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On the mechanism underlying the cobalt-induced inhibition of slow inward current in mammalian ventricular myocardium
3ournal of Molecular
and Cellular
Cardiology
(1980)
12, 1075-1090
On the Mechanism Underlying the Cobalt-induced Slow Inward Current in Mammalian Ventricular M. KOHLHARDT Physiologisches (Received
Institut
der Universitiit
29 January
1980,
AND
Freiburg,
Inhibition of Myocardium
K. HAAP D-78
accepted in revised
FreiburglBr.,
form
2 April
F.D.R. 1980)
M. KOHLHARDT AND K. Inhibition of Slow Inward
HAAP. On the Mechanism Underlying the Cobalt-induced Current in Mammalian Ventricular Myocardium. Journal of Molecular and Cellular CardioloD (1980) 12, 1075-1090. The effect of Cobalt (Co) ions on the slow inward current (Zsi) was studied in voltage clamp experiments and by analysing the slow response action potential in mammalian ventricular myodcardium. (1) Co (1 mu) reduced the maximum peak Zsi by 40 to 50% and shifted the current voltage relationship to weaker currents but left the time course of Zai virtually unchanged. (2) Co (1 mu) diminished the maximum Zsi tail current by about 40 to 50%, thereby shifting its current voltage characteristics to weaker currents. The kinetics of deactivation of Zsi tail current remained unaffected. There was no detectable shift of the d, curve along the voltage axis. (3) In accordance‘with the Co-induced reduction of Zsi, the overshoot and ri,,, of the slow response action potential declined and, in the presence of 2 rn~ Co, a complete blockade occurred. (4) Excess Ca abolished both the blockade and the depression of the slow response action potential. The Ca concentration necessary for neutralization of the inhibitory Co effect was higher at 2 IIN Co than at 1 rn~ Co. The G-induced neutralization of the Co effect was accompanied by an increase of the change in overshoot per tenfold variation of external Ca, exceeding the control values (28 to 32 mV) by far. (5) At 2 tn~ Ca, there occurred a strongly restricted response of the Z,i-mediated action potential towards isoproterenol after treatment with 1 rn~ Co; isoproterenol became virtually ineffective in the presence of 2 rn~ Co. The promoting isoproterenol action on V,,, and overshoot proved to be dependent on the external Ca concentration and was more pronounced in the presence of Co ions with increasing Ca concentration. KEY WORDS: muscle.
Slow
inward
current;
Slow
response
action
potential;
Co;
Ca;
Heart
1. Introduction Numerous studies during recent years have widely clarified the properties and biological significance of the slow inward current (Isi) in heart muscle [25, 41. This membrane current differs from the excitatory fast Na current by its kinetic features consisting of comparatively low rates of activation, inactivation and rerecovery from inactivation and by a specific susceptibility towards certain membrane-active agents. In contrast to the fast inward current, Isi was found to 0022-2828/80/101075+16
$02.00
0
1980
Academic
Press
Inc.
(London)
Limited
1076
M. KOHLHARDT
AND
K.
HAAP
be TTX insensitive but can be increased or inhibited by p-adrenergic catecholamines or verapamil, respectively, without concomitant changes of the fast Na system [13, 261. This justifies the assumption of the existence of a separate conductance system for Isi, the slow channel, which can be described by and obeys fairly well the Hodgkin-Huxley formalism [la]. Isi is involved in maintaining the plateau of the cardiac action potential and can also generate in the working myocardium a special type of excitation, namely the slow response action potential, in the absence of the fast Na system. Divalent cations are of crucial importance. Ca ions are the main charge carriers of 1si, at least in mammalian ventricular myocardium [Z, 201 and can be substituted by Sr and Ba [1.5, 271. Another group of divalent cations, however, among them, Mn, Ni and Co, exert an entirely opposite action and inhibit Isi [.24, 211. But the mode of action of these inhibitory cations has remained an unanswered question and the suggestion of Kohlhardt et al. [14] that the Co-induced inhibition of Isi arises from a decreased conductance of the 1si system of the cardiac membrane has yet to be proved. It was, therefore, the aim of the present study to elucidate further the nature of the suppression of Isi evoked by Co ions. 2. Materials
and
Methods
Methods Voltage clamp experiments were performed on isolated trabeculae and papillary muscles from the right ventricle of cats using the double sucrose gap technique. The preparations used had a minimum length of 7 mm and a diameter of 0.6 to 0.8 mm. Under deep ether anaesthesia the hearts of the animals were rapidly removed. In a dissection chamber continuously perfused with oxygenated Tyrode solution the right ventricle was opened and suitable trabeculae and papillary muscles were excised. After an equilibration period of 20 to 30 min the preparations were transferred into the voltage clamp bath. Using the same liquid partition system as originally described by Haas et al. [7], the central test compartment (continuously perfused with solution 1) was separated by two sucrose gaps each of 1.5 mm in width (continuously perfused with solution 4) from the neighbouring right and left KC1 compartment (continuously perfused with solution 3). The width of the test compartment was 0.2 mm at a maximum. Extracellular Ag-AgCl electrodes of low resistance ( <500 Q) were used in order to record membrane potential and to inject current into the preparation under voltage clamp conditions. The feedback factor of the voltage clamp circuit was 1: lo*. The current issued from the clamp amplifier had a maximum of 5 mA and a maximum voltage swing of f 12 V. Variable RC combinations were provided in the clamp circuit in order to avoid any tendency to swing. The clamp programme described below was rhythmically applied at a constant rate of 12 min-l. Membrane potential and membrane currents were displayed on a Tektronix storage oscilloscope 564B.
COBALT-INDUCED
INHIBITION
OF
SLOW
INWARD
CURRENT
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The extracellular (X,,) and the intracellular (Rig) resistance between the KC1 and the test compartment were monitored periodically in an arrangement described by New and Trautwein [XI]. The preparation was discarded if the ratio an intracellular washout and/or a -Rig/‘&g exceeded a factor of 0.4, indicating change in the resistance of the nexus of those parts of the preparation that were exposed to the sucrose solution. Despite of the very small test compartment, severe errors and complications can arise in voltage clamp experiments on cardiac muscle because of the multicellular nature of the preparation. Thus, due to a spatial inhomogeneity and an insufficient voltage control, in about 70% of all the preparations notches in the current record and/or a steep rise of the current-voltage relationship curve of 181 appeared. These preparations were rejected and not considered for analysis of membrane currents. Only those experiments were taken for the study of the Co effects in which such artifacts were completely absent and in which the maximum Isi appeared at least 25 mV above threshold potential. Slow response action potentials were analysed on papillary muscles of guineapigs (male and female, weight 250 to 300 g). The animals were killed by a blow on the neck. The hearts were rapidly removed and brought into a dissection chamber continuously perfused with oxygenated Tyrode solution. The right ventricle was opened and suitable papillary muscles (diameter less than 1 mm) were excised. In a continuously perfused (12 ml~min) muscle chamber (volume 1.2 ml) the base of the preparation was fixed by a small forceps and the valvular end was connected with the core of a linear displacement transducer. The papillary muscles were stretched to 110% of their initial length and electrically driven (stimulation frequency 1 mini, voltage twice threshold, duration 2 ms). Action potentials were recorded by means of conventional 3 M KCl-filled microelectrodes (resistance 10 to 30 MS2) and differentiated by an analogue differentiator. Both action potential and rate of rise ( pmax) were displayed on a Tektronix storage oscilloscope 564B. The muscles were equilibrated initially in normal Tyrode solution (K concentration 2.7 mM) for at Ieast 30 min. Only those experiments were considered for analysis in which a continuous impalement of the mict-oelectrode could be maintained. All values are given as mean values & standard deviation.
(1) Tyrode solution (mM): NaGI 137; KC1 2.7; CaCl, 2; Tris (hydrox~ethyl)aminomethane 2; glucose I I. The pH of 7.4 was adjusted with I-ICI. Equilibrated with 0,. (2) K-rich Tyrode solution (mM): NaCl 137; KC1 18; CaCl, 2; Tris(hydroxymethyl)-aminomethane 2; glucose Il. The pH of 7.4 was adjusted with HCl. Equilibrated with 0,.
1078
M.
KCIHLHARDT
AND
K.
HAAP
(3) KC1 solution (mM): KC1 181; CaCl, 0.55; glucose 1. (4) Sucrose solution (mM): Sucrose (enzymic grade) 292; CaCl, 0.05; glucose (enzymic grade) 5. Dissolved in highly purified water (specific resistance 20 Ma cm). Specific resistance of the sucrose solution > 2 M&2 cm. Temperature in the voltage clamp experiments 33.0 & 0.5”C, in the microelectrode experiments 34.5 f 0.5”C. 3; Results In the voltage clamp experiments of the present study the resting potential was lowered to about -40 mV by a conditioning prepulse of 600 ms duration in order to eliminate the fast inward current. Depolarizations starting from that holding potential triggered membrane currents consisting of two different components, 1si and an outward current (I,&) (see Figure I}. The latter overlaps somewhat the
500
ms
0 Time
(ms)
FIGURE 1. Registration of I,% and Iout (a) prior to and (b) after Co treatment. The upper beam in each panel is the membrane current; the lower beam registers the membrane potential. Eh plot of the currents demonstrated means holding potential (about -40 mV). (c) S emilogarithmic in (a), taking the current attained at the end of the 4 s Iasting clamp pulse as asymptote. (d) Semilogarith~~ plot of the currents demonstrated in (b), taking the current level attained 300 ms after peak Isi as asymptote. 0, Controls; 0 in the presence of 1 mM Co.
COBALT-INDUCED
INHIBITION
OF
SLOW
INWARD
CURRENT
1079
slow inward current which can lead to serious problems in the determination of the magnitude of Zsi, particularly when the influence of some physiological or pharmacological interventions are analysed which could affect ZO”t. A reasonable reference level for Zsi could be obtained by discrimination of the time-dependent outward current from Zsi. In fact, if the current record during a clamp pulse lasting 4 s is plotted semilogarithmically against time after peak Zsi, two different current components become evident [Figure 1 (c)l. Taking the current at the end of the 4 s clamp pulse in Figure 1 as asymptote, the current decay does not fit a single exponential during the first 250 to 300 ms after peak Zsi. Thereafter, a second component develops which can be approximated fairly well by a straight line having a time constant in the range between 500 and 700 ms. The latter is thought to represent the activation of the time-dependent (II) outward current [I??]. If the current attained 300 ms after peak Zsi is taken as another asymptote the first component proves to be single exponential too. It reflects inactivation of Zsi and time constants of 70 to 80 ms (at 0 mV) were obtained. Although desirable, the current analysis just described could not be extended to all experiments, mainly for two reasons: (1) the majority of the preparations responded to depolarizing clamp pulses longer than 1 to 1.5 s with a significant decrease of resting potential and, particularly, with a mostly irreversible loss of action potential plateau. (2) In the presence of very small amplitudes of Zsi, i.e. near threshold or at membrane potentials more positive than about +30 mV, this graphical analysis becomes increasingly inaccurate. Because Co ions left the kinetics of the time-dependent outward current unaffected (see Figure l), it seems justified to take the current level 300 ms after peak Zsi throughout as reference for the slow inward current whilst the difference between the current at the end of a 800 ms clamp pulse and the current at Eh reflects a comparable fraction of total outward current prior to and after Co treatment. Co ions depressed Zsi. Within 10 min, 1 mM Co led to a reduction of peak magnitude of 40 to 50%. This Co-induced inhibition was found to be completely reversible and the initial control values were obtained 2 to 3 min after return to a Co-free solution. In some cases, the reduction appeared without any detectable changes of the time course of Zsi. One of them is demonstrated in Figure 1, where time to peak current as well as the kinetics of inactivation remained virtually unchanged. As expected, Co induced a shift of the current-voltage relationship curve of Zsi to weaker currents. Such an experiment is demonstrated in Figure 2. In the presence of 1 mM Co as well as under control conditions the respectively maximum current appeared at a membrane potential of around 0 mV but exhibited a magnitude of only 60% of control. Extrapolation of the currentvoltage relationship curves of Figure 2 to the voltage axis revealed a slight decline of the reversal potential of Zsi of 3 mV in the presence of Co ions. Similar small changes were obtained in three other experiments. It seems, therefore, to be unlikely that the Co-induced suppression of Zsi results predominantly from a
1080
PA I M.
KOHLHARDT
AND
K.
HAAP
2
I out 0
I
0 l I I
-40
.q
8 I I
I l
I I
:
I
I
-20
0
I
8 + 20
1
12PA
I
I
I
+40
mV
140 mV Eh
0
0
0 0
()‘O
0ISi
O
l 0
0 0
.
500
(a)
ms
(b)
FIGURE 2. (a) Current-voltage relationship characteristics of Z,i and Zout prior to (a) and after (0) Co treatment obtained from that experiment demonstrated sectionally in (b). Eh means holding potential (about -40 mV).
reduction of Esi, although the determination of Esi applied represents only a rough estimate. In most of the cases, the outward current proved to be insensitive towards Co ions. A characteristic example is demonstrated in Figure 2. In spite of the significant depression of Isi, the shape of the current-voltage relationship for Iout remained unchanged, suggesting a rather selective action of Co ions on the Isi system of the cardiac membrane. On the basis of the Hodgkin-Huxley model [ 101, and on the presupposition that Isi will be carried mainly by Ca ions, the magnitude of this ionic current arises from the equation Isi =
j&f
(E--Eta))
(1)
where gsi is the limiting maximum conductance of the membrane, d and f are voltage- and time-dependent dimensionless activation and inactivation variables, respectively, of Hodgkin-Huxley type ranging from 0 to 1 [23] and the term (E-Eta) represents the Ca driving force across the membrane. Because E,i and, consequently, Eta were found to be only slightly affected by Co ions, the Coinduced depression of 1si could predominantly result from a decrease of gsi, d or f.
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1081
In order to analyse the variable d, Isi tail current measurements were carried out. When the membrane is depolarized from a given potential to another, the activation (d) gate opens and attains its steady state after some 10 ms. Consequently, Isi develops. The state of the d-gate also determines the &i tail current [23], which occurs at termination of short depolarizations by clamping back to the holding potential. Figure 3 demonstrates 1si tail currents prior to and after treatment with 1 rnM Co at .Ea (about -40 mV). After an initial fast current component there appears a slow deactivation of the tail inward current. In contrast to the control conditions, the latter is less pronounced in the presence of Co ions. To obtain the magnitude of the JBi tail current the whole current run was plotted semilogarithmically against time after attaining Eh. As depicted in the graph of Figure 3, the decay of the 1.i tail currenot follows a singfe exponential except for the first few milliseconds. This first early current component probably reflects the disappearance of the capacitive current [IS]. Time constants for the &g tail current deactivation of35 to 50 ms were found. Co ions left the deactivation kinetics unchanged (see Figure 3), suggesting that the closing of the d-gate is
O*O’ %i??++& Time
(ms)
FlCUkE 3. Registrations of tail currents in response to the termination of a 26 ms depolarizing clamp pulse (a) prior to and (b) after Co treatment. Both tail currents are exclusively inwarddirected, reflecting Zsi tail currents. El, means holding potential (about -48 mV). (c) Semilogarithmic plot of the Zsi tail currents of the upper part.
1082
M.
KOHLHARDT
AND
K.
HAAP
Co-insensitive. However, Co caused a significant decrease of the 1ai tail current amplitude, This becomes evident from the graph of Figure 3, where the deactivation phase was extrapolated to zero time, i.e. that time at which the holding potential had been attained. This procedure revealed a decline of the maximum Isi tail current from 0.66 PA under control conditions to 0.39 PA in the presence of 1mM Co. Three other experiments yielded a similar decrease of 40 to 50% at the same membrane potential of +20 mV. Accordingly, the current-voltage relationship for the maximum 1st tail current was shifted to weaker currents [Figure 4(a) 1. In order to obtain the d, curve, the experimental data shown in Figure 4(a) were normalized because the variable d ranges between 0 and 1. The resulting d, curve is given in Figure 4(b). Obviously, Co did not alter the shape of the curve, indicating that the voltage dependence of d, remained unaffected. The decrease of the 1ei tail current suggests, therefore, a diminished maximum conductance, Bi.
(al
-40
r
(b)
8 I
-20
I
I
I
0
I
I
I
20
-40 Voltage
I
I -20
I
I 0
I
I 20
(mV 1
Isi tail current FIGURE 4. (a) Current-voltage relationship c-haracterirtics o f the maximum prior to ( l ) and after (0) Co treatment. (b) The dependence of d, cn membrane potentiai prior to (0) and after (0) Co treatment. The values in (b) were obtained by normalization of the data depicted in (a).
The almost identical quantitative response of both .lsi and gsi towards Go ions already suggests that the decline of 1st arises mainly from the decrease of gsi. A shift of the steady state inactivation curve along the voltage axis to more negative potentials as another reason can hardly be .expected because the kinetics of inactivation of Isi remained unaltered in most of the Co experiments. Nevertheless, this theoretical postulate was tested by an experimental analysis of the fm curve in one of those Go experiments in which Tinactivation had not changed. It
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1083
yielded a voltage dependence offm after Co treatment which did not differ from that obtained under control conditions. After the voltage clamp analysis of the Isi system in the presence of Co ions had been accomplished the present study was extended to the &i-dependent slow response action potential in order to elucidate in more detail possible interactions of Co with Ca ions. According to Pappano [Z?], slow response action potentials can be elicited after inactivation of the fast inward current. The latter was achieved by decreasing the resting potential to about -45mV using K-rich (18 mM) Tyrode solutions. It would be an unjustified simplification to assume that the rising phase depends exclusively on the 1st system because of the possible overlapping with background outward currents. The compound 4-aminopyridine, known as an inhibitor of K conductance in several excitable membranes [19] and of the early outward current in cardiac Purkinje fibres [12], actually proved to be capable of augmenting pm,, and overshoot. However, in the case of an unaltered may be considered as a rough estimate of I out, the slow response action potential 1si for the following reasons: (I) consistent with observations of Weidmann [Z&J in Purkinje fibres and of Gettes and Reuter [S] in ventricular myocardium, hm was found to be zero between -62 mV and -58 mV; (2) the overshoot behaves as an almost perfect Ca electrode with slope factors of 28 to 31 mV per tenfold variation of external Ca concentration; (3) catecholamines increase like Isi, V,,, and overshoot. As expected from and consistent with the voltage clamp data, 1 rnM Co depressed the slow response action potential. Within 10 min, a gradual decrease of both pmaX and overshoot developed, accompanied by a shortening of action potential duration. In some cases a complete blockade appeared, which was the regular finding if the preparations were superfused with a 2 mM Co-containing solution. This reflects the inhibition of Isi. With regard to the voltage clamp results, an increase of Iout as an alternative explanation can be excluded. The effect of Co ions occurred without detectable changes of the resting potential and was not abolished by increasing the stimulus strength. The degree of the Co-induced depression depends crucially on the extracellular Ca concentration (Figure 5). After the Ca concentration had been increased from 2 to 4 mM, the blockade disappeared within 3 to 4 min. Consequently, slow response action potentials could be elicited again, having an overshoot of 41.0 f 1.7 mV and a maximum rate of rise of 19.3 -& 1.2 V/s (n = 3). Both values are significantly smaller than those obtained at 4 mM Ca prior to the Co treatment (50.7 f 2.6 mV and 28.3 f 3.8 Vi s, respectively). With increasing external Ca concentrations this deviation of overshoot and li,,, from the contro1 values became progressively smaller and amounted at 10 mM only 2.7 & 0.5 mV and 4.2 & 0.4 V/s. As shown in Figure 5, this was accompanied with a rise of slope factor of overshoot per tenfold change of external Ca concentration that rose from 28 mV under control conditions to 47 mV in the presence of Co ions.
1084
M.
KOHLHARDT
AND
Ca concentration FIGURE on external
5. The dependence Ca concentration.
of overshoot (a) 0-0, Control;
K.
HAAP
(mM)
action and pmimax (b) of the slow response O-0, in the presence of 2 rn~ Co.
potential
Two other experiments revealed the same result, i.e. after Co treatment the overshoot no longer behaved as Ca electrode. Extrapolation of the regression lines characterizing the dependence of overshoot on external Ca prior to and after treatment with 2 mM Co to Ca concentrations higher than 10 mM reveals an intercept of both straight lines at about 15 mM (see Figure 5). The question arose “which slope factor would appear in the presence of Co ions at Ca concentrations above 15 mM. In other words, does the overshoot regain its Nernstian behaviour in spite of the continued presence of Co ions or not?” Because of possible osmotic effects and the fact that the overshoot tends to saturate at very strong external Ca concentrations (50 mM or more [S]}, experiments in that range might yield ambiguous results. We decided, therefore, to test this at a lowered cobalt concentration, namely 1 mM. The theoretical background was the hypothesis that the intercept of both regression lines is due to the interaction of Co with Ca ions and will be shifted to the left, i.e. to lower Ca concentrations if a cobalt concentration lower than 2 mM is applied. Figure 6 gives the evaluation of such an experiment. Although the treatment with 1 mM Co had even evoked a blockade of the slow response action potential, the overshoot attained its control value at 6 mM Ca. In the range from 4 to 6 mM Ca, the overshoot did not behave as a Ca electrode. Above 6 mM Ca, however, a Nernstian behaviour was found and the same slope factor as under control conditions
COBALT-INDUCED
INHIBITION
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SLO\V
INlVARD
1085
CI‘RRENT
60 -
SOL
2
4
5 6
Co concentration
FIGURE 6. The dependence Ca concentration prior to (0)
0
IO
15
(mr.4)
of the overshoot of the slow response action and after (0) treatment with 1 mM Co.
potential
on external
(31 mV) per tenfold change of external Ca appeared. Three other experiments of this type yielded the same results and, in the presence of 1 mM Co, the overshoot reached its control value at 6 to 8 mM Ca and regains Nernstian properties above that Ca concentration. Co ions diminished the catecholamine-induced increase of vm,, and overshoot of the slow response action potential. Whilst rm,, and overshoot responded under control conditions, i.e. in the absence of Co ions, to 9.2 x 10m6 M isoproterenol with an increase of 12.0 * 1.5 VI s and 12.4 f 0.8 mV, respectively, after treatment with 1 mM Co the same isoproterenol concentration produced an augmentation of only 2.0 & 0.65 V/s and 7.5 & 0.9 mV (n = 3), respectively. In accordance with this significantly restricted response is the observation that at higher Co concentrations (2 mM) isoproterenol became virtually ineffective since isoproterenol concentrations even as high as 9.2 x 10e4 M failed to overcome the Co-induced blockade of the slow response action potential. Only after return to a Co-free medium did the promoting effect of isoproterenol occur (see Figure 7). Both the restricted quantitative response of v,,, and overshoot towards isoproterenol at 1 mM Co and the lack of any promoting catecholamine action in the presence of 2 mM Co might arise from an interference of Co ions with some steps in the chain of events leading ultimately (probably via cyclic AMP) to the catecholamine-induced increase of conductance of the Isi system of the cardiac membrane or from the interaction of Co with Ca. To test this, the effect of isoproterenol (9.2 x 10d6 M and 1.38 x 10~~ M) was analysed in the presence of 2 mM Co at external Ca concentrations higher than 2 mM. Above 2 mM Ca, the
FIGURE 2 rn~ Co
and
7. The effect of isoproterenol after switching to a. Co-free
on the medium.
slow
response
5
6 7 8910
action
potential
in the
presence
of
60
50
40
4
Ca concentration
FIGURE on external 9.2 X 10e6
8. The dependence of (a), overshoot Ca concentration in the presence M isoproterenol (0).
(rnht)
and (b), vm’,,, of the slow of 2 WIM Co prior to, (0)
response action potential and after application.
of
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1087
slow response action potential regained its sensitivity towards isoproterenol. As demonstrated in Figure.& the increase of 3 lllLX and overshoot produced by any isoproterenol concentration was the stronger the higher the external Ca concentration was which led to a further rise of slope factor of overshoot per tenfold change of external Ca, in this case from 42 to 60 mV. Isoproterenol concentrations higher than 9.2 x lO-6 M did not cause a further increase of slope factor but evoked a parallel shift of the regression lines describing the dependence of overshoot on logarithm of external Ca concentration and describing the dependence of external Ca concentration to higher values for overshoot of I’,,x on logarithm respectively. At 10 mM Ca, there appeared an almost normal quantiand limax, tative response towards 9.2 x 10M6 M isoproterenol since pm,, increased by 12.2 + 1.6 V/s and overshoot by 16.1 & 7.2 mV (n :- 3). Needless to say, isoproterenol failed to produce any alteration of slope factor of overshoot per decade of Ca concentration in the absence of Co ions. 4. Discussion According to earlier observations by Kohlhardt et al. [14J Co ions reduced Isi. This inhibitory effect occurred without detectable changes of the time course of Isi. It is, therefore, unlikely that the suppression of Isi is caused by a decreased rate of activation or by an increased rate of inactivation. Rather the diminution of gsi seems to be the principal action of Co ions underlying the depression of 1si. It suggests that Co ions reduce the number of isi channels that open on depolarization of the cardiac membrane, leading consequently to a depression of both pm,, and overshoot of the slow response action potential. Despite the continued presence of Co ions, their inhibitory effect could be abolished by excess Ca, which restored the &mediated action potential. The Ga concentration needed for restoration of the 1si system depends on the Co concentration and proved to be smaller in the presence of I mM Co than at 2 mM Co, which might reflect an interaction of Co with Ca at certain sites of the cardiac membrane. This assumption is further supported by the observation that, after Co treatment, the 1simediated action potential loses its property as a Ca electrode in that range of external Ca concentration in which neutralization of the inhibitory Co effects occurs. Under control conditions, namely in the absence of Co ions, the I,i-mediated action potential behaved as an almost perfect Ca electrode in a Ca concentration range between 2 mM and 15 mM, although a rise of external Ca leads to an increase of K outward currents in Purkinje fibres and ventricular myocardium [I, 111. The variation of overshoot is predicted by the Nernst equation:
1088
M. KOHLHARDT
AND
K.
HAAP
where R, I and F have their conventional meanings and [Car] and [Gas] are two different external Ca concentrations. In the presence of Co, however, the slope factors of overshoot per decade of Ca variation experimentally obtained considerably exceeded the theoretical Nernst value by some 10 mV, indicating that Px, Pea or [Cali no longer remained unaffected on a Ca concentration change. Considering the specific situation in the case of an increase of external Ca, there are no realistic reasons to suppose a decline of 2’~ or [Cali, but it is most plausible to attribute this rise of slope factor, i.e. the supernormal Ca sensitivity of overshoot, to a rise of Pea. This increase of Pea could be interpreted as being indicative for an augmentation of gsi and might be basically involved in the removal of the Co-induced inhibition of the Isi system by excess Ca. The present results agree with earlier observations of Hagiwara and Takahashi [R] demonstrating suppression of the Ca spike in barnacle muscle by Co ions and attributing this inhibition to a competitive interaction of Co with Ca ions. However, the nature of this process cannot be definitely judged and it remains, furthermore, an unanswered question where this interaction finally leading to the decrease of @St of the cardiac membrane happens. Suppose that the cardiac slow membrane channel bears, like other ionic channels [5.9], negatively charged groups accessible for cations and involved in the process of cationic inward movements. The interaction of Co with Ca at these groups might cause finally the blockade of the number of channels per unit area of the membrane. On the other hand, a rather superficial structure capable of binding certain cations, among them Ca [3], could be taken into consideration, namely the glycocalyx, the surface coat of which represents an integral part of the cardiac sarcolemma. Studies by Langer and his co-workers [17] revealed a high afhnity of this structure for Ca, which can be displaced from its binding sites by La, Mn and other cations. This Ca pool might be a possible source of that Ca passing the slow channel and carrying Jsi during excitation [l6]. Because Co exerts its inhibitory action without a significant decrease of Esi, it is unlikely that a displacement of Ca from binding sites within the glycocalyx is involved in the blockade of Isiventricular myocardium, As shown by Reuter and Scholz [24] m . mammalian P-adrenergic catecholamines augment gsi. This augmentation of gsi is the reason for the well-known increase of Isi in the presence of catecholamines and very probably reflects a rise in the number of slow channels per unit area of the cardiac membrane. It is conceivable that Co ions interfere with the de noun formation of slow channels, which offers a possible explanation for the diminished isoproterenol effect. However, the Isi system regains its usual sensitivity towards isoproterenoi with increasing Ca concentrations despite the continued presence of CO ions. It seems to be, therefore, more plausible to attribute the restricted response towards isoproterenol at low Ca (<4 mM) to the interaction of Co with Ca, which might also operate at deLOUDformed channels, thereby making them unable to allow the inward movements of Ca.
COBALT-INDUCED
INHIBITION
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INWARD
CURRENT
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8. 9. 10.
11. 12. 13.
BASSNGTHWAXGHTE, J. B., FRY, C. H. & MCGUIGAN, J. A. S. Relationship between internal calcium and outward current in mammalian ventricular muscle; a mechanism for the control of the action potential duration? Journal of Physiology, London 262, 15-37 (1976). BEELER, G. W. & REUTER, H. Membrane calcium cur’rent in ventricular myocardial fibres. Journal of Physiology, Lo&on 207, 191-209 (1970). BENNETT, II. S. Morpbolo~ical aspects of extracellular polysaccharides. Journal of Histochemistry and Cytochemistry 11, 111-3 ( 1963). CORABOEUF, E. Ionic basis of electrical activity in cardiac tissues. American Journal of Physiology 234, HlOlH116 (1978). DROUIN, H. & THE, R. The effect of reducing extracellular pH on the membrane currents of the Ranvier node. P@igers Archiv f& die gesamte PhysioLogie des it4e92iscfren und der Tiere 313, 80-88 (1969). GETTES, L. S. & REUTER, H. Slow recovery from inactivation of inward currents in mammalian myocardial fibres. Journal qf Physiology, London 240, 703-724 (1974). HAAS, H. G., KERN, 5. & EINWACHTER, H. M. Electrical activity and metabolism in cardiac tissues. An experimental and theoretical study. Jowxal of Membrane Biology 3, 180-209 (1970). HAGIWARA, S. & TAK~HASHI, K. Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. Journal of General Phys~olo~ 50, 583-601 (1967). HILLE, B. Charges and potentials at the nerve surface. Divalent ions and pH. Journal of General Physiology 51, 22 l-236 (1968). HODGKIN, A. L. & HUXLEY, A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. Journal of Physiology, London 117, 500 (1952). KASS, R. S. & TSIEN, R. W. Control of action potential duration by calcium ions in cardiac Purkinje fibers. Journal of General Physiology 67, 599-617 (1976). KENYON, J. L. & GIBBONS, W. R. 4-aminopyridine and the early outward current of sheep cardiac Purkinje fibres. Jourlaal of General Physio~o~ 73, 139-157 (1979). KOHLHARDT, M., BAUF,R, B., KRAUSE, H. & FLECKENSTEIN, A. Differentiation of the transmembrane Na and Ca channels in mammalian cardiac fibres by the use of specific inhibitors. PJlilgers Archiu die gesaml Physiologic des Menschen und der Tiere 335,309-322 (1972). KOHLHARDT, M., BAUER, B., KRIIUSE, H. & FLECKENSTEIN, A. Selective inhibition of the transmembrane Ca conductivity of mammalian myocardial fibres by Ni, Co and Mn ions. PJiigers Archivftir die gesamte Physiologie des Menschen und der Tiere 338, 115-123 (1973). KOHLHARDT, M., HAA~TERT, H. P. & KRAUSE, H. Evidence of non-specificity of the Ca channel in mammalian myocardial fibre membranes. Substitution of Ca by Sr, %a or Mg as charge carriers. Pjtigers Archiv die gesamte Physiologzie des Menschen und der Tiere 342, 125-136 (1973). KOHLHARDT, M., MNICH, Z., WAIS, U. & MAIER, G. Studies on the time-dependent response of the cardiac Ga-mediated action potential due to variations of the external Ca concentration. Basic Research in Cardiology 73, 257-272 (1978). I.ANGER, G. A. The structure and function of the myocardial cell surface. Americaa Journal of Physiology 235, H461-H468 (1978). MCDONALD, 'I?. F. & TRAUTWEIN, W, Membrane currents in cat myocardium: separation of inward and outward currents. Joyful of Phys~lo~, Loladon 274,193-216 (1978).
fdr
14.
15.
fi?r
16.
17. 18.
1090 19.
20. 21.
22.
23. 24. 25. 26.
27.
28.
M.KOHLHARDTAND
K.HAAP
MEVES, H. & PICHON, Y. The effect of internal and external 4-aminopyridine on the potassium currents in intracellularly perfused squid axons. Journal of PhysioloQ, London 268, 511-532 (1977). NEW, W. & TRAUTWEIN, W. Inward membrane currents in mammalian myocardium. Pjtigers Archiv fir die gesamte Physiologie des Menschen und der Tiere 334, I-23 (1972). OCHI, R. The slow inward current and the action of manganese ions in guinea-pig myocardium. PJiTiigers Archiu die gesanate Physiologic des Menscha und der Tiere 316, 81-94 (1970). PAPFANO, A. J. calcium-dependent action potentials produced by catecholamines in guinea-pig atria1 muscle depolarized by potassium. G~~c~l~~~o~ Research 27, 379-390 (1970). REUTER, H. Divalent cations as charge carriers in excitable membranes. Progress ia Biophysics and Molecular Biolopy 26, 3-43 (1973). REUTER, H. & SCHOLZ, H. The regulation of the calcium conductance of cardiac muscle by adrenaline. Journal of Physiology, London 264, 49-62 (1977). TRAUTWEIN, W. Membrane currents in cardiac muscle fibres. Physiological Reviews 53, 793-835 (1973). VASSORT, G., ROUGIER, O., GARNIER, D., SAUVIAT, M. P., CORABOEUF, E. & GARGOUIL, Y. M. Effects of adrenaline on membrane inward currents during the cardiac action potential. ~~~~ers Arch~~~~r die gesamte Phy~~log~ des Me~~~~ und der Tim-e 309, 70-81 (1969). VEREEGKE, J. & CARMELIET, E. Sr action potentials in cardiac Purkinje fibres. 1. Evidence for a regenerative increase in Sr conductance. PjZgers Arch& fir die gesamte Physiologic des Mescherz und der Tiere 322, 60-72. (197 1). WEIDMANN, S. The effect of the cardiac membrane potential on the rapid availability of the sodium carrying system. Journal of Physiology, London 127, 213-224 (1955).
fik
and Cellular
Cardiology
(1980)
12, 1075-1090
On the Mechanism Underlying the Cobalt-induced Slow Inward Current in Mammalian Ventricular M. KOHLHARDT Physiologisches (Received
Institut
der Universitiit
29 January
1980,
AND
Freiburg,
Inhibition of Myocardium
K. HAAP D-78
accepted in revised
FreiburglBr.,
form
2 April
F.D.R. 1980)
M. KOHLHARDT AND K. Inhibition of Slow Inward
HAAP. On the Mechanism Underlying the Cobalt-induced Current in Mammalian Ventricular Myocardium. Journal of Molecular and Cellular CardioloD (1980) 12, 1075-1090. The effect of Cobalt (Co) ions on the slow inward current (Zsi) was studied in voltage clamp experiments and by analysing the slow response action potential in mammalian ventricular myodcardium. (1) Co (1 mu) reduced the maximum peak Zsi by 40 to 50% and shifted the current voltage relationship to weaker currents but left the time course of Zai virtually unchanged. (2) Co (1 mu) diminished the maximum Zsi tail current by about 40 to 50%, thereby shifting its current voltage characteristics to weaker currents. The kinetics of deactivation of Zsi tail current remained unaffected. There was no detectable shift of the d, curve along the voltage axis. (3) In accordance‘with the Co-induced reduction of Zsi, the overshoot and ri,,, of the slow response action potential declined and, in the presence of 2 rn~ Co, a complete blockade occurred. (4) Excess Ca abolished both the blockade and the depression of the slow response action potential. The Ca concentration necessary for neutralization of the inhibitory Co effect was higher at 2 IIN Co than at 1 rn~ Co. The G-induced neutralization of the Co effect was accompanied by an increase of the change in overshoot per tenfold variation of external Ca, exceeding the control values (28 to 32 mV) by far. (5) At 2 tn~ Ca, there occurred a strongly restricted response of the Z,i-mediated action potential towards isoproterenol after treatment with 1 rn~ Co; isoproterenol became virtually ineffective in the presence of 2 rn~ Co. The promoting isoproterenol action on V,,, and overshoot proved to be dependent on the external Ca concentration and was more pronounced in the presence of Co ions with increasing Ca concentration. KEY WORDS: muscle.
Slow
inward
current;
Slow
response
action
potential;
Co;
Ca;
Heart
1. Introduction Numerous studies during recent years have widely clarified the properties and biological significance of the slow inward current (Isi) in heart muscle [25, 41. This membrane current differs from the excitatory fast Na current by its kinetic features consisting of comparatively low rates of activation, inactivation and rerecovery from inactivation and by a specific susceptibility towards certain membrane-active agents. In contrast to the fast inward current, Isi was found to 0022-2828/80/101075+16
$02.00
0
1980
Academic
Press
Inc.
(London)
Limited
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M. KOHLHARDT
AND
K.
HAAP
be TTX insensitive but can be increased or inhibited by p-adrenergic catecholamines or verapamil, respectively, without concomitant changes of the fast Na system [13, 261. This justifies the assumption of the existence of a separate conductance system for Isi, the slow channel, which can be described by and obeys fairly well the Hodgkin-Huxley formalism [la]. Isi is involved in maintaining the plateau of the cardiac action potential and can also generate in the working myocardium a special type of excitation, namely the slow response action potential, in the absence of the fast Na system. Divalent cations are of crucial importance. Ca ions are the main charge carriers of 1si, at least in mammalian ventricular myocardium [Z, 201 and can be substituted by Sr and Ba [1.5, 271. Another group of divalent cations, however, among them, Mn, Ni and Co, exert an entirely opposite action and inhibit Isi [.24, 211. But the mode of action of these inhibitory cations has remained an unanswered question and the suggestion of Kohlhardt et al. [14] that the Co-induced inhibition of Isi arises from a decreased conductance of the 1si system of the cardiac membrane has yet to be proved. It was, therefore, the aim of the present study to elucidate further the nature of the suppression of Isi evoked by Co ions. 2. Materials
and
Methods
Methods Voltage clamp experiments were performed on isolated trabeculae and papillary muscles from the right ventricle of cats using the double sucrose gap technique. The preparations used had a minimum length of 7 mm and a diameter of 0.6 to 0.8 mm. Under deep ether anaesthesia the hearts of the animals were rapidly removed. In a dissection chamber continuously perfused with oxygenated Tyrode solution the right ventricle was opened and suitable trabeculae and papillary muscles were excised. After an equilibration period of 20 to 30 min the preparations were transferred into the voltage clamp bath. Using the same liquid partition system as originally described by Haas et al. [7], the central test compartment (continuously perfused with solution 1) was separated by two sucrose gaps each of 1.5 mm in width (continuously perfused with solution 4) from the neighbouring right and left KC1 compartment (continuously perfused with solution 3). The width of the test compartment was 0.2 mm at a maximum. Extracellular Ag-AgCl electrodes of low resistance ( <500 Q) were used in order to record membrane potential and to inject current into the preparation under voltage clamp conditions. The feedback factor of the voltage clamp circuit was 1: lo*. The current issued from the clamp amplifier had a maximum of 5 mA and a maximum voltage swing of f 12 V. Variable RC combinations were provided in the clamp circuit in order to avoid any tendency to swing. The clamp programme described below was rhythmically applied at a constant rate of 12 min-l. Membrane potential and membrane currents were displayed on a Tektronix storage oscilloscope 564B.
COBALT-INDUCED
INHIBITION
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INWARD
CURRENT
1077
The extracellular (X,,) and the intracellular (Rig) resistance between the KC1 and the test compartment were monitored periodically in an arrangement described by New and Trautwein [XI]. The preparation was discarded if the ratio an intracellular washout and/or a -Rig/‘&g exceeded a factor of 0.4, indicating change in the resistance of the nexus of those parts of the preparation that were exposed to the sucrose solution. Despite of the very small test compartment, severe errors and complications can arise in voltage clamp experiments on cardiac muscle because of the multicellular nature of the preparation. Thus, due to a spatial inhomogeneity and an insufficient voltage control, in about 70% of all the preparations notches in the current record and/or a steep rise of the current-voltage relationship curve of 181 appeared. These preparations were rejected and not considered for analysis of membrane currents. Only those experiments were taken for the study of the Co effects in which such artifacts were completely absent and in which the maximum Isi appeared at least 25 mV above threshold potential. Slow response action potentials were analysed on papillary muscles of guineapigs (male and female, weight 250 to 300 g). The animals were killed by a blow on the neck. The hearts were rapidly removed and brought into a dissection chamber continuously perfused with oxygenated Tyrode solution. The right ventricle was opened and suitable papillary muscles (diameter less than 1 mm) were excised. In a continuously perfused (12 ml~min) muscle chamber (volume 1.2 ml) the base of the preparation was fixed by a small forceps and the valvular end was connected with the core of a linear displacement transducer. The papillary muscles were stretched to 110% of their initial length and electrically driven (stimulation frequency 1 mini, voltage twice threshold, duration 2 ms). Action potentials were recorded by means of conventional 3 M KCl-filled microelectrodes (resistance 10 to 30 MS2) and differentiated by an analogue differentiator. Both action potential and rate of rise ( pmax) were displayed on a Tektronix storage oscilloscope 564B. The muscles were equilibrated initially in normal Tyrode solution (K concentration 2.7 mM) for at Ieast 30 min. Only those experiments were considered for analysis in which a continuous impalement of the mict-oelectrode could be maintained. All values are given as mean values & standard deviation.
(1) Tyrode solution (mM): NaGI 137; KC1 2.7; CaCl, 2; Tris (hydrox~ethyl)aminomethane 2; glucose I I. The pH of 7.4 was adjusted with I-ICI. Equilibrated with 0,. (2) K-rich Tyrode solution (mM): NaCl 137; KC1 18; CaCl, 2; Tris(hydroxymethyl)-aminomethane 2; glucose Il. The pH of 7.4 was adjusted with HCl. Equilibrated with 0,.
1078
M.
KCIHLHARDT
AND
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HAAP
(3) KC1 solution (mM): KC1 181; CaCl, 0.55; glucose 1. (4) Sucrose solution (mM): Sucrose (enzymic grade) 292; CaCl, 0.05; glucose (enzymic grade) 5. Dissolved in highly purified water (specific resistance 20 Ma cm). Specific resistance of the sucrose solution > 2 M&2 cm. Temperature in the voltage clamp experiments 33.0 & 0.5”C, in the microelectrode experiments 34.5 f 0.5”C. 3; Results In the voltage clamp experiments of the present study the resting potential was lowered to about -40 mV by a conditioning prepulse of 600 ms duration in order to eliminate the fast inward current. Depolarizations starting from that holding potential triggered membrane currents consisting of two different components, 1si and an outward current (I,&) (see Figure I}. The latter overlaps somewhat the
500
ms
0 Time
(ms)
FIGURE 1. Registration of I,% and Iout (a) prior to and (b) after Co treatment. The upper beam in each panel is the membrane current; the lower beam registers the membrane potential. Eh plot of the currents demonstrated means holding potential (about -40 mV). (c) S emilogarithmic in (a), taking the current attained at the end of the 4 s Iasting clamp pulse as asymptote. (d) Semilogarith~~ plot of the currents demonstrated in (b), taking the current level attained 300 ms after peak Isi as asymptote. 0, Controls; 0 in the presence of 1 mM Co.
COBALT-INDUCED
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SLOW
INWARD
CURRENT
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slow inward current which can lead to serious problems in the determination of the magnitude of Zsi, particularly when the influence of some physiological or pharmacological interventions are analysed which could affect ZO”t. A reasonable reference level for Zsi could be obtained by discrimination of the time-dependent outward current from Zsi. In fact, if the current record during a clamp pulse lasting 4 s is plotted semilogarithmically against time after peak Zsi, two different current components become evident [Figure 1 (c)l. Taking the current at the end of the 4 s clamp pulse in Figure 1 as asymptote, the current decay does not fit a single exponential during the first 250 to 300 ms after peak Zsi. Thereafter, a second component develops which can be approximated fairly well by a straight line having a time constant in the range between 500 and 700 ms. The latter is thought to represent the activation of the time-dependent (II) outward current [I??]. If the current attained 300 ms after peak Zsi is taken as another asymptote the first component proves to be single exponential too. It reflects inactivation of Zsi and time constants of 70 to 80 ms (at 0 mV) were obtained. Although desirable, the current analysis just described could not be extended to all experiments, mainly for two reasons: (1) the majority of the preparations responded to depolarizing clamp pulses longer than 1 to 1.5 s with a significant decrease of resting potential and, particularly, with a mostly irreversible loss of action potential plateau. (2) In the presence of very small amplitudes of Zsi, i.e. near threshold or at membrane potentials more positive than about +30 mV, this graphical analysis becomes increasingly inaccurate. Because Co ions left the kinetics of the time-dependent outward current unaffected (see Figure l), it seems justified to take the current level 300 ms after peak Zsi throughout as reference for the slow inward current whilst the difference between the current at the end of a 800 ms clamp pulse and the current at Eh reflects a comparable fraction of total outward current prior to and after Co treatment. Co ions depressed Zsi. Within 10 min, 1 mM Co led to a reduction of peak magnitude of 40 to 50%. This Co-induced inhibition was found to be completely reversible and the initial control values were obtained 2 to 3 min after return to a Co-free solution. In some cases, the reduction appeared without any detectable changes of the time course of Zsi. One of them is demonstrated in Figure 1, where time to peak current as well as the kinetics of inactivation remained virtually unchanged. As expected, Co induced a shift of the current-voltage relationship curve of Zsi to weaker currents. Such an experiment is demonstrated in Figure 2. In the presence of 1 mM Co as well as under control conditions the respectively maximum current appeared at a membrane potential of around 0 mV but exhibited a magnitude of only 60% of control. Extrapolation of the currentvoltage relationship curves of Figure 2 to the voltage axis revealed a slight decline of the reversal potential of Zsi of 3 mV in the presence of Co ions. Similar small changes were obtained in three other experiments. It seems, therefore, to be unlikely that the Co-induced suppression of Zsi results predominantly from a
1080
PA I M.
KOHLHARDT
AND
K.
HAAP
2
I out 0
I
0 l I I
-40
.q
8 I I
I l
I I
:
I
I
-20
0
I
8 + 20
1
12PA
I
I
I
+40
mV
140 mV Eh
0
0
0 0
()‘O
0ISi
O
l 0
0 0
.
500
(a)
ms
(b)
FIGURE 2. (a) Current-voltage relationship characteristics of Z,i and Zout prior to (a) and after (0) Co treatment obtained from that experiment demonstrated sectionally in (b). Eh means holding potential (about -40 mV).
reduction of Esi, although the determination of Esi applied represents only a rough estimate. In most of the cases, the outward current proved to be insensitive towards Co ions. A characteristic example is demonstrated in Figure 2. In spite of the significant depression of Isi, the shape of the current-voltage relationship for Iout remained unchanged, suggesting a rather selective action of Co ions on the Isi system of the cardiac membrane. On the basis of the Hodgkin-Huxley model [ 101, and on the presupposition that Isi will be carried mainly by Ca ions, the magnitude of this ionic current arises from the equation Isi =
j&f
(E--Eta))
(1)
where gsi is the limiting maximum conductance of the membrane, d and f are voltage- and time-dependent dimensionless activation and inactivation variables, respectively, of Hodgkin-Huxley type ranging from 0 to 1 [23] and the term (E-Eta) represents the Ca driving force across the membrane. Because E,i and, consequently, Eta were found to be only slightly affected by Co ions, the Coinduced depression of 1si could predominantly result from a decrease of gsi, d or f.
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1081
In order to analyse the variable d, Isi tail current measurements were carried out. When the membrane is depolarized from a given potential to another, the activation (d) gate opens and attains its steady state after some 10 ms. Consequently, Isi develops. The state of the d-gate also determines the &i tail current [23], which occurs at termination of short depolarizations by clamping back to the holding potential. Figure 3 demonstrates 1si tail currents prior to and after treatment with 1 rnM Co at .Ea (about -40 mV). After an initial fast current component there appears a slow deactivation of the tail inward current. In contrast to the control conditions, the latter is less pronounced in the presence of Co ions. To obtain the magnitude of the JBi tail current the whole current run was plotted semilogarithmically against time after attaining Eh. As depicted in the graph of Figure 3, the decay of the 1.i tail currenot follows a singfe exponential except for the first few milliseconds. This first early current component probably reflects the disappearance of the capacitive current [IS]. Time constants for the &g tail current deactivation of35 to 50 ms were found. Co ions left the deactivation kinetics unchanged (see Figure 3), suggesting that the closing of the d-gate is
O*O’ %i??++& Time
(ms)
FlCUkE 3. Registrations of tail currents in response to the termination of a 26 ms depolarizing clamp pulse (a) prior to and (b) after Co treatment. Both tail currents are exclusively inwarddirected, reflecting Zsi tail currents. El, means holding potential (about -48 mV). (c) Semilogarithmic plot of the Zsi tail currents of the upper part.
1082
M.
KOHLHARDT
AND
K.
HAAP
Co-insensitive. However, Co caused a significant decrease of the 1ai tail current amplitude, This becomes evident from the graph of Figure 3, where the deactivation phase was extrapolated to zero time, i.e. that time at which the holding potential had been attained. This procedure revealed a decline of the maximum Isi tail current from 0.66 PA under control conditions to 0.39 PA in the presence of 1mM Co. Three other experiments yielded a similar decrease of 40 to 50% at the same membrane potential of +20 mV. Accordingly, the current-voltage relationship for the maximum 1st tail current was shifted to weaker currents [Figure 4(a) 1. In order to obtain the d, curve, the experimental data shown in Figure 4(a) were normalized because the variable d ranges between 0 and 1. The resulting d, curve is given in Figure 4(b). Obviously, Co did not alter the shape of the curve, indicating that the voltage dependence of d, remained unaffected. The decrease of the 1ei tail current suggests, therefore, a diminished maximum conductance, Bi.
(al
-40
r
(b)
8 I
-20
I
I
I
0
I
I
I
20
-40 Voltage
I
I -20
I
I 0
I
I 20
(mV 1
Isi tail current FIGURE 4. (a) Current-voltage relationship c-haracterirtics o f the maximum prior to ( l ) and after (0) Co treatment. (b) The dependence of d, cn membrane potentiai prior to (0) and after (0) Co treatment. The values in (b) were obtained by normalization of the data depicted in (a).
The almost identical quantitative response of both .lsi and gsi towards Go ions already suggests that the decline of 1st arises mainly from the decrease of gsi. A shift of the steady state inactivation curve along the voltage axis to more negative potentials as another reason can hardly be .expected because the kinetics of inactivation of Isi remained unaltered in most of the Co experiments. Nevertheless, this theoretical postulate was tested by an experimental analysis of the fm curve in one of those Go experiments in which Tinactivation had not changed. It
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1083
yielded a voltage dependence offm after Co treatment which did not differ from that obtained under control conditions. After the voltage clamp analysis of the Isi system in the presence of Co ions had been accomplished the present study was extended to the &i-dependent slow response action potential in order to elucidate in more detail possible interactions of Co with Ca ions. According to Pappano [Z?], slow response action potentials can be elicited after inactivation of the fast inward current. The latter was achieved by decreasing the resting potential to about -45mV using K-rich (18 mM) Tyrode solutions. It would be an unjustified simplification to assume that the rising phase depends exclusively on the 1st system because of the possible overlapping with background outward currents. The compound 4-aminopyridine, known as an inhibitor of K conductance in several excitable membranes [19] and of the early outward current in cardiac Purkinje fibres [12], actually proved to be capable of augmenting pm,, and overshoot. However, in the case of an unaltered may be considered as a rough estimate of I out, the slow response action potential 1si for the following reasons: (I) consistent with observations of Weidmann [Z&J in Purkinje fibres and of Gettes and Reuter [S] in ventricular myocardium, hm was found to be zero between -62 mV and -58 mV; (2) the overshoot behaves as an almost perfect Ca electrode with slope factors of 28 to 31 mV per tenfold variation of external Ca concentration; (3) catecholamines increase like Isi, V,,, and overshoot. As expected from and consistent with the voltage clamp data, 1 rnM Co depressed the slow response action potential. Within 10 min, a gradual decrease of both pmaX and overshoot developed, accompanied by a shortening of action potential duration. In some cases a complete blockade appeared, which was the regular finding if the preparations were superfused with a 2 mM Co-containing solution. This reflects the inhibition of Isi. With regard to the voltage clamp results, an increase of Iout as an alternative explanation can be excluded. The effect of Co ions occurred without detectable changes of the resting potential and was not abolished by increasing the stimulus strength. The degree of the Co-induced depression depends crucially on the extracellular Ca concentration (Figure 5). After the Ca concentration had been increased from 2 to 4 mM, the blockade disappeared within 3 to 4 min. Consequently, slow response action potentials could be elicited again, having an overshoot of 41.0 f 1.7 mV and a maximum rate of rise of 19.3 -& 1.2 V/s (n = 3). Both values are significantly smaller than those obtained at 4 mM Ca prior to the Co treatment (50.7 f 2.6 mV and 28.3 f 3.8 Vi s, respectively). With increasing external Ca concentrations this deviation of overshoot and li,,, from the contro1 values became progressively smaller and amounted at 10 mM only 2.7 & 0.5 mV and 4.2 & 0.4 V/s. As shown in Figure 5, this was accompanied with a rise of slope factor of overshoot per tenfold change of external Ca concentration that rose from 28 mV under control conditions to 47 mV in the presence of Co ions.
1084
M.
KOHLHARDT
AND
Ca concentration FIGURE on external
5. The dependence Ca concentration.
of overshoot (a) 0-0, Control;
K.
HAAP
(mM)
action and pmimax (b) of the slow response O-0, in the presence of 2 rn~ Co.
potential
Two other experiments revealed the same result, i.e. after Co treatment the overshoot no longer behaved as Ca electrode. Extrapolation of the regression lines characterizing the dependence of overshoot on external Ca prior to and after treatment with 2 mM Co to Ca concentrations higher than 10 mM reveals an intercept of both straight lines at about 15 mM (see Figure 5). The question arose “which slope factor would appear in the presence of Co ions at Ca concentrations above 15 mM. In other words, does the overshoot regain its Nernstian behaviour in spite of the continued presence of Co ions or not?” Because of possible osmotic effects and the fact that the overshoot tends to saturate at very strong external Ca concentrations (50 mM or more [S]}, experiments in that range might yield ambiguous results. We decided, therefore, to test this at a lowered cobalt concentration, namely 1 mM. The theoretical background was the hypothesis that the intercept of both regression lines is due to the interaction of Co with Ca ions and will be shifted to the left, i.e. to lower Ca concentrations if a cobalt concentration lower than 2 mM is applied. Figure 6 gives the evaluation of such an experiment. Although the treatment with 1 mM Co had even evoked a blockade of the slow response action potential, the overshoot attained its control value at 6 mM Ca. In the range from 4 to 6 mM Ca, the overshoot did not behave as a Ca electrode. Above 6 mM Ca, however, a Nernstian behaviour was found and the same slope factor as under control conditions
COBALT-INDUCED
INHIBITION
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SLO\V
INlVARD
1085
CI‘RRENT
60 -
SOL
2
4
5 6
Co concentration
FIGURE 6. The dependence Ca concentration prior to (0)
0
IO
15
(mr.4)
of the overshoot of the slow response action and after (0) treatment with 1 mM Co.
potential
on external
(31 mV) per tenfold change of external Ca appeared. Three other experiments of this type yielded the same results and, in the presence of 1 mM Co, the overshoot reached its control value at 6 to 8 mM Ca and regains Nernstian properties above that Ca concentration. Co ions diminished the catecholamine-induced increase of vm,, and overshoot of the slow response action potential. Whilst rm,, and overshoot responded under control conditions, i.e. in the absence of Co ions, to 9.2 x 10m6 M isoproterenol with an increase of 12.0 * 1.5 VI s and 12.4 f 0.8 mV, respectively, after treatment with 1 mM Co the same isoproterenol concentration produced an augmentation of only 2.0 & 0.65 V/s and 7.5 & 0.9 mV (n = 3), respectively. In accordance with this significantly restricted response is the observation that at higher Co concentrations (2 mM) isoproterenol became virtually ineffective since isoproterenol concentrations even as high as 9.2 x 10e4 M failed to overcome the Co-induced blockade of the slow response action potential. Only after return to a Co-free medium did the promoting effect of isoproterenol occur (see Figure 7). Both the restricted quantitative response of v,,, and overshoot towards isoproterenol at 1 mM Co and the lack of any promoting catecholamine action in the presence of 2 mM Co might arise from an interference of Co ions with some steps in the chain of events leading ultimately (probably via cyclic AMP) to the catecholamine-induced increase of conductance of the Isi system of the cardiac membrane or from the interaction of Co with Ca. To test this, the effect of isoproterenol (9.2 x 10d6 M and 1.38 x 10~~ M) was analysed in the presence of 2 mM Co at external Ca concentrations higher than 2 mM. Above 2 mM Ca, the
FIGURE 2 rn~ Co
and
7. The effect of isoproterenol after switching to a. Co-free
on the medium.
slow
response
5
6 7 8910
action
potential
in the
presence
of
60
50
40
4
Ca concentration
FIGURE on external 9.2 X 10e6
8. The dependence of (a), overshoot Ca concentration in the presence M isoproterenol (0).
(rnht)
and (b), vm’,,, of the slow of 2 WIM Co prior to, (0)
response action potential and after application.
of
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1087
slow response action potential regained its sensitivity towards isoproterenol. As demonstrated in Figure.& the increase of 3 lllLX and overshoot produced by any isoproterenol concentration was the stronger the higher the external Ca concentration was which led to a further rise of slope factor of overshoot per tenfold change of external Ca, in this case from 42 to 60 mV. Isoproterenol concentrations higher than 9.2 x lO-6 M did not cause a further increase of slope factor but evoked a parallel shift of the regression lines describing the dependence of overshoot on logarithm of external Ca concentration and describing the dependence of external Ca concentration to higher values for overshoot of I’,,x on logarithm respectively. At 10 mM Ca, there appeared an almost normal quantiand limax, tative response towards 9.2 x 10M6 M isoproterenol since pm,, increased by 12.2 + 1.6 V/s and overshoot by 16.1 & 7.2 mV (n :- 3). Needless to say, isoproterenol failed to produce any alteration of slope factor of overshoot per decade of Ca concentration in the absence of Co ions. 4. Discussion According to earlier observations by Kohlhardt et al. [14J Co ions reduced Isi. This inhibitory effect occurred without detectable changes of the time course of Isi. It is, therefore, unlikely that the suppression of Isi is caused by a decreased rate of activation or by an increased rate of inactivation. Rather the diminution of gsi seems to be the principal action of Co ions underlying the depression of 1si. It suggests that Co ions reduce the number of isi channels that open on depolarization of the cardiac membrane, leading consequently to a depression of both pm,, and overshoot of the slow response action potential. Despite the continued presence of Co ions, their inhibitory effect could be abolished by excess Ca, which restored the &mediated action potential. The Ga concentration needed for restoration of the 1si system depends on the Co concentration and proved to be smaller in the presence of I mM Co than at 2 mM Co, which might reflect an interaction of Co with Ca at certain sites of the cardiac membrane. This assumption is further supported by the observation that, after Co treatment, the 1simediated action potential loses its property as a Ca electrode in that range of external Ca concentration in which neutralization of the inhibitory Co effects occurs. Under control conditions, namely in the absence of Co ions, the I,i-mediated action potential behaved as an almost perfect Ca electrode in a Ca concentration range between 2 mM and 15 mM, although a rise of external Ca leads to an increase of K outward currents in Purkinje fibres and ventricular myocardium [I, 111. The variation of overshoot is predicted by the Nernst equation:
1088
M. KOHLHARDT
AND
K.
HAAP
where R, I and F have their conventional meanings and [Car] and [Gas] are two different external Ca concentrations. In the presence of Co, however, the slope factors of overshoot per decade of Ca variation experimentally obtained considerably exceeded the theoretical Nernst value by some 10 mV, indicating that Px, Pea or [Cali no longer remained unaffected on a Ca concentration change. Considering the specific situation in the case of an increase of external Ca, there are no realistic reasons to suppose a decline of 2’~ or [Cali, but it is most plausible to attribute this rise of slope factor, i.e. the supernormal Ca sensitivity of overshoot, to a rise of Pea. This increase of Pea could be interpreted as being indicative for an augmentation of gsi and might be basically involved in the removal of the Co-induced inhibition of the Isi system by excess Ca. The present results agree with earlier observations of Hagiwara and Takahashi [R] demonstrating suppression of the Ca spike in barnacle muscle by Co ions and attributing this inhibition to a competitive interaction of Co with Ca ions. However, the nature of this process cannot be definitely judged and it remains, furthermore, an unanswered question where this interaction finally leading to the decrease of @St of the cardiac membrane happens. Suppose that the cardiac slow membrane channel bears, like other ionic channels [5.9], negatively charged groups accessible for cations and involved in the process of cationic inward movements. The interaction of Co with Ca at these groups might cause finally the blockade of the number of channels per unit area of the membrane. On the other hand, a rather superficial structure capable of binding certain cations, among them Ca [3], could be taken into consideration, namely the glycocalyx, the surface coat of which represents an integral part of the cardiac sarcolemma. Studies by Langer and his co-workers [17] revealed a high afhnity of this structure for Ca, which can be displaced from its binding sites by La, Mn and other cations. This Ca pool might be a possible source of that Ca passing the slow channel and carrying Jsi during excitation [l6]. Because Co exerts its inhibitory action without a significant decrease of Esi, it is unlikely that a displacement of Ca from binding sites within the glycocalyx is involved in the blockade of Isiventricular myocardium, As shown by Reuter and Scholz [24] m . mammalian P-adrenergic catecholamines augment gsi. This augmentation of gsi is the reason for the well-known increase of Isi in the presence of catecholamines and very probably reflects a rise in the number of slow channels per unit area of the cardiac membrane. It is conceivable that Co ions interfere with the de noun formation of slow channels, which offers a possible explanation for the diminished isoproterenol effect. However, the Isi system regains its usual sensitivity towards isoproterenoi with increasing Ca concentrations despite the continued presence of CO ions. It seems to be, therefore, more plausible to attribute the restricted response towards isoproterenol at low Ca (<4 mM) to the interaction of Co with Ca, which might also operate at deLOUDformed channels, thereby making them unable to allow the inward movements of Ca.
COBALT-INDUCED
INHIBITION
OF SLOW
INWARD
CURRENT
1089
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