0022-3565/97/2831-0375$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:375–383, 1997
Vol. 283, No. 1
Printed in U.S.A.
The Novel Calcium Sensitizer Levosimendan Activates the
ATP-Sensitive K1 Channel in Rat Ventricular Cells1
HISASHI YOKOSHIKI, YASUHIRO KATSUBE, MASANORI SUNAGAWA and NICHOLAS SPERELAKIS
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
Accepted for publication June 16, 1997
The treatment of heart failure is very important in the field
of cardiovascular medicine. Recent clinical trials with angiotensin-converting enzyme inhibitors demonstrated the improvement of quality of life and the reduction of mortality in
patients with severe chronic heart failure (CONSENSUS
Trial Study Group, 1987; SOLVD Investigators, 1991). However, results with positive inotropic agents, such as PDE
inhibitors and beta 1 agonists, were disappointing in terms of
excess morbidity and mortality without producing important
clinical benefits (Packer, 1988; Packer et al., 1991).
A new class of cardiovascular drugs, the myofilament
Ca11 sensitizers, has been developed for the treatment of
both acute and chronic heart failure (Packer, 1988; NielsenKudsk and Aldershvile, 1995). Compared with previous positive inotropic agents that enhance intracellular Ca11, the
Ca11-sensitizing drugs, including pimobendan, EMD 53998,
MCI-154 and levosimendan, increase myocardial contractility by producing more force for a given amount of intracellu-
Received for publication March 25, 1997.
1
This study was supported in part by a gift from Orion Pharma (Espoo,
Finland).
However, levosimendan did not stimulate the KATP channels
that exhibited high spontaneous activity in ATP-free solution.
Levosimendan also could not stimulate KATP channels that had
rundown in ATP-free solution. However, levosimendan could
stimulate rundown KATP channels that were reactivated by
nucleotide diphosphates. KATP channels inhibited by 0.5 mM
AMP-PNP, a nonhydrolyzable ATP analog, were not stimulated
by levosimendan; however, the channels were stimulated by
levosimendan in the presence of 30 to 50 mM ADP. Levosimendan stimulates cardiac KATP channels that are suppressed by
intracellular ATP. It appears that levosimendan acts synergistically with nucleotide diphosphates. These properties of levosimendan may help protect ischemic myocardium because activation of KATP channels by levosimendan would likely occur in
ischemic regions in which intracellular ADP concentration is
increased and intracellular ATP concentration is decreased.
lar Ca11 (Nielsen-Kudsk and Aldershvile, 1995). Among
these agents, levosimendan has a unique property in that it
binds to cardiac cTnC in a Ca11-dependent manner
(Pollesello et al., 1994; Haikala et al., 1995a, 1995b; NielsenKudsk and Aldershvile, 1995). The positive inotropic effect,
due to increased Ca11 sensitivity of the contractile proteins,
has been reported to be exerted at concentrations of 0.03 to
10 mM in skinned fibers from guinea pig papillary muscles
(Edes et al., 1995; Haikala et al., 1995b). In addition, levosimendan causes vasodilation in both experimental animal
models (Harkin et al., 1995; Pagel et al., 1995; Vegh et al.,
1995) and clinical studies (Lilleberg et al., 1995; Sundberg et
al., 1995; Vegh et al., 1995). Although levosimendan (0.1– 0.3
mM) was suggested to act as a PDE inhibitor (Edes et al.,
1995), this drug had favorable preventive effects against
ventricular tachyarrhythmias induced by ischemia-reperfusion (Kaszala et al., 1994).
Although levosimendan (5–10 mM) increased the ICa(L) in
guinea pig ventricular cells, presumably by the PDE inhibition (Virag et al., 1996; Boknik et al., 1997), little data are
available concerning the electrophysiological effects of levosimendan. In the present study, we explored the effects of
ABBREVIATIONS: KATP, ATP-sensitive K1; NDP, nucleotide diphosphate; cTnC, cardiac troponin C; PDE, phosphodiesterase; APD, action
potential duration; RP, resting potential; APA, action potential amplitude; ICa(L), L-type Ca21 current; KCO, K1 channel-opening drug.
375
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ABSTRACT
Levosimendan, a new Ca11-sensitizing and positive inotropic
agent, was reported to act as a coronary vasodilator and protect ischemic myocardium. To elucidate the mechanisms of
these actions, the possible electrophysiological effects of levosimendan on isolated rat ventricular cells were examined by the
patch-clamp technique with whole-cell and single-channel recordings. Levosimendan (3 and 10 mM) markedly shortened
action potential duration and activated an outward current at
potentials positive to 270 mV. The increased current was abolished by glibenclamide, a blocker of the ATP-sensitive K1
(KATP) current. Stimulation of KATP current was dose dependent, with an EC50 value of 4.7 mM; a maximal effect occurred
at 30 mM. The L-type Ca11 current was not affected by levosimendan (0.2–10 mM). In single-channel current recording in
open cell-attached patches, KATP channels, which had been
inhibited by 0.3 mM ATP, were activated by levosimendan.
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Yokoshiki et al.
Vol. 283
levosimendan on membrane currents and action potentials in
rat ventricular cells using the patch-clamp technique of
whole-cell and single-channel recordings. Levosimendan
shortened APD, and our data suggest this occurred through
opening of KATP channels.
Methods
Young (10 –18 days old) Sprague-Dawley rats were used for this
study. The rats were handled in accordance with the Declaration of
Helsinki and the Guiding Principles in the Care and Use of Animals.
Cell preparation. Freshly isolated single cells were prepared
from ventricles of young rats as previously described (Yokoshiki et
al., 1996). In brief, the rats were decapitated while under CO2
anesthesia, and the hearts were removed and rinsed in oxygenated
Tyrode’s solution and, then immersed in Ca11-free Tyrode’s solution
for 20 min. After the spontaneous beatings had ceased, the ventricles
were dissected, and small pieces were enzymatically digested for 50
min (37°C) in Ca11-free Tyrode’s solution containing collagenase (1
Fig. 2. Stimulation of K1 current by levosimendan in rat ventricular
cells. A, Superimposed current traces evoked by voltage-clamp ramps
(240 mV/sec) between 140 mV and 2100 mV. The outward current
was rapidly increased by levosimendan (b) and intersected with the
abscissa at ;275 mV. The reversal potential (Vrev) was close to the EK
(283 mV), suggesting that most of the current stimulated by levosimendan was a K1 current. Glibenclamide (1 mM) abolished the levosimendan-stimulated currents (c). B, Time courses of the currents measured at 0 and 2100 mV. a– c, Time points at which the currents in A
were recorded.
mg/ml; Wako Chemicals, Osaka, Japan). The cells were mechanically
dispersed in the modified KB solution at room temperature using a
Pasteur pipette. The cell suspensions were stored in a refrigerator
(4°C) and were used for 2 to 6 hr after isolation. Usually, 40% to 60%
of the isolated cells were rod shaped in Tyrode’s solution (containing
1.8 mM Ca11). Rod-shaped cells with smooth surfaces and clear
cross-striations were selected for experiments. All experiments were
performed at room temperature (20–22°C).
The composition (in mM) of the normal Tyrode’s solution was NaCl
TABLE 1
Summary of changes produced by levosimendan in action
potentials in rat ventricular cells
The action potential parameters after exposure to 10 mM levosimendan were
assessed from the action potentials 10 sec before the cells became inexcitable.
The rats were 10 to 18 days old.
n
RP
APA
APD260
119 6 4.4
115 6 5.9
119 6 4.6
100 6 5.9a
132 6 29
61 6 29a
110 6 35
11 6 3.4a
mV
Control
Levosimendan (3 mM)
Control
Levosimendan (10 mM)
APD260, APD at 260 mV.
a
P , .05 vs. control.
6
6
7
7
274.4 6 0.7
275.3 6 0.5
276.3 6 0.5
277.1 6 0.5a
msec
Fig. 3. Dose-dependent activation of K1 currents by levosimendan. A,
Current-voltage relations for the currents stimulated by different doses
of levosimendan. The currents were evoked by voltage-clamp ramps
(240 mV/sec) between 140 and 2100 mV. B, Dose-response relation
for stimulation of K1 currents by levosimendan. Currents were evoked
by ramp pulses and measured at 0 mV when the currents were maximally activated. Difference in currents between the maximal activated
and control (before application of levosimendan) were plotted against
each concentration. Number of cells studied are shown in parentheses.
Data points were fitted by the Hill equation. The EC50 value was 4.7 mM,
and the Hill coefficient (nH) value was 2.3.
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Fig. 1. Shortening of APD by levosimendan in young (days 10 –18) rat
ventricular cells in current-clamp mode. A, Action potentials recorded
in current-clamp. Extracellular application of 10 mM levosimendan
caused shortening of APD; this effect could be reversed after washing
out (W.O.). Arrows, peak levels of the action potentials in d and e. B,
Time courses of changes in APA, APD at 260 mV (APD260) and resting
potential (RP). a– h, Time points at which action potentials in A were
recorded.
1997
Levosimendan Activates K1 Channel
377
Fig. 4. Lack of effects of levosimendan on rat cardiac ICa(L). ICa(L) was
evoked every 15 sec by a test potential to 110 mV from a holding
potential of 240 mV. Levosimendan (10 mM) had no effects on ICa(L),
which was markedly stimulated by the subsequent application of 1 mM
isoproterenol (ISO).
143, KCl 5.4, NaH2PO4 0.33, MgCl2 0.5, CaCl2 1.8, glucose 5.5 and
HEPES 5, pH adjusted to 7.4 with NaOH. The modified KB solution
was K-glutamate 50, KOH 20, KCl 40, taurine 20, KH2PO4 20, MgCl2
3, glucose 10, EGTA 0.5 and HEPES 10, pH adjusted to 7.4 with
KOH.
Whole-cell recordings. The standard patch-clamp technique
was applied in the whole-cell configuration with a patch-clamp amplifier (Axopatch-1D; Axon Instruments, Foster City, CA). Action
potentials and whole-cell currents were measured under currentclamp or voltage-clamp mode, respectively. Voltage-clamp experiments were performed by applying either voltage ramp or step
pulses. The patch electrodes (2–5 MV) were made from borosilicate
glass capillary tubing (World Precision Instruments, Sarasota, FL).
The cell suspension was placed into a small chamber (0.5 ml) on the
stage of an inverted microscope (TMD-Diaphot; Nikon, Tokyo, Japan). The bath was superfused with the normal Tyrode’s solution.
The composition of the internal (pipette) solution for the whole-cell
experiments was (in mM) KCl 140, MgCl2 1, HEPES 5, EGTA 10 and
ATP-Na2 5, pH adjusted to 7.2 with KOH.
To isolate the ICa(L), the pipette was filled with high Cs1 solution
of the following composition (in mM): CsOH 110, CsCl 20, glutamic
acid 112, MgCl2 5, ATP-Na2 5, EGTA 10, HEPES 10 and CaCl2 0.44
(pCa 5 7), pH adjusted to 7.2 with CsOH. The bath solution contained (in mM) tetraethylammonium (TEA) chloride 150, MgCl2 0.5,
CaCl2 1.8, glucose 5.5, 4-aminopyridine (4-AP) 3 and HEPES 5, pH
adjusted to 7.4 with HCl.
Current and voltage signals were filtered at 1 kHz, digitized by an
A/D converter (TL-1, Axon Instruments) and analyzed on a personal
computer (IBM-compatible computer system) with pCLAMP (version
5.05, Axon Instruments). The membrane capacitance was determined from the current amplitude elicited in response to a hyperpolarizing voltage ramp pulse of 0.2 V/sec from a holding potential of 0
mV (duration, 25 msec; peak amplitude, 2 5 mV) to avoid interference by any time-dependent ionic currents. Average membrane capacitance of the cells (10–18 day) was 24.9 6 0.9 pF (n 5 37).
Single-channel recordings. Single-channel current recordings
were made in the open cell-attached patch configuration (Kakei et
al., 1985; Ohya and Sperelakis, 1989) with the same patch-clamp
amplifier used in the whole-cell experiments. In brief, after making
cell-attached patch with recording pipette, one end of the cell was
mechanically disrupted using another glass pipette containing the
Results
Shortening of APD. Action potentials in young (days
10–18) rat ventricular cells were recorded in current-clamp
at a stimulation rate of 0.1 Hz (fig. 1). Bath application of 10
mM levosimendan produced shortening of APD with slight
hyperpolarization of RP (table 1). This effect usually started
1 to 5 min after exposure to levosimendan and reached a
maximum within 1 to 2 min from the onset of the response.
As shown in traces d and e of figure 1A, the cells exposed to
10 mM levosimendan became inexcitable at its maximal effect. This effect could be reversed after washing out of the
levosimendan (fig. 1A, right). Levosimendan (3 mM) also abbreviated the APD (similar to trace c) but did not abolish
excitability [i.e., the overshoot of the action potential (above
the zero potential) remained]. These effects on the action
potentials are summarized in table 1.
Stimulation of KATP current. Superimposed current
traces evoked by voltage-clamp ramps are shown in figure
2A, and the time courses of the currents (measured at 0 and
2100 mV) are illustrated in figure 2B. The ramp pulses were
applied every 15 sec from 140 to 2100 mV (at 40 mV/sec).
The outward current was rapidly increased by levosimendan.
The average reversal potential (Vrev) was 276.3 6 0.7 mV
(n 5 5), which was close to the equilibrium potential for K1
(EK) value (283 mV) [EK 5 259 mV 3 log (140/5.4)], suggesting that most of the current stimulated by levosimendan
was a K1 current. This effect usually started 1 to 5 min after
exposure to levosimendan and reached a maximum within 1
to 2 min from the onset of the response. Glibenclamide (1
mM), a relatively specific inhibitor of KATP channels, abolished the levosimendan-stimulated current (n 5 4), as shown
in figure 2. Therefore, the levosimendan-stimulated current
is similar to a KATP current.
Figure 3 gives the current-voltage relations for the currents produced by different doses of levosimendan (fig. 3A)
and the dose-response relation (fig. 3B). The maximal activated current at 0 mV was measured, and the difference
between the maximal-activated and control currents (before
application of levosimendan) was plotted against each concentration (fig. 3B). Data points were fitted to the Hill equation: current increase 5 [xnH/(xnH 1 EC50nH)] 3 Emax, where
Emax is the maximal stimulatory effect, nH is the Hill coeffi-
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bath solution. The tip of the patch electrode was coated with Sylgard
(Dow Corning, MI), and its resistance ranged from 5 to 8 MV when
it was filled with the following pipette (extracellular) solution (in
mM): KCl 150, MgCl2 0.5, CaCl2 1.8 and HEPES 5, pH adjusted to
7.4 with KOH. The bath (intracellular) solution contained (in mM)
KCl 150, MgCl2 0.5, HEPES 5, and EGTA 1, pH adjusted to 7.3 with
KOH. Current signals were filtered at 1 or 2 kHz, sampled at 1 or 10
kHz and stored in a personal computer. Data recorded from only one
functioning channel with a sampling rate of 10 kHz and a cutoff
frequency of 2 kHz were used for the channel kinetic analysis (see fig.
5C). The storing of the digitized signals was carried out using AxoTape (version 2, Axon Instruments), and analysis was performed
with pCLAMP (version 6.02, Axon Instruments).
Drugs and chemicals. Levosimendan was a gift from OrionFarmos (Espoo, Finland). Various NDPs, nucleotide triphosphates
and glibenclamide were purchased from Sigma Chemical (St. Louis,
MO).
Data analysis. All data are given as mean 6 S.E.M. Statistical
significance was evaluated by Student’s paired t test, and P , .05
was considered to be significant.
378
Yokoshiki et al.
appeared within 30 sec after the open cell-attached patches
were made by mechanical disruption of one end of the cell in
bath solution containing no ATP. The opening of the channels
appeared in bursts, and the flickerings within bursts decreased when the membrane was depolarized, as previously
reported (Kakei et al., 1985; Tung and Kurachi, 1991). The
current-voltage relation for this channel is shown in figure
5B. The conductance of the unitary inward current was 80 pS
(n 5 4), and a slight inward rectification was observed at
positive membrane potentials. The open-time histograms (at
280 mV) was fitted by a single exponential curve with a time
constant of 1.3 msec (fig. 5C). The conductance and kinetic
properties of this K1 channel are similar to those of the KATP
channel previously reported for cardiac cells (Kakei et al.,
1985; Tung and Kurachi, 1991).
The effects of levosimendan on these KATP channels were
examined in various conditions. Membrane potential was
held at 280 mV. Bath application of 10 mM levosimendan
stimulated the KATP channels, which had been inhibited by
0.3 mM ATP, and the channel activity was abolished by 10
Fig. 5. Conductance and kinetic properties of the KATP channel currents in open cell-attached patch. A, KATP channel currents recorded in open
cell-attached patch (symmetrical 150 mM K1) from young rat ventricular cells at various membrane potentials are given. Bath solution contained
no ATP. B, Current-voltage relationship of the KATP channel. The slope conductance of unitary inward current was 80 pS (n 5 4). C, Open-time
histograms of the unitary KATP channel currents at 280 mV. The histogram at 280 mV was fitted by a single exponential curve with a time constant
of 1.3 msec. Data were filtered at 2 kHz and sampled at 10 kHz.
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cient and EC50 is the concentration for half-maximal effect.
The EC50 value was 4.7 mM, and the nH value was 2.3. The
Emax was calculated to be 46.8 pA/pF.
No effect on ICa(L). ICa(L) was recorded under the condition in which all K1 currents were blocked by extracellular
TEA and 4-AP and intracellular Cs1. Fast Na1 current was
also blocked by substitution of extracellular Na1 with TEA.
ICa(L) was evoked every 15 sec by a test potential to 110 mV
from a holding potential of 240 mV. As shown in figure 4,
bath application of 10 mM levosimendan had no effect on
ICa(L); peak current amplitude after a 5-min exposure to
levosimendan was 96.5 6 0.8% of control (n 5 4). In contrast,
isoproterenol (1 mM) markedly stimulated ICa(L) (204 6 16%
of control). Lower doses of levosimendan [0.2 mM (n 5 5) and
1 mM (n 5 4)] also had no effect on ICa(L) (0.2 mM, 94.4 6 1.8%
of control; 1 mM, 97.7 6 1.0% of control).
Stimulation of KATP channels. Single-channel K1 currents were recorded in the open cell-attached patch mode
(symmetrical 150 mM K1) from rat ventricular cells at various membrane potentials (fig. 5A). The K1 channel activities
Vol. 283
Levosimendan Activates K1 Channel
1997
mM glibenclamide (fig. 6). However, KATP channels exhibiting high spontaneous activity in ATP-free solution were not
stimulated by levosimendan (fig. 7A). Levosimendan also
could not stimulate KATP channels that had rundown (i.e.,
channel activity almost disappeared) in ATP-free solution
(fig. 7B). However, levosimendan could stimulate rundown
KATP channels that were reactivated by 50 to 100 mM ADP
(fig. 8A) or 3 mM UDP (fig. 8B). In three of 13 patches tested,
these NDPs did not stimulate the rundown channels,
whereas subsequent application of levosimendan reactivated
the channels. Although KATP channels inhibited by 0.5 mM
AMP-PNP, a nonhydrolyzable ATP analog, were not stimulated by levosimendan (fig. 9A), the channels were activated
in the presence of 30 to 50 mM ADP (fig. 9B). A summary of
the relative open probability values of the KATP channels is
given in table 2.
In the present study, we demonstrated that the new Ca11sensitizing positive inotropic agent levosimendan activated
KATP channel currents in rat ventricular myocytes under
physiological conditions. In contrast, levosimendan (0.2–10
mM) had no effect on ICa(L). Single-channel recordings (in
open cell-attached patches) showed that levosimendan stimulated the KATP channels in the presence of ATP, whereas
stimulation was not observed in channels exhibiting a high
degree of spontaneous activity in ATP-free condition. Levosimendan also could not stimulate rundown KATP channels.
Although the channels inhibited by AMP-PNP, a nonhydrolyzable ATP analog, could not be activated by levosimendan,
the presence of NDPs restored the ability of levosimendan to
potentiate the channel activity. Furthermore, levosimendan
synergistically stimulated the rundown KATP channels when
NDPs were present.
In addition to the Ca11-sensitizing positive inotropic effect, levosimendan (0.1–100 mM) increased the cAMP level in
guinea pig cardiomyocytes (Edes et al., 1995; Boknik et al.,
1997). On the basis of these findings, levosimendan was
suggested to produce PDE inhibition, and levosimendan was
found to stimulate ICa(L) (Virag et al., 1996; Boknik et al.,
1997). On the contrary, in the present study, ICa(L) of rat
cardiomyocytes was not affected by levosimendan (0.2–10
mM). This discrepancy might arise from the different species
studied, which have different PDE isozymes (Polson, 1996).
For example, in isolated rat ventricular cells, several selective PDE III inhibitors (to which milrinone belongs) had little
or no effect on cAMP level, but a PDE IV inhibitor, Ro
20-1724, had a potent action in increasing cAMP (Kelso et al.,
1993). Thus, cAMP could still be hydrolyzed by the PDE IV
isozyme (shown to be abundant in rat ventricle; Bode et al.,
1991), even when the PDE III was inhibited. In addition, the
basal level of cAMP could be different in the two cases.
The activation of KATP channels by levosimendan is not
due to any metabolic impairment of the cell because levosimendan (30 mM) did not change the activity of the myofibrillar ATPase that consumes ATP for cross-bridge cycling
(Haikala et al., 1995b). Furthermore, ICa(L), which is metabolically regulated in several tissues (Sperelakis and Schneider, 1976; Irisawa and Kokubun, 1983; O’Rourke et al., 1992;
Yokoshiki et al., 1997), was not affected by levosimendan
(0.2–10 mM) in the present study.
The Ca11-sensitizing effect of levosimendan in skinned
Fig. 6. Stimulation of KATP channels by levosimendan in the presence of ATP. After formation of open-cell attached patches in ATP-free solution,
spontaneous variation of KATP channel activities was observed. KATP channel activities were inhibited by 0.3 mM ATP in the bath. Levosimendan
(10 mM) activated the KATP channels that had been inhibited by ATP, and the channels were abolished by the bath application of 10 mM
glibenclamide (Glib). Membrane potential (Vm) was held at 280 mV. Top trace, sampling rate of 0.25 kHz; expanded traces, 10 kHz. The cutoff
frequency of the filter was 2 kHz.
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Discussion
379
380
Yokoshiki et al.
Vol. 283
fiber was reported to increase dose-dependently from 0.03 to
10 mM (Edes et al., 1995; Haikala et al., 1995b). However, in
intact muscles (guinea pig papillary muscles), high doses (.1
mM or 10 mM) of levosimendan produced lesser stimulation or
even negative inotropy (Haikala et al., 1995b; Boknik et al.,
1997). Therefore, stimulation of KATP channels observed in
the present study may account for both the lesser stimulation
(or negative inotropy) at the higher doses and the vasodilatory action.
Although levosimendan produced PDE inhibition at the
doses of 0.1 to 0.3 mM (Edes et al., 1995), this drug prevented
ventricular fibrillation induced by ischemia-reperfusion in
anesthetized dogs (Kaszala et al., 1994), thought to be due to
Ca11 overload and subsequent triggered activity (Janse and
Wit, 1989). Levosimendan also reduced ischemic damage induced by ligation of the coronary artery in isolated rabbit
hearts (Rump et al., 1994a, 1994b), an effect observed even at
0.1 mM (Rump et al., 1994b). At this concentration, the increase in the coronary flow was relatively small, and the
authors speculated that factors beyond coronary dilation
might contribute to the anti-ischemic effects of levosimendan. On the other hand, opening of KATP channels is generally considered to be cardioprotective against ischemia-related events (Hearse, 1995). Therefore, our results may in
part account for the antiarrhythmic and anti-ischemic effects
reported in the previous studies (Kaszala et al., 1994; Rump
et al., 1994a, 1994b) because activation of KATP channels by
levosimendan would likely occur in ischemic regions in which
intracellular ADP concentration ([ADP]i) is increased and
intracellular ATP concentration ([ATP]i) is decreased.
The therapeutic concentration of levosimendan as a positive inotropic agent is thought to be ;50 ng/ml (i.e., 0.18 mM)
in patients with left ventricular dysfunction (Lilleberg et al.,
1995; Sandell et al., 1995). This value is lower than that
required for KCO action of levosimendan in the present
study. Therefore, levosimendan at this therapeutic concentration may produce little or no activation of KATP channels
in human hearts under normal conditions. However, levosimendan might more readily activate the KATP channels in
ischemic myocardium because the ratio of [ADP]i to [ATP]i
([ADP]i/[ATP]i) may be increased, as mentioned above. Because the synergistic action of levosimendan with ADP could
be observed at low concentrations (30 –50 mM ADP), levosimendan might protect ischemic myocardium more effectively
than nicorandil, which required higher ADP concentrations
[0.5 mM (Shen et al., 1991) to 1 mM (Jahangir et al., 1994)]
for activation of KATP channels. However, one must be careful in extrapolating these experimental results to the clinical
setting.
The site of action of KCOs remains unclear (Henry and
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Fig. 7. Lack of effect of levosimendan on KATP channels in the absence of ATP. KATP channels exhibiting high spontaneous activity (A) or run-down
(B) in ATP-free solution were not stimulated by 10 mM levosimendan.
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Levosimendan Activates K1 Channel
381
Escande, 1994). It might be simplest to assume that there is
competition between KCOs and ATP at the ATP-binding site
that closes the channel. The parallel shift produced by KCOs
in the concentration-response curve for ATP inhibition of the
channels (Thuringer and Escande, 1989; Nakayama et al.,
1990; Ripoll et al., 1990) may support this hypothesis. However, the possibility exists that ATP and KCOs act at different sites with opposing effects on the channel. Because levosimendan was not able to stimulate the channel that had
been inhibited by AMP-PNP, the competition hypothesis at
the ATP-binding site (inhibitory) would not account for the
K1 channel-opening action of levosimendan.
A synergistic activation of KATP channels by the KCO drug
diazoxide and ADP was reported in pancreatic b cells (Larrson et al., 1993). The authors concluded that channel stimulation by diazoxide is dependent on the binding of Mg-ADP to
a cytosolic regulatory constituent of the channel. Therefore,
the synergistic action with NDPs might be shared by other
KCOs as in the case of levosimendan. In addition, the functional classification of KCOs has recently been postulated
based on their mechanisms of action (Terzic et al., 1995).
According to their model, KCOs were classified into three
types because they seem to have at least three sites of action:
(1) the ATP-binding unit (inhibitory), (2) the NDP-binding
unit (stimulatory) and (3) the transducer unit. Nicorandil, a
NDP-acting drug (“type 3” in their classification), is considered to act in the presence of NDP by both enhancing the
maximal channel activity and decreasing sensitivity of KATP
channels to inhibition by ATP (Shen et al., 1991; Terzic et al.,
1995). With respect to mechanism of action, levosimendan
may act similarly to nicorandil. One difference is that activation of the channel by nicorandil was not observed in the
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Fig. 8. Levosimendan synergistically activates run-down KATP channels with nucleotide diphosphates (NDPs). After run-down of KATP channels,
100 mM ADP (A) and 3 mM UDP (B) could stimulate the channels. Subsequent application of levosimendan (10 mM) produced further stimulation
of the channels.
382
Yokoshiki et al.
Vol. 283
Relative Po values were calculated by dividing the Po value by that obtained in
ATP-free solution. The Po values were obtained from 45 to 60 sec of continuous
recordings. Rats were 10 to 18 days old.
Relative Po
n
ATP-free
ATP 0.3 mM
AMP-PNP 0.5 mM
AMP-PNP 1 ADP 30–50 mM
Rundown
Stimulation of activity after
rundown
ADP 50–100 mM
UDP 3 mM
Control
Levosimendan 10 mM
6
6
5
6
5
1.00 6 0.00
0.10 6 0.06
0.03 6 0.02
0.01 6 0.01
0.00 6 0.00
0.87 6 0.25
1.45 6 0.27a
0.01 6 0.01
0.20 6 0.07a
0.00 6 0.00
7
6
0.28 6 0.17
0.34 6 0.15
1.08 6 0.31a
1.17 6 0.25a
Po, open probability; n, number of patches tested.
a
P , .05 vs. control (before application of levosimendan).
could stimulate the channel in the presence of ATP alone,
that is, in the absence of added ADP; however, some endogenous ADP was undoubtedly present. Because levosimendan
could antagonize the channel inhibition by AMP-PNP if ADP
(30 –50 mM) were present, it might be possible that levosimendan synergistically activated the channels with a small
amount of ADP generated by ATP hydrolysis in the vicinity of
the channels. For example, hydrolysis of ATP occurs continuously within the sarcolemmal membrane in association
with cellular activities such as Na1-K1 pump, Ca11-ATPase
(Sperelakis, 1995) and actin filament assembly (Stossel,
1993) to maintain homeostasis.
In summary, levosimendan shortened APD in rat ventricular cells, presumably through opening of KATP channels.
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 1, 2016
Fig. 9. Effect of levosimendan on KATP channels in the presence of AMP-PNP, a nonhydrolyzable ATP analog. A, Levosimendan (10 mM) had no
effect on KATP channels inhibited by 0.5 mM AMP-PNP. However, subsequent application of 10 mM levosimendan in the presence of 0.3 mM ATP
was capable of stimulating the channels. B, KATP channels inhibited by AMP-PNP were activated by levosimendan when ADP (30 –50 mM) was
present in the bath solution.
TABLE 2
presence of ATP (0.5 mM) unless ADP (0.5 mM) was also
Summary of effects of levosimendan on KATP channels in rat
present (Shen et al., 1991). On the other hand, levosimendan
ventricular cells recorded in open-cell attached patches
1997
This stimulation of KATP channels by levosimendan may
result from the synergistic action with NDPs.
Acknowledgments
We wish to acknowledge Mrs. Sheila Blanck for excellent technical
help and Mr. George Sfyris for setting up the computer system for
the single-channel recording.
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