1124
Interrelation Between Pinacidil and
Intracellular ATP Concentrations on
Activation of the ATP-Sensitive K' Current in
Guinea Pig Ventricular Myocytes
Keiko Nakayama, Zheng Fan, Fumiaki Marumo, and Masayasu Hiraoka
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
The patch-clamp technique was used to study the relation between pinacidil and intracellular
ATP concentration ([ATP]i) on the activation of the outward K' current in guinea pig
ventricular myocytes. Pinacidil shortened the action potential duration, exhibiting stronger
effect at 2 mM [ATP]i than at 5 mM [ATP]i. Pinacidil at 5 ,M or higher concentrations
activated the time-independent outward current at potentials positive to -80 mV, and the
pinacidil-activated current was suppressed by increasing [ATP]i from 2 to 5 mM. The
dose-response curve of pinacidil at different [ATP]i showed a shift to the right and a depression
of the maximum response at increased [ATP]j. The pinacidil-induced shortening of the action
potential duration and outward current were inhibited by application of 0.3-1.0 JpM glibenclamide. In single-channel current recordings, pinacidil activated the intracellular ATP-sensitive
K' channel current without changing the unitary amplitude, and increased open probability of
the channel, an effect dependent on [ATP]i. The pinacidil-activated single-channel current was
blocked by glibenclamide. These results prove the notion that pinacidil activates the ATPsensitive K' channel current, which explains the action potential shortening in cardiac cells
after application of pinacidil. (Circulation Research 1990;67:1124-1133)
R ecently, considerable attention has been paid
to a group of agents -which includes cromakalim (BRL 34915), nicorandil, and
pinacidil -because of their unique abilities to produce a potent vasodilatory action and to promote
action on the potassium channel of vascular smooth
muscle cells. Because of the latter action, these
agents are called potassium channel openers.' All of
these agents also affect cardiac membranes to
shorten the action potential duration (APD) and,
sometimes, to hyperpolarize the resting membrane
potential.2-5 Therefore, they are assumed to modulate the potassium channel activity of cardiac membranes as well. An actual target of potassium channel
openers has not been known until recently, when two
of them, cromakalim and nicorandil, were shown
with the single-channel recording technique6,7 to
activate the ATP-sensitive K' channel current
From the Department of Cardiovascular Diseases Medical
Research Institute, (Z.F., M.H.), and the Second Department of
Medicine (K.N., F.M.), Tokyo Medical and Dental University,
Tokyo, Japan.
Address for correspondence: Masayasu Hiraoka, MD, PhD,
Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima,
Bunkyo-ku, Tokyo-113, Japan.
Received August 23, 1989; accepted July 2, 1990.
(IK.ATP) of ventricular myocytes. As pinacidil has an
effect similar to the others on the APD' and the
background potassium current,8 its target is assumed
to be the same K' channel. Indeed, Arena and Kass9
showed that pinacidil activated the time- and voltageindependent outward K' current, which was blocked
by glibenclamide, a potent blocker of IKATP.10-13
From these observations, they suggested that the
target for pinacidil in the heart is IK.ATP. A preliminary study by Escande et al14 with the single-channel
recording technique indicated that pinacidil activated this channel, but the detailed mechanism of its
action on IKATP remains unclear.
The ATP-sensitive K' channel is characterized by
a strong inhibition of activity when the intracellular
ATP concentration ([ATP]1) is increased above the
millimolar level.15 Therefore, modulation of this
channel by any agent depends on the level of [ATP]i.
Our previous study7 demonstrated that nicorandil
produced a prominent enhancement of IK.ATP at low
[ATP]j, whereas its effect was antagonized by increasing [ATP]i. The present study was done to
characterize the pinacidil-activated K' current in
intact cells under the condition of different [ATP]i.
Direct evidence of the pinacidil action on K.ATP was
further explored by means of the single-channel
recording technique.
Nakayama et al Pinacidil Activates ATP-Sensitive K' Current
Methods
Preparation
Single ventricular myocytes were isolated from
both ventricles of adult guinea pigs that weighed
250-350 g. Our technique to isolate single myocytes
has been described in a previous report.16
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
Solutions
The composition of Tyrode's solution was (mM)
NaCI 144, NaH2PO4 0.33, KCl 4.0, CaCl2 1.8, MgCl2
0.53, glucose 5.5, and HEPES 5.0, with pH adjusted
to 7.3-7.4 by addition of NaOH. The high K+ low
Cl- solution had the following composition (mM):
glutamic acid 70, taurine 15, KCl 30, KH2P04 10,
MgCl2 0.5, glucose 11, EGTA 0.5, and HEPES 10,
with pH adjusted to 7.4 by addition of KOH. Pinacidil, a gift of Shionogi Pharmaceutical Co. (Osaka,
Japan), was dissolved as a stock solution in 1% 0.1N
HCl and diluted appropriately before study. Glibenclamide, a gift of Hoechst Japan (Tokyo), was
dissolved as 0.2 mM stock solution in 2% dimethyl
sulfoxide (DMSO) and diluted into the test solution
to obtain the final concentration indicated in the text.
The final concentration of DMSO contained in the
test solution was less than 0.01%.
Whole-Cell Current Recordings
The patch-clamp technique of the whole-cell configuration17 was used to record membrane potentials
and currents by using a patch-clamp amplifier (model
8900, Dagan Corp., Minneapolis). Details of the
recording technique and the data-acquisition systems
are described in our previous reports.7'16 When the
ramp voltage-clamp method was used in some experiments, an intelligent arbitrary function synthesizer
(model 1731, NF Instruments, Yokohama, Japan)
was used to supply the command pulse. The composition of the pipette solution was (mM) KCI 120,
K2ATP (Sigma Chemical Co., St. Louis) 5.0, HEPES
5.0, and K4BAPTA (Dojin Co., Tokyo) 5.0, with pH
adjusted to 7.2 with KOH. In pipette solutions having
different ATP contents, the concentration of K2ATP
was changed, and at the same time, the concentration
of KCI was varied so as to maintain the final K'
concentration constant at 150 mM. The internal
perfusion technique as originally described by Soejima and Noma18 was used for changing [ATP]i.
Series resistance compensation was adjusted to minimize the duration of the capacitive surge on the
current trace. At the end of each experiment, liquid
junction potential was checked, and if more than +2
mV, the value of the membrane potential was corrected accordingly. The temperature of the perfusion
chamber was maintained at 33-35° C. The solution
in the chamber flowed continuously by gravity with a
speed of 1.2-1.5 ml/min. The chamber volume of 0.8
ml was completely exchanged 1 minute after a solution change. Control data were taken 5 minutes after
changing to the appropriate solution. Drug effects
1125
were tested 5-10 minutes after the drug-containing
solution was changed.
Single-Channel Current Recordings
Single-channel current recordings were made at
room temperature in the inside-out patch configuration'7 with the same patch-clamp amplifier used in
the whole-cell experiments. Currents and voltage
signals were recorded with a videocassette recorder
(HR-S 5500, Victor Co., Tokyo) through a pulse code
modulation converting system (RP- 882, NF Instruments). Recorded signals were filtered off-line
through an eight-pole Bessel low-pass filter (48 dB/
octave, FV- 665, NF Instruments) at 500 Hz and
digitized at 5 kHz using an analog-to-digital convertor (CED 1401, Cambridge Electronic Design Ltd.,
Cambridge, UK) to store into the disk of an IBM-AT
personal computer for later analysis. When the single-channel recording with the inside-out patch configuration was done, the bath solution (intracellular
solution) contained (mM) KCl 140, glucose 5.5,
EGTA 2, and HEPES 5, with pH adjusted to 7.3 with
KOH. The drug was dissolved in the bath solution at
the concentration indicated in the text. The composition of the pipette solution (extracellular medium)
was (mM) KCI 140, CaCl2 1.8, MgC12 0.53, glucose
5.5, and HEPES 5, with pH adjusted to 7.3 by
addition of KOH. The drug effects usually were
examined 2-3 minutes after the test solutions were
changed, except for calculation of open probability at
different [ATP]i as described below.
Calculation of Open Probability (ATP-Sensitive K+
Channel Current)
Open probability (PO) was calculated as
NT
where ti is the duration of an nith number of open
channels, N is the available channel in the patch, and
T is the total recording time (30 seconds in this
study). The numbers of available channels were
estimated as the maximum open channels at free
[ATP]i. To avoid possible influence from the rundown of the channel, total exposure times to different
[ATP]i with or without the presence of pinacidil were
limited to be less than 4 minutes in any patch. The
availability of the channels was reevaluated after
washout of pinacidil and ATP. Only those patches
showing PO> 0.98 at the second exposure to ATP-free
solution were selected for the calculation (assuming
Po=1.0 at the first exposure to 0 mM [ATP]i).
Measurements of Cell Surface Area
Cell surface areas were estimated from the cell
capacitance assuming a specific membrane capacitance of 1 ,uF/cm2. Cell capacitance was calculated
from current responses induced by 1-mV depolarizing pulses from a holding potential of 90 mV.19
1126
Circulation Research Vol 67, No 5, November 1990
A
5-ATP
o~~~
-\
1
-
0
,.
x
mV
A
**
.
(%
°-100
B
2-ATP
O
:control
O
:P.
A
:P1OuM
*PM1 OM
0
5M
1Os*
5S^l
1
D
50
11
\O
wO
100*
100
5.mA
I
0
-
rD~ ~ ~C
A
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
Lk
55Sa
L100
200ms
2-ATP
1O
CaM
5-ATP
*p<O.05
**p<0.01
FIGURE 1. Effects ofpinacidil on action potential duration (APD) ofguinea pig ventricular myocytes in two different intracellular
ATP concentrations ([A TP],). Panel A: Effect of 5 and 10 p.Mpinacidil (P) on action potentials recordedffrom a cell dialyzed with
an intemal solution containing 5 mMATP (5-A TP). Panel B: Effects of 5 and 10 pMpinacidil on action potentials recorded from
a cell dialyzed with an intemal solution containing 2 mMATP (2-ATP). Panel C: Summarized data of the effects ofpinacidil on
APD. Values of APD are expressed by relative APD at 90% repolarization (APD90) compared with APD90 in the control as 100%.
Multiple comparisons among data in the control, 5, and 10 pMpinacidil were done using the Student-Newman-Keuls method.
Unpaired t test was used to judge statistical significance between the data of 2 and 5 mM [ATP]i at the same pinacidil
concentration. Action potentials were elicited at a stimulation frequency of 0.067 Hz.
Statistical Analysis
Data are expressed as mean± SD. Comparisons
between two groups of data were evaluated by paired
or unpaired t test. Comparisons between more than
two groups were evaluated by Student-NewmanKeuls method for critical difference. A value of
p<0.05 was considered significant.
Results
Effect of Pinacidil on Membrane Potentials
Effects of pinacidil on membrane potentials of ventricular myocytes were examined at two different
[ATP]1, a normal (5 mM) and a low (2 mM) level, while
the preparations were stimulated at a constant frequency of 0.067 Hz. At 5 mM [ATP]i, 10 gM pinacidil
decreased action potential amplitude and shortened
APD both at 20% (APD20) and 90% repolarization
(APDgO), whereas 5 ,M pinacidil did not produce any
changes in these parameters. At 2 mM [ATP]i, however, 5 ,M pinacidil decreased action potential amplitude and shortened both APD20 and APDg, as did 10
,uM pinacidil. Furthermore, the degree of the APD
shortening by the same concentration of pinacidil was
significantly larger at the low than at the normal [ATP]i
(Figure 1, Table 1). The resting potential was not
changed by pinacidil at either [ATP]i.
Effect of Pinacidil on Steady-State Current
Figure 2A illustrates membrane current responses
to depolarizing or hyperpolarizing voltage steps from
the holding potential of -30 mV before and during
the application of 10 ,uM pinacidil using a pipette
solution containing 2 mM ATP. Pinacidil increased
outward current at a potential positive to around -80
mV, and this increase was time-independent. At
potentials negative to -90 mM, pinacidil decreased
inward current during pulses (see "Discussion"). The
steady-state current-voltage relation, in which current was measured at the end of the 2-second test
pulses, showed increased outward current with a loss
of negative slope region at voltages between -60 and
-20 mV (Figure 2B). There was no change in the
reversal potential. Similar results as shown in Figure
2 were confirmed in four cells, although the degree of
increased current by pinacidil differed among different preparations.
Effect of Intracellular ATP Concentration on the
Pinacidil-Activated Outward Current
The effects of pinacidil on membrane potentials
indicated that pinacidil had a stronger effect on APD
at the low [ATP]i than at the normal [ATPji. Therefore, we examined the pinacidil-activated outward
Nakayama et al Pinacidil Activates ATP-Sensitive K' Current
1127
TABLE 1. Effects of Pinacidil on Membrane Potentials of Guinea Pig Ventricular Myocytes at Two Different Intracellular
ATP Concentrations
[ATP]i
APD%o (msec)
2 mM
Control
10
-87.6±4.1
134.7±4.4
507.2±218.1
713.2±299.9
Pinacidil 5 ,uM
9
-85.6±5.5
128.7±7.0*
300.6±168.6*
434.5±212.5*
Pinacidil 10 ,uM
4
-86.5±3.6
123.4±11.2*
163.1±138.0*
245.8±187.1*
5 mM
Control
5
-89.8±0.7
136.3±2.9
491.8±185.8
689.1±249.9
Pinacidil 5 ,uM
5
-88.0±0.9
135.4±2.0
521.4±209.4
719.6±207.9
Pinacidil 10 tM
5
-87.6±1.0
133.5±1.7*
405.2±202.5*
571.8±253.2*
Values are mean±SD. [ATP]i, intracellular ATP concentration; RMP, resting membrane potential; APA, action potential amplitude;
APD20, action potential duration at 20% repolarization; APD%, action potential duration at 90% repolarization.
*p<0.05 vs. control.
n
RMP (mV)
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
current at different [ATP]i. First, antagonistic action
of increasing [ATP]i on pinacidil-activated outward
current was tested using the internal perfusion technique18 (Figure 3). Under the condition of 2 mM
[ATP]i, 10 ,uM pinacidil markedly increased the
time-independent outward current at potentials positive to -50 mV, and the current-voltage curve lost its
negative slope region. When [ATP]i was increased to
5 mM in the presence of pinacidil, the increased
outward current was nearly halved, and the currentvoltage curve regained a slight negative slope. In six
myocytes, the outward current at 0 mV increased
significantly from 0.13±0.07 nA in the control to
1.06+±0.63 nA after the pinacidil application at 2 mM
ATP in the pipette solution (p<O.OS). An increase in
[ATP]i to 5 mM in the presence of pinacidil decreased the current to 0.61+0.40 nA.
The dose-response curve of pinacidil-activated
current was examined at different [ATP]i. A ramp
voltage-clamp method was used to produce a slow
depolarization (200 mV/30 sec) from -120 to 80 mV.
Figure 4 presents typical results obtained with different [ATP]i. The pinacidil dose-dependently activated the outward current at a voltage positive to
-80 mV in each [ATP]i, and the degree of the
increase also was dependent on [ATP]i. The pinacidil
APA (mV)
APD20 (msec)
effect was the most prominent at 2 mM [ATP]i among
the three conditions. A quantitative estimation of the
type of experiments shown in Figure 4 was achieved
by expressing the dose-response curve as the current
density. The current density induced by pinacidil was
measured as the current level at 0 mV, because it was
nearly zero during the control (Figure 5). At 2 mM
[ATP]i, the current density showed a sigmoidal dependence on the pinacidil concentration, with a
nearly maximal response at 100 gM (159.4+±14.8
,NA/cm2; n=5). We could not judge whether the
response at 100 ,uM pinacidil was really saturated or
not, because all the cells died after application of the
drug at higher concentrations. At 5 mM [ATP]i, a
sigmoidal dependence of the current density on the
pinacidil concentration shifted to the right, and at the
same time, the maximal response at 100 ,uM pinacidil
was markedly suppressed to 16.8±4.5 tuA/cm2 (n=4).
The response was saturated when the concentration
of pinacidil was higher than 100 ,uM. At 10 mM
[ATP]i, the maximal current induced by pinacidil was
reduced further (15.8+4.0 gANcm'; n=6) and shifted
to develop at the higher concentration (300 ,uM).
The current density decreased to 10.3+2.5 gA/cm' at
500 ttM. The decrease of the current density response to 500 ,iM pinacidil in comparison to that at
B
A
VT mv
0
11[ nA
+60
-O
0
o
01~~~
----°
-60
-100HP=-30
*
o
VT
29
O:control
*:P. 10pM
*--4
:control
:P.1OyM
FIGURE 2. Effect ofpinacidil (P) on
membrane current. Panel A: Superimposed current traces before (o) and
during (0) the application of 10 pM
pinacidil at four different test voltages
(VT). Voltage protocol is shown at the
bottom. Panel B: Effect of 10 pM
pinacidil on current-voltage relation.
Current values were measured as the
level at the end of 2-second depolarizing or hyperpolarizing test pulses. HP,
holding potential.
Circulation Research Vol 67, No 5, November 1990
1128
A
B
nA
S
5.01
0-O :control,2-ATP
*---:2-ATP + P
-----A:5-ATP + P
l.OnA
o
z~
'l
X0~~~~-U
o :control,2-ATP
*:2-ATP + P
* :5-ATP + P
-e.-
- t 0of
1
-60 -4o -20
-30mV
-1.0f
is
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
300 ,uM was observed in four of six cells. By these
sigmoidal curves, the following Kd values were calculated: 77.0 and 69.0 ,M at 2 and 5 mM [ATP]i,
respectively, and 158 gM at 10 mM [ATP]i.
Block of the Pinacidil-Induced Changes in Membrane
Potential and Current by Glibenclamide
We examined the effects of glibenclamide on the
changes in action potentials and outward current
induced by pinacidil because Arena and Kass9 described the suppression of the pinacidil-activated
current by glibenclamide. Figure 6A illustrates the
effects of glibenclamide on the pinacidil-induced
action potential change at 2 mM [ATP]i. Application
of 0.3 and 1.0 ,M glibenclamide prolonged APD,
which was shortened by the pretreatment of 100 ,M
pinacidil. APD90 was shortened by 100 ,M pinacidil
from 481+77 (n =5) to 177 ±70 msec. The shortened
APDgO by pinacidil was prolonged by 0.3 ,uM glibenclamide to 292±167 msec (p<0.01 versus APD%0
in 100 ,M pinacidil) and further by 1.0 ,M to
416±135 msec (p<0.01) (Figure 6B). Therefore, 1.0
4M glibenclamide completely antagonized the shortening of APD induced by 100 ,uM pinacidil. The
pinacidil-activated outward current also was almost
2-ATP
|
40 600 80
80 mVV
completely suppressed by 0.3 jM glibenclamide (Figure 7). This result was confirmed in three myocytes.
Effect of Pinacidil on the Single-Channel Current
The effect of pinacidil on the single-channel current was studied using the inside-out patch configurations. Figure 8 shows a typical example of the
records at a membrane potential of +40 mV (Figure
8A), as well as amplitude histograms (Figure 8B).
When the intracellular solution did not contain ATP,
prominent openings of the single channels were
observed (Figure 8A, recording a); the openings were
largely inhibited by 2 mM [ATPJi (recording b).
Application of 30 j.M pinacidil in the presence of 2
mM [ATP]i activated the current again (recording c).
The amplitude of the pinacidil-activated channels at
2 mM [ATP]i (2.53 pA) was not different from that in
the ATP-free solution (2.48 pA, calculated by the
difference between the third and fourth open level in
recording a), confirming our recent report.20 Addition of 0.01% DMSO did not influence the pinacidilactivated channel activity (recording d), whereas 0.3
,uM glibenclamide dissolved in 0.01% DMSO completely inhibited channel activity (recording e). Values of PO before and after the addition of DMSO in
1O-ATP
5-ATP
nAA
|,
20
FIGURE 3. Effect of different intracellular ATP concentrations ([ATP]1)
on the pinacidil-activated outward current. Panel A: Current traces recorded
in response to depolarization to 0 mV
for 2 seconds from -30 mVat different
stages of treatment. o, Control with 2
mM [ATP]i (2-A TP); *, application of
10 M pinacidil (P) in 2 mM [ATP],,
A, application of 10 /Mpinacidil in 5
mM [ATTPFi (5-A TP). Panel B: Effect
of 10 pMM pinacidil on the currentvoltage relation at different ATP concentrations in the pipette solution. Data
were taken from the same experiment
shown in panel A. Current values were
measured as the level at the end of
2-second test voltages from -30 mV.
20
AL
-100
-5.
80mV
-
1 20mV
O control
*:P.50pM
A
30sec
P.loopM
FIGURE 4. Effects of pinacidil on the background current induced by 30-second depolarizing ramp from -120 to +80 mV at different
intracellular ATP concentrations ([ATP]i).
[ATP]i is indicated at the top of each graph as
2-ATP, 2 mM [ATP]i; 5-ATP, 5 mM [ATP]i;
and 10-A TP, 10 mM [ATP]li. o, Control current
before the application ofpinacidil (P); *, current
in the presence of 50 p!pinacidil; A, current in
the presence of 100 puM pinacidil.
Nakayama et al Pinacidil Activates ATP-Sensitive K' Current
1129
A
60
CO
cC4Q
40
0)
-100
O:Controi
*A:P+G0.3pM
A:P+GI.OMM
:20
0)
o
a
a
1
10
100
a
200ms
B
500
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
Pinacidiil
FIGURE 5. Dose-response cune of the pinacidil-activated
current at different intracellularATP concentrations ([A TP]J.
Ordinate indicates current density, and abscissa is the pinacidil concentration. Current density was calculated by the
background current at 0 mV induced by 30-second depolarizing ramp pulses from -120 to +80 mV Each value represents
offour to six measurements from different preparations; bars indicate±SD. Maximum current density activated
by pinacidil was depressed with increasing [ATP]I. 2 mM
[ATP]i (2-ATP); *, 5 mM [ATPfi (5-ATP); o, 10 mM
[ATP]I (10-A TP).
a mean
[mS]
500o
"
400-
300-1
lovv
200-
100-
e,
the presence of 2 mM [ATP]i and 30 ,uM pinacidil
were 12.6+5.1% and 9.7+3.2%, respectively (n=3).
Further application of 0.3 ,uM glibenclamide reduced
PO to less than 0.1% in five preparations. At 2 mM
[ATP]i, activation of the single-channel current sensitive to the ATP-free solution also was observed in
eight of 10 patches in 30 ,uM pinacidil, and two of five
patches in 10 ,M pinacidil. With either pinacidil
concentration, the amplitude of the single-channel
current was not changed from that with the ATP-free
solution. PO of IK.ATP in different [ATP]i was estimated with and without the presence of 30 .M
pinacidil. Figure 9A shows typical recordings of the
single-channel current; Figure 9B summarizes data
from four to six patches. At each [ATP]i (0.1-2.0
mM), pinacidil significantly increased PO. Furthermore, PO of the channel in the presence of pinacidil
was dependent on [ATP]i, because an increase in the
latter decreased the former.
Discussion
The present study demonstrated that a vasodilator,
pinacidil, shortened APD and increased the timeindependent outward current in ventricular myocytes. The effects of pinacidil were dependent on
[ATP]i, being accentuated by lowering [ATP]i and
attenuated by increasing it or by application of
glibenclamide. Single-channel current recordings
confirmed an activation of the [ATP]i-sensitive current by pinacidil without changing the unitary amplitude of the current and the dependence of the
activation on [ATP]i. These results indicate that
O-
Control
P
P
+
P
+
G
G
0.3PM 1.0MM
FIGURE 6. Effects of glibenclamide on the pinacidil-induced action potential shortening. Panel A: Superimposed
action potentials. o, Action potential in control with 2 mM
intracellularATP concentration ([A TPJd, *, application of 10
pMpinacidil (P); A, application of 10 tMpinacidil and 0.3
pMglibenclamide (G); A, application of 10 ,Mpinacidil and
1.0 iM glibenclamide. Panel B: Effects of pinacidil and
glibenclamide on action potential duration at 90% repolarization (APD90). Data show mean ±SD collected from five
preparations.
pinacidil increases PO of the ATP-sensitive K' channel, which explains the shortening of APD of heart
cells after drug treatment.
Pinacidil has been shown to shorten the APD of
heart muscles and to modify the shape of the T wave
on surface electrocardiograms. The effects are attributed to an increase in K' permeability of cardiac
membranes.5,21 lijima and Taira8 actually showed an
increase in the outward current using isolated ventricular myocytes, and they interpreted this action as
an activation of the background K' current. Arena
and Kass9 recently explored the target of the membrane current system activated by pinacidil and confirmed that pinacidil actually activated the K' current
in guinea pig ventricular myocytes, but they interpreted the pinacidil-activated current as IK.ATP on the
basis of its time- and voltage-independent properties,
as well as its pharmacological characteristics.
1130
Circulation Research Vol 67, No 5, November 1990
-100
+BOmV
-12OmV,V30sec,
0: control
*:
P.30,0M
A:
P.30,M+G.0.3pM
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
FIGURE 7. Effect of glibenclamide (G) on the pinacidil
(P)-induced background current. Current was induced by
30-second depolarizing ramp from -120 to +80 mV Note
that 0.3 ,M glibenclamide (A) completely suppressed the
pinacidil-activated background current (e).
A
a. 0-ATP
.... b
o1-1
...^..-
C-.
-1001
bill 01 W-1
11 w-T
-1-7
-
.
b. 2-ATP
1
C-_
c. 2-ATP + P 30uM
A-0-
~
a
A
--
m
---
I
c-~~~~~~~~~~~~~~-e. 2-ATP + P 3OpM + ool%MO + G 03PM
---
ONR o
1-
l
JL__---
B
0
x
CO)
z
W
*RO#Wb~
-1-
1 ---&- $_,;_j
d. 2-ATP + P 30MM + o.ol%DMSO
C~
S-
Our study confirmed that pinacidil shortened APD
of ventricular myocytes and activated the time-independent outward current. These effects were antagonized by raising [ATP]i. All of the above actions by
pinacidil were easily blocked by a low concentration
of glibenclamide, which has been claimed as a specific inhibitor of IK.ATP.10-13 Furthermore, the singlechannel recordings in this and the previous study20
proved the actual activation of IK.ATP by pinacidil.
Therefore, our data presented the confirming evidence that the target of the pinacidil action in heart
is indeed K.ATP.
The mode of channel activation by pinacidil appears to increase the PO Of IKATP, because marked
openings were seen without changing the unitary
amplitude. An increase in [ATP)i decreased the
channel opening without affecting the unitary current
amplitude, and the decreased PO of the channel
activity was restored by application of pinacidil (Figures 8 and 9). The results may indicate that ATP and
pinacidil share the same binding site or process for
channel modulation. Kinetic analysis of channel activity showed an antagonistic action by ATP and
WU
Ic
%W~
h
SpA
FIGURE 8. Effects of pinacidil (P) and glibenclamide (G) on the ATP-sensitive single-channel current. Panel A: Current
recordings obtained from a single patch. Membrane potential is held at +40 mV in the inside-out patch configuration. C, Closed
leveL Recording a: Presence of 0 mMATP (0-ATP) in the intracellular solution. There are marked openings ofthe channel activity
up to four levels. Recording b: Presence of 2 mMATP (2-ATP) in the intracellular solution. Most of the channel activity seen in
recording a is now inhibited. Recording c: Application of 2 mMATP and 30 pMpinacidil in the intracellular solution. Marked
openings of the channel again are seen. Recording d:Application of2 mMATP and 30 MMpinacidil and 0.01% dimethyl sulfoide
(DMSO) in the intracellular solution. There is no apparent change in the channel activity compared with that in recording c.
Recording e: Further application of 0.3 pMglibenclamide in the internal solution. Channel activity is completely suppressed. Panel
B: Amplitude histograms recorded from the same patch in panel A.
Nakayama et al Pinacidil Activates ATP-Sensitive K' Current
1131
A
ro.1-AI
0-ATP
T 'I
__1
7
_
1
VPin.1 3O-IM
~~~~~~~~~~~~~~~~~~~~~~~~~~l
cCTwU~~~~~ ~~
O.1-ATP
L
1
. .--
.5-ATP
-
C-~~~~~~~~~~~~~~~~~~~~7
ePf?hnacil3O>M&
|0-ATP
0.5-ATP
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
5 pA
C-
.....
B
1.Oro
I*
PO
0.5
FIGURE 9. The open probability (Po) of the
A TP-sensitive channel at different A TP concentrations in the presence and absence ofpinacidil
(P). Panel A: Typical examples ofthe continuous
current record. Holding potential is +40 mV
Panel B: Summary data of Po obtained from
four to six patches held at +40 mV The paired t
test between the data at the same intracellular
ATP concentration ([ATPJ) and Student-Newman-Keuls method among different [ATP]i are
used to judge statistical significance.
*
O:control
*:P.301*M
0
1-A-,.
*p<0.05
0.1
0.5
A T P
2.0
mM
pinacidil, where the channel was partially closed by
ATP.20 However, the dose-response curve of the
pinacidil action did not reveal a competitive inhibition by ATP, and the maximal current activated by
pinacidil was suppressed by the higher [ATP]i in
addition to a shift of the curve to the right (Figure 5).
Partially, this shift may be due to experimental
imperfection because of too few points on the curve
near the maximal response, especially at 2 mM
[ATP]i. Another possibility is that pinacidil may have
at least two sites or processes to interact with the
openings of IK.ATP. High concentrations of pinacidil
may block the channel opening, in addition to its
activation, or accelerate the "rundown" phenomenon
of this channel activity.22 Further study is necessary
to delineate an exact mechanism, as well as sites of
action for pinacidil to activate lK.ATPThe effects of pinacidil on the current-voltage
curve exhibit two characteristic changes worth mentioning in terms of the drug-Induced alteration in
membrane potential, other than a marked increase of
outward current. First, the increased outward current
positive to around -60 mV caused a loss of the
negative slope region in the current-voltage curve.
Second, pinacidil produced little change in the current at the resting potential level around -80 mV
and decreased inward current at potentials negative
to -90 mV. These two factors could explain a
marked shortening of APD without alternation of the
resting potential (see Table 1). These effects were
similar to those produced by cromakalim and nicorandil, which also were shown to activate IK.ATP.67
The result that pinacidil enhanced outward but not
inward current may be related to the intrinsic properties of IK.ATP, because it has a sigmoidal currentvoltage curve, showing outward-going rectification at
negative potentials and inward-going rectification at
positive potentials.23 Consequently, activating IK.ATP
will produce very little inward current negative to its
reversal. This factor, however, cannot explain a decrease of the inward current negative to K' equilibrium potential (EK) after pinacidil, and therefore, the
decrease of the current may be explained by several
other possibilities. First, the activation of the current
1132
Circulation Research Vol 67, No 5, November 1990
by pinacidil may be voltage-dependent. Second, pinacidil may produce inhibition of other currents, such
as the inward rectifier K' current (IK1). Third, pinacidil may display inhibition on IKATP in addition to
activating action. A voltage-dependent activation is
not likely because single-channel recording revealed
that pinacidil activated K.ATP at potentials both negative and positive to EK.20 As to inhibitory action on
IK.ATP, our preliminary study indicated that pinacidil
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
produced a suppression of channel activity at positive
voltages to EK but no such action at negative voltages.
Therefore, inhibitory action seems not to be the right
explanation. Although we have not examined the
drug effects on outside-out patches, the most likely
explanation is that pinacidil inhibits 1K1 from the
outer surface of the membrane, because internal
application of the drug did not affect the channel
activity of 1K120
The results of the present study point out a common mechanism of action on cardiac membranes
shared by the three agents cromakalim, nicorandil,
and pinacidil. These agents are claimed as K' channel openers,' which possess the ability to increase K'
permeability in vascular smooth muscle cells and to
produce a vasodilatory action. The exact mechanism
of the three agents to induce relaxation of vascular
smooth muscles is not known, but it is implicated that
increased K' permeability has some crucial role for
vasodilation.24 Recently, the existence of IKATP as
well as its activation were reported.25 Pinacidil also
was suggested to activate IK.ATP of vascular smooth
muscles because of inhibition of the vasorelaxant
effect of IK.ATP by glibenclamide.25 Although the
properties of IK.ATP between cardiac and smooth
muscle cells were not exactly the same, the present
results obtained from cardiac myocytes may help to
solve the mechanism of a vasodilatory action by K'
channel openers. Study in this area continues,2627
and it is an interesting area for future studies.
The physiological and clinical significance of the
present finding is not known. It would be interesting to
know why pinacidil, cromakalim, and nicorandil consistently are found to activate K.ATP even though their
chemical structures differ. Information regarding the
structure-function relation of these compounds may
be helpful in clarifying the regulatory mechanism of
IK.ATP itself and may lead to an understanding of the
basic mechanism of the drug action on the heart.
Pinacidil would be expected to be more effective in the
ischemic heart than in the normal heart, because the
ischemic heart would have less [ATP]i than the normal. Whether or not the action of pinacidil could bring
about beneficial effects for the function of the ischemic heart is to be evaluated further.
Acknowledgment
The authors thank Ms. N. Fujita for her secretarial
assistance.
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1133
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KEY WORDS * ATP-sensitive K' current * intracellular ATP
K' channel opener * pinacidil
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Interrelation between pinacidil and intracellular ATP concentrations on activation of the
ATP-sensitive K+ current in guinea pig ventricular myocytes.
K Nakayama, Z Fan, F Marumo and M Hiraoka
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
Circ Res. 1990;67:1124-1133
doi: 10.1161/01.RES.67.5.1124
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