Identification of Cardiotonic Sodium Channel Activators by Potassium Depolarization in Isolated Guinea-pig Atria
HEINER BERTHOLD, GRUNTERSCHOLTYSIK,AND ANDREAS SCHAAD
The inotropic actions of various drugs known to increase force of contraction in isolated mammalian cardiac muscle were investigated in electrically driven (1 Hz) guinea-pig left atria under both normal [K+], (4.7 mM) and high [K+], (22 mM). Under normal [K+], a concentration-dependent increase in force of contraction could be confirmed with the p-adrenoceptor agonist, isoprenaline, the cyclase activator, forskolin, the inhibitors of the cyclic AMP-phosphodiesterase (PDE), amrinone, IBMX, and OPC 8212, the Na+ channel activators, DPI 201-106, SDZ 210-921, veratridine, and ATX II, the Na’-ionophore monensin, the inhibitor of Nat/K+-ATPase, ouabain, and the Ca2+ channel activators, Bay K 8644, CCP 28 H 392, and SDZ 202-791. Partial depolarization of the muscle preparations by increasing [K+], in the organ bath to 22 mM completely abolished the positive inotropic action of the Na+ channel-activating drugs. In contrast, the effects of the other compounds were still present, although changes in the maximal force development were observed. The efficacy of the PDE inhibitors amrinone and IBMX were slightly increased; the maximal effects of isoprenaline, monensin, forskolin, and OPC 8212 were unchanged; the effect of ouabain decreased to about half maximal values; while the efficacy of the Ca2+ channel activators were either unchanged (CGP 28 392) or decreased (Bay K 8644 and SDZ 202-791). The results suggest that inactivation of cardiac fast Na+ channels by partially depolarizing isolated, electrically driven atria is a suitable model to distinguish between cardiotonic agents acting through activation of Na+ channels and those with other mechanisms of action. Key Words: Cardiotonic agents; Sodium channel activators; DPI 201-206; lated guinea-pig atria
Iso-
INTRODUCTION Drugs that increase myocardial contractility are a heterogeneous group of compounds with widely differing chemical structures and mechanisms of action. Besides the digitalis glycosides, the P-adrenoceptor agonists, the stimulants of cyclic adenosine monophosphate (AMP) formation, the inhibitors of cyclic AMP-phosphodiesterase (PDE), ionophores and the activators of Ca” channels, synthetic acti-
From Preclinical Research, Sandoz Ltd., Base1and Institute of Veterinary Pharmacology, University of Berne, Switzerland. Address reprint requests to: Professor G. Scholtysik, University of Berne, Institute of Veterinary Pharmacology, CH-3012 Berne, Switzerland. Received July 1989; revised and accepted February 1990. 121 journal
of Pharmacological
Q 1990 Elsevier
Methods
SciencePublishing
24, 121-13s
(1990
Co., Inc., 655 Avenue of the Americas, NW
York, NY 10010
122
H. Berthold et al. vators of cardiac Na+ channels new class of positive inotropic of the prototype
have recently been shown to represent an entirely agents (Scholtysik, 1989). The mechanism of action
of this class, DPI 201-106,
includes
prolongation
of the open
state
of Na+ channels (Buggisch et al., 1985) and a sensitization of contractile proteins to Ca2+ (Hajjar et al., 1988) and is independent from changes in cyclic AMP levels either
as a consequence
from inhibition
of PDE inhibition
of the Na’/K’-ATPase
or of P-adrenoceptor
(Scholtysik
activity is closely related with the activation
of myocardial
of intracellular Na+ traction (Gwathmey
increase
With
the aim to evaluate
tivating iments
levels with secondary et al., 1988).
mechanisms, performed
involving
in isolated
the above
pared their effects in normal
currents
to recognize
our accumulated
atria with various
mechanisms
polarized
Na’
and
and increase
in free Ca2+ available
test procedure
guinea-pig
mentioned
As the result, the described compounds and, therefore, mechanisms
a simple
we have now surveyed
stimulation
et al., 1985). Thus, its cardiotonic
of action.
and in partially
Na+
for con-
channel
results from
positive
inotropic
For this purpose,
Kc-depolarized
ac-
experdrugs
we com-
preparations.
method selectively identifies the Na+ channel activating is suitable to distinguish them from agents with other
of action.
METHODS
Isometric Force Measurement Guinea-pigs Their tically
of either
hearts were
of Isolated Atria
sex weighing
rapidly excised,
in a IOO-mL two-chambered
250-500
g were
and left atria were organ
bath.
stunned dissected
Internal
and exsanguinated. and suspended
circulation
ver-
was maintained
by continuous gassing with 95% O2 and 5% CO,. The atria were preloaded with IO mN, and isometric force development was measured by connecting the preparations at one end to a Statham
UC3 transducer
and recording
the contractions
on a Schwar-
zer polygraph.
Electrical Stimulation
and Potassium Depolarization
At the beginning of the experiment, a modified Krebs-Henseleit following composition was used (mM): NaCl 118, KCI 4.7, MgS04
solution of the 1.2, CaC12 2.5,
KH2P04 1.2, NaHC03 25, glucose IO. The temperature was kept constant at 31°C. The atria were stimulated at 1 Hz either via bipolar platinum-iridium punctate elecat the basal part ([K+l,
trodes
placed
ulated
by use of platinum
4.7 mM),
plate electrodes
or they were
([K+l,
22 mM).
electrically Square
wave
field stimpulses of
3-ms duration and 50 mA were generated by an HSE type C 215 stimulator (Hugo Sachs Elektronik, Freiburg, West Germany). For partial depolarization, [K+], in the organ bath was increased from 4.7 mM to 22 mM. The changes in osmolarity were not corrected, since they are reported to have no influence on force of contraction (Bachmaier et al., 1985). Since under these conditions, the atria ceased to respond to electrical stimulation, the stimuli were increased to 5 ms and 100 mA, and a new steady state was reached after 30 min. Figure 1 shows the cessation of the contraction response to electrical stimuli shortly after increasing [K+l, to 22 mM. Increasing
Cardiotonic Na+ Channel Activators
4.7
7x7
22 mM [K+]a
I lHz3ms50mA
lHz5mslOOmA I
f7'
t25
t30
t45
FIGURE 1. Representative tracing of the contractile force development by a driven guineapig left atrium before (t,) and after K+ depolarization over a period of 45 min. For experimental detail, see text.
the stimulus intensity restores the response, and baseline force of contraction is comparable to that obtained with [K+l, 4.7 mM. Each muscle was used for one concentration-response curve only. Drugs and Concentration-response
Curves
After an equilibration period of at least 60 min in normal Krebs-Henseleit solution and of 30 min after K+-depolarization, concentration-response curves were established by cumulatively increasing the drug concentrations in the organ bath. Measurements were made at each concentration step when steady state conditions were reached. The concentrations tested can be seen in Table 1. The following drugs were used in the experiments: isoprenaline sulphate (Sigma), DPI 201-106 ((S)-( -)-4-[3-(4-diph.enylmethyl-l-piperazinyl)-2-hydroxypropoxy)IH-indole-2-carbonitrile; Sandoz), SDZ 210-921 f(R)-(+)-4-[3-(4-diphenylmethyl-lpiperazinyl)-2-hydroxypropylaminollH-indole-2-carbonitrile; Sandoz), veratridine (Sigma), ATX II (A nemonia sulcata toxin II; Ferring), amrinone (synthetized at Sandoz), IBMX (3-isobutyl-I-methylxanthine; Janssen), OPC 8212 (3,4-dihydro-6-[4-(3,4dimethoxybenzoyl)-l-piperazinyll-2-(lH)-quinolinone; synthetized at Sandoz), Bay K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate; synthetized at Sandoz), CGP 28 392 (4-(2-difluoromethoxyphenyh-2-methyl-5-oxo-1 ,4,5,7-tetrahydro-furo-[3,4-blpyridine-3-carboxylic acid ethyl ester; Ciba-Geigy), SDZ 202-791 (4-2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6dimethyl-5-nitro-3-pyridine carboxylatecarbonic (Sandoz), Ouabain (synthetized at Sandoz), monensin (Serva). Isoprenaline, veratridine, monensin and, ouabain were dissolved in distilled water; DPI 201-106, amrinone, and OPC 8212 were dissolved in N-methyl-2-pyrrolidone at acidic pH values; SDZ 201-921, IBMX, Bay K 8644, CGP 28 392, and SDZ 202-791 were dissolved in a few drops of EtOH and further diluted in distilled water.
123
channel
DPI 201-106
Sodium
OPC 8212
IBMX
Amrinone
Phosphodiesterase
Forskolin
CAMP formation
lsoprenaline
P-Adrenoceptor
DRUG
activators
1.89
6
1.36
*
2.08
2 1.66
”
-+ 0.65
2 1.81
1.49
9.7 2 0.41
13.3
11.1
6
5
10.8
13.3
6
4
16.7
9.2 *
6
6
6 2 0.81
8.3 -t 1.54 11.0
a.7 2 0.71
6
+ 1.83
2
6
9.8 11.0
4
(mN)
BASELINE FC
6
inhibitors
stimulant
agonist
ATRIA
NUMBER OF
._ .
+71 +81
l-100
l-loo
-11
0.1-I
-
+55
0.1-10
+66
+35
I-100
3-100
+31 +81
l-100
10-100
+143 +115
0.001-1
+99
21
33
7’
5
19*
7
2 3**
? 15
f
2 10
t
*
f
2 6
*
2 22
*
+80’25
BASELINE)
FC (% CHANGE FROM
0.01
1
0.13
0.051 n.d.
*
13 ? 3.2**
100 1
28 5 3.9
5 0.45
8
7.1
2 2.9
-+ 0.008
14 f 2.4
0.0021 2 0.028
t
+ 0.0062
18 t
8.3
0.016
0.12
0.0044
(PM)
FC (EGO)
._
MAXIMAL INCREASE IN
PRODUCE HALF-
CONCENTRATION TO
100
30
100
100
100
1
1
0.1
0.1
(KM)
INCREASE IN FC
PRODUCE MAXIMAL
CONCENTRATION TO
in Electrically Driven
MAXIMAL INCREASE IN
0.001-1
0.001-1
0.001-0.3
(PM)
RANGE TESTED
CONCENTRATION
TABLE 1 Results of Experiments Performed with Positive lnotropic Compounds Isolated Guinea-pig left Atria
channel
The asterisks
FC = force
1.54
*
11.7
*
1.90
= no determination.
+85 +1
0.01-3 0.1-I
+100 f44 +53 +44
0.001-1 0.001-1 0.1-30 0.1-10
between
normal
Krebs-Henseleit
f44
normal
+I06
0.01-I
+48
IO**
9
14**
4.7 mM), and partially
([K’],
polarized
solution
+ 11
-I- 8
*
-r- 11
2 5**
? 20
*
+97 f
” 15 5 4***
0.3-3 0.01-10
13
4***
2 9***
f
t
? 7
0.1-3
0.003-0.03
+98 -6
+2
0.01-0.3
0.001-0.1
f56
0.01-I
differences
with
significant
the data obtained statistically
n.d.
denote
represents
9.0
6
2 0.90
0.50
9.9
10
*
2 1.61
10.8
7 13.8
? 1.69
12.7
6
6
? 1.46
10.1
6
1.64
‘- 2.00
12.5
2 2.13
6
11.5
1.09
6
4
of contraction;
* p < 0.05. ** p < 0.01. *** p < 0.001.
to 22 mM.
!I 1.30
11.0
7 9.0 *
f
10.3
6
6
f
10.3
6
1.09
? 0.97
13.8
IO
activators
inhibitor
The first row always
CGP 28 392
Bay K 8644
SDZ 202-791
Calcium
Ouabain
Na+/K+-ATPase
ATX II
Veratridine
SDZ 21 O-921
0.16
0.46
0.05
0.0043***
depolarized
preparations.
increasing
? 0.083 the second
2 0.091 0.48 3
[K’l,
-c 0.0076
2 0.0031
*
+ 0.036
2 0.1
0.56
0.012
0.014
0.026
row after
5 0.0014 n.d. 0.4 *
0.011
n.d.
10
0.3
1
0.3
3
3
3
0.03
0.03
0.24
2 0.035
1
1
2 0.0081 n.d.
0.036
0.3
0.3
126
H. Berthold et al. Data Analysis and Statistics The following parameters of the isometric contraction curve were evaluated: peak force of contraction (IX,,,) and half-maximally effective concentrations to exert the positive inotropic effect (EC&. Force of contraction data are presented as mean (+ SEM) percent change from the respective baseline value. The number of atria in each experimental group can be seen in Table 1. Comparisons between the maximal increase in FC in normal and partially depolarized atria were performed using unpaired two-tailed t tests. Concentration-response curves between no response and maximal response were fitted using the Nelder-Mead simplex minimization method followed by the Marquardt-Levenberg method according to the equation f(x) = E - E/(1 + (X/EC&~), where E is the maximal response and P’ is the slope (Hill coefficient). The obtained ECsOvalues were compared using unpaired two-tailed t tests. RESULTS Stability of K+-depolarized
Preparations
Stability of partially depolarized atria was confirmed over 45 min in six control experiments. At the end of this period, the baseline values of force of contraction were comparable to those of normal polarized atria. The new steady state was reached after 30 min (see Figure 1). The solvent N-methyl-2-pyrrolidone used to dissolve DPI 201-106, amrinone and OPC 8212 was ineffective in guinea-pig myocardium both at normal and high potassium (Scholtysik et al., 1985). Contractility
in Normal and Partially Depolarized
Preparations
As can be seen in Table 1, the force of contraction baseline values were slightly smaller in partially depolarized in comparison to normal atria. The differences were not statistically significant in the individual respective pairs of experimental groups. However, taking all experiments together, there was an overall significant difference (p = 0.029) between normal (11.7 -+ 0.41 mN; N = 74) and partially depolarized atria (10.4 ? 0.41 mN; N = 87). Comparison
of Drug Effects in Normal and Partially Depolarized
Atria
The results of compounds involving cyclic AMP in their mechanism of inotropic action are shown in Figure 2. The maximal positive inotropic effects and the EC5,,
FIGURE 2. The effects of the B-adrenoceptor agonist isoprenaline (a), the stimulant of cyclic AMP formation, forskolin (b), and of the cyclic AMP phosphodiesterase inhibitors amrinone (c), IBMX (d), and OPC 8212 (e) on isometric force development in electrically stimulated isolated guinea-pig left atria. Solid lines represent preparations in normal [K+l, (4.7 mM) and broken lines are partially depolarized preparations ([K+l, 22 mM). All responses are expressed as mean change (%) of baseline force of contraction (for absolute values, see Table 1). The vertical lines indicate the SE mean; n = 4-6. The concentration-response curves were established by cumulatively increasing the drug concentrations.
Cardiotonic Na’ FCA% 120 a
1
T
loo-
_______
80-
i FC A% 1601 b
-log [Isoprenaline]
M T
9
7 8 -log (Forskolin] M
FC A% IOOc
6
806040-
6 FC A% 801 d
5 -log [Amrinone]
4 M _+_-_-___
60-
/‘-,
6
5 -log [IBMX] M
4
6
5 -log [OFT 82121 M
4
FC A% 1001 a 806040-
Channel Activators
127
128
H. Berthold et al.
values of isoprenaline and of forskolin were not different in normal and in partially depolarized atria (Table 1). The cyclic AMP phosphodiesterase inhibitors had maximal positive inotropic effects between +31% and +81% (range order: OPC 8212 > IBMX = amrinone) with E&values between 8 and 28 ~.LM(range order: amrinone > IBMX > OPC 8212). In partially depolarized atria, FL,,, for amrinone and IBMX was significantly increased and for OPC 8212 almost unchanged. Figure 3 shows the concentration-response curves of the Na’ channel activators. Under normal [K+l,, these drugs exerted maximal positive inotropic effects between +55% and +98% mean increase in force of contraction with the range order ATX II > veratridine > SDZ 210-921 = DPI 201-106. The molar potency was in the range order ATX II > SDZ 210-921 > DPI 201-106 > veratridine with ECso values between 11 and 240 nM (Table 1). The positive inotropic effect was completely
FC A%
FC A% 807
80
a
1
b
60-
-20
’
I
I
I
7
6
5
-20
’
-20
I 6
FCA% c
( 8
I 7 -log [SDZ 210-9211 M
-log [DPI 201-1061 M FC A% 1001
I 8
I 7 -log [Veratridine]
M
1
120
I 6
-20
1
I 9
d T
1 8
, 7
-log [ATX II] M
FIGURE 3. The effects of the Naf channel activators DPI 201-106 (a), SDZ 210-921 (b), veratridine (c), and ATX II (d) on isometric force development in electrically stimulated isolated guinea-pig left atria. Solid lines represent preparations in normal [K+], (4.7 mM) and broken lines are partially depolarized preparations ([K’], 22 mM). All responses are expressed as mean change (%) of baseline force of contraction (for absolute values, see Table 1). The vertical lines indicate the SE mean; n = 4-10. The concentration-response curves were established by cumulatively increasing the drug concentrations.
Cardiotonic Na+ Channel Activators FC A% 120-
FC A%
a
b
80
loo60 1
80-
T
60-
9
8
7
6 -log [CGP 28 3921 M
-log [Bay K 86441 M FC A%
FC A% 1201
T
c
1201
d
7
-log [SD2 202-7911 M
6
5
-log [Ouabain] M
FIGURE 4. The effects of the Ca*+ channel activators Bay K 8844 (a), CGP 28 392 (b) and SDZ 202-791 (c) and of the inhibitor of the Na+/K+-ATPase, ouabain (d) on isometric force development in electrically stimulated isolated guinea-pig left atria. Solid lines represent preparations in normal [K+], (4.7 mM) and broken lines are partially depolarized preparations ([K+], 22 mM). All responses are expressed as mean change (%) of baseline force of contraction (for absolute values, see Table 1). The vertical lines indicate the SE mean; n = 410. The concentration-response curves were established by cumulatively increasing the drug concentrations.
abolished in partially depolarized preparations, and, therefore, the computation of best fit concentration-response curves was not feasible. Comparisons with the maximal response in normal atria were performed using the corresponding drug concentrations, and they were found to be highly significant. Figure 4 shows the results of the Ca*’ channel activators and of ouabain. The Ca2+ channel activators showed positive inotropic effects between +53% and +106% (range order: SDZ 202-791 = Bay K 8644 > CGP 28 392) with ECso values between 14 and 560 nM (range order: Bay K 8644 > SDZ 202-791 > CGP 28 392). Partial depolarization decreased the effects of these drugs, although this was only significant for Bay K 8644 and SDZ 202-791. The efficacy of ouabain (+97 + 9%)
129
130
H. Berthold et al.
was significantly decreased to f48 leaving the EC5,, value unaltered.
‘_ 10% by partially depolarizing
Effect of Catecholamine-depletion IBMX, Bay K 8644, and Monensin
by Reserpine Pretreatment
the preparations,
on the Action of
Drugs acting via a mechanism in which CAMP is involved exert stronger cardiotonic effects in high potassium at field stimulated atria when compared to normal potassium and local depolarization (Figure 2). In order to evaluate the possible influence of the release of autonomic neurotransmitters caused by field stimulation on force of contraction (Blinks, 19661, we pretreated animals with a single i.p. injection of 3 mg/kg reserpine 18 hr prior to sacrifice. This dose is known to efficiently deplete endogenous catecholamine stores. IBMX
In a separate series of experiments in these reserpinized animals, concentrationresponse curves for the PDE-inhibitor IBMX (l-100 FM) were established in normal and in partial depolarized preparations, both electrically field stimulated. Baseline force of contraction values were 12.4 -+ 0.7 mN ([K’l, 4.7 mM; N = 6) and 10.8 + 0.7 mM ([K+l, 22 mM; N = 6), respectively. The obtained ECSOvalues were 3.9 + 0.52 FM and 1.7 & 0.13 FM and FC,,, values at 30 PM IBMX were +94 t 11% and +85 ? 13%, respectively. These data were not significantly different and demonstrate that the increased effectiveness of IBMX in field stimulated depolarized atria (Figure 2d) was due to stimulation induced catecholamine liberation.
FC A% 1001
i
4 -log [Monensin] M
FIGURE 5. The effects of the Na+ ionophore monensin on isometric force development in electrically field stimulated isolated left atria from reserpinized guinea-pigs. Solid line represents preparations in normal [K+], (4.7 mM), and broken line shows partially depolarized preparations ([K+ I, 22 mM). Responses are expressed as means + SE mean percent changes from the following baseline values: normal potassium 11.4 + 1.29 mN, n = 7; high potassium 10.33 f 1.09 mN, n = 6.
Cardiotonic Na+ Channel Activators
Bay K 8644 In reserpinized field stimulated sod partially depolarized atria (N = 5), Bay K 8644 increased FC from a pretreatment value of 4.2 + 0.5 mN by 18.9 + 5.6, 65.9 + 8.4, and 114.2 + 12.9% after 0.01,0.03, and 0.1 FM, respectively. This result demonstrates that the positive inotropic action of Bay K 8644 in nonreserpinized atria (Figure 4a) was not elicited by stimulation induced catecholamine liberation but by direct Ca2+ channel activation. Monensin Monensin is a Na+ ionophore that independently from ionic channels loads the cell with Na+ (Sutko et al., 1977) and consequently increases cytosolic Ca2+. Monensin leads to virtually superimposable concentration-response curves in reserpinized field stimulated atria at normal and high potassium conditions (Figure 5). This observation demonstrates the effectiveness of the Na+ ionophore independent from Na’ channels that is detectable in reserpinized atria. DISCUSSION In the present experiments, the positive inotropic effects of compounds with different mechanisms of action have been investigated under both normal and elevated [K+],. The observed increases in contractility induced by the tested positive inotropic drugs under normal [K+], confirm general pharmacological experience. As the main result, the positive inotropic effects of the Na’ channel activators have been found to be completely abolished in K+-depolarized preparations. In contrast, the effects of the other investigated compounds were only slightly changed but still clearly present. Partial Depolarization
Slightly Decreases Baseline Force of Contraction
The basic tension development in partially depolarized atria was slightly smaller in some experimental groups, but these differences were not statistically significant. The overall difference between 11.7 +- 0.41 ([K+l, 4.7 mM) and 10.4 t 0.41 ([K+]], 22 mM) seems rather small and, therefore, cannot be claimed to exert a major influence on changes in the force development caused by test compounds in these preparations. The slightly decreased baseline force of contraction can be explained by the dependency of the state of the Ca2+ channel on the resting potential of the cell: In partially depolarized cells, the number of channels opening simultaneously is decreased, resulting in a smaller amplitude of the Ca2+ current (McDonald, 1982; Reuter, 1979). Na+ Channel Activators Become Ineffective Preparations
in Partially Depolarized
Two distinct inward membrane currents are responsible for excitation of the cardiac tissue, I,i and INa (Reuter, 1979). Partial depolarization of the membrane of the myocardial cell from about -90 mV at resting state to about -50 mV by increasing [K+l, to 22 mM inactivates the fast Na+ channels (Pappano, 1970). These cells can
131
132
H. Berthold et al. be externally
stimulated
to reach the threshold
of action
potential
ious toxins, like ATX II (Ravens, 1976) and veratridine (Horackova increase the Na’ permeability of the membrane of the cardiac thetic organic compounds been shown to represent
that are cardioselective activators a new class of positive inotropic
formation.
Var-
and Vassort, 1974), cell. Recently, syn-
of Na’ channels have agents, of which the
prototype is DPI 201-106 (Scholtysik, 1989; Scholtysik et al., 1985). The mechanism of action of DPI 201-106 includes prolongation of the open state of Na+ channels and a sensitization of contractile proteins to Ca2+ without involvement of cyclic AMP and Na+/K+-ATPase (Scholtysik et al., 1985). The Na’ channel activators DPI 201-106,
ATX II, and veratridine
used in the present
study bind to different
sites at
the cardiac Na+ channel, which are allosterically coupled (Scholtysik et al., 1986). The property they have in common is their ability to inhibit inactivation of the channels, and, therefore, the expected consequence of partial depolarization of the preparations
is loss of positive
inotropic
activity.
The results are in accordance
with qualitatively similar findings in K’-depolarized guinea-pig atria with DPI 201106 (Holck and Osterrieder, 1988) and with the cardiotonic striatoxin, a novel polypeptide from a marine snail, which is believed to act through Na+ channels (Ohizumi et al., 1988). The decrease in FC by DPI 201-106 rations in the present experiments may indicate properties
in partially additional
depolarized prepaCa*’ antagonistic
(Siegl et al., 1988).
Ouabain The
inhibitory
steroids
effect
is a well-known
of elevated
[K+l,
phenomenon,
on the inotropic which
is discussed
action
of cardioactive
in terms
of either
an
interference with drug binding and, on the other hand, as a result of its influence on the potential-dependent driving force for Nat entry (for review, see Reiter, 1981). Several reports showed that increased [K+l, inhibits receptor binding of cardiac glycosides in various membrane preparations (Akera et al., 1978; Erdmann and Schoner,
1973; Hansen,
1976). Bachmaier
et al. (1985) demonstrated
also that modi-
fication of other processes like increase in the ratio of Na+ efflux interference with Ca*+ elimination may be involved in the antagonism
to influx and of K+ against
the positive inotropic effects of glycosides. These authors found evidence that inhibition of drug binding by K+ caused about one-half of the antagonistic effect of K+. The highest [K+l, used in their study was 12 mM, and maximal increase in force of contraction was always achieved by increasing the concentration of ouabain. However, in the present study, the effect of ouabain at 22 mM [K+l, was only half of that seen with 4.7 mM [K+l,.
Ca’+ Channel Activators The dihydropyridine
derivative
Bay K 8644, which
is chemically
closely
related
to
the calcium channel antagonist nifedipine, has been shown to possess potent positive inotropic properties (Schramm et al., 1983). Bay K 8644 is thought to act as a Ca*+ channel activator, enhancing the slow inward current (Isi) by prolonging the mean open time of individual voltage gated calcium channels (Brown et al., 1984; Kokubun and Reuter, 1984). Cyclic AMP appears not to be involved in the mech-
Cardiotonic Na+ Channel Activators anism of action
investigated
in reserpinized
drugs like CGP 28 392 (Loutzenhiser
heart muscle
(BGhm et al., 1985). Other
et al., 1984) and SDZ 202-791
(Hof et al., 1985;
Kokubun et al., 1986) have been shown to have similar effects on divalent cation influx, increase in contractility (Freedman and Miller, 1984; Hess et al., 1984) and are selective Ca’ channel agonists also in depolarized heart muscle (Scholtysik and Schaad, 1988). From the results of the present experiments, the question arises why Bay K 8644 and SDZ 202-791 rations
than
under
normal
show weaker
activity
in partially
depolarized
prepa-
conditions. Sanguinetti and Kass (1984) have reported Ca*+ channel current in calf Purkinje fibers when the
that Bay K 8644 increases holding potential is more negative
than
-50
potential is positive to -40 mV. Additional activity have confirmed these observations
mV, but reduces
it when
the holding
experiments investigating the contractile and characterize Bay K 8644 as a partial
agonist. Similar effects of holding potential on the action of Bay K 8644 in single ventricular cells have been reported by Hess et al. (1984). These findings show that the effects
of Bay K 8644 on cardiac
membrane
potential
contractility
and might therefore
explain
is very dependent the decreased
on the cellular
activity of the com-
pound in partially depolarized atria. Kokubun et al. (1986) have shown that the Ca2+ channel activating effects of the (S)-enantiomer of SDZ 202-791 turn over into blocking effects at holding potential of positive to -20 mV. CGP 28 392 was also described as a partial Ca2+ channel agonist in potassium depolarized guinea-pig papillary muscles (Kamp creased activity fluence
et al., 1985). These circumstances may be responsible for the deof these compounds in depolarized preparations. However, an in-
of sympathetic
neurotransmitter
release as described
for Bay K 8644 (Mun-
dina et al., 1988) can be ruled out in the present experiments, did not change the effectiveness of Bay K 8644 in high [K+l,. Drugs with
since reserpinization
Effects on Cyclic AMP
In experiments using drugs with an influence on cyclic AMP, we observed greater values in FC,,, in partial depolarized atria after isoprenaline, amrinone, and IBMX or smaller ECso values after forskolin and OPC 8212. Since the results were obtained with punctate electrode stimulation in normal polarized preparations and field stimulation in partial depolarized preparations, it cannot be excluded that the difference is due to the more pronounced liberation of noradrenaline after field stimulation (Blinks, 1966). This hypothesis could be confirmed in a separate series of experiments with
the PDE inhibitor
IBMX,
where
no difference
in FC,,,
and ECso could
be seen between preparations of animals that had previously been reserpinized or not, indicating that endogenous catecholamines contribute to the increased efficacy. Moreover, the increase in [K’l, per se may contribute to slightly enhanced force development, as can be seen in Figure 1 shortly after changing the bathing solution
to high [K+],.
In conclusion, the positive inotropic effects of CAMP dependent drugs, of Ca2+ channel activators and of monensin are present after partial depolarization in contrast to the neutralization of the actions of Na’ channel activators, and, thus, these drugs can be identified The authors
gratefully
selectively
acknowledge
by means of the described
the skillful
technical
assistance
simple test procedure.
of Mme.
Michele
Bretz.
133
134
H. Berthold
et al.
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