(Circulation. 1997;95:1937-1944.)
© 1997 American Heart Association, Inc.
Articles |
Unanticipated Lessening of the Rise in Extracellular Potassium During Ischemia by Pinacidil
From the Experimental Cardiology Group, Cardiology Division, Department of Medicine (A.K., I.W., M.L.W., C.L.E., L.S.G.), and the Department of Biostatistics, School of Public Health (S.L., G.C.K.), University of North Carolina at Chapel Hill.
Correspondence to Leonard S. Gettes, MD, UNC School of Medicine, CB #7075, 349 Burnett-Womack Bldg, Chapel Hill, NC 27599-7075. E-mail lsgettes{at}vmax.card.unc.edu
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Abstract |
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Background The efflux of potassium (K) through the ATP- sensitive K channel is considered an important cause of the rise in extracellular K ([K+]e ) during no-flow ischemia. We postulated that agents that enhance K conductance in this channel would enhance the rise in [K+]e.
Methods and Results We studied the effects of 10 and 25 µmol/L pinacidil, an ATP-sensitive K channel opener that provides metabolic protection to the ischemic myocardium, on the rise in [K+]e recorded by K-sensitive electrodes, the change in action potential duration (APD) recorded by microelectrodes, and the changes in activation during ischemia in in situ pig hearts and Tyrode-perfused rabbit interventricular septa. Pinacidil 25 µmol/L unexpectedly lessened the rise in [K+]e and the activation delay in both preparations. Pinacidil 10 µmol/L had no effect in the rabbit and only a slight effect in the pig. Both concentrations significantly exaggerated the APD shortening induced by ischemia. By varying stimulation frequency, we demonstrated that the rise in [K+]e during ischemia, both before and after pinacidil, correlated with the time that the action potential was at its plateau voltage.
Conclusions Our results indicate that the rise in [K+]e during ischemia is due to multiple factors, including K conductance across membrane channels, K driving force as reflected by the time that the action potential is at its plateau voltage, and the metabolic effects of ischemia. The unanticipated lessening of the rise in [K+]e by pinacidil reflects the balance of its effects on these several parameters.
Key Words: potassium • pinacidil • ischemia • action potentials • electrophysiology
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Introduction |
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The rise in extracellular potassium ([K+]e) during acute ischemia1 2 3 is a major cause of the electrophysiological changes and ventricular arrhythmias that occur in this setting.4 5 The precise cause of this rise has not been well defined, although both anionic- and nonanionic-linked factors are believed to be important.6 7 8 Studies using the sulfonylureas glibenclamide and tolbutamide suggest that K+ efflux via the ATP-sensitive K+ channel8 9 10 11 may be one such factor. Thus, agents that enhance K+ conductance via this channel might be expected to enhance the rise in [K+]e during ischemia. To test this possibility, we studied the effects of 10 and 25 µmol/L pinacidil, a drug known to open ATP-sensitive K+ channels,12 on the rise in [K+]e and the associated changes in action potential duration (APD) and activation induced by acute no-flow ischemia in the in situ pig heart and the perfused rabbit interventricular septum. Pinacidil unexpectedly caused a decrease in the rise in [K+]e. However, analysis of the factors contributing to this unanticipated result provides insight into the causes of the ischemia-induced rise of [K+]e.
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Methods |
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The care of animals used in this study conformed to the Position of the American Heart Association on Research Animal Use and was done in accordance with accepted guidelines for the care and treatment of experimental animals at the University of North Carolina.
In Vivo Pig Experiments
Domestic swine of either sex weighing 30 to 50 kg were
anesthetized with thiopental sodium (25 mg/kg) followed by
-chloralose as needed. Mechanical ventilation and supplemental
oxygen were supplied via an endotracheal tube and a Harvard respirator.
Arterial blood gas samples were monitored and appropriate ventilator
adjustments made to maintain arterial PO2
>80 mm Hg and pH at 7.35 to 7.45. Heating blankets were used to
maintain body temperature at 36° to 37°C.
The heart was exposed via a median sternotomy and suspended in a pericardial cradle. A site midway in the left anterior descending coronary artery (LAD) was dissected and briefly occluded to define the epicardial margin between ischemic and nonischemic tissue. Multiple ion-selective electrode groups were placed in the center of the ischemic zone and in the normal (nonischemic) zone, and in some experiments, pairs of piezoultrasonic crystals were placed in the mid-myocardial wall as previously described3 to record changes in segment length with each beat. After electrode placement, systemic heparin (10 000 U followed by 2000 U/h) was administered, and a carotid-to-LAD shunt was created as previously described.3 13 Perfusion of the distal LAD was maintained at 1.2 mL/kg body wt per minute, which provides 1.2 to 1.5 mL of blood per gram of heart tissue per minute.3 13 A sidearm in the shunt was used for infusion of pinacidil. Flow through this sidearm was controlled by a second roller pump.
The heart rate was maintained between 100 and 120 bpm by atrial pacing in the experiments using a single rate. In those designed to determine the effects of increasing rates, rates of 100 and 150 bpm were used. Arterial blood pressure and a lead II ECG were continuously monitored on a 12-channel strip-chart recorder (WR3101, Western Graphtec Inc).
Ion-Selective Electrodes
K+- and pH-sensitive electrodes were constructed and
calibrated as previously described.14 Only electrodes
demonstrating a stable baseline (drift <1 mV/h) and 95% to 105% of
the predicted nernstian slope (56 to 62 mV shift per decade change in
K+ activity or pH at room temperature) were used. Pairs of
Teflon-coated silver wires, used to record bipolar electrograms, were
placed with one or two K+ or pH electrodes and a reference
electrode in a 19-gauge needle and inserted into the mid-myocardium.
The needle was then withdrawn, leaving the electrode group embedded in
the myocardium. As many as six such groups were placed at various
locations within the ischemic zone, and one or two were placed
outside of the ischemic zone. After electrode insertion, the in
vivo performance of the K+ electrodes was tested as
previously described.14 At the end of each experiment, the
electrodes were removed from the hearts and recalibrated.
Microelectrodes
Transmembrane action potentials were recorded by
"floating" 3 mol/L KCl–filled microelectrodes fabricated as
previously described,15 using the tips of standard
microelectrodes mounted on a tungsten wire 0.002 mm in diameter
connected to a high-input resistance buffer amplifier. The reference
electrode consisted of a silver/silver chloride wire placed as close as
possible to the recording site. The action potentials were continuously
monitored on an oscilloscope and recorded on the strip-chart recorder
at a paper speed of 50 mm/s.
Experimental Protocol
No intervention was performed for 50 minutes after electrode
placement. The first in a series of 8-minute episodes of myocardial
ischemia was induced by the abrupt cessation of flow through
the LAD shunt. Each episode was separated by 50 minutes of reperfusion.
We first performed two control ischemic periods because our
earlier studies showed that the rise in [K+]e
and the changes in activation in the first ischemic episode
differed from those occurring during subsequent ischemic
episodes.16 We then infused a solution of 500
µmol/L pinacidil into the sidearm of the shunt at a rate calculated
to produce a drug concentration of 10 or 25 µmol/L in the blood
supplying the LAD. The infusion of pinacidil was initiated after 30
minutes of reperfusion and maintained for 20 minutes, at which time
coronary flow was discontinued for 8 minutes. After the pinacidil
ischemic period, the LAD was reperfused with pinacidil-free
blood for 20 minutes and a post–pinacidil ischemic period was
performed.
Data Collection and Analysis
The signals from all electrodes were individually amplified by
high-impedance amplifiers, digitized (Phoenix Data analog-to-digital
converter), and simultaneously sampled (1000 samples per second) every
60 seconds during the pinacidil infusion and every 15 seconds during
ischemia by a Micro Vax II/GPX computer (DEC). Values of
[K+]e and pH were calculated from measured
millivolt changes using the calibration curve of each electrode, the
systemic [K+]e or pH determined from an
arterial blood sample obtained immediately before the event and, for
the K+ electrode, an activity coefficient of
.746.17 APD was determined at 50% and 90% repolarization
(APD50, APD90). APD50 served as a
measure of the action potential plateau or phase 2, and the difference
between APD90 and APD50
(APD90-APD50) served as a measure of phase 3.
Local activation delay was determined from the high-frequency
deflection of the bipolar electrogram filtered between 50 and 500 Hz
and referenced to an electrode in the nonischemic zone. Local
activation delay was determined only at sites where data from the
K+ electrodes were acceptable. The number of acceptable
K+ and pH electrodes in each experiment ranged from two to
four. Mean values for [K+]e, pH, and
activation delay were determined for each experiment and then used to
calculate a mean for the experimental group as a whole.
In Vitro Rabbit Experiments
Preparations of the arterially perfused rabbit interventricular
septum were adapted from previously reported
studies.2 18 19 New Zealand White rabbits (weight, 2 to 3
kg) of either sex were heparinized (200 U/kg IV) and anesthetized with
sodium thiopental (70 mg/kg IV). The heart was rapidly excised and
placed in cold (4°C) Tyrode solution. The atrial and left ventricular
free wall were removed and the ventricular septum was attached, left
side down, to a wax platform containing a large silver-silver chloride
grounded electrode. The septal branch of the left coronary artery was
cannulated and perfused with solution containing the following
(mmol/L): Na+ 149, K+ 4.5, Mg2+
0.49, Ca2+ 1.8, Cl- 133,
HCO3- 25, HPO42- 0.4,
glucose 20, albumin 2 g/L, dextran (MW, 70 000) 40 g/L, insulin 1 U/L,
heparin 400 U/L. The total ischemic time before starting
perfusion was <5 minutes. The perfused preparation was placed in a
recording chamber having a temperature of 36° to 37°C and
surrounded by a humidified atmosphere consisting of 95%
O2–5% CO2. Perfusion pressure, monitored by a
transducer (Millar Instruments Inc), was maintained between 40 and
50 mm Hg by adjustment of the perfusion flow rate (0.8 to 1
mL-1·min-1 · g-1)
via a roller pump (Digistaltic Cole, Parmer Instrument Co). The
preparation was stimulated by 2-ms stimuli of twice diastolic threshold
strength from a bipolar stainless steel electrode. The stimulation
frequency was 2 Hz in experiments using only a single rate and 1 Hz and
3 Hz in those designed to study the effects of increasing rate. The
partial pressures of O2 and CO2 in the
perfusate were controlled with a membrane gas exchanger and
continuously monitored by a pH glass electrode (Fisher Scientific)
connected to the perfusion line. The relative amounts of
O2, CO2, and N2 were adjusted by
individual gas flowmeters (Cole Palmer Instrument Co) to yield a
PO2 of 470±7.2 mm Hg (mean±SEM) and a
pH of 7.35 to 7.45. The PO2,
PCO2, and [HCO3-] in
the perfusate were measured with a blood gas analyzer (system 1304
pH·Blood Gas Analyzer Instrumentation Laboratory, Lexington,
Mass).
Microelectrodes and K+-Sensitive Electrodes
Transmembrane action potentials were measured with a 3 mol/L
KCl–filled floating glass microelectrode referenced to a closely
spaced extracellular electrode as described above. The action
potentials were continuously monitored on an oscilloscope (Tektronix)
and recorded on a Graphtec strip-chart recorder (WR3310, Western
Graphtec Inc) at a paper speed of 250 mm/s. The
K+-sensitive electrodes were fabricated using a
polyethylene tube filled with 150 mmol/L KCl and coated with a
polyvinylchloride-valinomycin–based membrane.19 In vitro
and in situ calibration procedures were as described above. The tip of
the K+-sensitive electrode was positioned on the surface of
the myocardium as close as possible to the microelectrode. In some
experiments, peak developed tension during isometric contraction was
determined by attaching the tip of the papillary muscle to a force
transducer, as previously described.19
Experimental Protocol
In each experiment, the preparation stabilized in the chamber
for at least 50 minutes before collection of control data. No-flow
ischemia was induced by stopping perfusion and by replacing the
95% O2–5% CO2 gas mixture inside the chamber
with 95% N2–5% CO2. After 6 minutes of
ischemia, the preparation was reperfused and the normoxic
surrounding atmosphere reestablished. After 40 minutes of reperfusion
the perfusate was changed to one containing 1, 10, or 25 µmol/L
pinacidil. After 20 minutes of perfusion with the pinacidil perfusate,
a second 6-minute period of no-flow ischemia was created.
Data Collection and Analysis
Determination of [K+]e from
K+ electrodes was as described for the pig. APD was
measured at 50% and 90% repolarization. Activation delay was
determined as the interval from the stimulus artifact to the peak of
the action potential upstroke. Although more than one microelectrode
penetration was often required in each experiment, the region from
which action potentials were recorded in each experiment was very
small, and no differences in activation time were noted with different
penetrations before the onset of ischemia.
Statistical Analysis
The statistical significance of the effect of pinacidil on the
rise in [K+]e, shortening of APD, and the
activation delay associated with no-flow ischemia was assessed
for averages over time using a repeated-measures ANOVA with a two-way
model for animal and treatment. We compared minutes 2 to 8 versus time
zero for the pigs and minutes 2 to 6 versus time zero for the rabbits.
The two-way repeated-measures ANOVA for averages over time as the
primary focus enabled potential issues concerning multiple comparisons
to be addressed appropriately through global assessments. To assess the
effects of stimulation frequency on the rise in
[K+]e, the shortening of APD, or the
cumulative APD, we used the Wilcoxon signed ranks test on the averaged
paired differences between the two stimulation frequencies. All results
are expressed as mean±SEM. Statistical significance was considered at
a value of P
.05.
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Results |
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We performed 14 experiments in the pigs and 21 in the rabbit interventricular septa. Pinacidil 10 µmol/L was the only concentration tested in four pigs and 25 µmol/L the only concentration tested in six. In the remaining four animals, both concentrations were tested. In the rabbit we tested only a single concentration in each preparation. A concentration of 1 µmol/L was tested in five experiments, 10 µmol/L in seven, and 25 µmol/L in eight. Pinacidil 1 µmol/L had no effect on the ischemia-induced changes in the rise in [K+]e, the change in APD, or the change in activation.
Fig 1
illustrates the changes in [K+]e,
APD90, and activation associated with no-flow
ischemia before and after the administration of 10 and 25
µmol/L pinacidil in both experimental
models. In the pig,
[K+]e rose from 3.3±0.1 to 10.1±0.3
mmol/L after 8 minutes of ischemia before pinacidil. Pinacidil
10 µmol/L caused a slight but significant lessening in the rise
in [K+]e (P<.05). After 25
µmol/L pinacidil, the rise in [K+]e was
significantly less than control or after 10 µmol/L
(P<.04). In the rabbit, [K+]e
rose from 4.5 (the value in the perfusate) to 9.4±0.3 mmol/L
after 6 minutes of ischemia before pinacidil. Pinacidil 10
µmol/L did not significantly affect the
[K+]e rise. After 25 µmol/L, the rise
in [K+]e from 4.6±0.1 to 7.6±0.4
mmol/L was significantly less than the control (P<.01).
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Before pinacidil, APD lengthened during the first 2 minutes of
ischemia in both the rabbit and the pig (Figs 1
and 2). This was due to lengthening of phase 3 of
the action potential as reflected by an increase in the difference
between APD90 and APD50
(APD90-APD50). In the examples shown in Fig 2,
APD90-APD50 increased from 30 to 60 ms in the
pig and from 29 to 49 ms in the rabbit. Thereafter, APD shortened due
primarily to a shortening of the plateau as reflected by the shortening
of APD50 from 300 to 200 ms in the pig and from 125 to 108
ms in the rabbit.
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Pinacidil 10 µmol/L caused only minimal shortening of the APD
before ischemia. However, it prevented the initial lengthening
of APD induced by ischemia and exaggerated the subsequent
shortening (P<.01) in both preparations. In the pig
experiment shown in Fig 2, 25 µmol/L pinacidil administered for
20 minutes before the onset of no-flow ischemia shortened
APD50 from 260 to 200 ms but had no effect on
APD90-APD50. These effects were similar in the
rabbit. After ischemia, APD shortened immediately in both
species, due in large part to an effect on the plateau. In the pig,
APD50 shortened from 200 to 60 ms while phase 3
(APD90-APD50) was unchanged. In the rabbit,
the plateau of the action potential was virtually nonexistent after 6
minutes of ischemia, and APD90-APD50
shortened from 33 to 16 ms. The shortening of APD was significantly
greater after 25 µmol/L pinacidil and then after 10
µmol/L in the pig (P<.01) but not in the rabbit.
The effect of pinacidil on the activation delay induced by no-flow
ischemia paralleled its effect on the rise in
[K+]e. Before pinacidil in the pig,
activation began to change 
90 seconds after the onset of no-flow
ischemia and lengthened by a maximum of 72±5 ms. Pinacidil
10 µmol/L did not significantly influence this activation delay.
However, after 25 µmol/L pinacidil, activation did not change
until 4 minutes after the initiation of ischemia and lengthened
by only 22±4 ms. This difference was statistically significant.
Similar results were observed in the rabbit. Neither 10 nor 25
µmol/L pinacidil influenced the incidence of ventricular fibrillation
in the pigs (2 of 14 in control animals; 1 of 8 after 10 µmol/L
pinacidil and 2 of 10 after 25 µmol/L pinacidil).
We next determined the effect of increasing stimulation frequency on
the rise in [K+]e during ischemia. We
reasoned that the accentuated shortening of the action potential
plateau during ischemia induced by pinacidil might have
lessened the time available for K+ efflux to occur. By
increasing stimulation frequency, the total time during
ischemia when the action potential would be at its plateau
level, and thus the total time for K+ efflux to occur via
the various plateau K+ channels, would increase. Fig 3
illustrates the effect of increasing driving rate from 100 to 150 bpm
in 6 pig experiments. Panels A show the rise
in [K+]e; panels B, the change in
APD50; and panels C, the cumulative time in seconds, during
which the transmembrane voltage was at the plateau level. This value
was determined by multiplying APD50 by the number of beats
in each 30-second interval throughout the ischemic period and
then summing the results.
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APD50). Data in upper panels were obtained in the
absence of pinacidil; lower panels after administration of 25
µmol/L pinacidil.Before pinacidil (top panels), the ischemia-induced rise in [K+]e was similar at both driving rates; ie, there was no rate-dependent effect (panel A). APD50 shortened from 331±31 to 193±34 ms when the rate was 100 bpm and from 234±15 to 160±24 ms when the rate was 150 bpm (panel B). There was no significant difference in the cumulative time during which the transmembrane potential was at the plateau level during ischemia at the two rates (panel C).
After pinacidil (bottom panels), the rise in [K+]e during ischemia was less at each rate than before the drug and the rise in [K+]e at 150 bpm was significantly greater than at 100 bpm (P<.05); ie, there was a rate-dependent effect. Pinacidil shortened APD before and during ischemia at both rates, but after pinacidil there was no significant difference between APD50 during ischemia at the two rates. The cumulative number of seconds during ischemia when the transmembrane voltage was at the plateau level was less at each rate than before pinacidil, and the cumulative value at 150 bpm was significantly greater than at 100 bpm (P<.05).
Fig 4 illustrates these effects in the rabbit
(n=5). Before pinacidil, the
[K+]e rise during ischemia was
significantly greater at 3 Hz than at 1 Hz (P<.05); ie,
there was a rate-dependent effect (panel A, top). APD50
shortened from 102±3 to 71±5 ms during 6 minutes of ischemia
at 1 Hz and from 99±5 to 45±5 ms at 3 Hz (P=.0625, the
minimum possible two-sided probability value with 5 animals) (panel B,
top). The cumulative number of seconds during which the transmembrane
voltage was at that plateau level during ischemia was also
consistently greater at 3 Hz (P=.0625, the minimum possible
two-sided probability value) (panel C, top).
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After pinacidil, the ischemia-induced rise in [K+]e at each rate was less than before pinacidil, but the rate-dependent effect persisted (P<.03) (panel A, bottom). Pinacidil shortened APD50 before ischemia at both frequencies. During ischemia, APD50 decreased from 65±6 to 15±4 ms at 1 Hz and from 52±9 to 8±2 ms at 3 Hz (panel B, bottom). The cumulative number of seconds during which the transmembrane voltage was at the plateau level during ischemia remained greater at 3 Hz than at 1 Hz (P=.0625).
Examination of the rabbit data reveals that the
ischemia-induced rise in [K+]e at 1
Hz before pinacidil and at 3 Hz after 25 µmol/L pinacidil were
similar (Fig 5, upper panel). The cumulative
number of seconds during which the transmembrane voltage was at the
plateau level was also similar at 1 Hz before pinacidil and at 3 Hz
after pinacidil. In the pig, examination of the data obtained during
similar conditions, ie, at a rate of 100 bpm before and 150 bpm after
25 µmol/L pinacidil (Fig 5, lower panels), reveals that both the
[K+]e rise and the cumulative number of
seconds at the plateau voltage were significantly greater
(P<.05) at the slower rate before pinacidil than at the
more rapid rate after pinacidil. The results, shown in Figs 3, 4, and 5, indicate that the total time during ischemia when the
transmembrane voltage was at the plateau level of the action potential
correlates with the rise of [K+]e. This
correlation is further illustrated in Fig 6, which shows correlation
coefficients of .6 to .9 under the various conditions explored in these
studies.
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[K+]e) to the cumulative time in seconds
(s) when the action potential was at its plateau voltage
(
The significantly greater decrease in the rise in
[K+]e after 25 µmol/L, then 10
µmol/L pinacidil, despite only a slightly greater decrease in
cumulative plateau duration, suggests that the higher concentration may
also have provided greater metabolic protection. Support for this
hypothesis is shown in Fig 7, which illustrates the recorded fall in
pHe during ischemia before and after pinacidil
(solid lines) and the fall anticipated in the second of the two
sequential pinacidil-free occlusions (broken
line). These data are derived from earlier
studies16 that showed that the decrease in pHe
decreased progressively in sequential ischemic periods although
the rise in [K+]e remained constant. The
figure illustrates that the fall in pHe after 10
µmol/L was similar to that anticipated by our earlier
studies,16 whereas the fall in pHe after
25 µmol/L was less than anticipated. This suggests that in the
in situ pig heart, 25 µmol/L pinacidil afforded greater
metabolic protection than did 10 µmol/L.
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Discussion |
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The triphasic rise in [K+]e that characterizes no-flow ischemia1 has been attributed to anionic-linked factors involving the movement of K+ ions out of the cell in association with anions generated during ischemia6 and to nonanionic factors, which include K+ efflux via voltage and ligand-gated K+ channels, ionic pumps, and transporters.5 The voltage-gated K+ channels include the channels responsible for the transient outward current (Ito), the delayed rectifying K+ current (IK), and the inward rectifying K+ current (IK1). The ligand-gated K+ channels include the ATP-sensitive K+ channel,20 the sodium-activated K+ channel,21 and the K+ channel activated by arachidonic acid.22 K+ efflux through these channels is determined by K+ conductance and K+ electrical driving force, which, in turn, is determined by the difference between the transmembrane potential and the K+ equilibrium potential (Em-Ek).
Inhibition of the ATP-sensitive K+ channel with glibenclamide and tolbutamide lessens the rise in [K+]e during acute ischemia.8 9 10 11 Thus, agents that enhance conductance of the ATP-sensitive K+ channel might reasonably be expected to accentuate the rise in [K+]e. Venkatesh et al23 found that 5 µmol/L cromakalin accelerated the shortening of the APD induced by ischemia in the perfused rabbit interventricular septum but did not significantly modify the initial rise in [K+]e. They postulated that the decreased driving force for K+ associated with the shortening of the action potential plateau balanced the increased efflux of K+ through the ATP-sensitive K+ channel. Mitani et al24 reported that in the rat ventricle, nicorandil caused an initial increase in the rise in [K+]e. However, these investigators did not measure APD, and it is possible that the shorter APD of the rat ventricle may have led to a different relationship between APD shortening and the rise in [K+]e.
The purpose of our study was to determine whether pinacidil would enhance the rise in [K+]e during acute ischemia by increasing K+ conductance or would decrease the rise in [K+]e by shortening APD and by providing metabolic protection. We used two concentrations of pinacidil (10 and 25 µmol/L) and two preparations, the in situ porcine heart and the isolated, perfused rabbit interventricular septum, to determine which if any of our observations might be concentration or species dependent. Our results demonstrate that 10 µmol/L pinacidil does not significantly alter the initial rise in [K+]e in the rabbit but that 25 µmol/L pinacidil significantly lessens the rise in [K+]e during 8 minutes of no-flow ischemia in the pig and 6 minutes of ischemia in the rabbit.
The primary effect of pinacidil in both preparations was to shorten the action potential plateau before ischemia, to prevent the lengthening of APD during the first 2 minutes of ischemia, and to accentuate the subsequent shortening of the action potential plateau. This effect on the plateau would have lessened the time for K+ efflux via the ATP-sensitive K+ channel to occur and was the mechanism proposed to explain the lack of effect by cromakalin on the rise in [K+]e.23 The importance of plateau duration on the rise in [K+]e during ischemia is suggested by the correlation of the rate-related changes in the ischemia-induced [K+]e rise to the cumulative time during which the transmembrane potential was at its plateau voltage both in the presence and absence of pinacidil.
The role of plateau duration on K+ efflux during ischemia should also apply to K+ channels other than the ATP-sensitive K+ channel and influence the rise in [K+]e, if in fact K+ efflux via these other channels contributes to the rise. Mitani and Shattock25 showed that inhibition of the sodium-activated K+ channel in the rat lessened the rise in [K+]e during ischemia, and Hicks and Cobbe26 showed that d-sotalol, a putative blocker of IK, lessened the ischemia-induced rise in [K+]e in the rabbit. Studies in our laboratory suggest that inhibition of Ito by 4-aminopyridine and IKr by E-4031 also lessens the rise in [K+]e during ischemia.27 Thus, we believe that the shortening of the plateau duration by pinacidil lessened the time during which K+ efflux occurred not only through the ATP-sensitive K+ channel but also through other voltage- and ligand-gated K+ channels.
The shortening of the plateau induced by 10 µmol/L pinacidil was only slightly less than that induced by 25 µmol/L pinacidil, suggesting that the increase in K+ conductance in the ATP-sensitive K+ channel may have been only slightly greater at the higher dose. An increase in K+ conductance would be expected to enhance the rise in [K+]e during ischemia. However, 10 µmol/L pinacidil had no effect on the ischemia-induced rise in [K+]e in the rabbit and only a slight effect in the pig, while 25 µmol/L pinacidil lessened the rise in [K+]e in both. This suggests that factors other than the increase in K+ efflux through the ATP-sensitive K+ channel and plateau duration contributed to the [K+]e rise.
Noma20 postulated that activation of the ATP-sensitive
K+ channel, with its effect on APD, would decrease the time
available for calcium to enter the cell, reduce contractility, lessen
the rate of decline of high-energy phosphates, thereby providing
metabolic protection, and lessen the onset of irreversible
ischemic injury. Such effects have been demonstrated in the
arterially perfused guinea pig right ventricular
wall.28 29 Our studies of contractility in a few animals
are consistent with this scenario. Evidence of the metabolic protection
afforded by 25 µmol/L pinacidil is provided by our studies of
the changes in extracellular pH, illustrated in Fig 7. This metabolic
protection would not only have lessened the rise in
[K+]e as a result of its effect on
anion-linked factors but might also have led to a reduced increase in
intracellular sodium and thereby reduced K+ efflux via the
sodium-activated K channel. Additional nonspecific effects of pinacidil
might also have contributed to its effect on the rise in
[K+]e.
In many ways, the effects of 25 µmol/L pinacidil on the rise in [K+]e13 and the depletion in high-energy phosphates30 resemble the effects of the L-type calcium channel–blocking agents verapamil and diltiazem.13 30 The effects of the calcium channel–blocking agents and pinacidil on intracellular pH and intracellular calcium might also be similar, even though verapamil has been reported to block the ATP-sensitive K+ channel.31 The similarity of the effects of verapamil and pinacidil on the rise in [K+]e provides additional support for the concept that this rise is multifactorial, influenced by both anionic- and nonionic-linked mechanisms, and that the ATP-sensitive K+ channel is but one of these mechanisms. The influence of any intervention on the rise in [K+]e will be determined by the balance of its effects on K+ conductance through the various voltage- and ligand-gated K+ channels, on K+ driving force as reflected by the time during which the action potential is at its plateau voltage, and on the metabolic changes induced by ischemia. Pinacidil appears to influence all of these parameters. In a concentration of 25 µmol/L, its effects on action potential plateau duration and on the metabolic changes of no-flow ischemia appear to overwhelm its effects on the conductance of the ATP-sensitive K+ channel and cause an unexpected lessening of the ischemia-induced rise in [K+]e.
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Acknowledgments |
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This research was supported in part by grants PO1-HL-27430 and 5R-37-HL-38885 from the National Heart, Lung, and Blood Institute, Bethesda, Md. Pinacidil was provided by the Eli Lily Co, Indianapolis, Ind. We are grateful to Dr Wayne Cascio for his insightful comments and criticisms, to Zoe Sherman, Weili Sun, and James Holmes for their technical assistance, and to Anne Middleton for her expert secretarial support.
Received July 23, 1996; revision received November 14, 1996; accepted November 25, 1996.
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