J Mol

Cell

Effect

Alain

Cardiol22,921-932

(1990)

of 2,SButanedione IL-Monoxime Outward Currents in Rat Coulombe, Laboratoire

Isabel

on Slow Ventricular

A. Lefevre,

Edith Deroubaix, Edouard Coraboeuf

de Physiologie Cornparke associe’ au GNRS ((IRA 91405 Orsay Cedex, France

Inward and Myocytes Dominique lIZI),

(Received 21 November 1989, accepted in revisedform

Transient

Thuringer

and

Universite’ Paris-&d,

10 April 1990)

I. A. LEFEVRE, E. DEROUBAIX, D. THURINCER AND E. CORABOEUF. Effect of 2,3-butanedione 2monoxime on slow inward and transient outward currents in rat ventricular myocytes. Journal of Molecular and Cellular Cardiology (1990) 22,921-932. The effect of 2,3-butanedione P-monoxime (BDM), a substance possessing phosphatase-like activity, was studied on action potentials of isolated rat heart and on the slow inward calcium current and outward current (including the 4-aminopyridine (4-AP) -sensitive transient outward component), in rat ventricular myocytes. In contrast to what was observed by other authors in different species and cardiac tissues, BDM increased markedly the amplitude and duration of the rat ventricular action potential plateau. On the other hand, in the presence of 4-AP and ryanodine BDM shortened action potential duration. BDM decreased in a dose dependent manner the amplitude of both the slow inward calcium current and the transient outward current, accelerated their inactivation and shifted their steady-state inactivation-voltage relationships towards negative potentials. BDM also depressed other components of outward current. It is suggested that the lengthening effect of BDM on action potential duration results mainly from the simultaneous reduction of both the slow inward calcium current and the transient outward current, two antagonistic currents with unequal influences on action potential plateau development. The similarity of effect ofBDM on these two currents also suggests that ionic channels generating them might require similar phosphorylation for their functioning. A. COULOMBE,

KEY WORDS:

aminopyridine;

Cardiac action potential; Phosphatase activity.

Rat cardiac

myocytes;

Introduction Oximes belong to a class of nucleophilic agents that are generally considered as “chemical phosphatases”. Their mode of action has been proposed as a result of the observation that oximes are able to remove phosphate groups from cholinesterase poisoned by organophosphorus compunds (Green and Saville, 1956). For almost 15 years the activity of the calcium channel has been thought to be regulated by phosphorylation. The /3-adrenergic agonists have been shown to enhance the slow calcium current (Reuter, 1967; Vassort et al., 1969) via an increase in CAMP production (Reuter, 1974) and protein kinase activation, an effect which has been more recently directly demonstrated at cellular (Osterrieder et al., 1982; Bean et al., 1984) and single channel levels Please address all correspondence Batiment 443, 91405 Orsay Cedex, 0022-2828/90/080921

+ 12 $03.00/O

to: Alain France.

Coulombe,

Slow inward

current;

Outward

current;

Oxime;

4-

(Flockerzi et al., 1986; see also Catterall et al., 1988) although complex cGMP-induced regulation also occurs (Levi et al., 1989!. One oxime that has been particularly studied, the 2,3-butanedione 2-monoxime (BDM) has been shown to reversibly shorten and depress the plateau phase of the cardiac action potential in embryonic chick heart (Sada et al., 1985), in adult ventricular myocardium (Wiggins et al., 1980; Li et al., 1985) and in dog Purkinje fibers (Bergey rt al., 1981). These sarcolemmal effects were interpreted as a consequence of calcium current inhibition resulting from removal of phosphate groups from the slow calcium channel protein, whereas the inhibition of contractile activity was shown to be related to an apparent decrease in sensitivity towards calcium, Laboratoire

de Physiologie

Comparee,

Lniversitc

:g 1990 Academic

Paris-Sud, Press Limited

922

A. Conlombe

involving either a direct effect at the myofibril level or an effect on the sarcoplasmic reticulum or both (Li et al., 1985). In cardiac tissues,the slow calcium current is not the only ionic current to be considered asregulated by channel phosphorylation. Recently, it has been suggested(Nakayama and Fozzard, 1988) that in canine Purkinje cells, the transient outward current responsiblefor the early repolarization of the action potential is also modulated by CAMP-dependent phosphorylation whereas in rat ventricular cells this current is reduced by al-adrenoceptor agonists (Apkon and Nerbonne, 1988). In the present paper we show that in the perfused rat heart the plateau of the action potential is not shortened by BDM but rather considerably lengthened and we demonstrate that this effect results from the simultaneous reduction of antagonistic currents, namely the slow inward calcium current and outward current components including the Carindependent transient outward current. A preliminary report of this work has already appeared (Coulombe et al., 1989).

et al.

95% Oz-5% CO,; pH 7.4. The temperature was maintained at 24f0.5”C. The following drugs were used: 2,3-butanedione 2-monoxime (BDM, Sigma); 4-aminopyridine (4AP, Sigma); ryanodine (Penick) . The shape of the rat ventricular action potential plateau at low temperatures is generally complex (Coraboeuf et al., 1956) and showsa repolarization which develops in two phases:a narrow initial plateau and a lower and slower one. For this reason we measured action potential duration at -3OmV and at - 65 mV. Data are expressedas mean + S.D. Isolated myocyte experiments

Adult rat ventricular myocytes were isolated according to the procedures of Powell et al. ( 1980) and Irisawa and Kokubun ( 1983). The digested ventricles were maintained in “KB” medium (Isenberg and Klockner, 1982) and stored at 4°C before use. Cells were mechanicalIy dispersedby gently shaking a small piece of tissue cut from either right or left ventricular myocardium in a plastic Petri dish containing the appropriate extracellular medium (see below). All experiments were Materials and Methods conducted at room temperature (19-22°C). Isolated perfused hearts Cell membrane currents were recorded Rats weighing 150 to 250 g were used. Experiusing the whole-cell configuration of the ments were performed on isolated hearts per- patch-clamp technique (Hamill et al., 1981). fused according to the Langendoti technique Patch pipettes were pulled from Pyrex capilwith constant perfusion pressure(75 cm water laries (Corning 7740) and were not firehigh). Transmembrane action potentials were polished before use. Pipettes with a resistance recorded from the epicardial surface of the of l-3 Mf2 were usedroutinely. A Dagan 8900 right ventricle by means of standard floating patch-clamp amplifier with a 100MR feedmicroelectrodes (15-30 MQ). The apex of the back resistor was used without filtering (wide heart was attached to a strain gauge (Statham band). The electrode resistancein serieswith UCz) for tension recording. Action potentials the cell membrane was compensated by miniwere recorded on one beam of a Tektronix 565 mizing the duration of the capacitive surge on oscilloscopevia a M707 amplifier (WP Instruthe current trace. Leakage current was not ments). On the other traces of the oscilloscope compensated. A flow of solution from one of a were recorded the action potential first deriva- seriesof five piped outlets continuously supertive and the contraction. Hearts were electri- fused the cell from which recording was being cally driven (2-3 mspulses)at a frequency of 1 made. The flow rate of the superfusion soto 1.5 Hz by meansof bipolar silver electrodes lutions was 50-lOO~l/min. For recording of located at the base of the right ventricle both slow inward and outward currents, vol(frequency lo-20% higher than the sponta- tage pulses were applied at a frequency of neousrhythm). 0.2Hz. Cell currents and voltages were reThe normal Tyrode solution had the follow- corded and stored with a beta video cassette recorder (Sony) after 16-bit digitization at ing composition (mM): 130 NaCl; 5.6KCI; 2.15 CaClz; 1.1 MgClz ; 20 NaHCOs ; 22 kHz with a pulse code modulator (Sony PCM-70 1-ES). Macroscopic currents were 0.6 NaHz PO4; 11dextrose. It was gassedwith

ESect of BDM

on Cardiac

further digitized at 4 or 6 kHz with a microcomputer (Compaq, Deskpro 286) using an S200 interface (Cambridge Research Systems, UK) analysed and retrieved on a HP-7475A plotter. The kinetic analysis ofcurrent inactivation was performed by fitting a sum of exponential decays to experimental data, using non-linear regression program the DISCRETE (Provencher, 1976). Data concerning the steady-state inactivation process were fitted by the theoretical Boltzmann distribution function by means of the non-linear least squares gradient-expansion algorithm of Marquardt. The values are expressed as mean f S.D. of n experiments if not otherwise indicated. The standard extracellular medium in which the myocytes were maintained contained (mM): 135 NaCI; 5.4 KCl; 1 MgClz; 1.8 CaClz ; 1 ribose; 10 glucose; 0.001 ryanodine; 10 HEPES; pH7.4 (NaOH). However, for recording either the slow calcium current or the outward current, the sodium current by substituting choline was eliminated chloride (choline-Cl) for NaCl in the superfusion medium, the possible activation of muscarinic potassium currents by choline being prevented by the addition of atropine. In order to record calcium current, potassium currents were minimized by using K+free internal and external media, and by adding 3rn~ 4-AP to both the standard extracellular medium and the superfusion medium. The superfusion medium contained (mM): 105 choline-Cl; 20 tetraethylammonium (TEA)Cl; 1 MgC12 ; 2 CaCls ; 1 ribose; 10 glucose; 3 4-AP; 0.01 atropine-S04; 0.001 ryanodine; 10 HEPES; pH 7.4 (CsOH). The pipette medium was (mM): 105 Cs-aspartate; 20‘TEACl; 5 MgClz; 5 Naz-phosphocreatine; 10 glucose; 4 Mg-ATP; 15 EGTA; 10 HEPES; pH 7.2 (CsOH). In order to record the potassium current, calcium currents were suppressed by including 0.5 mMCdCl2 in both the standard extracellular medium and the superfusion medium. The superfusion medium contained (mM) : 135 choline-Cl; 1 MgC12; 1.8 CaC12; 0.5 CdClz; 1 ribose; 10 glucose; 0.01 atropine-Sod; 0.001 ryanodine; 10 HEPES; pH 7.4 (KOH). The pipette medium contained (mM): 115 Kaspartate; 5 KCl; 5 Na-pyruvate; 7 MgClz; 4 Naz ATP; 5 EGTA (free Mg2 + approximate-

Currents

923

ly 2.7 mM, calculated with program SPECS; Fabiato, 1988), 10 HEPES; pH7.2 (KOH). Under these conditions, the contribution of calcium-activated potassium currents to the total potassium current was minimized. In Purkinje fibers two components of transient outward current have been described. One is Cat-dependent (Siegelbaum and Tsien, 1980) and inhibited by caffeine (Coraboeuf and Carmeliet, 1982) and ryanodine (Sutko and Kenyon, 1983). In sheep Purkinje fibers stimulated at very low frequencies, low concentrations of 4-AP (0.5 mM) suppress (i) the totality of the long Iasting Cai-independent transient outward current (Coraboeuf and Carmeliet, 1982), a current generally labelled ii, or iA (more recently itot, Tseng and Hoffman, 1989) and (ii) part of the timeindependent outward current (Van Bogaert and Snyders, 1982). At the pulse frequency used in the present work, the inhibition of il, by 4-AP was generally incomplete because of the removal of block upon depolarization (Simurda et al., 1986).

Results

Effects of BDM onperfused

rat heart

The effects of 5 and 20m~ BDM on action potential and contraction are shown in Figure 1. Both concentrations increased the action potential plateau amplitude but the effect was much larger with 20m~ BDM than with 5 mM, specially during the initial phase of the plateau. The maximum rate of rise of the action potential (V,,,,,) was markedly reduced at the higher concentration, in spite of an unchanged resting potential (Ek). The efIects of BDM on ER, timaX, action potential duration and contraction are shown in Table 1. Contraction was reduced by about half at 5rn~ BDM, by 95O/, at 10mM and entirely suppressed at 20mM. The effects on ER, pm,,, and contraction were very similar to those previously described in guinea-pig papillary muscle (Li et al., 1985). In contrast, in this species 20m~ BDM depressed and shortened the action potential plateau instead of increasing it. A plausible reason for such a striking difference is the existence of a 4-AP sensitive transient outward current in rat ventricle (Mitchell et al., 1983; Josephson et al., 1984)

Bffect of BDM

924

on Cardiac

Currents

O-

oBDM

n

BDM

5 I-IYM

20

mM

n

FIGURE 1. Effect of BDM on ventricular action potential and contraction of rat perfused heart. Action potentials (upper traces), contraction (middle traces) and first derivative of the rising phase of the action potential (lower traces) in control conditions (C) and during 5 rn~ BDM perfusion (left panel) and 20 rn~ BDM perfusion (right panel).

whereas such a current has not been observed in guinea-pig ventricular muscle. Becausethis current is responsible for the fast early repolarization of the cardiac action potential plateau, we tested the possibility that it is inhibited by BDM. Figure 2 showsthe effects of 20 mM BDM (A) and 1 mM 4-AP (B) on the ventricular action potential of the same perfused heart. The experiment was performed in the presenceof 10m6M ryanodine to eliminate calcium release from the sarcoplasmic reticulum and resulting effects on Cat-dependent membrane currents. It can be seen that the effects of BDM and 4-AP were very similar. This suggests that BDM inhibits the Catindependent transient outward current. If this is true and if this inhibition is one of the determinant causesof the BDM-induced lengthening of the action potential plateau in rat ventricular myocardium, BDM should no longer lengthen the plateau when added in the presence of 4-AP but rather shorten it. This is indeed shown in Figure 2(C) [in the concomitant presenceof ryanodine as in Fig. TABLE

1. Effect

of BDM

on action

potential

2(A) and (B)]. In fact although the action potential duration was markedly shortened by BDM in the presence of 4-AP, in this and almost all similar experiments the initial amplitude of the plateau was sizeably increased. Efects of BDM on macroscopic slow inward current and outward currents

Figure 3 showsan example of the effects of BDM on the macroscopic slow calcium current [ (iSi, Fig. 3(A)] and on the outward current [iO, Fig. 3(B)]. In this example, chosen concentrations of BDM were maximal concentrations allowing complete reversibility of ii (8 mM) and almost complete reversibility of iO (16 mM). At 16 mMBDM, iSi did not recover more than 50-60% of its control value in our experimental conditions. This was possibly due in part to the acceleration of the wellknown phenomenon of rundown (seeBelleset al., 1988) or to the greater insensitivity of ig to BDM compared to that of iO. Current traces

parameters

and APD-30 h)

Control n= 11 BDM 5 mM n=5 BDM 1OmM n=3 BDM 20 mM n=3

82.7 k3.1 84.4 f0.9 82.3 f3.0 83.0 +2.0

95.3 f9.7 91.5 fa.9 87.5 +a.7 59.0 f9.4

peak

mechanical

tension

APD - 65 (ms)

Tension

57.8 *a.0 85.1 f5.a 86.8 * 7.4 87.3 * 7.0

100

13.6 f2.2 30.9 f2.1 37.6 +4.5 39.5 +3.5

Ea: resting potential: v,,,..: maximum rate of rise of action potential; APD - 30, APD-ss: -3OmV and -65 mV; tension is expressed in percent of control values.

action potential

%

45.6 f4.5 5.6 k4.0 0

durations

at

A. Coulombe

et al.

925

FIGURE 2. Effect of 20 rn~ BDM (A), 1 rn~ 4-AP (B) and 20 rn~ BDM in presence of 1 rn~ 4-AP (C) on venrricular action potential of isolated perfused rat heart. In A, B and C, 10w6M ryanodine was added to the Tyrode solution to prevent calcium-induced calcium release from the sarcoplasmic reticulum.

shown in Figure 3 indicate that BDM decreasesboth pi and i. and alter their kinetics. Lower concentrations of BDM (1 mM) reduced by -N lo:/6 the peak amplitude of ii with little effect on the current kinetics whereas the decay of & was slightly accelerated without any significant change in the peak amplitude (not shown). In rat ventricular myocytes l,i may be generally divided into two components, a timeand a timedependent component independent component, the former most often inactivating according to a doubleexponential process. The effects of 8 and 16m~ BDM on i,i are illustrated in Figure 4(A) and (B) respectively. 16mM BDM markedly decreased the two components of i,i. Superfusion with 0.5 mM Cd completely abolished i,i which fully recovered after washout (not shown). Table 2 gives the values of the parameters of the different kinetic components of the Cd-sensitive slow inward current at different potentials. At OmV, 8rn~ BDM reduced the amplitudes of both the fast and slow components of isi by about 20% and that of the steady-state component by 45%, whereas 16mM BDM reduced the three components by 76, 55 and 77% respectively. A

Figure 5(A) shows typical current-voltage curves of the Cd-sensitive i,i in control conditions (before and after BDM) and during perfusion with 16m~ BDM. In this and 4 other similar experiments BDM reduced the maximum current amplitude by 67 f 16% (n = 5). Figure 5(B) shows that the steadystate inactivation curve of i,i was slightly shifted towards negative membrane potential by 16mM BDM. Because run down of i,i developed with time the average VI/z values of control and recovery inactivation curves were calculated and compared with that in the presence of BDM. In the example shown in Figure 5(B) the shift was 2.7mV. For three different experiments, including that in the figure, it was 6.5 + 2.3 mV. No detectable shift occurred with 8 mM BDM. Outward currents which developed in our experimental control conditions are shown in Figure 6(A,a). As in the caseof isi, 1, can be divided into a steady-state component and a time-dependent component (transient outward current, il.,) which inactivates according to a double-exponential process.A large part, but not the totality of i, could be suppressed by 3rn~ 4-AP [Fig. 6(A,c)]. Compared with the inhibition exerted by 4-AP, that exerted i3

i: “r ‘,\

‘A\,

100 -. ms

washout

\\ i /I,I /-

BDM Control

and

washout

16 mM s

FIGURE 3. A: effect of8 rnM BDM on the slow inward calcium current. B: effect of 16m~ BDM on outward current. Currents were elicited by depolarizing pulses from - 60 to 0 mV of 350 ms duration in A and from - 80 to +60 mV of 700 ms duration in B. In both cases, steady-state effect ofBDM was reached within 10 s; BDM trace recorded after 30 s of BDM action; washout trace recorded after 3 min of recovery.

A. Coulombs

926 A

BDM

et al. B

BDM

FIGURE 4. Effect of BDM on the slow inward calcium current: effects of 8 mM (A) and 16 rnM BDM (B) on slow inward calcium current in control conditions (C), during BDM and in the presence of0.5 rnM cadmium (Cd) after BDM removal at different pulse potentials (- 20, 0 and + 30 mV) from a holding potential of -60 mV.

by BDM was weaker on the fast inactivation process of it, and stronger on its slow inactivation process whereas the inhibition of the steady-state component of Z, was about the same for the two substances [(Fig. 6(B) and (C)l. The stronger inhibition of i, shown in Figure 6(A,d) (16mM BDM + 4-AP) compared with that shown in Figure 6(A,c) (4AP) can explain in part the increase in plateau amplitude shown in Figure 2(C) during 20 mM BDM action. The effects of 8 mM BDM on i, were similar to but less pronounced than those of 16 mM. Table 2 gives the values of the different kinetic parameters calculated in the different experiments. The amplitudes of the two time-dependent components and the steady-state component of the outward current were depressed by BDM at all potentials in a dose-dependent manner. At +30mV, the time constants of the fast and the slow components were reduced by 40% at 8 mM BDM and 50-60% at 16m~ BDM. The current-voltage curves shown in Figure 7 correspond to the time-dependent and steady-state components of outward current before and during application of 16 mM BDM

(A) and 3 mM 4-AP (B) . The effects of 8 mM and 16m~ BDM on the steady-state inactivation of the time-dependent outward current are shown in Figure 7(C) and (D) respectively. Whereas 8mr+4 BDM produced only a small shift of the curve towards negative potentials (2.7 +_ 0.4mV, n = 3), 16mM BDM produced more complex effects on the voltagedependence of inactivation. After normalization of the right part of the curves (between -45 and + 5 mV) that corresponding to BDM was shifted by 5.3 f 2.2 mV in three different experiments. Discussion

The major findings of this study are the following (i) in the rat perfused ventricular myocardium BDM markedly and reversibly increasedaction potential duration and plateau amplitude (ii) in isolated rat ventricular myocytes BDM decreasedboth the slow inward calcium current, Z,i, and the outward current, t,, including the Car-independent transient outward current it, and accelerated the decay of thesecurrents.

Effect TABLE

2. Characteristics

V, imY

n

of slow inward BDM (4

of BDM calcium

+ 10

0

- 10

-2o*

7 3 7 3 4 7 3 4 7 3 4 7 3

C 8 C 8 16 C 8 16 C 8 16 C 8

26k

+50

f30

f20

fl0

10 5 5 10 5 1; 5 1; 5 1; 5 5

and outward

INWARD

10

8 16 C 8 16 C 8 16 C 8 16

The currents obtained under control different test membrane potentials (V,). equation:

927

current. AA., ( 0,“1

If (msi

& Ims)

9&l 12f4 16+ 15 * 10 + 23 & 17 * 16f4 27 + 27 + 13 + 25 f 21 *4

113 + 16 89 k 6 112 * 20 52* 13 71 f28 89 + 9 58f 17 58+ 12 114+ 19 101 & 18 66+ 10 187 + 60 134 + 16

CURRENT 5*4

60+

13

21 k8 80 + 20

26 + 7 60+ 15

60 + 9 85+ 12

19 + 13 76f 14

18 + 8 55i 15

45 * 10 77 i 16

20 * 7 88k 10

18+ 70&

45* 74+

18 k 10

C 8 16

Currents

AAs lo/n)

OUTWARD +60

current

A.% (%I SLOW

+ 30;

on Cardiac

11 19

352 POTASSIUM

15 12

40 + 20

1 3 3 2 3 5 6 3 2

CURRENT

31*5 51 k6

32+ 12 29 + 4

34 + 6 20 * 3

33 k 6 43 + 5

30 * 11 37 k 6

21 *5 34& 10

43 k 6 48 + 6

27 k 9 38k 12

17+4 41 k8

38 k 6 45 + 5

25 &- 8 30 + 4

28 + 5 39 * 5

33+ 11 39 + 6

40+6 46 + 8

26 k 4 34 & 3

63 35 21 57 39 22 57 33 24 46 34 27 54 38 29

& 7 * 3 +2 f 7 * 5 + 2 + 7 f 3 k 3 + 6 + 6 + 4 + 7 k 8 k 4

457 291 273 536 332 315 716 421 353 839 569 549 649 419 303

* k & k 2 + + * * k If: + k + &

40 39 18 56 11 14 60 79 37 72 87 78 65 60 63

conditions (C) and in presence of 8 and 16 rnM BDM were analysed for 5 A sum of two exponentials and a constant term, represented by the following

i(t) = Arexp(-t/sr)+A,exp(-t/r,)

+ A,,

was used to extract the amplitudes and time constants of the time-dependent components and the amplitude of the steady-state component. Af and A, are determined at the time of peak currents, of the fast and the slow components respectively. A,, is the amplitude of the steady-state current. AAf, AA, and A&, represent the inhibition of the amplitudes of the fast, slow and steady-state components respectively, in percent of control values. Q and T$ correspond to fast and slow time constants. For analysis of the calcium current, traces obtained under 0.5 rnM Cd have been subtracted from control of BDM traces. Values are expressed as mean + SEM of n experiments. *At these membrane potentials, 16 rnM BDM suppressed entirely the current, so that no data are given.

The observation that the rat ventricular action potential is prolonged and its plateau amplitude increased by BDM is at variance with results obtained in dog Purkinje fibers (Bergey et al., 198 1 ), embryonic chick heart (Sada et al., 1985) and guinea-pig papillary muscle (Li et al., 1985) whose normal and/or slow cardiac action potentials were generally sizeably shortened by 10-30 mM BDM. In all the cases it was suggested but not directly

demonstrated by current measurements that BDM inhibited the calcium current. The present paper fills this gap by showing that 8-16 mM BDM does indeed depress i,i and alters its time constants of inactivation. The fact that BDM also strongly depresses outward currents can explain the lengthening effect of this substance on the rat ventricular action potential. In this tissue and this species il, has a rapid recovery time course (Josephson et al.,

A. Coulombe

928

et al.

+ Control \/ -60

-20

0

BDM

x

washout

20

16 rn~

60

+

Control

0

BDM

16 mM

X washout

-40

-80 Membrane

0 potential

(mV)

FIGURE 5. Effect of 16 rn~ BDM on the current-voltage relationship curve (B) of the slow inward Cd-sensitive current (i.e. after subtraction Cd).

1984), is of large magnitude (larger than &i, Fig. 3) and is fully able to mask the inward calcium current. Indeed, in rat ventricular cells bathed in normal Tyrode solution, it has been observed that depolarizing stepsto 0 mV initiated inward peaks of current when starting from a holding potential of -4OmV, i.e. when ii, was completely inactivated whereas they initiated outward peaks of current when starting from - 60 or - 80 mV (Mitchell et al., 1983 their Fig. 8). In the latter case, addition of 1mM 4-AP unmasked the initial peak of inward current. Such transient outward currents have never been observed in guinea-pig papillary muscle. As a result a substance which acts as a blocker of both isi and i,, can very well lengthen the action potential

(A) and the steady-state of the current remaining

inactivation-voltage in presence of 0.5 mM

plateau of a cell which possesses the two types of current if the drug-induced depression of the repolarizing outward current is, in absolute value, larger than that of the depolarizing inward current. In contrast the samesubstance can only shorten the action potential of cells which possessonly i,i. The fact that in dog Purkinje fibers (which possess a transient outward current; Nakayama and Fozzard, 1988) BDM shortensthe action potential indicates that the relative participation of the two currents in the development of the plateau and/or their sensitivity to BDM are markedly different between this tissueand the rat ventricle. Our results confirm that after suppressionof the 4-AP sensitive outward currents (and in presence of ryanodine) BDM

Effect

of BDM

on Cardiac

Currents

929

A

(a)

(b) Control

4-AP

3 mM

8

BDM

16 ITIM

BDM+4-AP

(d)

C BOM 8mM

BDM

FIGURE 6. Effect of BDM on outward current. A: Families of superimposed current traces in four different conditions as indicated. Current traces were elicited from a holding potential of -8OmV by hyperpolarizing or depolarizing 700 ms pulses, applied in 10 mV increments, from - 110 to +60 mV. B and C: Effects of8 rnM (B) and 16 mM (C) on outward current in control condition (c), during BDM and in presence of 3 mM 4-aminopyridine (4.API after BDM removal. at different potentials (+ 10, +30 and + 60 mV) from a holding potential of -80 mV.

shortens the plateau of the rat ventricular action potential instead of lengthening it. The main effect of ryanodine, which was systematically used in our experiments on isolated myocytes, was to eliminate two currents: (i) an inward current induced by the release of calcium from sarcoplasmic reticulum which participates in the later part of the plateau of the rat ventricular action potential (Mitchell et al., 1984) and (ii) a transient component of outward current different from ito, also inhibited by caffeine and activated by internal calcium (Coraboeuf and Carmeliet, 1982; Sutko and Kenyon, 1983; Escande et al., 1987; Hiraoka and Kawano, 1989; Tseng and Hoffman, 1989). Our results demonstrate an interesting

similarity of BDM action on both the slow inward current and the Cat-independent transient outward current (depression of the amplitude and acceleration of decay of their components and shift towards negative potentials of their steady-state inactivation curves). This suggests the existence of some similarities in the structure of the corresponding channels particularly in the phosphorylated regions rcquired for their functioning, if one admits that oximes act mainly as chemical phosphatases. The effect of BDM on ionic channels is generally considered as occurring at the intracellular surface of the membrane because BDM is known to cross, for example, the brain barrier and because those quaternary amine oxime derivatives which cannot cross the membrane

930

A.Codombcctal.

0.6

-80

-40

0 Membrane

potentlol

-80 (mV)

FIGURE 7. Effect of BDM (A) and 4-AP (B) on current-voltage elicited by depolarizing (or hyperpolarizing) pulses. Open symbols: peak (or initial) currents; dotted lines: steady-state currents. C and D: control conditions and in the presence of 8 mM (C) and 16 mM (D) normalized data in the presence of 16 rnM BDM in the potential range control is - 2.9 mV.

do not exert electrophysiological effects (Bergey et al., 1981). Oximes have been reported not to act on phosphodiesters,but mainly on phosphotriesters and phosphomonoesters(for references,seeBergey et al., 1981). As a result oximes could attack the terminal phosphate of ATP and therefore depressATP content but this is apparently not the case because the ATP content of cardiac Purkinje fibers has been found to remain unchanged after 30 min of oxime action (Bergey et al., 1981). For this reason the main site of oxime action is generally thought to be phosphomonoesterssuch as those formed by kinase activity. However the recent observation that in squid axon the potassium current steady-state inactivation relationship is shifted towards positive potentials by internally applied ATP (Perozo ct al., 1989) points to a possiblerole of this substance in the interpretation of BDM effects. Part of

-40

0

relationships of outward (or inward) currents control; filled symbols: drugs. Continuous lines: Steady-state inactivation-voltage relationships in BDM. Filled squares and corresponding curve: -45 mV to +5 mV: the VI,2 shift with respect to

the effect of internal ATP has indeed been attributed to the additional negative charge carried by the phosphate group. The negative shift of the inactivation relationships of both isi and it, observed here in the presenceof BDM is therefore consistent with a reduction of negative charges at the intracellular part of the channels as a result of dephosphorylation. Curiously this effect is at variance with that induced on the sodium channel by B-receptor stimulation, i.e. a shift of the inactivation relationship towards negative potentials attributed to the phosphorylation of the channel (Ono et al., 1989). BDM action sites are multiple and are clearly not limited to the sarcolemma. This substance exerts a strong negative inotropic effect which has been attributed in cardiac (Li et al., 1985) and skeletal muscle (Horiuti et al., 1988) to a direct effect on the contractile

ESect of BDM

on Cardiac

system, and also, in skeletal muscle, to a reduction in calcium release from the sarcoplasmic reticulum, perhaps by dephosphorylation of some sites linking the transverse tubules to the reticulum (Fryer et al., 1988). These possibilities constitute additional reasons for having used ryanodine in the present work as an attempt to dissociate sarcolemmal sites of BDM action from endocellular ones. The effect of BDM shown in Figure 7(D) is surprising. The existence of a double S-shaped curve suggests that the channels can be divided into two populations. One population, about 75% of the total, behaves under 16 mM BDM as the totality of the channels under 8rn~ BDM. They undergo only a small shift (a few mV) in their inactivation curve towards negative potentials. On the other hand, the remaining channels undergo a large shift ( N 40 mV) in their inactivation curve. This is very similar to the effect produced by local anesthetics on the Na channel (Strichartz, 1973) and by calcium blockers on the Ca channel (McDonald et al., 1984, their Fig. 5), an effect which results from the blocking effect

Currents

Ylsl

of the molecule by its binding inside the channel, and its removal by negative potentials. Interestingly a local anesthetic effect of BDM on the fast sodium current, i+, is also suggested by the reduction of Vmax shown in Figure 1 (C) and Table 1. It is therefore possible that, in addition to strong inhibitory effects on i,i and il, and moderate shifts of the inactivation curves along the voltage axis resulting from phosphatase-like activity on some phosphorylated sites of the molecules, at high concentrations (16mM) BDM exerts a blocking effect of local anesthetic type on il, and &a but not on i,i. Acknowledgement We wish to thank Evelyne Ferrary for having done preliminary experiments on action potentials of isolated rat heart. We thank for excellent technical assistance, Paulette Richer for preparing the isolated cells and Gtrard Sadoc for computer programming. This work was supported by a grant from Association Franqaise contre les Myopathies.

References APKON M, NERBONNE JM (1988) aI-adrenergic agonists selectively suppress voltage-dependent Ii+ currents in rat ventricular myocytes. Proc Nat1 Acad Sci (USA) 85: 8756-8760. BEAN BP, NOWYCKY MC, TSIEN RW (1984) /?-adrenergic modulation ofcalcium channels in frog ventricular heart cells. Nature (Lond) 307: 371-375. BELLES S, MA&COT CO, HESCHELER J, TRAUTWEIN W. (1988) “Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium. Pfliigers Arch 411: 353-360. BERGEY JL, REISER J, WIGGINS JR, FREEMAN AR (1981) Oximes: ‘enzymatic’ slow channel antagonists in cardiac Purkinje fibers? Eur J Pharmacol71: 307-319. CATERRALL WA, SEAGER MJ, TAKAHASHI M (1988) Molecular properties of dihydropyridine-sensitive calcium channels in skeletal muscle. J Biol Chem 263: 3535-3538. CORABOEUF E, CARMELIET E (1982) Existence of two transient outward currents in sheep cardiac Purkinje fibres. Pfliigers Arch 392: 352-359. CORABOEUF E, KAYSER C, GARGOU~‘L YM (1956) La repolarisation du myocarde au tours de I’hypothermie chez trois especes de mammiferes: Cobaye, Spermophile (Citellus citellus) et Rat blanc. C R Acad Sci 243: 1673-1676. COULOMBE A, LEFEVRE IA, DEROUBAIX E, CORABOEUF E (1989) Effect of diacetyl monoxime on transient outward current in rat ventricular myocytes. Pfliigers Arch 414 (Suppl 1): S 173-S174. ESCANDE D, COULOMBE A, FAIVRE JF, DEROUBAIX E, CORABOE~F E (19871 Two types of transient outward currents in adult human atria1 cells. Am J Physiol 252: H142-148. FABIATO A (1988) Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. In: Methods in Enzymology, Biomembranes, Vol 157: ATP-driven pumps and related transport, S Fleischer, B Fleischer (Eds). Orlando, Academic Press, pp 378417. FRYER MW, NEERING IR, STEPHENSON DG (1988) Effects of 2,3-butanedione monoxime on the contractile activation properties of fast- and slow-twitch rat muscle fibers. J Physiol (Lond) 407: 53-75. FLOCKERZI V, OEKEN HJ, HOFMANN F, PELZER D, CAVALIER A, TRAUTWEIN W. (1986) Purified dihydropyridinebinding site from skeletal muscle t-tubules is a functional calcium channel. Nature (Lond) 323: 6&68. GREEN AL, SAVILLE B (1956) The reaction of oxime with isopropyl mcthylphosphonofluoridate (sarin). J Chrm SOC 756: 3887-3892. HAMILL OP, MARTY A, NEHER E, SAKMANN B, &WORTH FJ (1981) Improved patch-clamp techniqurs tbr highresolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391: 85-100.

932

A. Coulombe

et al.

HIRAOKA M, KAWANO S (1989) Calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes. J Physiol (Lond) 410: 187-212. HORIUTI K, HICUCHI H, UMAZUME Y, KONISHI M, OKAZAKI 0, KURIHARA S (1988) Mechanism of action of 2,3butanedione P-monoxime on contraction of frog skeletal muscle fibres. J Muscle Res Cell Motility 9: 156-164. IRISAWA H, KOKIJBUN S (1983) Modulation by ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of guinea-pig. J Physiol (Lond) 338: 321-337. ISENBERG G, KLGCKNER U (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”. Pfliigers Arch 395: 6-18. JOSEPHSON IR, SANCHEZ-CHAPULA J, BROWN AM (1984) Early outward current in rat single ventricular cells. Circ Res 54: 157-162. LEVI RC, ALLOATTI G, FISCHMEISTER R (1989) Cyclic-GMP regulates the &-channel current in guinea pig ventricular myocytes. Pflugers Arch 413: 885887. Lr T, SPERELAKIS N, TENEICK RE, SOLARO RJ (1985) Effects of diacetyl monoxime on cardiac excitation-contraction coupling. J Pharmacol Exp Ther 232: 688695. MCDONALD TF, PELZER D, TRAUTWEIN W (1984) Cat ventricular muscle treated with D600: characteristics of calcium channel block and unblock. J Physiol (Lond) 352: 217-241. MITCHELL MR, POWELL T, TERRAR DA, TWIST VW (1983) Characteristics of the second inward current in cells isolated from rat ventricular muscle. Proc R Sot Lond B 210: 447469. MITCHELL MR, POWELL T, TERRAR DA, TWIST VW (1984) The effects of ryanodine, EGTA and low-sodium on action potentials in rat and guinea-pig ventricular myocytes: evidence for two inward currents during the plateau. Br J Pharmacol81: 543-550. NAKAYAMA T, FOZZARD HA (1988) Adrenergic modulation of the transient outward current in isolated canine Purkinje cell. Circ Res 62: 162-I 72. ONO K, KIYOSUE T, ARITA M (1989) Isoproterenol, DBcAMP, and forskolin inhibit cardiac sodium current. Am J Physiol256: Cl 131-C1137. OSTERRIEDER W, BRUM G, HESCHELER J, HOFMANN F, FLOCKERZI V (1982) Injection of subunits of cyclic AMPdependent protein kinase into cardiac myocytes modulates Ca” current. Nature (Lond) 298: 576-578. PEROZO E, BEZANILLA F, DIPOLO R (1989) Modulation of K channels in dialized squid axons. ATP-mediated phosphorylation. J Gen Physiol93: 1195-1218. PROVENCHER SW (1976) A Fourier method for the analysis of exponential decay curves. Biophys J 16: 27-41. POWELL T, TERRAR DA, TWIST VW (1980) Electrical properties of individual cells isolated from adult rat ventricular myocardium. J Physiol (Lond) 302: 13 l-l 53. REUTER H (1967) The dependence ofthe slow inward current in Purkinje fibres on extracellular calcium concentration. J Physiol (Lond) 192: 479-492. REUTER H (1974) Localization ofbeta adrenergic receptors and effects ofnoradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle. J Physiol (Lond) 242: 429-45 I. SADA H, SADA S, SPERELAKIS N (1985) The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues. Can J Physiol Pharmacol63: 1267-1270. SIMURDA J, SIMURDOVA M, CLJPERA P (1988) 4-aminopyridine sensitive transient outward current in dog ventricular 6bres. Pfliigers Arch 411: 442449. SIECELBAUM SA, TSIEN RW (1980) Calcium-activated transient outward current in calf cardiac Purkinje fibres. J Physiol (Lond) 229: 485-506. STRICHARTZ GR (1973) The inhibition ofsodium currents in myelinated nerve by quaternary derivatives of lidocaine. J Gen Physio162: 37-57. SUTKO JL, KENYON JL (1983) Ryanodine modification ofcardiac muscle responses to potassium free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release. J Gen Physiol82: 385-404. TSENG G-N, HOFFMAN BF (1989) Two components of transient outward current in canine ventricular myocytes. Circ Res 61: 633-647. VAN BOCAERT PP, SNYDERS DJ (1982) Effect of 4-aminopyridine on inward rectifying and pacemaker currents of cardiac Purkinje fibres. Pfhigers Arch 391: 230-238. VASSORT G, ROUCIER 0, GARNIER D, SAUVIAT MP, CORABOEUF E, GARGOU~L YM (1969) Effects of adrenaline on membrane inward currents during the cardiac action potential. Pfliigers Arch 309: 70-81, WIGGINS JR, REISER J, FITZPATRICK DF, BERGEY JL (1980) Inotropic actions of diacetyl monoxime in cat ventricular muscle. J Pharmacol Exp Ther 212: 217-224.