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1-Adrenergic activation of
L-type Ca current in rat ventricular myocytes: perforated
patch-clamp recordings
Departments of 1 Biopharmaceutical Sciences and 2 Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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1-Adrenergic stimulation
has little effect on L-type Ca2+
channel current
(ICa,L) in
adult cardiac myocytes measured using conventional whole cell
voltage-clamp techniques. In this study using perforated-patch
techniques, we reevaluated the effect of 1-adrenergic stimulation on
ICa,L in adult
rat ventricular myocytes. Action potentials and
ICa,L were
examined in the presence of 1 µM nadolol, a 
-adrenergic
antagonist, in myocytes internally dialyzed with
Na+- and
K+-free solutions
(Cs+ and tetraethylammonium as
substitutes). Phenylephrine (PE; 30 µM) increased the action
potential duration measured at 25 and 70% of repolarization by 104 and
86%, respectively. In the perforated-patch configuration, PE elicited
a transient decrease followed by a ~60% increase in
ICa,L, whereas
only the transient decrease in ICa,L was
observed in myocytes when the conventional whole cell configuration was
used. The PE-induced increase in
ICa,L was
reversibly blocked by 1 µM prazosin, an
1-adrenergic antagonist. These
results suggest that
1-adrenergic stimulation
enhances cardiac
ICa,L and that
obligatory intracellular mediators for this action are lost during
whole cell recordings.
cardiac muscle cells; whole cell patch clamp; action potential; ion channel
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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STIMULATION of
1-adrenergic receptors has been
shown to elicit a sustained positive inotropic effect in cardiac muscle
(2-4, 10, 12, 15, 17) that is often accompanied by an increase in
action potential duration (APD) (2, 3, 12, 15). The mechanism
underlying the
1-adrenoceptor-mediated
increase in APD is often attributed to inhibition of a variety of
K+ currents including the
transient outward current
(Ito) and the inward rectifier (3, 5-7, 15, 16). Many studies have reported that
1-adrenergic stimulation has no
effect on the L-type Ca2+ channel
current (ICa,L)
(3, 5, 7, 12, 15). However, studies using
Ca2+-sensitive indicators have
demonstrated an increase in the intracellular Ca2+ concentration
([Ca2+]i)
transient in response to
1-adrenergic stimulation in
most intact cardiac cells (4). In addition, studies in rat atrial muscle with
45Ca2+
flux measurement suggested that phenylephrine (PE) increases Ca2+ influx through
Ca2+ channels and decreases
Ca2+ efflux via
Na+/Ca2+
exchange (9). This led us to believe that the relative absence of an
effect of 1-adrenergic
stimulation on
ICa,L in whole
cell patch-clamped myocytes results from a dilution or loss of
obligatory intracellular regulatory components. To test this
possibility we used perforated patch-clamp techniques to reevaluate the
effect of 1-adrenergic
stimulation on
ICa,L in adult
rat ventricular myocytes. Results indicate that activation of
1-adrenoceptors elicits
significant and sustained increases in
ICa,L and in APD under conditions that minimize K+
currents.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Myocyte isolation. Single adult ventricular myocytes were isolated from the hearts of male Sprague-Dawley rats (250-300 g) using protocols described previously (11). Briefly, hearts were rapidly excised and perfused at 37°C via the aorta with an oxygenated control buffer solution (see Solutions) followed by another 5-min perfusion with Ca2+-free buffer solution. Hearts were then perfused for 20 min with a buffer solution containing 125 µM CaCl2 plus 0.5 mg/ml collagenase. The ventricles were removed, minced, rinsed with control buffer solution, and shaken in a water bath at 37°C for two to three periods of 10 min each. Isolated ventricular myocytes were then plated into 60-mm culture dishes (Falcon) containing antibiotic-free, bicarbonate-buffered culture medium 199 (60%, GIBCO, Grand Island, NY) with 36% Earle's balanced salt solution composed of (in mM) 116 NaCl, 4.7 KCl, 0.9 NaH2PO4, 0.8 MgSO4, 26 NaHCO3, and 5.6 glucose and 4% fetal bovine serum (GIBCO) (pH 7.4 in 5% CO2-95% air at 37°C).
Electrophysiological measurements.
Ventricular myocytes were placed on the heated stage of an inverted
microscope (Nikon Diaphot) and perfused with a normal Tyrode solution.
Cells were patch-clamped in the whole cell configuration by
conventional techniques (8) or in the perforated-patch configuration (13) using a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA) as previously described (11). Briefly, patch electrodes were filled with a pipette solution and had a tip resistance of 2-5 M
. Recorded currents were filtered at 1-2 kHz
through a four-pole low-pass Bessel filter and sampled at 5 kHz with a PC/AT computer using pCLAMP 6.03 software (Axon Instruments) through an
Axon Digidata 2000A acquisition system.
40 mV and
superfused in the normal Tyrode solution at 37°C. After the formation of a gigaseal, a 5-mV test pulse was repetitively applied to
monitor the access resistance until it fell below 20 M. After formation of the perforated-patch or conventional whole cell
configuration, ICa,L was
elicited by a single pulse of 250 ms to +10 mV from the holding
potential. The peak current-voltage
(I-V) relationship of
ICa,L was
constructed by applying 250-ms voltage pulses to potentials between
60 and +70 mV in 10-mV increments from the holding potential of
40 mV at 0.1 Hz. The magnitude of
ICa,L was defined
by the difference between the peak current and the level at the end of
the 250-ms pulse. Because the access resistance improved with time
during the perforated-patch mode, some experiments monitoring the time
course of the effect of PE utilized a 120-ms voltage-ramp protocol to
potentials between 60 and +60 mV from the holding potential to
avoid the rupture of the patched membrane resulting from the
overcompensation that can occur when a step voltage pulse is applied.
Such a voltage-ramp protocol has been used by other investigators for
rapid recordings of the I-V
relationship of
ICa,L (1, 19).
Note that PE-induced changes in peak
ICa,L are usually
underestimated by ~20% when the voltage-ramp protocol is used;
however, this does not affect the conclusion of this study. Successful
formation of the perforated-patch configuration was justified by the
contraction of myocytes concomitant with the elicited action potential.
The success rate for acceptable perforated-patch recordings was
<30%, and the average time to achieve a stable perforated patch was
>15 min as described previously (13). Once adequate access (access
resistance < 10 M) was achieved, ICa,L was stable
for more than 1 h. In some experiments, after measurement of
ICa,L in the
perforated-patch configuration, the patched membrane was ruptured to
obtain the whole cell configuration (see, e.g., Fig.
3B). Once in the whole cell mode,
myocytes did not contract in response to voltage pulses because of the
high concentration of EGTA in the pipette solution. The
Na+ current was inactivated by
using the holding potential of 40 mV.
K+ currents were minimized by
using 20 mM tetraethylammonium (TEA)-Cl and
Cs+ to replace
K+ in the pipette solution.
Nadolol (1 µM) was added 3-5 min before the addition of PE in
all experiments. The selected concentration of PE (30 µM) was used in
this study because it produces maximum effects on developed tension in
rat papillary muscle (18) and on
Ito in rat
ventricular myocytes (15).
Solutions. The control buffer solution for isolation of myocytes contained (in mM) 110 NaCl, 3.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 0.2 CaCl2, and 11 glucose (pH 7.4 in 95% O2-5% CO2 at 37°C). Normal Tyrode solution consisted of (in mM) 145 NaCl, 5.4 KCl, 0.8 MgCl2, 1.0 CaCl2, 5.6 glucose, 5.8 HEPES, and 4.2 Tris-base (pH 7.4 at 37°C). The pipette solution for ICa,L measurement consisted of (in mM) 100 CsOH, 70 aspartic acid, 11 CsCl, 15 TEA-Cl, 2 MgCl2, 5 Mg-ATP, 10 EGTA, 0.1 CaCl2, 5 pyruvic acid, 5.6 glucose, 5 (Tris)2-phosphocreatine, 0.4 Li4-GTP, 5 HEPES, and 5 Tris-base (pH 7.2 at 37°C). The pipette solution for the perforated-patch also contained 100-200 µg/ml gramicidin D (Sigma, St. Louis, MO). Stock solutions of 10 mg/ml gramicidin were made in methanol (13), and stock solutions of 0.1 M nadolol and 10 mM PE were made in ethanol and Milli-Q water, respectively.
Statistics. Values are presented as means ± SE. Statistical significance was evaluated by the two-tailed paired Student's t-test or, when more than two conditions were compared, by one-way ANOVA with Duncan's multiple-range test. Differences with P < 0.05 were considered significant.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Prolongation of APD by PE. Figure 1 shows the effect of PE on the action potential of a ventricular myocyte. After formation of a perforated patch in normal Tyrode solution containing 1 µM nadolol, myocytes were current-clamped, and action potentials were elicited by a 1-ms depolarizing pulse (trace 1 in Fig. 1). Exposure to 30 µM PE caused a gradual increase in the duration of the action potential as shown by recordings taken at 6 min (trace 2 in Fig. 1) and at 15 min (trace 3, Fig. 1). At the steady-state response to PE, the APDs measured at 25 and 70% of repolarization (APD25 and APD70, respectively) were increased by 104 ± 11 (n = 4 myocytes, P < 0.05) and 86 ± 15 (n = 4, P < 0.05) %, respectively. After removal of PE for 9 min, the APD partially recovered (trace 4, Fig. 1). This PE-induced prolongation of APD is consistent with findings by others (2, 3, 15); however, the extent of prolongation is greater in the present conditions, in which K+ currents are minimized.
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Activation of ICa,L induced by PE in perforated-patch recordings. Figure 2 shows representative results from one myocyte that was exposed for 6 min to 30 µM PE. PE doubled peak ICa,L in this cell without a significant effect on the voltage dependence of its I-V relationship. The ICa,L recovered almost completely after removal of PE for 15 min (compared with control). Figure 2, inset, shows superimposed current traces of peak ICa,L before, during, and after exposure to 30 µM PE.
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1-adrenergic
activation of
ICa,L are
diminished during whole cell patch recordings.
Figure 3D shows that in
perforated-patch recordings the PE-induced increase in peak
ICa,L was
reversed by addition of 1 µM prazosin, an
1-adrenoceptor antagonist. On
removal of prazosin in the presence of PE, the increase in
ICa,L was again
observed. Similar results were found in three other experiments. These
data confirm that
1-adrenoceptors mediate the
PE-induced increase in peak
ICa,L.
Figure 4 summarizes the steady-state PE effects on the
peak I-V curves of
ICa,L when
examined in the perforated-patch and conventional whole cell patch
configurations. In the perforated-patch mode, PE elicited a 66%
increase in peak
ICa,L measured at
0 mV [29.0 ± 2.3 pA/pF
(n = 5 myocytes) compared with
17.4 ± 1.9 pA/pF in control
(n = 8)]. In contrast,
conventional whole cell patch recordings show no significant effect of
PE on peak ICa,L [13.9 ± 2.7 pA/pF (n = 5) compared with 16.4 ± 3.9 pA/pF in control
(n = 5)]. There was no apparent
effect of PE on the voltage-dependence of peak
ICa,L in either
condition.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Using perforated-patch techniques, we show in the present study that
1-adrenoceptor stimulation
results in a dramatic increase in
ICa,L accompanied
by a prolongation of APD in adult rat ventricular myocytes
intracellularly dialyzed with Na+-
and K+-free solutions. These
effects were not observed when conventional whole cell patch recordings
were used. The perforated-patch technique retains most intracellular
constituents including second messenger(s) for regulation of
ICa,L that can be
diluted during conventional whole cell patch recordings. Two lines of
evidence show that the perforated-patch technique per se does not alter
the properties of
ICa,L:
1) the control
I-V relationship of
ICa,L obtained
from perforated-patch recordings is not different from that recorded in
the conventional whole cell mode (Fig. 4); and
2) the responsiveness to PE in the
whole cell mode that was formed after a perforated-patch recording does
not differ from that in the conventional whole cell recordings (Fig.
3). Thus our data suggest that intracellular constituents play an
important role in the cellular mechanism underlying the regulation of
cardiac ICa,L by
1-adrenoceptors. This is
further supported by our results showing that after switching from a
perforated-patch to a conventional whole cell patch configuration, the
PE-induced increase in
ICa,L is
abolished.
1-Adrenergic stimulation has
been shown to elicit a three-phase inotropic response in rat
ventricular myocytes or papillary muscle: an initial brief positive
inotropy followed by a transient negative inotropic phase and a
sustained positive inotropic response (2, 3, 12, 15, 17). The
PE-induced inotropic effects are believed to be mediated through
multiple pathways (4, 10, 12, 17, 18). The PE-induced sustained
positive inotropic response has been suggested to result in part from
inhibition of Ito
(3, 5, 15) that leads to prolongation of APD and thereby an increased
Ca2+ influx (4). PE has been shown
to increase Ca2+ influx and
[Ca2+]i
transients (9), and Ca2+ channel
blockers such as verapamil and nifedipine have been shown to block the
sustained positive inotropy (12, 14). However, whether the PE-induced
inhibition (by 12-42%) of
Ito can account for the increased Ca2+ influx and
the sustained positive inotropic effect is still debatable. An effect
of PE on ICa,L
has not been completely ruled out because ICa,L has been
measured in the conventional whole cell mode. Our data indicate that
1-adrenergic activation does
enhance ICa,L and
prolong the APD under conditions where
K+ currents are minimized; such
results can account for the sustained positive inotropic effect of PE.
This indicates that the effects of PE on APD and Ca influx are mediated
at least in part by increase in
ICa,L and not
entirely by inhibition of
Ito.
In summary, results of this study suggest that
1) PE-induced activation of
ICa,L requires
dialyzable intracellular second messengers and
2) the perforated-patch technique is
required to delineate the cellular mechanisms by which
1-adrenergic stimulation modulates cardiac function.
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ACKNOWLEDGEMENTS |
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The authors thank Meei-Yueh Liu and Richard Wyeth for excellent technical assistance. This work was supported in part by grants from the American Heart Association, Arkansas Affiliate, the American Health Assistance Foundation, and the Office of Naval Research.
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FOOTNOTES |
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Address for reprint requests: S. J. Liu, Dept. of Biopharmaceutical Sciences, Univ. of Arkansas for Medical Sciences, 4301 West Markham St., MS#522-3, Little Rock, AR 72205.
Received 17 December 1997; accepted in final form 18 February 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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, see also inset of Fig.
).
I-V relationship recovered 15 min
after removal of PE (
). Inset,
superimposed current traces measured at 0 mV before, during, and after
exposure to PE. Dashed line, zero current level. Calibration bars, 10 pA/pF (vertical); 20 ms (horizontal).
,
n = 7).
D: inhibition of PE-enhanced
ICa,L by
prazosin. With the PP configuration, PE caused a sustained increase in
ICa,L (
). Removal of prazosin
in presence of PE revealed stimulatory effect of PE on
ICa,L. All
currents were normalized to the averaged current recorded 1 min before
each exposure to PE. Open symbols indicate currents in absence of PE.
