J Mol Cell Cardiol
22, 1371-1378
Antagonism Background
(1990)
by Adenosine and ATP Current in Guinea-pig
of an Isoprenaline-induced Ventricular Myocytes
A. C. Rankin,
and
R. Sitsapesan
K. A. Kane*
University Department of Medical Cardiology, Royal Infirmary, Glasgow *Department of Physiology and Pharmacology, lJniver.rit_v of Strathcl_yde, Glasgow.
CTII‘
(Received 26 October 1989, accepted in revised form 6 June 1990) C. RANKIN, R. SITSAPESAN AND K. A. KANE. Antagonism by Adenosine and ATP of an Isoprenaline-induced Background Current in Guinea-pig Ventricular Myocytes. Journal of Molecular and Cellular Cardiologv i 1990) 22. 1371-1378. A background current induced by isoprenaline, and its modulation by adenosine and ATP. have been studied using the whole cell patch clamp technique in guinea-pig ventricular myocytes. Isoprcnalinc I l-2000 nM) caused an inward shift of the holding current, in addition to increasing the inward calcium current jlc,). The effect on the background current was maximal earlier than the increase in I,,, but was of shorter duration. The magnitude of the background current was concentration dependent with a IX’s0 of 8 tlM. This currrnt was unaffected by tetrodotoxin 20 P(M, Cd 200 PM or verapamil, 10 ELM and potassium channel blockade ~intracellular Cs, extracellular Cs 20 mM, Ba 2 mM or tetraethylammonium 10 mM). Lowering the chloride content of the electrode solution reduced the magnitude of the background current. The background current was also induced by histamine (1 or 10 PM). Adenosine (10~1000 FM! and ATP (200 P(M) antagonised tht isoprcnaline induced background current and the increase in I,,. The histamine effects on these currents wrrr also reduced by adenosine. These results suggest that this background current may be carried by chloride ions and may be mediated via an increase in intracellular cyclic AMP concentration. Antagonism of this current ma) contribute to the antiarrhythmic actions of adenosine and ATP but their mrchanisms of action arc yet 10 br determined.
A.
KEY
WORDS:
Adenodnr;
ATP;
Ionic
currents;
Isoprenalinc;
Introduction Adenosine and adenosine triphosphate (ATP) are naturally occurring adenyl compounds which have been shown to have anti-arrhythmic properties. Both are of value in the treatment of paroxysmal supraventricular tachycardia [3] and adenosine may be effective in the termination of exercise-induced ventricular tachycardia in man [17]. In experimental adenosine and ATP have been animals, rrported to suppress ventricular arrhythmias induced by myocardial ischaemia [II, 221 or irl.jury [16]. The direct elcctrophysiological actions of the adenyl compounds on the atrio\,cantricular node underly their efficacy in the treatment of supraventricular tachycardia [I#] but adenosine has little direct effect on ventricular tissue [g]. The suppression ofventricular arrhythmias by adenosine may be related to its ability to modulate the effects of /?-adrrnocrptor stimulation [IR]. Isoprenaline Please Glasgow 0022-282X/90/
address all correspondence G31 2ER. VK. 12 137 1 + 08 SOS.OO/O
to: A. C. Rankin,
University
Ventricular
myocytrs;
Guinea-pig.
has recently been shown to induce a hackground current in ventricular cells [IO, 131. which may contribute to the arrhythmogenic effects of catecholamine stimulation [lo]. ‘The aims of this study were, firstly, to characterise this isoprenaline-induced current in guinea pig ventricular myocytes and, secondly, to assess its modulation by adenosinr and ATP. Methods Cell isolation Single ventricular myocytes were isolated from guinea-pig hearts by enzymatic dispersion using a technique similar to that prrviously described [19]. Male guinea-pigs (200-400 g) were killed by cervical dislocation, and the excised hearts were retrogradeh perfused with Tyrode’s solution at 37’(i. When clear of blood, the hearts wert‘ perfusc>d with Ca2+-free Tyrode for I min and then Department
of Medical
Cardiology, i‘
1990
Academic
Royal
Inhrmar->, Prrss
Limited
A. C. Rankin
1372
with the same solution with the addition of collagenase (1 mg ml-i), protease (0.1 mg ml-‘) and 80 /AM-ca ‘+ for a further 2-3 min. The ventricles were removed and incubated in enzyme solution for further variable periods (O-20 min). The digested tissue was placed in Tyrode’s solution containing bovine serum albumin (10 mg ml-‘) and Ca2+ (0.5 mM) and single cells were obtained by gentle mechanical agitation at 37°C. Cells were resuspended in Dulbecco’s modified Eagle’s medium (Gibco) and stored at room temperature, for use within 24 hours. Electrophysiological
recording
Myocytes were allowed to settle on the base of a chamber (0.4 ml volume) and superfused with Tyrode’s solution at a rate of 0.5-l ml/ min at 31-36°C. The composition of the Tyrode’s solution (mM) was NaCl 140; KC1 5.4; NaH2P04 0.3; MgClz 1 .O; Hepes 5.0. :$‘y~ 5.6; CaC12 1.8 and ascorbic acid 10 prevent oxidation of isoprenaline), gassed with 02, pH 7.4. Transmembrane potentials and currents were measured by the tight-seal whole cell recording method [IO] using a List EPC7 amplifier. Fire-polished electrodes coated with sylgard (l-3 M Q) were filled with a solution of the following composition (mM); KC1 110; KH2P04 10; MgCl2 2; CaC12 1; EGTA K2 5; ATP Naz 3 (pH = 7.2 at 34°C). In later experiments, KC1 was substituted by K Aspartate (80 mM) plus KC1 (30 mM) or CsCl (120 mM). Corrections were made for liquid junction potentials of - 5 mV (KC1 and CsCl) or -9 mV (Aspartate electrodes). Chemicals and drugs (-) Isoprenaline bitartrate, histamine dihydrochloride, adenosine, adenosine triphosphate (96-99o/o purity), propranolol hydroverapamil chloride, tetrodotoxin, hydrochloride, protease XIV and collagenase Type 1 were all obtained from Sigma. All drugs were dissolved in distilled water and diluted in Tyrode solution. Experimental protocol Voltage-clamp experiments were performed using one of two protocols in the majority of experiments. The cells were clamped near the
et al.
resting membrane potential (approximately - 80 mV) to study changes in the background current, or were depolarised to a holding potential of -40 or -50 mV (to inactivate the sodium current) with repetitive voltage steps (100 ms duration every 2-5 s) to study time- and voltage-dependent currents. Current-voltage relationships were determined by positive and negative potential changes in steps of 5-20 mV. Peak inward currents and the currents in the last 5 ms of the pulse were measured with reference to zero current. Series resistance compensation (40-80%) was utilised to optimise voltage clamping of the cells. With some cells, there was an increase in series resistance during experiments, particularly marked following the application of isoprenaline, due to partial re-sealing of the membrane patch. This could be corrected by applying repeated negative pressure, but, as the background currents under study were small and not greatly affected, negative pressure was often not applied to prevent disruption of the recording. Larger currents, however, were affected and, in consequence, . . quantrtative data on Zc, are limited. Two methods of applying drugs were used. Drugs were dissolved in Tyrode solution and the solutions perfusing the cells were changed by switching mechanically between reservoirs. Rapid, repeat drug testing was not possible using this system, and there was the additional problem of desensitisation of the cells to isoprenaline after prolonged exposure. An alternative method of drug application was therefore used in some experiments. Concentrated solutions of the drugs (isoprenaline 1 PM or histamine 10 PM) in Tyrode were applied by brief (lo-200 ms) pressure ejection from a micro-pipette tip placed in close proximity to the cell. The drug effect was rapid in onset and of short duration because the drug was removed in the superfusate. This technique had the disadvantage of being non-quantitative as the concentration of drug acting at the cell is unknown. The concentration can, however, be assumed to be constant if the pressure and duration of the ejection are unchanged. Data analysis Transmembrane potentials and currents were recorded continuously by a pen recorder
Adenosine
and
ATE’
(Gould) and also stored on an FM tape recorder (Racal) for subsequent analysis. Data analysis was performed using computer programmes written by J Dempster, University of Stratchclyde [I]. Mean values and standard error of the mean are quoted and “n” refers to the number of cells. Statistical significance of the difference between mean values was assessed using a Students t-test with P < 0.05 being regarded as significant. Results are presented from experiments on over 100 cells from 50 hearts.
Results
Effects of isoprenaline Isoprenaline (l-2000 nM) induced an inward shift in the holding current, and increased the calcium current (I,,) and delayed potassium current (1,) in voltage-clamped guinea-pig ventricular myocytes. The onsets of these effects on the background current and I,-, was identical but subsequent time courses differed (Fig. 1). Following bath superfusion [Fig. l(a)], the background current peaked earlier (54 + 6 s; n = 9) and tended to fade with time, while the peak increase in Zc, was slightly later (86 + 12 s; n = 8; P < 0.05) and was maintained. The differences were more apparent when isoprenaline was applied
on Cardiac
Currents
1373
briefly by pressure ejection [Fig. 1 (b)] when the effect on I,, was longer lasting than the transient inward shift in the holding current. The isoprenaline-induced background current was concentration dependent (Fig. 21, with a maximal inward current of around - 350 pA elicited by concentrations over 50 nM, at a holding potential of -50 mV. The EC,, concentration was 8 nM. The effects of isoprenaline on 1c, were also concentration dependent, with 10-2000 nM producing increases of up to 3000/,. Both isoprenaline effects were completely blocked by proprano101 (1 PM). The voltage dependence of the effects of isoprenaline are illustrated in Figure 3. Isoprenaline 10 nM caused an increase in I,, and an inward shift in the background current [Fig. 3(A)]. The current-voltage relationships for the peak inward current, &,, and the current at the end of the 100 ms pulse (which includes components of the background currents, a maintained proportion of I,-, and the delayed potassium current (Zk)), are shown in Figure 3(B) and (C) respectively. Subtraction of the control currents from those in the presence of isoprenaline gives the isoprenaline-induced currents [Fig. 3 (D)]. An approximation of the reversal potential of the isoprenaline-induced background current may he obtained by extrapolation from the current at
(A)
5s
b
FIGURE 1. The isoprenaline-induced background current and increase in inward calcium current, I,,, in guinea-pig ventricular myocytes. (A) Bath superfusion of isoprenaline (2 PM, start indicated by arrow) produced an inward shift of the holding current and an increase in I,-,. Individual current records (below) show the maximal background current (b) occurred prior to the maximal Zc, (c), but decreased with time while I,, was maintained (d), 100 s after (a). Holding potential -40 mV, with 1XLms steps to 5 mV, every 2 s; (B) Pressure ejection of isoprenalinc (1 PM, 50 ms pulse) produced an inward background current and an increase in I,,. Current records (below) show the maximal background current (b), which preceded maximal I,-,, but had returned to baseline while I,, was still increased (CI. Holding potential -50 mV, with 100 ms-steps to - 10 mV, every 2 s.
A. C. Rankin
1374
lsoprenaline FIGURE (n = 2-10
per
2. Concentration-response concentration),
from
curve 22 cells.
et al.
(nr.4)
for the isoprenaline-induced Holding potential -50
potentials negative to the threshold for Z,, and this was -6 f 5 mV in five control cells. The background current was unaffected by tetrodotoxin, 20 PM, calcium channel blockade with Cd, 200 PM, or verapamil, 10 PM, and potassium channel blockade with intra-cellular Cs, extracellular Cs 20 mM, Ba 2 mM or tetraethylammonium 10 mM. However, when
(A)
background
current.
Data
from
36 drug
tests
mV.
the chloride content of the electrode solution was reduced, 80 mM of KC1 being replaced by K Aspartate, the isoprenaline-induced background current was reduced, e.g. at -40 mV isoprenaline (100-1000 nM) produced a mean inward shift of 9 + 12 pA (n = 9), compared to 198 f 47 pA (n = 7) with high -Cl electrodes (P < 0.01).
20ms
-21nA
-2J
FIGURE 3. Antagonism by adenosine of the isoprenaline-induced currents. (A) Currents before (control) and in the presence of 10 nwisoprenaline (ISO), and after the addition of 100 pwadenosine (IS0 + ADENO), during 100 mssteps from -40 to 5 mV. (B) Voltage-dependence of peak inward currents, Z,--, and (C) currents at 100 ms. Control = filled circles; isoprenaline = shaded triangles; Isoprenaline and adenosine = open squares. (D) Subtraction of control currents from those in isoprenaline (filled triangles) and after addition of adenosine (open squares).
Adenosine
Effects
of adenosine
and
AIT
on Cardiac
and ATP
Adenosine (100-1000 PM) and ATP (200 PM), when applied alone, produced no effect on ionic currents in 18 and 8 cells respectively. Both antagonised the isoprenaline-induced background current and the increase in Zc,. Figure 3 shows the effects of isoprenaline (10 nM) on Z,, and the currents at 100 ms, and their antagonism by the addition of adenosine (100 PM) [Figs 3(A), (B), (C)l. Subtraction of control currents from those in isoprenaline before and after the addition of adenosine shows virtual abolition of the effect of isoprenaline [Fig. 3(D)]. This antagonism by adenosine,which was observed in 96% of cells (i.e. 50 cells from 30 guinea-pigs), was concentration dependent as shown in Figure 4. Pressure application of isoprenaline caused a brief inward current which was incompletely antagonisedby 10 PM-adenosinebut virtually abolished by 1 mM. Further quantitive analysis of the antagonist effects of adenosine was not possible due to desensitisation observed following prolonged or multiple applications of isoprenaline. ATP also antagonised the effects of isoprenaline in 78% of cells tested (i.e. 9 cells from 7 guinea-pigs) and the effect is illustrated in Figure 5. It was difficult to compare the quantitive effects of adenosine Control
Adenosme IO LLM
is0 7y *
Currents
1375
and ATP in the same cell due to the restrictions on repeat testing, but the degree of antagonism appeared to be similar. In six cells, histamine ( 1 or 10 PM) also induced an inward shift in the background current and an increase in Z,. These effects were similarly antagonised by adenosineand Figure 6 shows this effect on the background current. Discussion
Characterisation of the background current The isoprenaline-induced background current in guinea-pig ventricular myocytes observed using the whole cell patch clamp technique in the present study is similar to that originally reported by Egan et al., using conventional micro-electrodes [ZO]. It is unlikely to be secondary to changes in intracellular calcium since, in our experiments, the interior of the cells were dialysed with EGTA. The lack of effect of pharmacological blockade of calcium, fast sodium and potassium channels excludes these channels as mediators of the current. Egan et al. [Z0] reported a reversal potential around 0 mV, a lack of effect of alterations of external chloride, but an absenceof the current when external sodium was removed. They concluded, therefore, that it was sodium Wash
I rnhi
VA 20 pA
20s FIGURE 4. Antagonism by adenosine of the isoprenaline-induced background current; 10 pwadenosine reduced, and 1 rnM virtually abolished the effects of isoprenaline (1 PM) applied by pressure ejection (arrowed). Wash out of adenosine restored the effects of isoprenaline. Holding potential -80 mV.
1376
A. C. Rankin
et al.
ATP
-J 50
InA s
Control
‘74
-J
100pP
20s
FIGURE 5. Antagonism by ATP of the effects of isoprenaline. (A) Bath superfusion of ATP (200 PM) decreased the magnitude of Zc, (which was increased due to leakage of isoprenaline from the micro-pipette) and virtually abolished the responses to pressure applied isoprenaline (arrowed). Holding potential -40 mV, with 100 ms-steps to - 10 mV every 5 s. Low-Cl electrode solution; (B) Inward background current following pressure ejection of isoprenaline (1 PM) (arrowed) was reduced by ATP (200 PM). Holding potential -80 mV, high-Cl electrode solution.
dependent, although the nature of the charge carrier remained speculative. By contrast, Harvey and Hume [13] and Bahinski et al. [2] have reported that isoprenaline induced a background current which had a reversal potential which was dependent on changes in internal and extracellular chloride concentrations. The effects of altering internal chloride in our experiments support chloride* as the charge carrier for the isoprenaline-induced
Control
background current. Lowering the chloride content of the electrode reduced the magnitude of the background current, and the calculated reversal potential for a chloride current (i.e. - 7 mV with high Cl- electrode) agreed well with our measurement. Although Egan et al. [ZO] used conventional microelectrodes they were of relatively low resistance (8-12 MR) and were filled with l-2 M KCI. It is possible that their cells were also
Wash
Adenosine
Hlstomine
“L/
TLF
nv_,20p*
0.1 s FIGURE 6. Histamine-induced inward background current was antagonised P(M was applied by pressure ejection (arrowed). Holding potential -80 mV.
by adenosine
(200
PM).
Histamine
10
Adenosine
and ATP on Cardiac
chloride-loaded, resulting in a relatively positive reversal potential. The role of internal chloride in the production of this current may explain why it has only recently been reported, despite extensive study of the cardiac effects of isoprenaline. High resistance micro-electrodes may allow the maintenance of the normal internal chloride (around 20 mM) and hence little effect on resting potential would be observed. Much of the voltage-clamp work on the actions of isoprenaline on I,, used low-Cl internal solutions, and so would see little change in background current at the holding potentials usually used ( -40 or - 50 mV) [14, 151. It may also provide an explanation for the contradictory reports of the actions of isoprenaline on action potential duration. Action potential shortening has been reported with multi-cellular preparations [9] but prolongation has been observed with single ventricular myocytes [4, II]. When a low internal chloride was maintained, isoprenaline also shortened the action potential in single myocytes [13]. The effect on the background current is not specific to isoprenaline but is also produced by other agents which increase internal cyclic AMP such as histamine and phosphodiesterase inhibitors and forskolin [IO, 131. Whether it is mediated by channel phosphorylation, in the same way that the effects on Zc, are [15], is not known, but the different kinetics of the actions of isoprenaline on the two currents suggest differences in intracellular mechanisms.
Effects of Adenosine and ATP Adenosine was shown to antagonise the effects of isoprenaline and histamine on the background current and on the increase in Zc,, suggesting an intracellular site of action. It has been postulated that the anti-adrenergic action of adenosine is associated with a reduction in the levels of cyclic AMP [S] which is thought to be mediated by GMP-dependent inhibitory regulatory proteins [ZU] which reduce adenyl cyclase activity. There is evidence, however, that these cardiac effects of adenosine occur independently of may changes in cyclic AMP [.5j. It . may be that . . adenosine results in increased phosphatase
Currents
1377
activity with resultant channel dephosphorylation, as has been suggested to be the case with acetylcholine [I]. It is of interest to note that acetylcholine has a similar effect on this background current to that of adenosine and ATP [la]. The mechanism by which ATP antagonised the effects of isoprenaline is not certain. It may have a direct effect, or it may be broken down to adenosine by endothelial cells which have been shown to be present with the myocytes following the isolation procedure [7j, The antagonism by adenosine and ATP of the isoprenaline-induced background current contribute to their antiarrhythmic may action. The background current was ascribed arrhythmogenic potential by Egan et al. [IO] because the resultant depolarisation was associated with repetitive activity. The chloridedependence of the current, however, would indicate that in vivo, when internal chloride is low, it would not result in depolarisation. It would, by contrast, contribute to the isoprenaline-induced shortening of the action potential [13]. The resultant shortening of refractory periods would increase ventricular instability and contribute to the arrhythmic action of the catecholamines [21]. Antagonism of the background current, therefore, would produce prolongation of the action potential which thus confers the adenyl compounds with antiarrhythmic potential. Finally, additional antiarrhythmic action of adenosine and ATP ma) be due to their reduction of the increase in I,--, which may result in the abolition of slowly conducted action potentials which contribute to the arrhythmia substrate in myocardial ischaemia [.?I]. In conclusion, drugs that are known to raise the intracellular concentration of cyclic AMP induced a background current in guinea-pig ventricular myocytes, that may be carried by chloride ions. This current was accompanird by an increase in Ica and both were attenuated by adenosine and ATP. These actions may explain the reported anti-arrhythmic effects of the adenyl compounds in ventricular tissur.
Acknomledgement This work was supported by the Home and Health Department.
Scottish
1378
A.
C. Rankin
et al.
References F. J., SUBUHI, H. S., WATANABE, A. M. Autonomic regulation oftype I protein phosphatase in cardiac muscle. J Biol Chem 264, 3859-3863 (1989). BAHINSKI, A., NAIRN, A. C., GREENGORD, P., GADSBY, D. C. Chloride conductance regulated by cyclic AMPdependent protein kinase in cardiac myocytes. Nature 340, 718-721 (1989). BELHASSEN, B., PELLEG, A. Acute management of paroxysmal supraventricular tachycardia: Verapamil, adenosine triphosphate or adenosine? Am J Cardiol 54, 2255227 (1984). BELLARDINELLI, L., ISENBERG, G. Actions of adenosine and isoproterenol in isolated mammalian ventricular myocytes. Circ Res 53, 287-297 (1983). BOHM, M., BRUCKNER, R., HACKBARTH, I., HAUBITZ, B., LINHART, R.. MEYER, W., SCHMIDT, B., SCHMITZ, W., SCHOLZ, H. Adenosine inhibition ofcatecholamine-induced increase in force ofcontraction in guinea-pig atria1 and ventricular heart preparations. Evidence against a cyclic AMP- and cyclic GMP-dependent effect. J Pharmac Exp Ther 230, 483492 (1984). DEMPSTER, J. Computer analysis of electrophysiological signals. In: Microcomputers in Physiology: A practical approach, P. J. Fraser (Ed.), IRL Press, Oxford. pp 51-93 (1989). DENDORFER, A., LAUK, S., SCHAFF, A., REES, S. In: Topics and Perspectives in Adenosine Research, Gerlach, Becke (Eds). pp. 170-187 (1987). DOBSON, J. G. Reduction by adenosine of the isoproterenol-induced increase in cyclic adenosine 3’,5’-monophosphate formation and glycogen phosphorylase activity in rat heart muscle. Circ Res 43, 785-792 (1978). DUKES, I. D., VAUGHAN-WILLIAMS, E. M. Effects ofselective a, as, /l, and /?a-adrenoceptor stimulation on potentials and contractions in the rabbit heart. J Physiol 355, 5233546 (1984). EGAN, T. M., NOBLE, D., NOBLE, S. J,, POWELL, T., TWIST, V. W., YAMAOKA, K. On the mechanism ofisoprenaline and forskolin induced depolarization of single guinea-pig ventricular myocytes. J Physiol400, 299-320 (1988). FAGBEMI, O., PARRATT, J. R. Antiarrhythmic action of adenosine in the early stages of experimental myocardial ischaemia. Eur J Pharmacol 100, 243-244 (1984). HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B., SIGWORTH, J. Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391, 85-100 (1981). HARVEY, R. D., HUME, J. R. Autonomic regulation of a chloride current in heart. Science 244, 983-985 (1989). HESCHELER, J., KAMEYAMA, M., TRAUTWEIN, W. The mechanism of the muscarinic inhibition of the cardiac calcium current. Pfliigers Arch 407, 182-189 (1986). KAMEYAMA, M., HOFMAN. F., TRAUTWEIN, W. On the mechanism of /Y-adrenergic regulation of the Ca channel in guinea pig heart. Pfliigers Arch 405, 285-293 (1985). LAORDEN, M. D., HERNANDEZ, J,, RIBEIRO, J. A. The effects of adenosine, ATP and ADP on ventricular automaticity induced by local injury in the isolated right ventricle of the rat. Arch Internal de Pharmacodyn et de Therap 279, 258-267 (1986). Lcrman, B. B., Belardinelli, L., West, G. A., Berne, R. M., Dimarco, J. P. Adenosine sensitive ventricular tachycardia: Evidence suggesting cyclic AMP-mediated triggered activity. Circulation 74, 270-280 ( 1986). PELLEG, A. Cardiac electrophysiological actions of adenosine and adenosine 5’-triphosphate. In: Adenosine and Adenosine Nucbotides: Physiology and Pharmacology. D. M. Patton (Ed.), London: Taylor & Francis. pp 1433155 (1988). POWELL, T., TWIST, V. W. Isoprenaline stimulation of cyclic AMP production by isolated cells from adult rat myocardium. Biophys Res Commun 72, 1218-1225 (1976). RODBELL, M. The role ofhormone receptors and GTP-regulatory proteins in membrane transduction. Nature 2&4, 17-22 (1980). VAUGHAN WILLIAMS, E. M. Antiarrhythmic action and the puzzle of perhexiline. Academic Press, London (1980). \~AINWRIGHT, C. L., PARRATT, J. R. Antiarrhythmic effect of adenosine during myocardial ischaemia and repcrfusion. Eur J Pharmarol 145: 1833194 ( 1988). AHMAD,
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Z., GREEN,
22, 1371-1378
Antagonism Background
(1990)
by Adenosine and ATP Current in Guinea-pig
of an Isoprenaline-induced Ventricular Myocytes
A. C. Rankin,
and
R. Sitsapesan
K. A. Kane*
University Department of Medical Cardiology, Royal Infirmary, Glasgow *Department of Physiology and Pharmacology, lJniver.rit_v of Strathcl_yde, Glasgow.
CTII‘
(Received 26 October 1989, accepted in revised form 6 June 1990) C. RANKIN, R. SITSAPESAN AND K. A. KANE. Antagonism by Adenosine and ATP of an Isoprenaline-induced Background Current in Guinea-pig Ventricular Myocytes. Journal of Molecular and Cellular Cardiologv i 1990) 22. 1371-1378. A background current induced by isoprenaline, and its modulation by adenosine and ATP. have been studied using the whole cell patch clamp technique in guinea-pig ventricular myocytes. Isoprcnalinc I l-2000 nM) caused an inward shift of the holding current, in addition to increasing the inward calcium current jlc,). The effect on the background current was maximal earlier than the increase in I,,, but was of shorter duration. The magnitude of the background current was concentration dependent with a IX’s0 of 8 tlM. This currrnt was unaffected by tetrodotoxin 20 P(M, Cd 200 PM or verapamil, 10 ELM and potassium channel blockade ~intracellular Cs, extracellular Cs 20 mM, Ba 2 mM or tetraethylammonium 10 mM). Lowering the chloride content of the electrode solution reduced the magnitude of the background current. The background current was also induced by histamine (1 or 10 PM). Adenosine (10~1000 FM! and ATP (200 P(M) antagonised tht isoprcnaline induced background current and the increase in I,,. The histamine effects on these currents wrrr also reduced by adenosine. These results suggest that this background current may be carried by chloride ions and may be mediated via an increase in intracellular cyclic AMP concentration. Antagonism of this current ma) contribute to the antiarrhythmic actions of adenosine and ATP but their mrchanisms of action arc yet 10 br determined.
A.
KEY
WORDS:
Adenodnr;
ATP;
Ionic
currents;
Isoprenalinc;
Introduction Adenosine and adenosine triphosphate (ATP) are naturally occurring adenyl compounds which have been shown to have anti-arrhythmic properties. Both are of value in the treatment of paroxysmal supraventricular tachycardia [3] and adenosine may be effective in the termination of exercise-induced ventricular tachycardia in man [17]. In experimental adenosine and ATP have been animals, rrported to suppress ventricular arrhythmias induced by myocardial ischaemia [II, 221 or irl.jury [16]. The direct elcctrophysiological actions of the adenyl compounds on the atrio\,cantricular node underly their efficacy in the treatment of supraventricular tachycardia [I#] but adenosine has little direct effect on ventricular tissue [g]. The suppression ofventricular arrhythmias by adenosine may be related to its ability to modulate the effects of /?-adrrnocrptor stimulation [IR]. Isoprenaline Please Glasgow 0022-282X/90/
address all correspondence G31 2ER. VK. 12 137 1 + 08 SOS.OO/O
to: A. C. Rankin,
University
Ventricular
myocytrs;
Guinea-pig.
has recently been shown to induce a hackground current in ventricular cells [IO, 131. which may contribute to the arrhythmogenic effects of catecholamine stimulation [lo]. ‘The aims of this study were, firstly, to characterise this isoprenaline-induced current in guinea pig ventricular myocytes and, secondly, to assess its modulation by adenosinr and ATP. Methods Cell isolation Single ventricular myocytes were isolated from guinea-pig hearts by enzymatic dispersion using a technique similar to that prrviously described [19]. Male guinea-pigs (200-400 g) were killed by cervical dislocation, and the excised hearts were retrogradeh perfused with Tyrode’s solution at 37’(i. When clear of blood, the hearts wert‘ perfusc>d with Ca2+-free Tyrode for I min and then Department
of Medical
Cardiology, i‘
1990
Academic
Royal
Inhrmar->, Prrss
Limited
A. C. Rankin
1372
with the same solution with the addition of collagenase (1 mg ml-i), protease (0.1 mg ml-‘) and 80 /AM-ca ‘+ for a further 2-3 min. The ventricles were removed and incubated in enzyme solution for further variable periods (O-20 min). The digested tissue was placed in Tyrode’s solution containing bovine serum albumin (10 mg ml-‘) and Ca2+ (0.5 mM) and single cells were obtained by gentle mechanical agitation at 37°C. Cells were resuspended in Dulbecco’s modified Eagle’s medium (Gibco) and stored at room temperature, for use within 24 hours. Electrophysiological
recording
Myocytes were allowed to settle on the base of a chamber (0.4 ml volume) and superfused with Tyrode’s solution at a rate of 0.5-l ml/ min at 31-36°C. The composition of the Tyrode’s solution (mM) was NaCl 140; KC1 5.4; NaH2P04 0.3; MgClz 1 .O; Hepes 5.0. :$‘y~ 5.6; CaC12 1.8 and ascorbic acid 10 prevent oxidation of isoprenaline), gassed with 02, pH 7.4. Transmembrane potentials and currents were measured by the tight-seal whole cell recording method [IO] using a List EPC7 amplifier. Fire-polished electrodes coated with sylgard (l-3 M Q) were filled with a solution of the following composition (mM); KC1 110; KH2P04 10; MgCl2 2; CaC12 1; EGTA K2 5; ATP Naz 3 (pH = 7.2 at 34°C). In later experiments, KC1 was substituted by K Aspartate (80 mM) plus KC1 (30 mM) or CsCl (120 mM). Corrections were made for liquid junction potentials of - 5 mV (KC1 and CsCl) or -9 mV (Aspartate electrodes). Chemicals and drugs (-) Isoprenaline bitartrate, histamine dihydrochloride, adenosine, adenosine triphosphate (96-99o/o purity), propranolol hydroverapamil chloride, tetrodotoxin, hydrochloride, protease XIV and collagenase Type 1 were all obtained from Sigma. All drugs were dissolved in distilled water and diluted in Tyrode solution. Experimental protocol Voltage-clamp experiments were performed using one of two protocols in the majority of experiments. The cells were clamped near the
et al.
resting membrane potential (approximately - 80 mV) to study changes in the background current, or were depolarised to a holding potential of -40 or -50 mV (to inactivate the sodium current) with repetitive voltage steps (100 ms duration every 2-5 s) to study time- and voltage-dependent currents. Current-voltage relationships were determined by positive and negative potential changes in steps of 5-20 mV. Peak inward currents and the currents in the last 5 ms of the pulse were measured with reference to zero current. Series resistance compensation (40-80%) was utilised to optimise voltage clamping of the cells. With some cells, there was an increase in series resistance during experiments, particularly marked following the application of isoprenaline, due to partial re-sealing of the membrane patch. This could be corrected by applying repeated negative pressure, but, as the background currents under study were small and not greatly affected, negative pressure was often not applied to prevent disruption of the recording. Larger currents, however, were affected and, in consequence, . . quantrtative data on Zc, are limited. Two methods of applying drugs were used. Drugs were dissolved in Tyrode solution and the solutions perfusing the cells were changed by switching mechanically between reservoirs. Rapid, repeat drug testing was not possible using this system, and there was the additional problem of desensitisation of the cells to isoprenaline after prolonged exposure. An alternative method of drug application was therefore used in some experiments. Concentrated solutions of the drugs (isoprenaline 1 PM or histamine 10 PM) in Tyrode were applied by brief (lo-200 ms) pressure ejection from a micro-pipette tip placed in close proximity to the cell. The drug effect was rapid in onset and of short duration because the drug was removed in the superfusate. This technique had the disadvantage of being non-quantitative as the concentration of drug acting at the cell is unknown. The concentration can, however, be assumed to be constant if the pressure and duration of the ejection are unchanged. Data analysis Transmembrane potentials and currents were recorded continuously by a pen recorder
Adenosine
and
ATE’
(Gould) and also stored on an FM tape recorder (Racal) for subsequent analysis. Data analysis was performed using computer programmes written by J Dempster, University of Stratchclyde [I]. Mean values and standard error of the mean are quoted and “n” refers to the number of cells. Statistical significance of the difference between mean values was assessed using a Students t-test with P < 0.05 being regarded as significant. Results are presented from experiments on over 100 cells from 50 hearts.
Results
Effects of isoprenaline Isoprenaline (l-2000 nM) induced an inward shift in the holding current, and increased the calcium current (I,,) and delayed potassium current (1,) in voltage-clamped guinea-pig ventricular myocytes. The onsets of these effects on the background current and I,-, was identical but subsequent time courses differed (Fig. 1). Following bath superfusion [Fig. l(a)], the background current peaked earlier (54 + 6 s; n = 9) and tended to fade with time, while the peak increase in Zc, was slightly later (86 + 12 s; n = 8; P < 0.05) and was maintained. The differences were more apparent when isoprenaline was applied
on Cardiac
Currents
1373
briefly by pressure ejection [Fig. 1 (b)] when the effect on I,, was longer lasting than the transient inward shift in the holding current. The isoprenaline-induced background current was concentration dependent (Fig. 21, with a maximal inward current of around - 350 pA elicited by concentrations over 50 nM, at a holding potential of -50 mV. The EC,, concentration was 8 nM. The effects of isoprenaline on 1c, were also concentration dependent, with 10-2000 nM producing increases of up to 3000/,. Both isoprenaline effects were completely blocked by proprano101 (1 PM). The voltage dependence of the effects of isoprenaline are illustrated in Figure 3. Isoprenaline 10 nM caused an increase in I,, and an inward shift in the background current [Fig. 3(A)]. The current-voltage relationships for the peak inward current, &,, and the current at the end of the 100 ms pulse (which includes components of the background currents, a maintained proportion of I,-, and the delayed potassium current (Zk)), are shown in Figure 3(B) and (C) respectively. Subtraction of the control currents from those in the presence of isoprenaline gives the isoprenaline-induced currents [Fig. 3 (D)]. An approximation of the reversal potential of the isoprenaline-induced background current may he obtained by extrapolation from the current at
(A)
5s
b
FIGURE 1. The isoprenaline-induced background current and increase in inward calcium current, I,,, in guinea-pig ventricular myocytes. (A) Bath superfusion of isoprenaline (2 PM, start indicated by arrow) produced an inward shift of the holding current and an increase in I,-,. Individual current records (below) show the maximal background current (b) occurred prior to the maximal Zc, (c), but decreased with time while I,, was maintained (d), 100 s after (a). Holding potential -40 mV, with 1XLms steps to 5 mV, every 2 s; (B) Pressure ejection of isoprenalinc (1 PM, 50 ms pulse) produced an inward background current and an increase in I,,. Current records (below) show the maximal background current (b), which preceded maximal I,-,, but had returned to baseline while I,, was still increased (CI. Holding potential -50 mV, with 100 ms-steps to - 10 mV, every 2 s.
A. C. Rankin
1374
lsoprenaline FIGURE (n = 2-10
per
2. Concentration-response concentration),
from
curve 22 cells.
et al.
(nr.4)
for the isoprenaline-induced Holding potential -50
potentials negative to the threshold for Z,, and this was -6 f 5 mV in five control cells. The background current was unaffected by tetrodotoxin, 20 PM, calcium channel blockade with Cd, 200 PM, or verapamil, 10 PM, and potassium channel blockade with intra-cellular Cs, extracellular Cs 20 mM, Ba 2 mM or tetraethylammonium 10 mM. However, when
(A)
background
current.
Data
from
36 drug
tests
mV.
the chloride content of the electrode solution was reduced, 80 mM of KC1 being replaced by K Aspartate, the isoprenaline-induced background current was reduced, e.g. at -40 mV isoprenaline (100-1000 nM) produced a mean inward shift of 9 + 12 pA (n = 9), compared to 198 f 47 pA (n = 7) with high -Cl electrodes (P < 0.01).
20ms
-21nA
-2J
FIGURE 3. Antagonism by adenosine of the isoprenaline-induced currents. (A) Currents before (control) and in the presence of 10 nwisoprenaline (ISO), and after the addition of 100 pwadenosine (IS0 + ADENO), during 100 mssteps from -40 to 5 mV. (B) Voltage-dependence of peak inward currents, Z,--, and (C) currents at 100 ms. Control = filled circles; isoprenaline = shaded triangles; Isoprenaline and adenosine = open squares. (D) Subtraction of control currents from those in isoprenaline (filled triangles) and after addition of adenosine (open squares).
Adenosine
Effects
of adenosine
and
AIT
on Cardiac
and ATP
Adenosine (100-1000 PM) and ATP (200 PM), when applied alone, produced no effect on ionic currents in 18 and 8 cells respectively. Both antagonised the isoprenaline-induced background current and the increase in Zc,. Figure 3 shows the effects of isoprenaline (10 nM) on Z,, and the currents at 100 ms, and their antagonism by the addition of adenosine (100 PM) [Figs 3(A), (B), (C)l. Subtraction of control currents from those in isoprenaline before and after the addition of adenosine shows virtual abolition of the effect of isoprenaline [Fig. 3(D)]. This antagonism by adenosine,which was observed in 96% of cells (i.e. 50 cells from 30 guinea-pigs), was concentration dependent as shown in Figure 4. Pressure application of isoprenaline caused a brief inward current which was incompletely antagonisedby 10 PM-adenosinebut virtually abolished by 1 mM. Further quantitive analysis of the antagonist effects of adenosine was not possible due to desensitisation observed following prolonged or multiple applications of isoprenaline. ATP also antagonised the effects of isoprenaline in 78% of cells tested (i.e. 9 cells from 7 guinea-pigs) and the effect is illustrated in Figure 5. It was difficult to compare the quantitive effects of adenosine Control
Adenosme IO LLM
is0 7y *
Currents
1375
and ATP in the same cell due to the restrictions on repeat testing, but the degree of antagonism appeared to be similar. In six cells, histamine ( 1 or 10 PM) also induced an inward shift in the background current and an increase in Z,. These effects were similarly antagonised by adenosineand Figure 6 shows this effect on the background current. Discussion
Characterisation of the background current The isoprenaline-induced background current in guinea-pig ventricular myocytes observed using the whole cell patch clamp technique in the present study is similar to that originally reported by Egan et al., using conventional micro-electrodes [ZO]. It is unlikely to be secondary to changes in intracellular calcium since, in our experiments, the interior of the cells were dialysed with EGTA. The lack of effect of pharmacological blockade of calcium, fast sodium and potassium channels excludes these channels as mediators of the current. Egan et al. [Z0] reported a reversal potential around 0 mV, a lack of effect of alterations of external chloride, but an absenceof the current when external sodium was removed. They concluded, therefore, that it was sodium Wash
I rnhi
VA 20 pA
20s FIGURE 4. Antagonism by adenosine of the isoprenaline-induced background current; 10 pwadenosine reduced, and 1 rnM virtually abolished the effects of isoprenaline (1 PM) applied by pressure ejection (arrowed). Wash out of adenosine restored the effects of isoprenaline. Holding potential -80 mV.
1376
A. C. Rankin
et al.
ATP
-J 50
InA s
Control
‘74
-J
100pP
20s
FIGURE 5. Antagonism by ATP of the effects of isoprenaline. (A) Bath superfusion of ATP (200 PM) decreased the magnitude of Zc, (which was increased due to leakage of isoprenaline from the micro-pipette) and virtually abolished the responses to pressure applied isoprenaline (arrowed). Holding potential -40 mV, with 100 ms-steps to - 10 mV every 5 s. Low-Cl electrode solution; (B) Inward background current following pressure ejection of isoprenaline (1 PM) (arrowed) was reduced by ATP (200 PM). Holding potential -80 mV, high-Cl electrode solution.
dependent, although the nature of the charge carrier remained speculative. By contrast, Harvey and Hume [13] and Bahinski et al. [2] have reported that isoprenaline induced a background current which had a reversal potential which was dependent on changes in internal and extracellular chloride concentrations. The effects of altering internal chloride in our experiments support chloride* as the charge carrier for the isoprenaline-induced
Control
background current. Lowering the chloride content of the electrode reduced the magnitude of the background current, and the calculated reversal potential for a chloride current (i.e. - 7 mV with high Cl- electrode) agreed well with our measurement. Although Egan et al. [ZO] used conventional microelectrodes they were of relatively low resistance (8-12 MR) and were filled with l-2 M KCI. It is possible that their cells were also
Wash
Adenosine
Hlstomine
“L/
TLF
nv_,20p*
0.1 s FIGURE 6. Histamine-induced inward background current was antagonised P(M was applied by pressure ejection (arrowed). Holding potential -80 mV.
by adenosine
(200
PM).
Histamine
10
Adenosine
and ATP on Cardiac
chloride-loaded, resulting in a relatively positive reversal potential. The role of internal chloride in the production of this current may explain why it has only recently been reported, despite extensive study of the cardiac effects of isoprenaline. High resistance micro-electrodes may allow the maintenance of the normal internal chloride (around 20 mM) and hence little effect on resting potential would be observed. Much of the voltage-clamp work on the actions of isoprenaline on I,, used low-Cl internal solutions, and so would see little change in background current at the holding potentials usually used ( -40 or - 50 mV) [14, 151. It may also provide an explanation for the contradictory reports of the actions of isoprenaline on action potential duration. Action potential shortening has been reported with multi-cellular preparations [9] but prolongation has been observed with single ventricular myocytes [4, II]. When a low internal chloride was maintained, isoprenaline also shortened the action potential in single myocytes [13]. The effect on the background current is not specific to isoprenaline but is also produced by other agents which increase internal cyclic AMP such as histamine and phosphodiesterase inhibitors and forskolin [IO, 131. Whether it is mediated by channel phosphorylation, in the same way that the effects on Zc, are [15], is not known, but the different kinetics of the actions of isoprenaline on the two currents suggest differences in intracellular mechanisms.
Effects of Adenosine and ATP Adenosine was shown to antagonise the effects of isoprenaline and histamine on the background current and on the increase in Zc,, suggesting an intracellular site of action. It has been postulated that the anti-adrenergic action of adenosine is associated with a reduction in the levels of cyclic AMP [S] which is thought to be mediated by GMP-dependent inhibitory regulatory proteins [ZU] which reduce adenyl cyclase activity. There is evidence, however, that these cardiac effects of adenosine occur independently of may changes in cyclic AMP [.5j. It . may be that . . adenosine results in increased phosphatase
Currents
1377
activity with resultant channel dephosphorylation, as has been suggested to be the case with acetylcholine [I]. It is of interest to note that acetylcholine has a similar effect on this background current to that of adenosine and ATP [la]. The mechanism by which ATP antagonised the effects of isoprenaline is not certain. It may have a direct effect, or it may be broken down to adenosine by endothelial cells which have been shown to be present with the myocytes following the isolation procedure [7j, The antagonism by adenosine and ATP of the isoprenaline-induced background current contribute to their antiarrhythmic may action. The background current was ascribed arrhythmogenic potential by Egan et al. [IO] because the resultant depolarisation was associated with repetitive activity. The chloridedependence of the current, however, would indicate that in vivo, when internal chloride is low, it would not result in depolarisation. It would, by contrast, contribute to the isoprenaline-induced shortening of the action potential [13]. The resultant shortening of refractory periods would increase ventricular instability and contribute to the arrhythmic action of the catecholamines [21]. Antagonism of the background current, therefore, would produce prolongation of the action potential which thus confers the adenyl compounds with antiarrhythmic potential. Finally, additional antiarrhythmic action of adenosine and ATP ma) be due to their reduction of the increase in I,--, which may result in the abolition of slowly conducted action potentials which contribute to the arrhythmia substrate in myocardial ischaemia [.?I]. In conclusion, drugs that are known to raise the intracellular concentration of cyclic AMP induced a background current in guinea-pig ventricular myocytes, that may be carried by chloride ions. This current was accompanird by an increase in Ica and both were attenuated by adenosine and ATP. These actions may explain the reported anti-arrhythmic effects of the adenyl compounds in ventricular tissur.
Acknomledgement This work was supported by the Home and Health Department.
Scottish
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A.
C. Rankin
et al.
References F. J., SUBUHI, H. S., WATANABE, A. M. Autonomic regulation oftype I protein phosphatase in cardiac muscle. J Biol Chem 264, 3859-3863 (1989). BAHINSKI, A., NAIRN, A. C., GREENGORD, P., GADSBY, D. C. Chloride conductance regulated by cyclic AMPdependent protein kinase in cardiac myocytes. Nature 340, 718-721 (1989). BELHASSEN, B., PELLEG, A. Acute management of paroxysmal supraventricular tachycardia: Verapamil, adenosine triphosphate or adenosine? Am J Cardiol 54, 2255227 (1984). BELLARDINELLI, L., ISENBERG, G. Actions of adenosine and isoproterenol in isolated mammalian ventricular myocytes. Circ Res 53, 287-297 (1983). BOHM, M., BRUCKNER, R., HACKBARTH, I., HAUBITZ, B., LINHART, R.. MEYER, W., SCHMIDT, B., SCHMITZ, W., SCHOLZ, H. Adenosine inhibition ofcatecholamine-induced increase in force ofcontraction in guinea-pig atria1 and ventricular heart preparations. Evidence against a cyclic AMP- and cyclic GMP-dependent effect. J Pharmac Exp Ther 230, 483492 (1984). DEMPSTER, J. Computer analysis of electrophysiological signals. In: Microcomputers in Physiology: A practical approach, P. J. Fraser (Ed.), IRL Press, Oxford. pp 51-93 (1989). DENDORFER, A., LAUK, S., SCHAFF, A., REES, S. In: Topics and Perspectives in Adenosine Research, Gerlach, Becke (Eds). pp. 170-187 (1987). DOBSON, J. G. Reduction by adenosine of the isoproterenol-induced increase in cyclic adenosine 3’,5’-monophosphate formation and glycogen phosphorylase activity in rat heart muscle. Circ Res 43, 785-792 (1978). DUKES, I. D., VAUGHAN-WILLIAMS, E. M. Effects ofselective a, as, /l, and /?a-adrenoceptor stimulation on potentials and contractions in the rabbit heart. J Physiol 355, 5233546 (1984). EGAN, T. M., NOBLE, D., NOBLE, S. J,, POWELL, T., TWIST, V. W., YAMAOKA, K. On the mechanism ofisoprenaline and forskolin induced depolarization of single guinea-pig ventricular myocytes. J Physiol400, 299-320 (1988). FAGBEMI, O., PARRATT, J. R. Antiarrhythmic action of adenosine in the early stages of experimental myocardial ischaemia. Eur J Pharmacol 100, 243-244 (1984). HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B., SIGWORTH, J. Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391, 85-100 (1981). HARVEY, R. D., HUME, J. R. Autonomic regulation of a chloride current in heart. Science 244, 983-985 (1989). HESCHELER, J., KAMEYAMA, M., TRAUTWEIN, W. The mechanism of the muscarinic inhibition of the cardiac calcium current. Pfliigers Arch 407, 182-189 (1986). KAMEYAMA, M., HOFMAN. F., TRAUTWEIN, W. On the mechanism of /Y-adrenergic regulation of the Ca channel in guinea pig heart. Pfliigers Arch 405, 285-293 (1985). LAORDEN, M. D., HERNANDEZ, J,, RIBEIRO, J. A. The effects of adenosine, ATP and ADP on ventricular automaticity induced by local injury in the isolated right ventricle of the rat. Arch Internal de Pharmacodyn et de Therap 279, 258-267 (1986). Lcrman, B. B., Belardinelli, L., West, G. A., Berne, R. M., Dimarco, J. P. Adenosine sensitive ventricular tachycardia: Evidence suggesting cyclic AMP-mediated triggered activity. Circulation 74, 270-280 ( 1986). PELLEG, A. Cardiac electrophysiological actions of adenosine and adenosine 5’-triphosphate. In: Adenosine and Adenosine Nucbotides: Physiology and Pharmacology. D. M. Patton (Ed.), London: Taylor & Francis. pp 1433155 (1988). POWELL, T., TWIST, V. W. Isoprenaline stimulation of cyclic AMP production by isolated cells from adult rat myocardium. Biophys Res Commun 72, 1218-1225 (1976). RODBELL, M. The role ofhormone receptors and GTP-regulatory proteins in membrane transduction. Nature 2&4, 17-22 (1980). VAUGHAN WILLIAMS, E. M. Antiarrhythmic action and the puzzle of perhexiline. Academic Press, London (1980). \~AINWRIGHT, C. L., PARRATT, J. R. Antiarrhythmic effect of adenosine during myocardial ischaemia and repcrfusion. Eur J Pharmarol 145: 1833194 ( 1988). AHMAD,
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