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School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a Ba2+-sensitive outward current with the characteristics of muscarinic K+ current (IK,ACh); the presence of the muscarinic K+ channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward Na+/Ca2+ exchange current (INaCa). ACh also inhibited the background inward K+ current (IK,1). It is concluded that ACh activates IK,ACh, inhibits IK,1, and indirectly inhibits INaCa; this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of IK,1, minimum decrease in excitability.
heart; acetylcholine; muscarinic K+ channel
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PARASYMPATHETIC
NERVES extensively innervate the ventricles, and parasympathetic
stimulation has a negative inotropic effect on the ventricular muscle
(29, 36). Previously, the parasympathetic transmitter ACh
was thought only to have a negative inotropic effect on ventricular
muscle after the muscle had first been potentiated by a 
-agonist
(28). However, it is now well established that ACh does
have a negative inotropic effect in the absence of -stimulation in
ferret, rat, and dog ventricular cells (5, 30, 31, 39). In
the dog, ACh has a substantial effect on left ventricular subepicardial cells, a smaller effect on midwall cells, and little or no effect on
subendocardial cells (39). The negative inotropic effect of ACh has been suggested to be the result of the activation of the
muscarinic K+ current (IK,ACh)
despite the fact that the muscarinic K+ channel (a
heteromultimer of Kir3.1 or GIRK1 and Kir3.4 or GIRK4), although
detectable in the atrium, has not been detected in the ventricles
(26). It is postulated that the activation of
IK,ACh results in a shortening of the action
potential and thus a decrease in the contraction. An ACh-induced
decrease in the basal L-type Ca2+ current
(ICa,L) may also be involved in the ACh-induced
changes in the action potential and contraction (27, 39).
The action potential-clamp technique was devised by Starzak and Needle (37) and first used with cardiac tissue by Doerr et al. (13). Whereas the conventional voltage-clamp technique uses square-wave command pulses, the action potential-clamp technique involves the use of an action potential waveform as the voltage-clamp command. The action potential-clamp technique has been used to study the time course and amplitude of ionic currents during the action potential: current recorded during an action potential voltage-clamp command waveform after the application of a blocker has been subtracted from that under control conditions; ICa, the Na+/Ca2+ exchange current (INaCa), the delayed rectifier K+ current (IK), and the background inward rectifier K+ current (IK,1) have been studied in this way (1, 17, 19, 20). The principal aim of this study was to investigate the ACh-induced changes in current during the ventricular action potential. Adenosine was studied in the same way. This is the first study to investigate agonist-induced changes in membrane current during the action potential. Another aim of the study was to investigate the mechanisms underlying the negative inotropic effect of ACh during the action potential. In part, this involved the detection of Kir3.1 mRNA by screening ventricular tissue cDNA using the PCR technique and Kir3.1 and Kir3.4 proteins in ventricular cells by immunocytochemistry.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiments on single cells. Experiments were performed on isolated ferret ventricular cells. Ferrets were anesthetized by intraperitoneal injection of 90-150 mg pentobarbitone sodium. The heart was rapidly excised and placed in "isolation solution" (see below) containing 750 µM CaCl2. The aorta was cannulated and the heart retrogradely perfused at ~20-23 ml/min with isolation solution containing 750 µM Ca2+ for sufficient time to clear all the blood (~2-3 min). The perfusate was then switched to Ca2+-free isolation solution (containing 0.1 mM EGTA) for 4 min. Finally, the heart was perfused for 10 min with "enzyme solution": isolation solution containing 1 mg/ml collagenase (Worthington type II, Lorne laboratories), 0.1 mg/ml protease (Sigma; Poole, UK), and 50 µM CaCl2; this solution was recirculated through the heart. The ventricles were cut away from the atria, finely chopped, placed in a conical flask, and digested with the enzyme solution supplemented with 10% (wt/vol) bovine serum albumin for a further 5 min. The tissue was shaken gently during this period. This process was repeated four times, and the cells from each 5-min period were harvested by filtration and pelleted by centrifugation at 400 rpm for 40 s. Cells were washed by resuspending them in isolation solution containing 750 µM Ca2+ and then recentrifuging them. The cells were stored in isolation solution containing 750 µM Ca2+ at 4°C until required. The isolation procedure was performed at 37°C. Isolation solution contained (in mM) 130 NaCl, 5.4 KCl, 1.4 MgCl2, 0.4 NaH2PO4, 10 creatine, 20 taurine, 10 HEPES, and 10 glucose; pH 7.3 at room temperature. This solution was equilibrated with O2.
Cells were pipetted into a small tissue bath (volume, 0.2 ml) attached to the stage of an inverted microscope (Nikon Diaphot). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused at a rate of ~1.6 ml/min with Tyrode solution of the following composition (in mM): 136.9 NaCl, 5.4 KCl, 2 CaCl2, 0.57 MgCl2, 0.37 NaH2PO4, 5 HEPES, and 5.6 glucose; pH 7.4 at 37°C. In some experiments 5 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was added to the Tyrode solution to buffer intracellular Ca2+. A 1 mM BAPTA-AM stock solution was made by dissolving the drug in DMSO. Cells were perfused with Tyrode solution containing 5 µM BAPTA-AM (0.5% DMSO) for 5-10 min. During this period the size of the contraction dwindled as a result of the buffering of intracellular Ca2+. When the contraction was almost completely abolished, the BAPTA-AM was washed off. On washoff of BAPTA-AM, the contraction did not recover (BAPTA remained trapped within the cells as a result of the cleavage of the AM group by intracellular esterases). As a control, cells were exposed to Tyrode solution containing 0.5% DMSO only for 10 min; this had no effect (n = 4). Measurements were only made after more than 2 min of washoff of BAPTA-AM. We used the muscarinic and purinergic agonists acetylcholine chloride and adenosine (Sigma). A 10
2 M stock solution of each drug was prepared in
double-distilled water; this was added to the Tyrode solution in
appropriate quantities. The pH of Tyrode solution containing a drug was
checked and readjusted if necessary.
Miniature solenoid valves (Lee Products; Gerrards Cross,
Buckinghamshire, UK) controlled which of the four solutions flowed to
the chamber. The fluid level in the chamber was controlled using the system described by Cannell and Lederer (8). The temperature of the solution was maintained at 37 ± 0.5°C by a heating coil wrapped around the glass inflow tube immediately before
the chamber. Solution temperature was monitored by a thermistor mounted
in the side of the chamber and controlled by a feedback circuit, which
regulated the flow of current to the heating coil.
Ventricular cells were impaled with conventional microelectrodes
(15-30 M
) filled with 1 M KCl. The switch clamp technique was
used to voltage clamp cells (Dagan 8800 amplifier, Dagan; switching
frequency, ~4.5 kHz) to minimize intracellular dialysis and allow
stable recordings of up to and over 1 h. The capacitance of the
microelectrode was compensated just before impalement. The electrode
potential upon switching between current injection and voltage
recording was monitored on an oscilloscope throughout an experiment to
ensure that the electrode potential settled between current injection
pulses. Membrane current was typically filtered at 1 kHz (low-pass
filter). Membrane potential and current were also displayed on an
oscilloscope (Tektronix 5000 series) to assess the quality of the
voltage clamp. Timing units (Hi-Med; Reading, UK) were used to control
voltage-clamp protocols and synchronize other recording equipment. A
pulse generator (3) was used to drive the action
potential-clamp experiments. Briefly, action potentials at a
stimulation rate of 1 Hz were recorded from a cell, and an average of
five were transferred to the pulse generator as a text file. The pulse
generator was then used to apply the action potential voltage-clamp
command waveform (via the Dagan 8800 amplifier) repetitively at a rate
of 1 Hz.
Cell length was recorded using an optical system based on a photodiode
array (7); twitch shortening (the shortening of a cell
during a twitch contraction) was measured electronically (6). Action potential duration was measured electronically at 
65 mV (22). Membrane current and potential, cell
length, twitch shortening, and action potential duration were displayed on a six-channel chart recorder (Gould 2600S or RS 3600) and
simultaneously recorded on videotape using a pulse code modulator
(Neuro-Corder DR-890, Neuro Data Instruments) and a video recorder.
Membrane current and potential and cell length were also digitized
using an analog-to-digital converter (1401 plus, Cambridge Electronic Design; Cambridge, UK) and stored on an IBM-compatible computer running
the Cambridge Electronic Design voltage-clamp software.
Cells were stimulated to contract by either a 5-ms current pulse
(current clamp mode) or application of the action potential clamp, at 1 Hz.
Data are presented as means ± SE (number of cells).
Computer modeling.
Computer simulations were carried out using Version 7 of the Oxsoft
HEART program (33). The "simplified guinea pig
ventricular cell" model was modified to simulate the ferret
ventricular action potential. The modifications made were the
following: extracellular K+ concentration set to 5.4 mM
(same as experiments); conductance for tetrodotoxin-sensitive
Na+ current (INa), GNA = 1.05;
Ca2+ permeability for L-type Ca2+ current
(ICa,L), PCA = 0.07; conductance for
transient outward K+ current (Ito),
GTO = 0.03; speed of inactivation variable for Ito, SPEED[18] = 6; maximum
outward K+ current for IK, IKM = 0.8; conductance for background Na+ current
(Ib,Na) GBNA = 0.0012; voltage shift of the
voltage dependence of the background IK,1,
SHIFTK1 = 10; steepness of the voltage dependence of
IK,1, and STEEPK1 = 2; and conductance for
IK,1, GK1 = 1.
cDNA cloning and sequencing. Standard molecular biology techniques were carried out as described by Berger and Kimmel (2) and Sambrook et al. (35). Oligonucleotide primers 1 (bases 1240 to 1263) and 2 (bases 1501 to 1533) were designed by comparison with known Kir3.1 sequences (26): sense primer 1, 5'-AAACTCCTGAGGATGAGTTCT-3'; antisense primer 2, 5'-TGAAATAATGCCTCGAGGGGTGTTTTGCTATGT-3'.
Total RNA was purified from the ferret ventricle and atrium (10). Total RNA was also purified from the dog ventricle (obtained at the end of an experiment carried out by a colleague). Poly (A+) RNA, purified from total RNA by oligo(dT)-cellulose affinity chromatography (35), was used to generate cDNA by the rapid amplification of cDNA ends [RACE technique (14)] using a Marathon cDNA amplification kit according to the manufacturer's instructions (Clontech Laboratories; Palo Alto, CA). PCR amplification of ~500 pg cDNA (55°C annealing temperature, 50 cycles, Taq polymerase), using oligonucleotides 1 and 2 as primers, generated a single reproducible product when visualized under ultraviolet light after electrophoresis on a 1.5% (wt/vol) agarose gel, containing 50 µg/ml ethidium bromide, for 1 h at 5 V/cm in 40 mM Tris-acetate and 1 mM EDTA buffer (see Fig. 12). No products were generated when PCR was performed in the absence of template cDNA (data not shown). The size of the PCR product was estimated by comparison to fragments of DNA of known size (
-HindIII markers;
GIBCO-BRL; Paisley, UK). The PCR product obtained from the ferret
ventricle was cloned into pCR2.1 T/A cloning vector (Invitrogen;
Groningen, The Netherlands) and then sequenced on an Applied Biosystems
373 DNA Sequencer (Perkin Elmer) using fluorescent cycle sequencing.
Immunocytochemistry. Immunocytochemistry on ferret ventricular and atrial cells was carried out as previously described (12). After isolation, heart cells were plated on Bunsen burner flame-treated, polysine-coated slides (BDH; Poole, UK) and were single labeled with either anti-Kir3.1 (Alomone Labs; Jerusalem, Israel) or anti-Kir3.4 (gift from Dr. G. B. Krapivinsky, Harvard Medical School, Boston, MA) primary antibodies followed by an anti-rabbit secondary antibody conjugated to FITC and examined by a confocal laser scanning microscope (Leica; Heidelberg, Germany) as previously described (12). The images recorded were single optical sections of 10 atrial cells and 6 ventricular cells for each antibody used. The anti-Kir3.1 antibody was previously characterized by immunocytochemistry on Chinese hamster ovary and single rat heart cells (12), whereas the anti-Kir3.4 antibody was previously characterized by Western blotting on Sf9 cells (25). Various control experiments were carried out with these antibodies and are fully described (12). In this study, no labeling of heart cells was detected when the secondary antibody was applied only (i.e., primary antibodies not applied; not shown).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of ACh on the action potential and contraction under current
clamp conditions.
Typical effects of 1 µM ACh on a ferret ventricular cell under
current-clamp conditions are shown in Fig.
1A. The cell was stimulated at
1 Hz. Slow time-base records of contractions and action potential
duration are shown on the left. On application of ACh during
the period shown by the bar, there was an abrupt shortening of the
action potential by 66% accompanied by a decrease in the contraction
of 55%. Fast time-base records of action potentials and contractions
under control conditions and at the time of the peak effect of ACh are
shown on the right of Fig. 1A. In five cells,
contraction was maximally decreased by 75 ± 4% by 1 µM ACh
under current-clamp conditions (see Fig. 2, trace i). After a peak was reached, the effects of ACh faded with time during the
remainder of the 2-min exposure to ACh; the duration of the action
potential and the amplitude of the contraction both increased (Fig.
1A). This is attributed to desensitization to ACh. On
washoff of ACh, action potential duration and contraction rapidly
increased; action potential duration returned to its control value,
whereas there was a small rebound increase in the contraction beyond
its control value (Fig. 1A). The contraction declined to its
control value over ~2 min (not shown).
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Effect of ACh under action potential-clamp conditions. Typical effects of 1 µM ACh on the contraction of a ferret ventricular cell under action potential-clamp conditions are shown in Fig. 1B (all data in Fig. 1 were obtained from the same cell). Before the experiment shown, the cell was stimulated at 1 Hz under current-clamp conditions, and the steady-state action potential was recorded. Recording conditions were then switched from current clamp to voltage clamp. The control action potential was used as the voltage-clamp command waveform. This was repetitively applied to the cell at a rate of 1 Hz. Application of ACh under these conditions, of course, did not result in a shortening of the action potential, and the decrease in contraction (16%) was much reduced compared with that observed under current-clamp conditions (cf. Fig. 1, A and B). Figure 2 shows that in five cells, 1 µM ACh maximally decreased contraction by 7 ± 3% under action potential-clamp conditions (Fig. 2, bar iii), whereas under current-clamp conditions it decreased contraction by 75 ± 4% (Fig. 2, bar i; P < 0.001, paired t-test). This much reduced the inotropic effect of ACh under action potential-clamp conditions suggests that the inotropic effect under current-clamp conditions (Fig. 1A) is primarily the result of the action potential shortening caused by ACh. Notice that the small effect of ACh on contraction under action potential-clamp conditions faded with time as a result of desensitization (Fig. 1B).
The changes in the membrane current during the experiment in Fig. 1B are shown in Fig. 3. The voltage-clamp command waveform (the control action potential) is again shown in Fig. 3A. As expected, under control conditions, little or no current was recorded during the action potential command waveform (Fig. 3B). On application of 1 µM ACh, an outward current was activated during the action potential. Four seconds after the start of the application of ACh, the current was ~2 nA in amplitude in this example, but 60 s after the start, the current had declined to ~1 nA as a result of desensitization (Fig. 3B). The ACh-activated outward current explains the ACh-induced shortening of the action potential under current-clamp conditions. The current-voltage relationship of the ACh-activated outward current was obtained by plotting the current against the membrane potential during the action potential command waveform. Figure 3C shows current-voltage relationships 4 and 60 s after the start of the ACh application. The current-voltage relationships are those expected of an inward-rectifying K+ current such as IK,ACh (see DISCUSSION). Similar data were obtained from 14 cells.
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Simulation of the effect of ACh.
If the inotropic effect of ACh is primarily the result of the action
potential shortening, then it should be possible to simulate the
inotropic effect of ACh by using the shortened action potential recorded in the presence of ACh as the voltage-clamp command waveform. Figures 1 and 4 were obtained from the
same cell. Before the experiment shown in Fig. 4, the experiment in
Fig. 1A was carried out: the cell was stimulated at 1 Hz
under current-clamp conditions, 1 µM ACh was applied, and the
shortest action potential in the presence of ACh was recorded.
ACh was washed off, and the preparation was allowed to return to steady
state. The experiment in Fig. 4 was then started. Recording conditions
were switched from current clamp to voltage clamp, and the short action
potential recorded previously in the presence of ACh was used as the
voltage-clamp command waveform and repetitively applied to the cell at
1 Hz. Despite the fact that ACh was absent, there was a decrease in contraction comparable to that observed on application of ACh under
current-clamp conditions. In five cells, application of the short ACh
action potential command waveform resulted in a 74 ± 3% decrease
in contraction (Fig. 2, bar ii), which is not significantly
different (P = 0.526, paired t-test) from
the decrease in contraction (75 ± 4%; Fig. 2, bar i)
when ACh was applied under current-clamp conditions. This confirms that
the inotropic effect of ACh is primarily the result of the action
potential shortening rather than another action such as a decrease of
ICa,L. However, the inotropic effect of the ACh
action potential command waveform was not identical to that of
ACh; during an exposure to ACh under current-clamp conditions, the
inotropic effect faded as a result of desensitization (Fig.
1A), whereas during the application of the ACh action
potential command waveform, the inotropic effect did not fade (Fig. 4).
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Activation of IK,1 by ACh.
The shortening of the action potential in the presence of ACh is
attributable to the ACh-activated outward current shown in Fig.
3B. The current-voltage relationship of this current
suggests that it is an IK,1. To test this
possibility, we investigated the effects of Ba2+, a potent
blocker of inward-rectifying K+ channels. First, the effect
of Ba2+ alone was investigated. Figure
7A shows the effect of 2 mM
Ba2+ on a cell under current-clamp conditions. Under
control conditions, a stimulus resulted in a normal action potential.
After the application of Ba2+, there was a large
depolarization. The cell was no longer excitable, and instead there
were small spontaneous oscillations of the membrane potential
(accompanied by contractions; not shown). Figure 7B shows a
control action potential command waveform that was repetitively applied
to the same cell at a rate of 1 Hz under voltage-clamp control.
Application of Ba2+ under these conditions, of course, did
not result in a depolarization of the resting membrane. Instead it
resulted in the loss of an outward current. The
Ba2+-sensitive outward current was obtained by subtracting
the current after the application of Ba2+ from the current
before the application of Ba2+ and is shown in Fig.
7B, bottom. The current-voltage relationship of
this current was obtained by plotting the current against the membrane
potential during the control action potential command waveform and is
shown in Fig. 7C. The current displays inward rectification
more pronounced than that of the ACh-activated current (Fig.
3C). The current is a combination of all
Ba2+-sensitive currents but is likely to be primarily the
background IK,1. Similar results were obtained
from five cells.
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Inhibition of IK,1 by ACh.
To confirm that the ACh-activated outward current is a
K+ current, we attempted to measure its reversal potential.
This was not possible using the action potential-clamp technique,
because the membrane potential never exceeds the K+
equilibrium potential (EK) during the action
potential. Instead, we used a ramp-clamp protocol that ramped the
membrane potential from +33 to 120 mV (below
EK) over 200 ms (roughly similar to the duration
of the action potential) at a rate of 1 Hz (holding potential, 80
mV). Current-voltage relationships were obtained by plotting membrane
current against the membrane potential during the ramp clamp. Figure
9A shows the current-voltage
relationship of total membrane current before and early and late during
an application of ACh. ACh had the expected effect on membrane current at potentials more positive than EK; it caused
an outward shift in current that faded with time as a result of
desensitization as in the previous series of action potential-clamp
experiments. ACh also had little effect on current near
EK. However, at potentials more negative than
EK, early during the application of ACh there was an inward shift in current, but later during the application an
outward shift in current occurred. The two different effects of ACh can
be more clearly seen in Fig. 9B, which shows current-voltage relationships of the ACh-dependent current. The ACh-dependent current
was calculated by subtracting the current under control conditions from
the current in the presence of ACh. Early during the application of
ACh, the current-voltage relationship of the ACh-dependent current is
characteristic of an inward-rectifying K+ current such as
IK,1, and the current reversed close to the
suspected value of EK (see
DISCUSSION). However, later during the application of ACh
the current-voltage relationship was greatly altered, and the
ACh-dependent current was outward at potentials both more negative and
more positive than EK.
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Computer model of the activation of IK,ACh and
inhibition of IK,1 by ACh.
The consequences of the simultaneous activation of
IK,ACh and inhibition of
IK,1 by ACh were investigated in a modified
Oxsoft HEART model of the ventricular action potential (see
MATERIALS AND METHODS for details). Figure
10A shows calculated
current-voltage relationships for IK,ACh, and
Fig. 10B shows calculated current-voltage relationships for
IK,1; on application of ACh, it was assumed that
IK,ACh was activated (maximum current at
positive potentials, 1 nA) and IK,1 was
inhibited by 75%. Figure 10C shows calculated current-voltage relationships for IK,ACh plus
IK,1. These are assumed to be roughly equivalent
to the current-voltage relationships in Fig. 9, A and
C. As observed experimentally, on application of ACh, the
current was shifted in the outwards direction at potentials both
positive and negative to EK. The current-voltage
relationship of the ACh-dependent current had the characteristic
U-shape (cf. Figs. 10D and 9, B and
D). The reason why the activation of
IK,ACh dominates at potentials positive to
EK and the inhibition of
IK,1 dominates at potentials negative to
EK is further considered in the
DISCUSSION.
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1.5 nA stimulus was sufficient to trigger an
action potential (Fig. 10E, trace i), after
application of 1 µM ACh the stimulus was no longer sufficient and a
stimulus-evoked depolarization (Fig. 10E, trace
ii) only was seen. When the stimulus was increased to 1.8 nA, an
action potential (shortened compared with the control) was again
triggered (Fig. 10E, trace iii). This observation, which was
made in other cells, demonstrates that in the presence of ACh there was
a decrease in excitability. Figure 10F shows simulated
action potentials from the modified Oxsoft HEART model. In Fig.
10F, a 1.55 nA stimulus was used to trigger a control
action potential (Fig. 10F, trace i). After activation of
IK,ACh (maximum current, 1 nA), the stimulus was
no longer suprathreshold and a stimulus-evoked depolarization (Fig.
10F, trace ii) only was seen. This demonstrates that, in the
model at least, it is the activation of IK,ACh
that depresses excitability (in the model, the stimulus threshold was
increased from 1.15 to 1.80 nA after activation of
IK,ACh). After the activation of
IK,ACh, if the stimulus was increased to 1.80
nA, an action potential was again triggered, but it was short (not shown). Figure 10F, trace iii, shows that when
IK,1 was decreased by 75%
(IK,ACh was still activated), the 1.55 nA
stimulus was again able to trigger an action potential. Thus the
simultaneous inhibition of IK,1 was able to
restore excitability (the stimulus threshold fell to 1.55 nA). In
conclusion, it is proposed that the activation of
IK,ACh depresses excitability of ventricular cells. This is presumably undesirable. It is suggested that the effect
of ACh on excitability is blunted as a result of the twin actions of
ACh on IK,ACh and IK,1.
By the way, in the model the activation of
IK,ACh resulted in the shortening of the action potential (Fig. 10F), and the simultaneous inhibition of
IK,1 had little antagonistic effect on the
shortening. This proposal is considered further in the
DISCUSSION.
Adenosine.
In addition to activation by ACh, IK,ACh is
known to be activated by other agonists, including adenosine
(4). Adenosine has a negative inotropic effect on the
ferret ventricle (16). To test whether the conclusions of
the present study are restricted to ACh, we investigated the inotropic
effect of 100 µM adenosine using the action potential-clamp
technique. Figure 11 shows three experiments carried out on the same ferret ventricular cell. Figure 11A shows the typical effect of 100 µM adenosine under
current-clamp conditions. On application of adenosine there was a
shortening of the action potential accompanied by a decrease in
contraction, which can be seen in both the slow and fast time base
records. In eight cells, 100 µM adenosine caused a maximal reduction
of 42 ± 4% in contraction under current-clamp conditions (Fig.
2, bar iv). Like that of ACh, the effect of adenosine faded
with time (Fig. 11A). Figure 11B shows an
experiment in which a control action potential, recorded under
current-clamp conditions, was used as the voltage-clamp command
waveform. In this case, application of adenosine had no observable
effect on contraction. In four cells under action potential-clamp
conditions, adenosine caused a 5 ± 4% reduction in contraction
(Fig. 2, bar vi). The current-voltage relationship of the adenosine-activated current was the same shape as
the ACh-activated current (Fig. 3C) and as expected of
IK,ACh (not shown). The negative inotropic
effect of adenosine was simulated in the same way as before for ACh
(Fig. 4). Figure 11C shows that switching from current-clamp
to voltage-clamp mode, using the shortest action potential under
current-clamp conditions in the presence of adenosine (Fig.
11A) as the voltage-clamp command waveform, caused a
decrease in contraction comparable to the decrease in Fig.
11A (once again there was no fade in the effect). In five cells, application of the shortened action potential command waveform caused a 45 ± 5% reduction in contraction (Fig. 2,
bar v).
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Detection of Kir3.1 mRNA in ventricle.
The electrophysiological experiments above suggest that the inotropic
effect of ACh (and adenosine) on ferret ventricular cells is
principally the result of the activation of
IK,ACh. The muscarinic K+ channel is
known to be a heteromultimer of Kir 3.1 and Kir 3.4. Northern blot
analysis of Kir3.1 mRNA expression in the rat and guinea pig heart has
previously shown expression of Kir3.1 in the atria in the two species,
but not in the ventricles (26). Because of this conflict,
we have reexamined the issue of whether there is expression of Kir3.1
in the ferret ventricle. Total RNA from the ferret atrium, ferret
ventricle, and dog ventricle was purified to yield mRNA from which cDNA
was generated by the RACE technique. With use of Kir3.1-specific
oligonucleotide primers (see MATERIALS AND METHODS), the
Kir3.1 sequence was amplified by PCR. Figure
12E shows PCR products
subjected to agarose gel electrophoresis and shows that Kir3.1 was
detected in the ferret ventricle as well as the atrium. Kir3.1 was also
detected in the dog ventricle (Fig. 12E). We have previously
obtained electrophysiological evidence of the presence of
IK,ACh in the dog ventricle (39). In the case of the ferret ventricle, the PCR product was cloned into
pCR2.1 vector, and the sequence was determined to confirm that it was
Kir3.1.
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Detection of Kir3.1 and Kir3.4 proteins in ventricular and atrial cells. The muscarinic K+ channel protein Kir3.1 was not detected by immunocytochemistry in tissue sections through the ferret ventricle (Kir3.4 not tested). Immunocytochemistry is more sensitive when applied to isolated cells compared with tissue sections, because in isolated cells there is better access of the antibodies to the antigenic sites. Figure 12, A and B, shows ventricular cells labeled for Kir3.1 or Kir3.4. Both Kir3.1 (Fig. 12A) and Kir3.4 (Fig. 12B) labeling was observed (although it was weak) and was absent from the outer cell membrane and was present in the t tubules. Similar results were obtained from six Kir3.1 and seven Kir3.4 ventricular cells. Kir3.1 was detected in tissue sections through the ferret atrium. Figure 12, C and D, shows atrial cells labeled for Kir3.1 or Kir3.4. Both Kir3.1 (Fig. 12C) and Kir3.4 (Fig. 12D) labeling were strong and present in the outer cell membrane (including the intercalated disks). Similar results were obtained from 10 Kir3.1 and 10 Kir3.4 atrial cells. In summary, the immunocytochemical data show low expression of both Kir3.1 and Kir3.4 proteins in the ventricle and high expression of both Kir3.1 and Kir3.4 proteins in the atrium.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This study has proven that the negative inotropic effects of ACh and adenosine are the result of the action potential shortening in the presence of the agonists. It has also suggested that ACh activates IK,ACh and inhibits INaCa and IK,1. The first two effects cause the action potential shortening, whereas the third may preserve cell excitability.
Presence of Kir3.1 in the ventricle.
This study and previous studies (18, 24, 31, 39) have
provided electrophysiological evidence of IK,ACh
in ferret, rat, human, cat, guinea pig, and dog ventricular cells.
Previously, a Northern blot by Kubo et al. (26) failed to
show the presence of Kir3.1 mRNA in the rat ventricle. However, the PCR
technique is a more sensitive test because it amplifies cDNA derived
from low-abundance mRNAs. Using the PCR technique, we were able to show
the presence of Kir3.1 mRNA in the ferret and dog ventricle (Fig.
12E). However, the presence of Kir3.1 mRNA in the ferret ventricle does not prove that the Kir3.1 protein is present in ferret
ventricular cells and for this reason we examined Kir3.1 and Kir3.4
protein expression in ferret ventricular cells using immunocytochemistry. Labeling of Kir3.1 and Kir3.4 was detected in the
t tubules of ferret ventricular cells (Fig. 12). It is interesting that
other channels (
1C, Task-1, Kv4.2, Kir2.1) are also
found in the t tubules of ventricular cells (11, 21, 32,
38).
Agonist-induced changes in membrane current. ACh activated an outward current during a control action potential command waveform (Fig. 3B). This current may be IK,ACh for several reasons. First, it was activated by ACh. Second, the ACh-activated current displayed a typical current-voltage relationship of IK,ACh- (Figs. 3C and 8B). IK,ACh shows inward rectification, but the degree of rectification of IK,ACh is less than that of, for example, IK,1 (34, 41); this is illustrated by the calculated data in Fig. 10, A and B. The degree of rectification of the ACh-activated current (Figs. 3C and 8B) is typical of that of IK,ACh. Third, the ACh-activated current was blocked by 2 mM Ba2+ (Fig. 8). Ba2+ at 2 mM is known to block IK,ACh fully (40). Fourth, during an application of ACh, the ACh-activated outward current faded with time (Fig. 3B). Fade during an exposure to ACh is a characteristic feature of IK,ACh and is the result of desensitization to ACh (e.g., Ref. 41). Finally, the reversal potential of the ACh-activated current was close to EK. Using the ramp clamp, in one cell at least, we were able to observe reversal of the ACh-activated current close to the suspected value of EK early during an application of ACh (Fig. 9B).
In all cells studied, ACh eventually inhibited inward current at potentials negative to EK (Fig. 9). ACh has a similar action in dog left ventricular subepicardial cells (39). This may be the result of inhibition of IK,1. In support of this, the ACh-induced change in current was abolished in the presence of 2 mM Ba2+ (Fig. 9D); IK,1 is known to be abolished under these conditions (9). The voltage dependence of the ACh-induced changes in membrane current (Fig. 9, B and D) can be explained by a dual effect of ACh on IK,ACh and IK,1. At approximately80 mV, there was little change in membrane current;
this is expected, because this potential is close to
EK and at this potential both
IK,ACh and IK,1 are small
and, furthermore, it is possible that at potentials near EK any effects of ACh on
IK,ACh and IK,1 are equal
and opposite. At potentials positive to IK, the
results indicate that activation of IK,ACh was
more important than inhibition of IK,1. The
likely reason for this is shown by Fig. 10,
A-D. IK,1 shows
greater inward rectification than IK,ACh over
this range of potentials. For example, at plateau potentials at ~0
mV, IK,1 may be negligible, whereas IK,ACh, although it rectifies at more positive
potentials, does not decline below a maximum value (34,
40). Therefore, at the more positive potentials a decrease in
IK,1 will have little or no effect on total
current, whereas the activation of IK,ACh will.
At potentials negative to EK, the results
indicate that the activation of IK,ACh was less
important than the inhibition of IK,1, and the
likely reason for this result is again shown by Fig. 10,
A-D. At these potentials,
IK,1 steeply increases with hyperpolarization,
and Fig. 10, A-D, suggests that it is
greater in amplitude than IK,ACh. Therefore, the
change in total current may be dominated by the decrease in
IK,1 at these potentials. Figure 10,
E and F, suggests a possible reason for the
simultaneous inhibition of IK,1 by ACh: it may
blunt the depression of excitability caused by the activation of
IK,ACh. Excitability will be determined by the
slope conductance at the resting potential (close to
EK). Figure 10A shows that the
activation of IK,ACh alone will result in a
large increase in the slope conductance and thus decrease in
excitability. When the slope of the current-voltage relationship of the
control IK,1 at IK (this
will largely determine the slope conductance under control conditions)
is compared with that of IK,ACh (after
activation of IK,ACh), the two are comparable,
which shows that activation of IK,ACh alone will
result in a large fractional increase in the slope conductance at the
resting potential. The central panel of Fig. 10F confirms
that in the model, at least, activation of
IK,ACh does result in a decrease in
excitability. On the other hand, Fig. 10C shows that the
simultaneous activation of IK,ACh and inhibition
of IK,1 results in little change in the slope
conductance near EK. In this way, the
simultaneous inhibition of IK,1 blunts the
depression of excitability; in the model, this is confirmed by Fig.
10F.
When a short ACh action potential command waveform was applied, an
inward current was recorded (Fig. 5). During an application of ACh, the
ACh-activated outward current in Fig. 3B must be able to
overcome this current to be able to shorten the action potential (the
two currents are expected to be roughly equal and opposite). Strict
comparison of the currents in Figs. 3C and 5C is
not possible, because they were recorded from different cells and
during different action potential command waveforms, but the two
currents are roughly equivalent in amplitude. It is well known that
after a short square wave voltage-clamp pulse, an inward tail current
is recorded (e.g., Ref. 15). The inward tail current is
analogous to the current in Fig. 5B. It is also well known
that a substantial fraction of the inward tail current after a square
wave voltage-clamp pulse is inward INaCa
triggered by the Ca2+ transient. Figure 6 shows that this
is also likely to be true for the inward current recorded during the
short ACh action potential command waveform: after the suppression of
the Ca2+ transient by BAPTA-AM, the inward tail current was
greatly diminished. Figures 5B and 6A show that
during repetitive application of the ACh action potential command
waveform, the inward current diminished and Fig. 6B suggests
that it was the result of the expected reduction in the underlying
Ca2+ transient.
Figures 5C and 6A show that during repetitive
application of the ACh action potential command waveform, there was a
reduction in a transient outward current. Figure 6B suggests
that this too was a consequence of the expected reduction in the
Ca2+ transient. Janvier et al. (20) showed
that the suppression of the Ca2+ transient in ferret
ventricular cells by BAPTA-AM resulted in the loss of a transient
outward current as well as a transient inward current. Whereas the
transient inward current exhibited the properties of inward
INaCa triggered by the Ca2+
transient, the transient outward current exhibited the properties of
Ca2+-activated Cl current triggered by the
Ca2+ transient. The time course and amplitude of the
transient outward current in the study of Janvier et al.
(20) is very similar to those of the transient outward
current in the present study (Figs. 5C and 6A).
It is suggested that in the present study during the application of the
ACh action potential command waveform, a reduction in the
Ca2+ transient resulted in a reduction in the
Ca2+-activated Cl current.
Figure 7 shows a Ba2+-sensitive current during the action
potential. Inward rectifying K+ currents are most sensitive
to Ba2+ and, therefore, the Ba2+-sensitive
current is likely to be IK,1. Furthermore, the
current-voltage relationship of the Ba2+-sensitive current
shows the marked inward rectification characteristic of
IK,1 (Fig. 7C) (34).
Agonist-induced changes in the action potential.
The shortening of the ferret ventricular action potential in the
presence of ACh and adenosine is likely to be primarily the result of
the activation of IK,ACh. The current-voltage
relationship of IK,ACh (Figs. 3C,
8B, and 9) indicates that over the range of potentials
during the action potential plateau, a significant outward current
flows that will tend to shorten the action potential. The fade of the
action potential shortening in the presence of both agonists (Figs.
1A and 11A) can be explained by
IK,ACh, because IK,ACh
faded with time during application of both agonists (Fig. 3 and
unpublished observation). It is likely that the density of the
muscarinic K+ channel is less in the ventricle than the
atrium, and it is interesting that despite this, the effects on the
ventricular action potential are still substantial. A reduction in
ICa,L may also explain some of the action
potential shortening, although in dog ventricular cells 10 µM ACh
only caused an 8% reduction in ICa,L at a
holding potential of 80 mV (39). The results in
Figs. 5 and 6 suggest that on application of ACh the reduction of the
Ca2+ transient underlying the reduction of the contraction
leads to an indirect reduction in inward INaCa,
and this is also expected to contribute to the shortening of the action
potential. It is already known that suppression of inward
INaCa by buffering intracellular Ca2+ by BAPTA-AM leads to a shortened action potential
(20) (see also the control action potential and the action
potential after the application of BAPTA-AM in Fig. 6). The inhibition
of IK,1 by ACh may be expected to antagonize the
shortening of the action potential caused by ACh. However, in the
model, the simultaneous inhibition of IK,1 had
little effect on the shortening of the action potential caused by
IK,ACh (Fig. 10F). This is because at plateau potentials there is little or no IK,1
(Fig. 10B).
Agonist-induced changes in contraction. In ferret ventricular cells, the negative inotropic effect of ACh and adenosine is likely to be primarily the result of the shortening of the action potential. With the use a control action potential waveform as the voltage-clamp command, the negative inotropic effect of ACh and adenosine was almost abolished (Fig. 2). Furthermore, use of the agonist-shortened action potential as the voltage-clamp command waveform caused a reduction in contraction comparable to that observed on application of the agonist under current-clamp conditions (Fig. 2). With both agonists there could be a small reduction in contraction when the cell was under action potential-clamp control (Fig. 2). This may be the result of a small reduction in ICa,L by the agonists. The agonist-induced shortening of the action potential is expected to lead to a reduction in the Ca2+ transient and thus the contraction by reducing the Ca2+ content of the sarcoplasmic reticulum by: 1) promoting Ca2+ efflux on the Na/Ca exchanger and 2) reducing Ca2+ influx via ICa,L (because of the shortened action potential, ICa,L flows for a shorter time). Direct evidence of the stimulation of Ca2+ efflux via the Na/Ca exchanger is the inward current measured on application of the ACh action potential command waveform (Figs. 5B and 6A). The evidence that this is partly composed of inward INaCa (corresponding to Ca2+ efflux) has already been discussed.
During an application of ACh or adenosine, the slow increase in contraction under current-clamp conditions (Figs. 1A and 11A) can be explained by fade of the action potential shortening (primarily the consequence of the fade of IK,ACh as a result of desensitization) or a fade of a decrease in ICa,L (39). The slow increase in contraction during an application of ACh has also been suggested to be the result of a slow increase in intracellular Na+ (23). During action potential clamp the small inotropic effect faded with time (Fig. 1B), and this at least could not have been the result of a change in action potential duration. In summary, it is concluded that the negative inotropic effects of ACh and adenosine are primarily the result of the activation of IK,ACh and the consequent shortening of the action potential. The reduction in the intracellular Ca2+ transient (underlying the decrease in the contraction) causes a reduction in inward INaCa, and this also contributes to the action potential shortening. A small reduction in ICa,L may also play a role in the negative inotropic effect. ACh inhibits IK,1 and this may blunt a reduction in cell excitability following activation of IK,ACh. Although the present study was carried out in the ferret, the results are unlikely to be specific to the ferret, because, in ventricular cells from a range of species, IK,ACh has been recorded and ACh shown to have a negative inotropic effect in the absence of-stimulation (5,
18, 24, 30, 31, 39).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Luke Blumler, Andy O'Brien, and Dave Johannson for excellent technical assistance.
| |
FOOTNOTES |
|---|
This work was supported by grants from the British Heart Foundation and the Wellcome Trust. N. C. Janvier was a Wellcome Prize student.
Address for reprint requests and other correspondence: M. R. Boyett, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9JT, UK (E-mail: m.r.boyett{at}leeds.ac.uk).
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. Section 1734 solely to indicate this fact.
April 25, 2002;10.1152/ajpheart.00130.2002
Received 21 February 2002; accepted in final form 12 April 2002.
| |
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) in
current between the 1st and ss ACh AP command waveforms. Difference
current was calculated by subtracting the current at ss from the
current during the 1st ACh AP command waveform.
