Extract
Note: Please read the complete
full text with Figures and Tables at
7.3_7.4 with TEAOH. Resveratrol was dissolved in dimethylsulfoxide and
diluted in the external solution to achieve concentrations of
10, 20, and 40 µmol/L. The same amount of
dimethylsulfoxide was also added to the normal external solution as a control.
No change was observed during perfusion with the control
solution.
Statisticsal analysis Data are expressed as mean±SD.
Statistical analysis was performed using the
t-test and one-way ANOVA. P<0.05 was considered statistically significant.
Results
Effect of resveratrol on L-type calcium
current L-type Ca2+ current in rat ventricular myocytes was evoked by
using a depolarizing step pulse from a holding potential
(Eh) of -50 mV to 0 mV at a frequency of 0.1 Hz. The step pulse
dura-tion was 350 ms. The rundown of
ICa-L was minimized by adding Mg-ATP (3 mmol/L) and egtazic acid (10
mmol/L)[13]. Resveratrol at concentrations of 10, 20, and 40 µmol/L
inhibited the peak amplitude of
ICa-L in a concentration-dependent
manner (Figure 1). The inhibitory effect of resveratrol
disappeared after 15 min washout with control solution.
Effect of resveratrol on current-voltage relationship of
ICa-L The current-voltage
(I_V) curve of L-type Ca2+ current
was obtained by using a number of depolarizing step
pulses (350 ms) from an Eh of -50 mV to test potentials
between -40 mV and 50 mV in 10 mV increments. The pulse
frequency was 0.1 Hz. ICa-L was activated at -30 mV and the
peak amplitude occurred at a potential of 0 mV. Resveratrol
at concentrations of 10, 20, and 40 µmol/L shifted the
I_V curve upwards, and decreased the peak amplitude of
ICa-L from -14.2±1.5 pA/pF to -10.5±1.5 pA/pF
(n=6 cells from 5 hearts; P<0.05), _7.5±2.4 pA/pF
(P<0.01) and -5.2±1.2 pA/pF
(P<0.01), respectively (Figure 1).
Effects of resveratrol on activation of
ICa-L Steady-state activation of L-type calcium channels was obtained by
using depolarizing pulses from an Eh of -50 mV to the test
potential of +50 mV in 10 mV increments for 350 ms. The pulse
frequency was 0.1 Hz. The activation curves were fitted
according to the Boltzmann equation:
I/Imax=1/{1+EXP[(V-V
0.5)/k]}. V0.5 is the midpoint voltage of the activation
functions, and k is the Boltzmann slope parameter for
activation. Resveratrol at a concentration of 40 µmol/L
shifted the half-activation potential
(V0.5) from 19.4±0.4 mV
to -15.4±1.9 mV, and the slope parameter (k) from 5.4±0.8 mV
to 3.8±0.6 mV (n=6 cells from 5 hearts;
P<0.05; Figure 2).
Effects of resveratrol on inactivation of
ICa-L Steady-state inactivation of L-type channels was measured by using a double-pulse protocol[14]. Membrane potential was
first stepped from -50 mV to +20 mV in 10 mV increments for
1000 ms and then to +10 mV for 400 ms (test pulse) and
finally clamped back to the holding potential of -50 mV at a
pulse frequency of 0.1 Hz. The peak current elicited by test
pulses was normalized against the maximum current and
plotted against the conditioning potential. The inactivation
curves were also fitted according to the Boltzmann equation.
V0.5 is the midpoint voltage of the inactivation functions,
and k is the Boltzmann slope parameter for inactivation.
Resveratrol at a concentration of 40 µmol/L shifted
V0.5 from -29.9±2.3 mV to -31.1±3.6 mV and
k from 6.3±1.8 mV to 6.8± 3.6 mV
(P>0.05; n=6 cells from 5 hearts; Figure 2).
Effect of resveratrol on recovery of
ICa-L from inactivation The recovery of
ICa-L from inactivation was studied by
using a double-pulse protocol consisting of a 300 ms
prepulse to +10 mV (P1) followed by a 300 ms test pulse to
+10 mV (P2) after a variable P1_P2 coupling interval from 0 to
2500 ms at a holding potential of -60 mV. Double-pulse
stimulation was repeated every 6 s. Resveratrol at a concentration
of 40 µmol/L markedly shifted the recovery curve of
ICa-L to the right and changed the half-recovery time of the
Ca2+ channel from 169±19 ms to 197±19 ms
(n=6 cells from 6 hearts; P<0.05), indicating slower recovery of
ICa-L from inactivation (Figure 3).
Effects of sodium orthovanadate on resveratrol-induced
ICa-L change To further assess the mechanism underlying
the inhibitory effect of resveratrol on
ICa-L, we observed the effect of sodium orthovanadate
(Na3VO4; 1 mmol/L), a potent inhibitor of protein tyrosine phosphatase, on the
inhibitory effect of resveratrol. When cells were pretreated
with Na3VO4 (1 mmol/L), the resveratrol-induced reduction
in ICa-L was significantly attenuated.
(n=6 cells from 6 hearts; P<0.01; Figure 4).
Discussion
In the present study, we found that resveratrol (10, 20,
and 40 µmol/L) decreased
ICa-L and shifted the I_V curve
upward in a concentration-dependent manner. Furthermore,
the inhibitory effect of resveratrol disappeared after 15 min
washout. This indicates that resveratrol can block L-type
calcium channels in rat ventricular myocytes, but the
characteristic of I_V curve of
ICa-L was not significantly alerted
by resveratrol. Resveratrol (40 µmol/L) shifted the
steady-state activation curve of
ICa-L to the right, suggesting that it
can inhibit the steady-state activation of
ICa-L; but the inactivation kinetics of
ICa-L were not changed by resveratrol (40 µmol/L). In addition, resveratrol (40 µmol/L) markedly shifted
the recovery curve of ICa-L to the right, indicating that
resveratrol can slow down the recovery of
ICa-L from inactiva-tion. These findings indicate that resveratrol decreased
ICa-L in rat ventricular myocytes by markedly slowing down the
activation of L-type calcium channels and their recovery from
inactivation. Therefore, the myocardial
electrophysiological effects of resveratrol observed in our previous
studies[9,10] might be attributed to its inhibitory effect on
ICa-L.
Tyrosine kinase activation is thought to contribute to
cell survival, proliferation, and differentiation in many cell
types[15]. Furthermore, several lines of evidence show that
phosphorylation of tyrosine kinase modulates ion channel
activity[16,17]. Some researchers have reported that resveratrol
inhibites tyrosine kinase activity, and that many of effects of
the resveratrol are mediated by tyrosine
kinase[18,19]. Bruder et al reported that resveratrol-induced cellular phenotype
was dependent on intracellular calcium and tyrosine kinase
activity in bovine pulmonary artery endothelial
cells[20]. Furthermore, Conte et
al reported that resveratrol could inhibit tyrosine kinase activity in PC12
cells[21]. Sodium orthovanadate, an inhibitor of tyrosine phosphatase, can
enhance protein tyrosine
phosphorylation[22]. In the present study, we observed the influence of sodium orthovanadate
on the effect of resveratrol. We found that pretreatment with
sodium orthovanadate markedly antagonized the inhibitory
effects of resveratrol on L-type calcium current, suggesting
that the tyrosine kinase pathway might be involved in the
effects of resveratrol.
In conclusion, we found that resveratrol inhibited
ICa-L in rat ventricular myocytes mainly by inhibiting the
activation of L-type calcium channels and slowing down the recovery
of calcium channels from inactivation. These effects of
resveratrol might be mediated via the tyrosine kinase
path-way. The inhibitory effects of resveratrol on
ICa-L may contribute to its antiarrhythmic actions. This study provides an
electrophysiological basis for the use of
resveratrol in the treatment of cardiovascular diseases.
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