European Journal of Pharmacology, 220 (1992) 35-41
35
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52626
5-Hydroxydecanoate inhibits ATP-sensitive K + channel currents in guinea-pig single ventricular myocytes Tatsuto Notsu, Kiyokazu Ohhashi, Isao Tanaka, Hiroshi Ishikawa, Takeshi Niho, Katsuhiko Fukutake and Masahiro Mizota Fuji Central Research Laboratory, Mochida Pharmaceutical Co., Ltd., Gotemba 412, Japan
Received 19 December 1991, revised MS received 3 June 1992, accepted 16 June 1992
We investigated the effect of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the electrical activity of guinea-pig ventricular myocytes. The outward K + current increased by lowering the intracellular ATP concentration (0.5 raM) was efficiently blocked by 5-hydroxydecanoate when recording in the whole cell configuration with the application of voltage ramps. The increase in the time-independent outward K + current induced by reducing intracellular ATP to 0 mM was also blocked by 5-hydroxydecanoate (10 or 100 tzM) and by tolbutamide (1 mM). Using the single channel recording technique, we found that 5-hydroxydecanote blocked ATP-sensitive K + channels when its channel open probability was increased by 1 mM ATP together with 1 mM ADP or by an intracellular pH of 6.6. These conditions are well documented to reflect metabolic changes in the early stages of myocardial ischemic attack. These results suggest that 5-hydroxydecanoate could inhibit ATP-sensitive K + channels, resulting in an antiarrhythmic effect specifically on ischemic hearts. Ischemia; Metabolic changes; Patch clamp; Ventricular cells; 5-Hydroxydecanoate
1. Introduction There are several lines of evidence that ATP-sensitive K + channels in cardiac cells play an important role in the regulation of cellular energy metabolism in the control of m e m b r a n e excitability (Noma, 1983), and are responsible for the increase in outward currents and the shortening of the action potential duration under various anoxic conditions (Noma and Shibasaki, 1985). We have demonstrated that ATP-sensitive K + channels are involved in the pathogenesis of arrhythmias in the early stage of ischemia (Niho et al., 1987). During the myocardial ischemia, several metabolic changes occur in the myocardia: (i) a decrease in intracellular A T P synthesis (Allen et al., 1985; Allen and Orchard, 1987) and (ii) a decrease in p H in the cells (Allen and Orchard, 1987; Ellis and Noireaud, 1987), resulting in modulation of the activity of ATP-sensitive K + channels (Cuevas et al., 1991). The A T P concentration in
Correspondence to: T. Notsu, Fuji Central Research Laboratory, Mochida Pharmaceutical Co., Ltd., 722 Jimba-aza-Uenohara, Gotemba, Shizuoka 412, Japan. Tel. 81.550.89 7881, fax 81.550.89 8070.
such myocardia gradually decreases and ADP, which is shown to activate ATP-sensitive K + channels in the presence of Mg 2+ (Findlay, 1988; Lederer and Nichols, 1989), concomitantly increases to almost the same level as that of A T P (Meno et al., 1984; Neely and Grotyohann, 1984). Under these conditions ATP-sensitive K + channels can be activated even in the presence of a physiological level of A T P (Noma, 1983; Kakei et al., 1985 Ashcroft, 1988). Thus, it is likely that the ATPsensitive K + channel is involved in the ischemia-specific electrophysiological alteration in cardiac muscle. We previously reported that 5-hydroxydecanoate, a component of hydroxy fatty acids in milk (Wyatt et al., 1967), has significant effects on ventricular fibrillation in animals with myocardial ischemia (Niho et al., 1987) and also reduces the activity of ATP-sensitive K + channels in guinea-pig myocardial membranes under ATP-free conditions (Notsu et al., 1992). The first aim of the present study was to re-evaluate the effect of 5-hydroxydecanoate on m e m b r a n e currents under conditions of a depressed metabolic state in single cardiac cells in the whole cell configuration. Second, we investigated the effect of 5-hydroxydecanoate on ATP-sensitive K + channels under conditions reflecting the metabolic changes of early ischemia, namely in the
36
presence of ATP (1 raM) and ADP (1 raM) or in the presence of a lower intracellular pH (6.6).
2. Materials and methods
2.1. Preparation of single centricular myocytes Single cardiac cells were prepared as described previously (Powell et al., 1980). Briefly, Hartley guinea-pigs were anesthetized with sodium pentobarbital (30 m / k g i.p.) and the chest was opened under artificial respiration. The aorta was cannulated in situ to perfuse the coronary arteries with Tyrode solution equilibrated with 100% 0 2. The heart was then dissected out and mounted in a Langendorff apparatus (hydrostatic pressure was 65 cm), followed by perfusion with approximately 100 ml of Ca2+-free Tyrode solution. The perfusate was then switched to Ca2+-free Tyrode solution containing 0.04% (w/v) collagenase (Sigma, type I) or 0.017% (w/v) collagenase (Yakult). After collagenase treatment, the heart was washed with 100 ml of Kraftbriihe solution (Isenberg and K16ckner, 1982), and the ventricular tissues were excised and minced. Single cells were separated from the minced tissue by passing them through a 150-/xm mesh.
2.2. Electrical recording Whole cell and single channel recording techniques were essentially the same as described by Hamill et al. (1981). Isolated ventricular cells were dispersed in a recording chamber filled with Tyrode solution. Whole cell clamp recordings were obtained with micro-electrodes of a resistance of 1-2 M.O. After the establishment of the GY~ resistance seal, recordings were started after the rupture of the membrane in the pipette, which contained a low ATP concentration (0 or 0.5 raM), and the bath solution was changed to glucose-free Tyrode solution containing the test drug. In the control cell, glucose-free Tyrode solution was continuously perfused throughout the experiment. Whole-cell membrane currents were measured every 5 s during depolarization pulses of 300 ms, applied from a holding potential of - 40 mV, and voltage ramps from - 100 to + 100 mV at 1.6 V/s, applied from a holding potential of 0 mV. In single channel recording, the bath solution was changed from normal Tyrode to the internal solution after making the G ~ seal. The ATP-regulated K + channels were recorded in the inside-out or open-cell attached configuration. The open-cell attached patch technique was essentially the same as described in Kakei et al. (1985). After establishment of the cell-attached mode, cell membrane permeabilization was achieved with saponin (0.05% w/v) applied from the tip of a micropipette positioned near the cell. The
period of saponin application lasted 5-0 s. Membrane currents and voltages were recorded with a patch clamp amplifier (S-3666, Nihon Kohden or 8900, Dagan) at a low pass filter setting of 1 kHz. The signal from the patch clamp amplifier was recorded on a magnetic tape recorder (A-49, Sony or XR-510, Teac) for later computer analysis (MBC-18SJ, Sanyo). Open probability (Po) was calculated with the formula P o = 1/(Ni), where I is the mean patch current, N is the number of channels in the patch approximated as the maximum number of overlaps of openings in the ATP-free internal solution, and i is the unit amplitude of the single channel current. A '50% threshold' criterion was used to detect channel events.
2.3. Solutions The composition of the Tyrode solution was (in mM) NaCl 136.1, KCI 5.4, CaC12 1.8, MgCI 2 0.53, NaH2PO 4 0.33, glucose 5.5, HEPES 5.0 (pH 7.4). The composition of the Kraftbriihe solution was (in raM) taurine 10, oxalic acid 10, glutamic acid 80, KCI 25, KH2PO 4 10, HEPES 10, glucose 11, EGTA 0.5 (pH 7.4). The composition of the internal solution was (in mM) KC1 140, MgC12 1.0, KH2PO 4 0.5, EGTA 1.0, HEPES 5.0 (pH 7.4). Various amounts of ATP or ADP were added to the internal solution and the concentration of free Mg 2+ was adjusted to 0.5 mM according to the equilibrium binding between Mg 2+ and ATP, ADP or EGTA (Fabiato and Fabiato, 1979). The composi-
,°°2;
250ms B
Current (hA)
2F
/ . f l 0rain
5-HD 100/~M
Control
_,1o5o0 _.o.:t" 7 i
Current (nA) 2
C
~ 1 5
Voltage (mV)
/X III I
--107--500
1
j l O --
p5min
I
I
50
100
Voltage (mV)
,0,/
Fig. 1. Effect of 5-hydroxydecanoate on the current induced by voltage ramps from - 100 to + 100 mV while decreasing intracellular ATP to 0.5 mM. Pipette solution (in raM): aspartate 128, KCI 20, MgCI 2 l, E G T A 5, HEPES 5, ATP 0.5 pH ~ 7.4. Recordings were started after rupture of the membrane in the pipette and control or 5-hydroxydecanoate perfusion was started at the same time. (A) Voltage ramps were applied every 5 s from a holding potential of 0 mV. (B) Membrane currents of control cell 5 or 10 min after the start of recording. (C) Membrane currents of 5-hydroxydecanoatetreated cells. Bath solution was normal Tyrode solution.
37
tion of the standard pipette solution for ramp clamp recording was (in mM) aspartate 128, KC1 20, MgC12 1.0, ATP (dipotassium salt) 0.5, EGTA 5.0, HEPES 5.0 (pH 7.4). The ATP concentration was varied through replacement with aspartate. For whole cell clamp recording, ATP was omitted and 50 /~M 2,4-dinitrophenol (DNP) was added. In the single channel recording, the composition of the pipette solution was (in mM) KCI 5.4, NaC1 144.6, CaC12 2.0, HEPES 5.0 (pH 7.4). Experiments were performed at 35-37 o C, except single channel recordings, which were conducted at 24-26 o C. The sodium salt of 5-hydroxydecanoate was used in the experiment and was synthesized by Mochida Pharmaceutical Co. 5-Hydroxydecanoate was directly dissolved in bath solutions for each experiment. Tolbutamide (Sigma) was dissolved in 0.2 N NaOH at a concentration of 100 mM and diluted in the bath solution for the experiment.
2.4. Data analysis Results are expressed as means + S.E. and statistical difference was determined with Student's t-test or the paired t-test.
3. Results
3.1. Effect of 5-hydroxydecanoate on whole cell currents induced by lowering the intracellular A TP concentration Current-voltage curves obtained by applying voltage ramps from -100 to + 100 mV at an ATP concentration of 0.5 mM in the pipette solution are shown in fig. 1. In the control cell, the current-voltage relationship showed inward rectification 5 min after the start of whole cell recording by the perfusion of Tyrode solu-
A 300ms
B Control
6. 5 min
--.--o--• 5.
C 5-HD 10 I.tM
"
Control 5-HD 10 I.tM 5-H D 100 }.tM tolbutamide 100 I.tM tolbutamicle 1000 u.M
4. 8 min
35
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52626
5-Hydroxydecanoate inhibits ATP-sensitive K + channel currents in guinea-pig single ventricular myocytes Tatsuto Notsu, Kiyokazu Ohhashi, Isao Tanaka, Hiroshi Ishikawa, Takeshi Niho, Katsuhiko Fukutake and Masahiro Mizota Fuji Central Research Laboratory, Mochida Pharmaceutical Co., Ltd., Gotemba 412, Japan
Received 19 December 1991, revised MS received 3 June 1992, accepted 16 June 1992
We investigated the effect of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the electrical activity of guinea-pig ventricular myocytes. The outward K + current increased by lowering the intracellular ATP concentration (0.5 raM) was efficiently blocked by 5-hydroxydecanoate when recording in the whole cell configuration with the application of voltage ramps. The increase in the time-independent outward K + current induced by reducing intracellular ATP to 0 mM was also blocked by 5-hydroxydecanoate (10 or 100 tzM) and by tolbutamide (1 mM). Using the single channel recording technique, we found that 5-hydroxydecanote blocked ATP-sensitive K + channels when its channel open probability was increased by 1 mM ATP together with 1 mM ADP or by an intracellular pH of 6.6. These conditions are well documented to reflect metabolic changes in the early stages of myocardial ischemic attack. These results suggest that 5-hydroxydecanoate could inhibit ATP-sensitive K + channels, resulting in an antiarrhythmic effect specifically on ischemic hearts. Ischemia; Metabolic changes; Patch clamp; Ventricular cells; 5-Hydroxydecanoate
1. Introduction There are several lines of evidence that ATP-sensitive K + channels in cardiac cells play an important role in the regulation of cellular energy metabolism in the control of m e m b r a n e excitability (Noma, 1983), and are responsible for the increase in outward currents and the shortening of the action potential duration under various anoxic conditions (Noma and Shibasaki, 1985). We have demonstrated that ATP-sensitive K + channels are involved in the pathogenesis of arrhythmias in the early stage of ischemia (Niho et al., 1987). During the myocardial ischemia, several metabolic changes occur in the myocardia: (i) a decrease in intracellular A T P synthesis (Allen et al., 1985; Allen and Orchard, 1987) and (ii) a decrease in p H in the cells (Allen and Orchard, 1987; Ellis and Noireaud, 1987), resulting in modulation of the activity of ATP-sensitive K + channels (Cuevas et al., 1991). The A T P concentration in
Correspondence to: T. Notsu, Fuji Central Research Laboratory, Mochida Pharmaceutical Co., Ltd., 722 Jimba-aza-Uenohara, Gotemba, Shizuoka 412, Japan. Tel. 81.550.89 7881, fax 81.550.89 8070.
such myocardia gradually decreases and ADP, which is shown to activate ATP-sensitive K + channels in the presence of Mg 2+ (Findlay, 1988; Lederer and Nichols, 1989), concomitantly increases to almost the same level as that of A T P (Meno et al., 1984; Neely and Grotyohann, 1984). Under these conditions ATP-sensitive K + channels can be activated even in the presence of a physiological level of A T P (Noma, 1983; Kakei et al., 1985 Ashcroft, 1988). Thus, it is likely that the ATPsensitive K + channel is involved in the ischemia-specific electrophysiological alteration in cardiac muscle. We previously reported that 5-hydroxydecanoate, a component of hydroxy fatty acids in milk (Wyatt et al., 1967), has significant effects on ventricular fibrillation in animals with myocardial ischemia (Niho et al., 1987) and also reduces the activity of ATP-sensitive K + channels in guinea-pig myocardial membranes under ATP-free conditions (Notsu et al., 1992). The first aim of the present study was to re-evaluate the effect of 5-hydroxydecanoate on m e m b r a n e currents under conditions of a depressed metabolic state in single cardiac cells in the whole cell configuration. Second, we investigated the effect of 5-hydroxydecanoate on ATP-sensitive K + channels under conditions reflecting the metabolic changes of early ischemia, namely in the
36
presence of ATP (1 raM) and ADP (1 raM) or in the presence of a lower intracellular pH (6.6).
2. Materials and methods
2.1. Preparation of single centricular myocytes Single cardiac cells were prepared as described previously (Powell et al., 1980). Briefly, Hartley guinea-pigs were anesthetized with sodium pentobarbital (30 m / k g i.p.) and the chest was opened under artificial respiration. The aorta was cannulated in situ to perfuse the coronary arteries with Tyrode solution equilibrated with 100% 0 2. The heart was then dissected out and mounted in a Langendorff apparatus (hydrostatic pressure was 65 cm), followed by perfusion with approximately 100 ml of Ca2+-free Tyrode solution. The perfusate was then switched to Ca2+-free Tyrode solution containing 0.04% (w/v) collagenase (Sigma, type I) or 0.017% (w/v) collagenase (Yakult). After collagenase treatment, the heart was washed with 100 ml of Kraftbriihe solution (Isenberg and K16ckner, 1982), and the ventricular tissues were excised and minced. Single cells were separated from the minced tissue by passing them through a 150-/xm mesh.
2.2. Electrical recording Whole cell and single channel recording techniques were essentially the same as described by Hamill et al. (1981). Isolated ventricular cells were dispersed in a recording chamber filled with Tyrode solution. Whole cell clamp recordings were obtained with micro-electrodes of a resistance of 1-2 M.O. After the establishment of the GY~ resistance seal, recordings were started after the rupture of the membrane in the pipette, which contained a low ATP concentration (0 or 0.5 raM), and the bath solution was changed to glucose-free Tyrode solution containing the test drug. In the control cell, glucose-free Tyrode solution was continuously perfused throughout the experiment. Whole-cell membrane currents were measured every 5 s during depolarization pulses of 300 ms, applied from a holding potential of - 40 mV, and voltage ramps from - 100 to + 100 mV at 1.6 V/s, applied from a holding potential of 0 mV. In single channel recording, the bath solution was changed from normal Tyrode to the internal solution after making the G ~ seal. The ATP-regulated K + channels were recorded in the inside-out or open-cell attached configuration. The open-cell attached patch technique was essentially the same as described in Kakei et al. (1985). After establishment of the cell-attached mode, cell membrane permeabilization was achieved with saponin (0.05% w/v) applied from the tip of a micropipette positioned near the cell. The
period of saponin application lasted 5-0 s. Membrane currents and voltages were recorded with a patch clamp amplifier (S-3666, Nihon Kohden or 8900, Dagan) at a low pass filter setting of 1 kHz. The signal from the patch clamp amplifier was recorded on a magnetic tape recorder (A-49, Sony or XR-510, Teac) for later computer analysis (MBC-18SJ, Sanyo). Open probability (Po) was calculated with the formula P o = 1/(Ni), where I is the mean patch current, N is the number of channels in the patch approximated as the maximum number of overlaps of openings in the ATP-free internal solution, and i is the unit amplitude of the single channel current. A '50% threshold' criterion was used to detect channel events.
2.3. Solutions The composition of the Tyrode solution was (in mM) NaCl 136.1, KCI 5.4, CaC12 1.8, MgCI 2 0.53, NaH2PO 4 0.33, glucose 5.5, HEPES 5.0 (pH 7.4). The composition of the Kraftbriihe solution was (in raM) taurine 10, oxalic acid 10, glutamic acid 80, KCI 25, KH2PO 4 10, HEPES 10, glucose 11, EGTA 0.5 (pH 7.4). The composition of the internal solution was (in mM) KC1 140, MgC12 1.0, KH2PO 4 0.5, EGTA 1.0, HEPES 5.0 (pH 7.4). Various amounts of ATP or ADP were added to the internal solution and the concentration of free Mg 2+ was adjusted to 0.5 mM according to the equilibrium binding between Mg 2+ and ATP, ADP or EGTA (Fabiato and Fabiato, 1979). The composi-
,°°2;
250ms B
Current (hA)
2F
/ . f l 0rain
5-HD 100/~M
Control
_,1o5o0 _.o.:t" 7 i
Current (nA) 2
C
~ 1 5
Voltage (mV)
/X III I
--107--500
1
j l O --
p5min
I
I
50
100
Voltage (mV)
,0,/
Fig. 1. Effect of 5-hydroxydecanoate on the current induced by voltage ramps from - 100 to + 100 mV while decreasing intracellular ATP to 0.5 mM. Pipette solution (in raM): aspartate 128, KCI 20, MgCI 2 l, E G T A 5, HEPES 5, ATP 0.5 pH ~ 7.4. Recordings were started after rupture of the membrane in the pipette and control or 5-hydroxydecanoate perfusion was started at the same time. (A) Voltage ramps were applied every 5 s from a holding potential of 0 mV. (B) Membrane currents of control cell 5 or 10 min after the start of recording. (C) Membrane currents of 5-hydroxydecanoatetreated cells. Bath solution was normal Tyrode solution.
37
tion of the standard pipette solution for ramp clamp recording was (in mM) aspartate 128, KC1 20, MgC12 1.0, ATP (dipotassium salt) 0.5, EGTA 5.0, HEPES 5.0 (pH 7.4). The ATP concentration was varied through replacement with aspartate. For whole cell clamp recording, ATP was omitted and 50 /~M 2,4-dinitrophenol (DNP) was added. In the single channel recording, the composition of the pipette solution was (in mM) KCI 5.4, NaC1 144.6, CaC12 2.0, HEPES 5.0 (pH 7.4). Experiments were performed at 35-37 o C, except single channel recordings, which were conducted at 24-26 o C. The sodium salt of 5-hydroxydecanoate was used in the experiment and was synthesized by Mochida Pharmaceutical Co. 5-Hydroxydecanoate was directly dissolved in bath solutions for each experiment. Tolbutamide (Sigma) was dissolved in 0.2 N NaOH at a concentration of 100 mM and diluted in the bath solution for the experiment.
2.4. Data analysis Results are expressed as means + S.E. and statistical difference was determined with Student's t-test or the paired t-test.
3. Results
3.1. Effect of 5-hydroxydecanoate on whole cell currents induced by lowering the intracellular A TP concentration Current-voltage curves obtained by applying voltage ramps from -100 to + 100 mV at an ATP concentration of 0.5 mM in the pipette solution are shown in fig. 1. In the control cell, the current-voltage relationship showed inward rectification 5 min after the start of whole cell recording by the perfusion of Tyrode solu-
A 300ms
B Control
6. 5 min
--.--o--• 5.
C 5-HD 10 I.tM
"
Control 5-HD 10 I.tM 5-H D 100 }.tM tolbutamide 100 I.tM tolbutamicle 1000 u.M
4. 8 min
