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Circulation Research. 1997;81:211-218

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(Circulation Research. 1997;81:211-218.)
© 1997 American Heart Association, Inc.

Articles

Endothelin-1 Inhibits the Slow Component of Cardiac Delayed Rectifier K+ Currents via a Pertussis Toxin–Sensitive Mechanism

Takashi Washizuka, Minoru Horie, Masato Watanuki, , Shigetake Sasayama

From the Department of Cardiovascular Medicine, Kyoto (Japan) University Graduate School of Medicine.

Correspondence to Minoru Horie, Division of Cardiac Electrophysiology, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Shogoin, Kyoto 606-01, Japan. E-mail horie{at}kuhp.kyoto-u.ac.jp


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract Endothelin-1 (ET-1) is a 21–amino acid peptide hormone released from myocardial and endothelial cells, whose receptors (both ETA and ETB) are expressed in the myocardium. We report here that ET-1 inhibits the cardiac delayed rectifier K+ current (IK) via a pertussis toxin (PTX)–sensitive mechanism. Ventricular myocytes enzymatically isolated from guinea pig hearts were voltage-clamped by the conventional whole-cell and nystatin–perforated patch technique (intrapipette and extrapipette K+ concentrations, 150 and 5.4 mmol/L, respectively) in the presence of nifedipine (2 µmol/L). Amplitudes of tail and steady state (2-second pulse) currents were measured as IK. ET-1 suppressed the basal IK by 20.9±2.3% in a concentration-dependent manner, with an IC50 of 1.1±0.3 nmol/L (n=19), although it did not suppress the basal IK using the nystatin method. E-4031 (5 µmol/L), a blocker of the rapid component of IK (IKr), did not prevent the inhibitory action of ET-1. ET-1 reduced by 63.4±6.5% the slow component of IK (IKs) that had been enhanced to {approx}2-fold by isoproterenol (ISO, 20 nmol/L). The action was concentration dependent, with an IC50 of 0.7±0.4 nmol/L (n=22), and was also observed using the nystatin method. The effect of ET-1 appeared to be mediated by an ETA receptor, because it was prevented by FR139317, an ETA-selective antagonist (1 µmol/L, n=4), and sarafotoxin S6c, an ETB-selective agonist (100 nmol/L, n=4), could not inhibit the ISO-enhanced IK. ET-1 antagonized IKs enhanced by histamine (250 nmol/L, n=7) and forskolin (500 nmol/L, n=7) but did not inhibit IKs enhanced by the internal application of cAMP (100 µmol/L, n=6). Preincubation of myocytes with PTX (5 µg/mL for >60 minutes at 36°C) completely abolished the inhibitory action of ET-1 on the ISO-enhanced IKs (n=4). Thus, nanomolar ET-1 inhibits IKs via the ETA receptor/PTX–sensitive G protein/PKA pathway.


Key Words: endothelin-1 • delayed rectifier K+ current • endothelin A receptor • G protein • protein kinase A


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Endothelin-1 is a 21–amino acid peptide hormone originally isolated and characterized from the culture supernatant of porcine aortic endothelial cells,1 2 which is released from vascular and endocardial endothelial cells, especially under pathological conditions such as heart failure, myocardial ischemia, and hypertension.3 4 Endothelin receptors have been shown to be distributed in many organs,1 2 5 and at least two cDNA clones of the endothelin receptor have been isolated, encoding ETA and ETB subtypes of the endothelin receptor.6 7 Recently, both subtypes of receptor have been found to coexist in rat8 9 and guinea pig10 11 hearts.

ET-1 has been shown to regulate cardiac function through the modulation of ion channels. Two intracellular signaling pathways have been proposed: (1) activation of PKC12 13 and (2) accentuated antagonism of PKA.10 13 14 ET-1 has been reported to increase basal ICa,L in rabbit ventricular myocytes.15 PKC activation has been shown to mediate the ET-1–dependent inhibition of T-type Ca2+ channels in neonatal rat myocytes.16 In contrast, in guinea pig ventricular cells,17 18 ET-1 did not alter or slightly reduce ICa,L. ET-1 consistently decreased the ISO-enhanced ICa,L and PKA-dependent Cl- current in a PTX-sensitive manner.10 18 19 Activation of both PKA and PKC pathways should increase IKs in guinea pig ventricular cells.20 21 22 23 24 Indeed, Habuchi et al25 demonstrated that ET-1 increased IK through the PKC pathway. However, the action of ET-1 on IK remains unknown in terms of PKA activation.

In the rat heart-failure model involving experimental myocardial infarction, where the PKA pathway is thought to be activated, antagonism of the ETA receptor–mediated signal transduction was found to significantly suppress the postinfarct ventricular remodeling.26 Thus, ET-1 may have direct action on the development of cellular hypertrophy through the ETA receptor. Prolongation of APD and the resultant increase in Ca2+ influx were attributed with inducing the myocardial hypertrophy, but electrophysiological experiments yielded the opposite results: ET-1 was actually shown to reduce ventricular ICa,L in the presence of catecholamine18 and to enhance IK,25 both of which would work to shorten APD. Therefore, we focused on the action of ET-1 on ventricular IK, particularly in the presence of PKA activation, because IK plays a primary role in modulating the repolarization. We found that ET-1 inhibits ISO-enhanced IK, presumably through the inhibition of adenylate cyclase via a PTX-sensitive G protein, thereby antagonizing the ISO-induced shortening of APD.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
 down arrowReferences
 
Preparation of Single Myocytes and Solutions
Single ventricular myocytes were isolated from the left ventricle of adult guinea pig hearts (250 to 350 g) by using the enzymatic dissociation procedure as described previously.27 28 Briefly, after deep anesthesia with pentobarbital sodium ({approx}50 mg/kg IP), the chest was opened under artificial respiration, the aorta was cannulated using Langendorff's apparatus, and the heart was quickly excised. By retrograde perfusion, normal Tyrode's solution (36°C) was applied for {approx}5 minutes, followed by nominally Ca2+-free Tyrode's solution until contraction ceased. The latter solution supplemented with 0.036 mmol/L Ca2+ and 0.4 mg/mL collagenase (type 1, Sigma Chemical Co) was retrogradely perfused for 18 to 20 minutes. The composition of normal Tyrode's solution was (mmol/L) NaCl 145, KCl 5.4, CaCl2 1.8, NaH2PO4 0.3, MgCl2 0.5, glucose 5.5, and HEPES 5 (pH adjusted to 7.4 with NaOH). Finally, the heart was perfused with a KB medium28 at room temperature ({approx}21°C) to rinse away the collagenase. The composition of KB medium was (mmol/L) L-glutamic acid 70, KCl 25, taurine 20, KH2PO4 10, MgCl2 3, EGTA 0.5, glucose 11, and HEPES 10 (pH 7.3 with KOH). The partially digested heart was gently minced with scissors in the KB solution. After filtration through 105-µm mesh, cells were stored in the KB solution at room temperature.

The standard external solution contained (mmol/L) NaCl 145, KCl 5.4, CaCl2 1.8, NaH2PO4 0.3, MgCl2 0.5, glucose 5.5, and HEPES 5 (pH 7.4 with NaOH). In all experiments, ICa,L was inhibited by the addition of 2 µmol/L nifedipine (Bayer Pharmaceutical Co). Nifedipine was dissolved with ethanol for the 10 mmol/L stock solution. The control pipette solution contained (mmol/L) potassium aspartate 110, KCl 20, MgCl2 7.0, CaCl2 0.69, K2-ATP 5, Na2-GTP 0.1, creatine phosphate-K2 5, EGTA 5, and HEPES 5 (pH 7.4 with KOH). According to the stabilizing constants proposed by Fabiato and Fabiato,29 with the correction of Tsien and Rink,30 the pCa of these internal solutions was calculated to be 8.0. For the modified nystatin–perforated patch technique,31 32 the composition of the pipette solution was (mmol/L) potassium asparate 110, KCl 20, MgCl2 5.0, K2-ATP 5, and HEPES 5 (pH 7.4 adjusted with KOH). The pipette solution that was back-filled to the electrodes contained nystatin (250 µg/mL, Sigma) and fluorescein sodium (1 mg/mL, Nacalai Tesque Chemicals).

CCh, ISO, and histamine (all from Nacalai Tesque Chemicals) solutions were freshly prepared immediately before each experiment from stock solutions. Forskolin (Sigma) was dissolved in DMSO (10 mmol/L stock solution), and cAMP (Sigma) was dissolved in distilled water (10 mmol/L stock solution) for later use. E-4031 (a kind gift from Eisai Co, Ltd) was dissolved in distilled water (10 mmol/L stock solution) for later use. ET-1 (Peptide Institute) and SRTXc (Peptide Institute) were dissolved in 0.1% aqueous acetic acid and stored in 100 µL aliquots at -20°C until use. FR139317) (kind gift from Fujisawa Pharmaceutical Co) was dissolved in DMSO (1 mmol/L stock solution). DMSO (<0.1%) alone had no effects on membrane currents. PTX (Seikagaku Co) dissolved in a KB solution (50 µg/mL stock solution) was diluted to the myocyte suspension (KB solution) at a final concentration of 5 µg/mL. The PTX incubation was made at 36°C for >60 minutes.

Electrophysiology
A few drops of cell suspension were dispersed into a small chamber (volume, 0.5 mL) superfused with Tyrode's medium on the stage of an inverted microscope (Diaphot, Nikon). Whole-cell currents were measured with low-resistance pipettes ({approx}2 M{Omega}) by using a patch-clamp amplifier (model EPC-7, List). High-resistance seals were usually obtained on the center of the cells by applying negative pressure to the interior of the pipettes by gentle suction ({approx}-20 cm H2O). The patch membrane was then broken by a gentle increase in negative pressure ({approx}-50 cm H2O). Liquid junction potentials (-10 mV) were corrected by the voltage offset on the patch-clamp amplifier. Cell membrane capacitance was measured by using the internal circuit for capacitance-current compensation. The series resistance was compensated to minimize the duration of the capacitive surge. A gigaohm seal was attained while cells were perfused with normal Tyrode's solution. After the formation of whole-cell mode, the external solution was switched to a test solution. Chamber perfusates were continuously drained by suction, and complete exchange of the perfusates could be achieved within 30 seconds.

All the data were digitized on-line to an NEC computer (PC-9801RA) at a sampling frequency of 2 kHz through a 1-kHz Bessel filter (24 dB/octave, model NF FV624) and were simultaneously stored for backup to videotape via a PCM recorder (Sony, model PCM501ES). IK was usually activated by depolarizing step pulses of 2-second duration applied from a -40-mV holding potential to various test potentials every 10 seconds. Amplitudes of IK tail currents were measured after repolarization to -40 mV as the difference between the peak point of the tail and the holding current. IK steady state currents were defined as the difference between the peak point of the time-dependently activated component and the holding current. Numerical data are shown as mean±SE. Statistical significance was determined by Students' t test (P<.05).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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 down arrowReferences
 
CCh and ET-1 Reduce the IK Enhanced by ISO
Fig 1ADown shows four original traces of whole-cell currents elicited by step pulses from a holding potential of -40 to +40 mV before and after exposure to CCh and ET-1 on IKs enhanced by ISO. Labels a through d correspond to the arrows in the time course of the tail current amplitude obtained from the same cell (Fig 1BDown). ISO at 20 nmol/L produced a 2-fold increase (196±7.8%, n=22) in the amplitude of the tail current of IK (traces a->b); the subsequent addition of 20 µmol/L CCh reversibly inhibited the IK to 44% of the ISO-induced component in this particular myocyte (traces b->c). Inhibition of the PKA system via PTX-sensitive G proteins has been shown to cause this inhibitory response to CCh.21 33



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  		Figure 1. ET-1 inhibits the ISO-induced enhancement of IK. A, Original current traces corresponding to a through d are indicated by arrows in panel B. Test pulses of 2-second duration were applied at holding potentials from -40 to +40 mV. B, Amplitude of the tail current was measured every 10 seconds between the peak of the tail current and the holding current and is plotted as a function of time (in minutes). Horizontal bars above the graph indicate the application of ISO (20 nmol/L), carbachol (CCh, 20 µmol/L), and ET-1 (50 nmol/L). C, Competition between isoproterenol and ET-1 in activating IK is shown. Horizontal bars indicate exposure to 20 nmol/L and 1 µmol/L ISO and 50 nmol/L ET-1.

Exposure of the myocyte to ET-1 (50 nmol/L) also reduced the ISO-induced IK to 42% of the ISO-induced component (d in Fig 1Up). In most experiments, as typified in Fig 1Up, the inhibitory effect of ET-1 at 50 nmol/L was not reversed after washout of the peptide. Subsequent increase of ISO concentration from 20 nmol/L to 1 µmol/L could antagonize the ET-1–induced inhibition and produce a larger response in the tail current of IK than that obtained by 20 nmol/L ISO (Fig 1CUp), suggesting competition between ß-adrenoceptors and ET-1 receptors.

Fig 2ADown shows three sets of original traces elicited by depolarizing pulses between -40 and +60 mV with 10-mV steps from top to bottom as follows: control, open circles; exposure to 20 nmol/L ISO, solid circles; and after addition of 50 nmol/L ET-1, open inverted triangles. In these experiments, the IK tail currents were measured and plotted as a function of test potential in Fig 2BDown (symbols in panels A and B are corresponding). The threshold potential for IK activation appeared not to be altered, although ISO increased IK and ET-1 decreased the ISO-enhanced current component.



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  		Figure 2. ET-1 effects on current-voltage relationships and concentration-inhibition relationships for IK enhanced by ISO. A, Three sets of current traces at test potentials between -40 and +60 mV with 10-mV steps. Arrows indicate the zero current level. B, Tail current amplitudes plotted against test potentials. C, Activation curves obtained by normalizing the IK tail current amplitudes. The solid curves are expressed as a function of membrane potential (E) as follows: normalized IK tail current=1/{1+1/exp[(E-V0.5)/k]}, where k is the slope factor. {circ} indicates the control condition; {bullet}, exposure to 20 nmol/L ISO; and {bigtriangledown}, after addition of 50 nmol/L ET-1. Symbols and bars represent mean±SE. D, ET-1–dependent inhibitions accessed by normalizing amplitudes of IK tails after +40-mV test potential in the presence of various concentrations of ET-1 by that measured in the absence of the peptide and plotted as a function of ET-1 concentration. Symbols represent the mean percent inhibition; the smooth line, a best fit to the Hill equation: % inhibition=Bmax/{1+(IC50/[ET-1])h}, where Bmax indicates the maximal inhibition, and h, the Hill coefficient. Vertical bars represent SE. Numbers in parentheses indicate the number of experiments.

Experiments of the same protocol were carried out in five different myocytes, and IK tail currents were normalized by those measured after a +60-mV test potential. Fig 2CUp shows three IK tail current–test potential relations thus calculated. Smooth curves are best fit to the Boltzmann equation as described in the Fig 2Up legend. Membrane potentials for V0.5 were 16.9±1.0 mV in the control condition (open circles), 10.4±1.0 mV in the presence of ISO (solid circles), and 11.9±1.2 mV after the addition of ET-1 (open inverted triangles). Therefore, ISO shifted V0.5 by -6 to {approx}-7 mV, which was not reversed by subsequent ET-1.

Guinea pig ventricular IKs are composed of two components.24 34 IKr, which is activated at more negative potentials, shows rapid kinetics and strong inward rectification. In contrast, IKs is activated at more positive membrane potentials. Lacking the property of inward rectification, the steady state current after a prolonged depolarization to positive membrane potentials (+20 to {approx}+60 mV, 2 seconds in our experiments) consists of IKs. On the other hand, when the cell is clamped back to the holding potential (-40 mV) after a brief depolarization, the resulting tail current reflects IKr in addition to IKs. Since our experimental protocol of the IK measurement was designed for IKs and since this component is selectively potentiated by ISO,21 24 ET-1 appeared to inhibit the IKs.

Fig 2DUp summarizes the ET-1 concentration–IK inhibition relation in the continued presence of 20 nmol/L ISO (n=20). The smooth curve in the graph shows a best fit to the Hill equation as described in the Fig 2Up legend. ET-1 produced a maximal 63.4±6.5% inhibition of the ISO-enhanced component, with an IC50 of 0.7±0.4 nmol/L and a Hill coefficient of 1.1±0.7.

Since both ETA and ETB receptors6 7 were found in our preparation,10 11 we determined which type of receptor mediates the ET-1–dependent inhibition by using receptor-selective compounds. Fig 3Down shows two panels of time courses for the IK tail currents. ISO (20 nmol/L)–enhanced IK tail currents were not affected by SRTXc (100 nmol/L), an ETB-selective agonist,35 but consistently inhibited by ET-1 (50 nmol/L; Fig 3ADown, n=4). In contrast, FR139317 (1 µmol/L), an ETA-selective antagonist,35 36 completely prevented the inhibitory action of ET-1 (Fig 3BDown, n=4), suggesting that ETA receptors mediate the ET-1–induced inhibition of IK.



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  		Figure 3. The inhibitory effect of ET-1 is mediated with ETA receptors. Time course of the tail current of IK at -40 mV after a +40-mV test potential. A, Effect of 100 nmol/L SRTXc and 50 nmol/L ET-1. B, Loss of ET-1 inhibitory action after preincubation with 1 µmol/L FR139317. ET-1 inhibits the ISO-enhanced IK after washout of 1 µmol/L FR139317.

ETA receptors are members of the G protein–coupled receptor superfamily with seven transmembrane domains.35 37 To examine which type of G protein is actually involved during IK inhibition, the effect of the incubation with PTX was studied. As shown in Fig 4ADown, prior incubation of the myocytes with PTX abolished both CCh-dependent and ET-1–dependent inhibition of the ISO-enhanced IK component, which was consistently seen in control cells (see Fig 1Up). The percent change induced by ET-1 was 2.2±1.5% of the ISO-enhanced component (n=4). These results suggest that, similar to CCh, ET-1 stimulation couples to PTX-sensitive G proteins.



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  		Figure 4. The inhibitory effect of ET-1 is mediated with PTX-sensitive G proteins, presumably Gi, and ET-1 reduces the histamine- and forskolin- but not internal cAMP-induced IK. A, Preincubation with PTX (5 µg/mL at 36°C >60 minutes) abolished the inhibitory effects of both CCh (20 µmol/L) and ET-1 (50 nmol/L). B through D, Three time courses showing ET-1 actions on the tail current of IK potentiated by histamine (250 nmol/L [B]), forskolin (500 nmol/L [C]), and intrapipette cAMP (100 µmol/L [D]).

To examine the intracellular signal transduction underlying ET-1–induced IK inhibition, we used several pharmacological tools to enhance the IK. Histamine is known to increase cAMP and increase IKs via the stimulatory G protein/adenylate cyclase pathway in guinea pig ventricular myocytes.38 Submaximal concentrations of histamine (250 nmol/L) enhanced IK tail currents to 169.4±12.5% (n=7). As typically shown in Fig 4BUp, ET-1 (50 nmol/L) inhibited the histamine-induced component to 50.2±4.1% (n=7), suggesting that ET-1 antagonizes activation of the adenylate cyclase by stimulation of either histamine or ß-adrenergic receptors.

Forskolin, a diterpene plant alkaloid, has been shown to increase intracellular cAMP by directly stimulating adenylate cyclase.39 Forskolin at 500 nmol/L produced an enhancement of IK (214.9±13.5%, n=7) comparable to that induced by 20 nmol/L ISO and 250 nmol/L histamine. As shown in Fig 4CUp, ET-1 (100 nmol/L) also inhibited the forskolin-enhanced component by 48.6±10.3%.

Interaction between ß-adrenergic and muscarinic effects occurs at steps before the production of cAMP.21 22 23 Therefore, we tested whether ET-1 works after the production of cAMP. Direct dialysis of myocytes with intrapipette cAMP (100 µmol/L) enhanced IK soon after formation of the whole-cell patch-clamp mode to a comparable level induced by 20 nmol/L ISO22 23 (Fig 4DUp). ET-1 (50 nmol/L) failed to inhibit the IK thus potentiated (percent inhibition was 5.2±2.2%, n=6). These results support the idea that ET-1 may inhibit the IK by reducing the intracellular cAMP concentration through the inhibition of the adenylate cyclase.

Basal IK Is Inhibited by ET-1 but Not by CCh
Fig 5ADown depicts three original current traces (top of panel A) before (trace a) and after CCh (trace b) and ET-1 (trace c) and the time course of the IK tail current (bottom of panel A). Labels a through c are corresponding in both panels. IK was not changed after 20 µmol/L CCh (traces a and b) but was consistently decreased by 50 nmol/L ET-1 (traces a and c). In this particular myocyte, IK tail currents were reduced from 5.0 to 3.6 pA/pF (27.5%).



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  		Figure 5. The effect of ET-1 on basal IK. A, Three original current traces of whole-cell currents at +40-mV test potential before (trace a) and after exposure to CCh (20 µmol/L, trace b) and ET-1 (50 nmol/L, trace c) on basal IK (top) and the time course of the tail current of IK from the same cell (bottom) are shown. B, The effect of ET-1 on the current-voltage relation of the basal IK is shown. {circ} indicates control; {bullet}, 50 nmol/L ET-1. Symbols and bars represent mean±SE (n=5). C, Activation curves were obtained by normalizing the IK tail current amplitudes. The solid curves were drawn by fitting to the Boltzmann equation. Symbols and bars are the same as those in panel B. D, ET-1 concentration dependence for inhibition of basal IK ET-1–dependent inhibitions of IK tail current at -40 mV after +40-mV test potential was expressed as a proportion of the basal current, as in Fig 2DUp. Smooth curve in the graph was drawn by fitting to the Hill equation. Symbols and bars are the same as those in panel B. Numbers in parentheses indicate the number of observations. E, Incubation with PTX reduced the inhibitory effects of ET-1 (50 nmol/L).

Fig 5BUp shows IK tail current–test potential relations obtained from another myocyte. Experiments of the same protocol were carried out in five different myocytes, and IK tail currents were normalized by those after a +60-mV test potential. In Fig 5CUp, relative IK tail currents thus calculated are plotted against test potentials: control, open circles; after 50 nmol/L ET-1, solid circles. Smooth curves are best fits to the Boltzman equation. Values for V0.5 were 14.0±1.2 mV in the control condition and 12.7±1.1 mV in the presence of ET-1. Therefore, ET-1 reduced basal IK without any change in the shape of the current-voltage curve. Reductions in IK at various ET-1 concentrations were estimated by normalization with those measured in the absence of ET-1 (Fig 5DUp). The smooth curve was drawn by the Hill equation with a maximal 20.9±2.3% inhibition of the basal IK, with an IC50 of 1.1±0.2 nmol/L and a Hill coefficient of 1.0±0.2.

As shown in Fig 5EUp, prior incubation of the myocytes with PTX decreased ET-1–dependent inhibition of basal IK tail current, which was consistently seen in control cells (see Fig 5AUp). The percent inhibition induced by ET-1 was 6.1±2.0% of the basal component (n=4). These results suggest that ET-1 inhibition of IK is mediated by PTX-sensitive G proteins.

Although ET-1 inhibited the IKs in the presence of ISO, it was not known which component of basal IK was affected by ET-1. Therefore, we used E-4031, an IKr blocker, and brief test pulses (0.2 seconds) to separate the two components.34 E-4031 (5 µmol/L) reduced IK tail currents (Fig 6ADown; from a to b, the percent reduction was 24.3±5.4%; n=4), whereas the reduction in steady state currents was minimal. Subsequent application of 50 nmol/L ET-1 inhibited both steady state and tail currents (Fig 6ADown, trace c). In the prolonged presence of E-4031, the percent reduction of IK tail currents by ET-1 was 31.7±2.8% (n=4). These values were comparable to, or even larger than, the value obtained in the absence of E-4031 (21.2±1.8%, n=4). Therefore, it was concluded that ET-1 mainly inhibits the IKs component of basal IK.



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  		Figure 6. ET-1 inhibits an E-4031–insensitive component of IK. A, Three original current traces of whole-cell currents. Square test pulses of 200-millisecond duration were applied from a holding potential of -40 to +40 mV. The reduction of IK tail and steady state currents was observed after exposure to 5 µmol/L E-4031 and 50 nmol/L ET-1. Traces are as follows: a, control; b, after E-4031 (5 µmol/L); and c, after E-4031 (5 µmol/L)+ET-1 (50 nmol/L). Difference currents are shown in panel B (a-b) and in panel C (b-c).

ET-1 Modulates Basal and ISO-Enhanced IK in the Nystatin–Perforated Patch Method
ET-1 has been shown by Habuchi et al25 to increase IK through the PKC pathway in guinea pig ventricular myocytes. Therefore, there was a discrepancy in the experimental results of Habuchi et al and the present study. The PKC pathway appeared to be inactivated in our preparations. Since intracellular Ca2+ concentrations play a key role in activating the PKC pathway, we conducted nystatin–perforated patch experiments in which the intracellular condition, including Ca2+ concentration, was minimally disturbed.

In the perforated-patch whole-cell mode, depolarizing pulses from a holding potential of -40 to +40 mV produced IK, as depicted in Fig 7ADown-i (trace a). Exposure of the myocyte to ET-1 (50 nmol/L) first decreased to 82% of the control IK tail current (trace b) and then increased to 105% (trace c) in this particular experiment. Labels a through c correspond to the arrows in the time course of the tail current amplitude obtained from the same cell (Fig 7ADown-ii). In a total of five cells, ET-1 showed a biphasic effect on the amplitude of IK tail currents: Within 2 minutes of exposure to ET-1, IK decreased maximally to 80.3±2.1% and then in 10 minutes increased to 106.8±5.7% of the control current. The latter increasing action of ET-1 was similar to that previously demonstrated by Habuchi et al25 and was probably mediated via the PKC pathway.



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  		Figure 7. Responses of IK to ET-1 are different in nystatin–perforated patch experiments. A-i, Current traces corresponding to traces a through c indicated by arrows in panel A-ii. Test pulses of 2-second duration were applied from a -40-mV holding potential to +40 mV. A-ii, The time course of the tail current of IK from the same cell before (trace a) and after exposure to ET-1 (50 nmol/L, traces b and c). Horizontal bars above the graph indicate the application of ET-1 (50 nmol/L). B-i, Current traces corresponding to traces a through c indicated by arrows in panel B-ii using same protocol as in panel A-i. B-ii, The time course of the tail current of IK from the same cell before (trace a) and after exposure to ISO (20 nmol/L, trace b) and addition of ET-1 (50 nmol/L, trace c).

Fig 7BUp-i depicts the action of ET-1 in the presence of ISO (20 nmol/L). ISO enhanced the IK tail current to 166.2% (traces a and b), and subsequent ET-1 (50 nmol/L) inhibited the ISO-enhanced IK to 59.2% of the ISO-induced component in this myocyte (traces b and c). In five myocytes, ISO produced a 191.0±20.4% increase of the control IK tail current (n=5), which was comparable to that observed in the whole-cell experiment (Fig 1Up). The ISO-enhanced IK component was reduced to 60.4±1.9% by subsequent addition of ET-1 (50 nmol/L). Thus, the combined balance of both PKA and PKC pathways determines the effect of ET-1 on IK, but in the presence of the preactivated PKA pathway, ET-1 indeed inhibits IK in an accentuated antagonistic manner.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
ET-1 Suppresses ISO-Enhanced IKs via a PTX-Sensitive Mechanism
The present study demonstrated for the first time that nanomolar concentrations of ET-1 inhibit ISO-enhanced IKs in guinea pig ventricular myocytes. The action appeared to be analogous to that of muscarinic agonists.33 ET-1–induced inhibition of IKs appeared to be mediated by the ETA receptor/inhibitory G protein/adenylate cyclase/PKA pathway, because the effect of the peptide was prevented by an ETA-selective antagonist (Fig 3BUp) or by preincubation with PTX (Fig 4AUp). Furthermore, ET-1 suppressed the IKs enhanced by both histamine (250 nmol/L, Fig 4BUp) and forskolin (500 nmol/L, Fig 4CUp).

Although the peptide inhibited the IK enhanced by these agonists, it failed to suppress the IK that had been potentiated by the internal dialysis with 100 µmol/L cAMP (Fig 4DUp). The activation level of IK by this concentration of cAMP was comparable to that induced by the above-mentioned agonists ({approx}10 pA/pF). Therefore, the abolition of inhibitory action on cAMP-enhanced IK does not reflect the maximal activation of IK, as seen in the experiment by using 1 µmol/L ISO (Fig 1CUp) where ET-1 action was blunted. Thus, the inhibitory action of ET-1 on IK appeared to be due primarily to the inhibition of adenylate cyclase by PTX-sensitive G proteins. In this regard, we have demonstrated that 200 nmol/L ET-1 inhibited both basal and ISO-increased levels of cAMP content in guinea pig ventricular slices by radioimmunological assay.10

Since ETA receptors are known to link to membrane phosphoinositide breakdown by phospholipase C, thereby generating inositol trisphosphate and diacylglycerol, involvement of the PKC pathway by ET-1 stimulation must be taken into consideration, especially because PKC activation by {alpha}1-adrenergic agonists has been shown to increase IK40 and phorbol esters in single guinea pig ventricular myocytes.19 20 21 Activation of IK by {alpha}1-adrenoceptor stimulation was smaller (+23% [Reference 4040 ]) than that induced via the PKA mechanism and could be prevented by pretreatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor.40 Phorbol esters could mimic the {alpha}1-adrenergic action, indicating that the PKC pathway is presumably involved in increasing the IK. This increase by PKC activation was present after the IK enhancement by maximal PKA activation, suggesting that the stimulatory signal transduction pathways of these two protein kinases are distinct.

More recently, in guinea pig ventricular myocytes, ET-1 has also been shown to enhance IK via this PKC pathway.25 Our findings in nystatin–perforated patch experiments (Fig 7AUp) were not necessarily opposite to theirs. We saw that ET-1 had dual actions on basal IK: first decrease and then increase. The latter increasing action was similar to that demonstrated by Habuchi et al25 and was probably mediated via the PKC pathway. This stimulatory effect of ET-1 on IK appeared to be masked when ISO was present (Fig 7BUp). In this condition, on the contrary, ET-1 was found to antagonize the ISO-enhanced IK. These findings suggest that IK is regulated predominantly by the PKA pathway in more physiological conditions, especially because there is a tonic stimulation of ß-adrenoceptors in the in situ heart.

In terms of conventional whole-cell experiments (Fig 5Up of the present study and Fig 1Up of Habuchi et al25 ), there are at least two experimental differences that may explain the different results. First, in the present study, the Ca2+ concentration in the pipette solution was lower (pCa 8.0) than theirs (pCa 7.6). We used relatively lower-resistance pipettes to obtain a smaller access resistance ({approx}2 M{Omega} versus 2 to 4 M{Omega}). Since the activities of phospholipase C and PKC are highly Ca2+ sensitive, a lower Ca2+ concentration and smaller access resistance in the present study would result in tighter chelation of intracellular Ca2+ ions, thereby minimizing the PKC-mediated signal transduction. This was also proved by our nystatin–perforated patch experiments as mentioned above.

The extracellular K+ concentration was higher in the present study (5.4 mmol/L) than in the study of Habuchi et al25 (2 mmol/L). The lower concentration of extracellular K+ attenuates the Na+-K+ pump activity and produces the accumulation of intracellular Na+, which in turn leads to the increase of subsarcolemmal Ca2+ concentration through Na+-Ca2+ exchange. This may promote the PKC pathway, which is primarily Ca2+ sensitive, particularly in the absence of tight chelation of intracellular Ca2+ ions.

Moreover, although muscarinic agonists did not decrease the "basal" IK in the present study, the agonists have been reported to inhibit basal ICa,L in frog atrial and rabbit nodal cells.21 The inhibition of basal IK by ET-1 was also observed in the nystatin–perforated patch experiment (Fig 7AUp). The mechanism for this ET-induced IK suppression remains to be elucidated.

Clinical Implications and Limitations
ET-1 has been shown to prolong the APD in canine isolated papillary muscle.41 In the acute animal models, ET-1 that had been directly administered into the coronary artery has been shown to prolong the APD as monitored by monophasic action potentials, followed by the development of early afterdepolarizations (dogs42 ) and induced ventricular dysrhythmias (pigs and rats43 44 ). Moreover, BQ123, an ETA receptor antagonist, prevented these types of tachyarrhythmia (rats44 ). In a rat chronic heart failure model involving myocardial infarction, BQ123 was also found to significantly suppress the postinfarct ventricular remodeling.26 Thus, ET-1 may have direct action on APD prolongation or development of cellular hypertrophy through the ETA receptor.

As for an ionic basis for the prolongation of APD by ET-1 observed in the above-mentioned literature, ICa,L appeared not to contribute, because ET-1 actually reduced ventricular ICa,L in the presence of catecholamine.18 However, Lauer et al15 demonstrated that in adult rabbit ventricular myocytes ET-1 increased basal ICa,L, suggesting the presence of species differences in the effect of ET-1 on ICa,L. On the basis of the findings of the present study, ET-1–induced inhibition of IK appeared to prolong the APD, especially in the presence of ß-adrenergic stimulation.

Since APD is determined as a result of a subtle balance between numerous ion currents and since ET-1 modulates both inward and outward currents, the peptide may also cause the dispersion of APD in the in situ heart, which would give another arrhythmogeneity under pathological conditions, as reported previously in animal models.42 43 44 Finally, the APD prolongation may favor the increase in the Ca2+ influx through the reverse mode of Na+-Ca2+ exchange and partially explain the development of cellular hypertrophy.26 Specific antagonists for the ETA type of endothelin receptor would therefore counteract these undesired actions of ET-1.


*    Selected Abbreviations and Acronyms
 
APD = action potential duration
CCh = carbachol
DMSO = dimethyl sulfoxide
E-4031 = 1-[2-(6-methyl-2-pyridyl)ethyl]-4- (4-methylsulfonylaminobenzoyl)piperidine
ET-1 = endothelin-1
ETA, ETB = endothelin receptor subtypes A and B
FR139317 = (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl] amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl)propionic acid
ICa,L = L-type Ca2+ current
IK = delayed rectifier K+ current
IKr = rapidly activating component of IK
IKs = slowly activating component of IK
ISO = isoproterenol
PKA, PKC = protein kinases A and C
PTX = pertussis toxin
SRTXc = sarafotoxin S6c
V0.5 = half-maximum amplitude


*    Acknowledgments
 
This study was partially supported by a grant-in-aid for scientific research on the priority area of "Channel-Transporter Correlation" from the Ministry of Education, Science, and Culture, Japan. The authors would like to thank Prof A. Noma (Kyoto University School of Medicine), Dr A.F. James (King's College London), and K. Salter (Oxford University) for critical comments on the manuscript and continuous encouragement. Thanks are also due to Prof Y. Aizawa (Niigata University School of Medicine) for providing Dr Washizuka with the opportunity to work at Kyoto University and continuous encouragement.

Received March 1, 1997; accepted May 13, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
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 *References
 

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