as 100 mM stock solutions in DMSO. Appropriate amounts of stock solutions were added to external solutions, and the mixture was sonicated to ensure proper dispersion. Corresponding amounts of DMSO (
0.2% vol/vol) were also added to the control external solutions.
These concentrations of DMSO have little effect on
ICa,L in guinea
pig ventricular myocytes (23). Aqueous stock solutions (100 mM) of
Na3VO4
(Fisher Scientific, Nepeon, ON, Canada) were freshly prepared, and the
pH was adjusted to ~10 (8, 24). Appropriate amounts of stock solution
were added to the superfusate just before use, and the pH was
readjusted to 7.4 with NaOH.
Statistics
Values are means ± SE; n represents the number of experiments. Single comparisons were made using Student's t-test. ANOVA followed by Bonferroni's test was used for multiple comparisons. Differences were considered significant when P < 0.05. A nonlinear least-squares method was used for theoretical curve fitting.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Isoflavones on ICa,L
The effects of three isoflavones (TK-active Gst and "inactive" daidzein and genistin) were examined on guinea pig ventricular myocytes superfused with K+-free Tyrode solution and dialyzed with Cs+ solution. The results obtained with Gst are presented first and include justification of the method used for measurement of ICa,L in the study.Effects of Gst
The results shown in Fig. 1 indicate that Gst had two major effects on membrane currents elicited by 200-ms depolarizations from40 mV: it reduced peak inward current
(Iin) and
shifted the end-of-pulse current
(I200) in the
outward direction. These effects were concentration dependent (10-200 µM), complete within 4 min, and largely reversed on removal of the
drug (Fig. 1A).
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In six myocytes exposed to 50 µM Gst,
Iin at 0 mV
declined from 1.12 ± 0.22 to 0.50 ± 0.12 nA
(P < 0.01), and
I200 increased from 2 ± 17 to 111 ± 27 pA (P < 0.01). Because (concentration-dependent) increases in
I200 ruled out
the use of Iin to
estimate changes in
ICa,L, we
examined the validity of using a different measurement, 
I (=
I200 Iin), by
determining the effects of Gst on 1)
the time courses of decay of the inward transient and
2) the current in the absence of
ICa,L.
The decays of inward transients at 0 mV were fitted with the sum of two
exponential functions: I = Afexp(t/
f) + Asexp(t/s) + Iss, where
f and
s are fast and slow time
constants with amplitudes Af and
As, respectively,
and Iss is the
amplitude of steady-state current. Gst (50 µM) had no
significant effect on f,
s, and relative
Af
(n = 6); pre-Gst values were
8.7 ± 0.8 ms, 54.9 ± 4.7 ms,
and 0.67 ± 0.05 for
f,
s, and relative
Af, respectively, whereas test values were 10.0 ± 0.8 ms, 61.4 ± 6.6 ms, and 0.64 ± 0.06. With the assumption of full inactivation of
ICa,L at longer times, the degree of inactivation at the end of 200-ms pulses [100 × (Iin I200)/(Iin Iss)] was
also unaffected by Gst (control = 99 ± 0.4%, Gst = 97 ± 1%). These results suggest that the Gst-induced changes in
I200
were primarily due to activation of a time-independent current. This
point was examined in experiments on myocytes that were treated with
0.2 mM Cd2+ (to suppress
ICa,L) but
otherwise bathed, dialyzed, and pulsed as before.
In Cd2+-treated myocytes, Gst
rapidly increased the amplitude of outward currents elicited by 200-ms
pulses from 40 to 0 mV. The currents induced by Gst were
essentially time independent, and their amplitudes were dependent on
the concentration of the drug (Fig.
1B). In addition, the reversal
potential of Gst-activated current (Gst control) was sensitive
to dialysate Cl
concentration. The current-voltage relationships in Fig.
1D indicate that the reversal
potential in a myocyte dialyzed with standard 40 mM
Cl dialysate was 32
mV [calculated Cl
equilibrium potential
(ECl) = 36 mV], whereas that in a myocyte dialyzed with 130 mM
Cl solution was +2 mV
(calculated ECl = 3 mV). Thus we conclude that the outward shifts in
I200 caused by
Gst in myocytes under non-Cd2+
conditions are primarily due to activation of
Cl conductance (9, 24).
On the basis of the foregoing, we adopted
I as the best method for estimation
of the effects of Gst on the amplitude of
ICa,L. Figure
2, A and
B, illustrates this approach and
indicates that maximal peak
ICa,L was reduced
by 38 ± 8% (n = 6) after exposure to 50 µM Gst.
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The dependence of
ICa,L on the
concentration of Gst was evaluated in myocytes that were pulsed from
40 to 0 mV and treated with a single concentration of Gst for
3-4 min. The data from these experiments (Fig.
2C) are fitted with the equation
ICa,L (%control) = Emax/{1 + ([Gst]/IC50)nH} + (100 Emax) where
Emax is the maximal percent
inhibition caused by the drug,
IC50 is the Gst concentration for
half-maximal inhibition, and
nH is the Hill
coefficient. The best-fit parameters are
Emax = 79%,
IC50 = 47 µM, and
nH = 1.1.
Effects of Daidzein and Genistin
Representative results from myocytes treated with these analogs at 100 µM (Fig. 3, A and B) and a summary of the effects of these analogs at 10-200 µM (Fig. 3C) indicate that 1) daidzein caused significant 16-22% inhibition at 50-200 µM (P < 0.05-0.005), 2) inhibition by daidzein was significantly smaller (P < 0.05-0.005) than inhibition by Gst at each concentration tested, and 3) ICa,L was unaffected by genistin.
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Effects of Tyrphostins
T25 is a broad-spectrum TK inhibitor, the action of which is frequently contrasted with that of (negative control) "TK-inactive" T1 (1, 3, 7). Myocytes were pulsed from40 to 0 mV and treated with 50 or 200 µM T25 or T1. These concentrations of T25 reduced
ICa,L
(I) by 18 ± 4%
(n = 6) and 71 ± 6%
(n = 3), respectively (Fig.
4). The lower concentration of T1 had no
significant effect (inhibition of 3 ± 2%,
n = 4), but 200 µM reduced the
current by 27 ± 5% (n = 5, P < 0.05;
P < 0.05-0.005 vs. A25; Fig.
4). Inhibition by 200 µM T1 was reversed during 6- to 8-min
washout periods, whereas inhibition by 200 µM T25 appeared to be
irreversible (Fig. 4A).
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TK-inhibitory T23 (1, 3, 7) inhibited
ICa,L at 0 mV in
a reversible manner (Fig. 5,
A and
B). Data obtained after 3- to 5-min
treatments with a single concentration of T23 (Fig. 5C) are fitted with a Hill equation
that has an IC50 of 76 µM, projected maximal inhibition of 87%, and
nH of 1.6. T23-induced inhibition of
ICa,L (50 µM:
31 ± 5%, n = 12; 200 µM: 72 ± 6%, n = 6) was
significantly (P < 0.01-0.001)
larger than inhibition by T1 (50 µM: 3 ± 2%,
n = 4; 200 µM: 27 ± 6%,
n = 5).
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Antagonistic Effects of Orthovanadate on the Inhibition of ICa,L by Gst and T23
At commonly used bath concentrations of 0.1-10 mM, PTP-inhibiting orthovanadate (14, 27) has been shown to antagonize various Gst-induced cellular responses (4, 5, 24). Figure 6A shows the effects of 1 mM orthovanadate on membrane currents in a myocyte treated with 50 µM Gst for 3 min. The Gst-induced reduction in Iin and elevation of I200 were rapidly antagonized by the PTP inhibitor. Similarly, the Gst-like changes induced by 50 µM T23 were quickly reversed after addition of 1 mM orthovanadate (Fig. 6B).
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Figure 7A
summarizes the antagonistic effect of 1 mM orthovanadate on the
inhibition of
ICa,L by Gst and
T23. In eight myocytes treated with 50 µM Gst, addition of
orthovanadate increased the current from 64 ± 2% control to 92 ± 2% control (P < 0.005).
ICa,L reduced by
3- to 5-min exposures to 100 µM Gst or 100 µM T23 was also restored
to near control amplitude by application of the PTP inhibitor, whereas
inhibition by 200 µM Gst was only weakly antagonized (Fig.
7A). In additional experiments,
myocytes that were treated with 1 mM orthovanadate + 50 µM GST for 5 min responded to removal of orthovanadate with a 35 ± 3%
(n = 5) reduction in ICa,L (not
shown).
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To evaluate whether the orthovanadate-induced recovery of ICa,L in myocytes treated with a TK inhibitor was simply due to a general stimulatory effect of the PTP inhibitor, we examined the effects of a short application of the drug on basal ICa,L. In 12 myocytes, 1 mM orthovanadate increased ICa,L by 6 ± 2% (Fig. 7, B and C). Although this increase was statistically significant (P < 0.05), it was up to 14 times smaller than the increases produced by orthovanadate in myocytes that were pretreated with 50-100 µM Gst or T23 (Fig. 7C).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The objective of this study was to determine the effects of modulators of tyrosine phosphorylation on ICa,L in guinea pig ventricular myocytes. We applied three TK inhibitors (the isoflavone Gst and tyrphostins T23 and T25) and used three appropriate negative controls (the isoflavones daidzein and genistin and T1). The three TK inhibitors depressed ICa,L in a concentration-dependent manner, and this action was antagonized by PTP-inhibiting orthovanadate. Two of the three TK-inactive compounds (daidzein and T1) also inhibited ICa,L, but the inhibition was substantially smaller than that elicited by the corresponding TK inhibitors. We compare the Gst and daidzein findings with earlier ones on ICa,L in cardiac and noncardiac cells and then discuss whether the present data are consistent with acute regulation of L-type channels by the TK/PTP system and/or with other mechanisms.
Comparison With Earlier Findings on Gst and Daidzein
Yokoshiki et al. (34) and Katsube et al. (17) examined the effects of Gst and daidzein on rat ventricular myocytes, and Chiang et al. (6) performed a similar study on guinea pig ventricular myocytes. Maximally effective 100 µM Gst reduced ICa,L by 40-43% (17, 34) in neonatal rat myocytes and by a smaller amount in adult myocytes (17). In the study on guinea pig myocytes, the IC50 for Gst was 17.5 µM and projected maximal inhibition was ~70% (6). Surprisingly, ICa,L in each of these studies was as potently inhibited by daidzein as by Gst. Our results are different in three respects: 1) the IC50 for Gst was higher (47 µM), 2) high Gst was strongly inhibitory [e.g., 66 ± 7% (n = 8) at 200 µM], and 3) inhibition by daidzein [22 ± 2% (n = 10) at 200 µM] was considerably smaller. The reason for these differences is unknown. One difference in methodology is that the present experiments were conducted at 36°C vs. room temperature in the other studies. Excessive rundown of ICa,L during our experiments is not an explanation, because 1) low concentrations of Gst and T23 had minimal effects, 2) low and high concentrations of genistin had negligible effects, and 3) strong inhibition by high Gst and T23 was reversible.Wang and Lipsius (29) recently reported on the responses of
ICa,L in feline
atrial myocyte to Gst and daidzein. Points of agreement with the
present study (also see below) are that 50 µM daidzein caused little
inhibition of atrial
ICa,L, whereas 
10 µM GST caused a rapid concentration-dependent reduction. The present results are also similar in many respects to those reported in
studies on noncardiac
ICa,L. Uterine
smooth muscle
ICa,L was inhibited by Gst with IC50 = 50 µM (18), and vascular smooth muscle
ICa,L was nearly
completely inhibited by 300 µM Gst:
IC50 = 36 µM (32) and 55 µM
(19). Because smooth muscle
ICa,L was little
affected by daidzein up to 300 µM, the authors of those studies
proposed that endogenous TK plays an important role in the regulation
of L-type Ca2+ channel activity.
The same conclusion was reached by Cataldi et al. (5); they found that
25 and 50 µM GST inhibited
Ba2+-carried L-type current in
pituitary GH3 cells by 25 and
50%, respectively, whereas 100 µM daidzein reduced the current by
only 10%.
Evidence That Points to a Regulatory Role for TK/PTP
Two lines of evidence point to the participation of the TK/PTP system in the regulation of L-type Ca2+ channels in guinea pig ventricular myocytes: 1) TK inhibitors reduced ICa,L to a much greater extent than inactive analogs, and 2) PTP-inhibiting orthovanadate antagonized this action.Relative inhibition by TK-active and TK-inactive compounds. One approach to interpreting the data obtained with these compounds is to assume that the analogs have TK-independent effects, whereas the active compounds have TK-independent and TK-dependent effects. For example, Morikawa et al. (22) used the difference in inhibition caused by 100 µM daidzein and 100 µM Gst to conclude that inhibition of TK reduces N- and T-type Ba2+ currents in differentiated NG108-15 cells by ~30%. Subtraction of mean daidzein data from mean Gst data in the present study suggests that TK inhibition by 50, 100, and 200 µM Gst reduced ICa,L by 25, 35, and 45%, respectively. Similar subtraction of genistin data from Gst data gives 37, 48, and 66% inhibition at the three concentrations. T1 and T23 differences suggest TK-related inhibition of 28 and 45% at 50 and 200 µM, respectively.
Antagonism by orthovanadate. Orthovanadate has been shown to inhibit PTP with an inhibition constant of 30-80 µM (10, 16) and (along with other vanadates) has therefore been widely employed to enhance tyrosine phosphorylation and antagonize the effects of TK inhibitors in intact cells (10, 12). In the present study it rapidly reversed the inhibition of ICa,L induced by Gst or T23. Antagonism of Gst and T23 effects by orthovanadate has been observed in earlier studies on cation currents in noncardiac cells. Wang and Salter (30) reported that the vanadate reversed the Gst-induced inhibition of currents carried by N-methyl-D-aspartate receptors in neuronal cells, and Cataldi et al. (5) found that it largely prevented Gst-induced inhibition of Ca2+ influx in depolarized GH3 cells.
Orthovanadate had only a minor stimulatory effect on basal ICa,L in guinea pig ventricular myocytes. In contrast, it increased N-methyl-D-aspartate receptor open probability by 97 ± 18% (31) and increased basal ICa,L in arterial smooth muscle cells by ~50% (33). In the latter study, Wijetunge et al. (33) found that basal ICa,L was also increased by three other PTP inhibitors (100 µM orthovanadate, 100 µM phenylarsine oxide, and 50 µM dephostatin). Because these stimulations were prevented by pretreatment with 100 µM T23 and Western blot analysis showed that peroxyvanadate increased tyrosine phosphorylation of several endogenous proteins in these cells, the authors concluded that ICa,L stimulation was due to increased tyrosine phosphorylation and that this phosphorylation plays an important role in Ca2+ channel opening in arterial smooth muscle. The finding that orthovanadate had a relatively minor stimulatory effect on guinea pig ventricular myocytes suggests that tyrosine phosphorylation relevant to Ca2+ channel activity is near maximum under basal conditions. Two of our key results with 1 mM orthovanadate (i.e., minor effect on basal ICa,L and antagonism of inhibition by Gst) correspond with findings on feline atrial myocytes by Wang and Lipsius (29). However, these investigators also described an additional orthovanadate-sensitive mechanism, secondary stimulation of ICa,L by Gst, that they attributed to inhibition of cytoplasmic (vs. membrane) TK. This response was absent in the myocytes investigated here, as well as in the rat ventricular myocytes investigated previously (17, 25, 34), either because of species- or cell-type differences or changes in the cytoplasm of guinea pig and rat myocytes examined with the ruptured-patch technique (29).Other Interpretations of the Data
The finding that the inhibition of ICa,L by Gst was substantially larger than that caused by equimolar daidzein or genistin points to an involvement of TK. However, there is a need for caution in the interpretation of reductions in ICa,L caused by drugs that are thought to affect intracellular enzymatic pathways, because L-type Ca2+ channels are known to be blocked by a wide variety of organic compounds, especially when externally applied at high micromolar concentrations (21). Thus the finding that TK-inhibitory isoflavone and tyrphostin are more potent inhibitors of ICa,L than inactive analogs may be fortuitous; i.e., the active compounds may simply be more potent direct blockers of Ca2+ channels. The antagonism by orthovanadate is more difficult to place in a non-TK/PTP context unless one postulates that it 1) modifies the chemical structure of the TK inhibitors, 2) causes their displacement from channel binding sites, or 3) stimulates the activity of Ca2+ channels that are bound with TK inhibitor. A common factor in these seemingly unlikely schemes is the presence of TK inhibitor. This requirement arises because orthovanadate was only slightly stimulatory in control myocytes. Consequently, any "nonspecific" effects of the compound arising from its activity as an inorganic phosphate analog (8) and inhibitor of non-PTP (14, 28) appear to have been of minor importance in the absence of TK inhibitor. There remains the possibility that an ICa,L-modulatory action (such as inhibition of serine/threonine phosphatase) only comes to the fore when TK is inhibited.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Tatsuya Asai for performing exploratory experiments and Gina Dickie for technical assistance.
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FOOTNOTES |
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L. M. Shuba was supported by an award from the Dalhousie Medical Research Foundation. This study was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Nova Scotia.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. F. McDonald, Dept. of Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada B3H 4H7 (E-mail: terence.mcdonald{at}dal.ca).
Received 26 August 1998; accepted in final form 13 January 1999.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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] or 130 mM
Cl
). Cli, intracellular
Cl
P < 0.05 and
P < 0.005 vs. control;
* P < 0.05 and
§ P < 0.005 vs. Gst.
