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Am J Physiol Heart Circ Physiol 273: H1745-H1753, 1997;
0363-6135/97 $5.00
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Vol. 273, Issue 4, H1745-H1753, October 1997

Dual effects of endothelins on the muscarinic K+ current in guinea pig atrial cells

Hiroshi Yamaguchi, Naoya Sakamoto, Yasuhide Watanabe, Toshihiro Saito, Yoshiaki Masuda, and Haruaki Nakaya

Department of Pharmacology and Third Department of Internal Medicine, Chiba University School of Medicine, Chiba 260, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of endothelins (ETs) on the acetylcholine receptor-operated K+ current (IK ACh) were examined in isolated guinea pig atrial cells using patch-clamp techniques. ET-1 or ET-3 produced a transient activation of IK ACh in atrial cells held at -40 mV. When IK ACh was preactivated by 1 µM carbachol, however, both ETs produced a transient potentiation followed by a sustained inhibition of the current. When IK ACh was maximally activated by 10 µM carbachol or 100 µM adenosine, these ETs produced only a sustained inhibition of the IK ACh. Their inhibitory effects on the preactivated IK ACh were concentration dependent, and the half-maximal effective concentrations were 314 pM for ET-1 and 1.13 nM for ET-3. The inhibitory effect of ET-1 was antagonized by BQ-485, a specific ETA receptor antagonist, but not by BQ-788, a specific ETB receptor antagonist, indicating that the ET-1 effect is mediated by ETA receptors. On the other hand, the inhibitory effect of ET-3 was antagonized by BQ-788 and more effectively by BQ-485, suggesting the involvement of "atypical" ET receptors. Both ETs partly reversed the carbachol-induced shortening of the action potential recorded in the current-clamp mode. Inhibitory effects of ET-1 and ET-3 on the preactivated IK ACh may contribute to the positive inotropic and chronotropic effects of ETs in atrial tissues.

inositol 1,4,5-trisphosphate; protein kinase C; action potential; patch clamp

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ENDOTHELIN (ET), a potent vasoconstrictor polypeptide consisting of 21 amino acids, was originally isolated from the culture medium of porcine aortic endothelial cells (36). Three distinct ET-related genes were cloned by genomic DNA analysis, and encoded peptides were named ET-1, ET-2, and ET-3 (8). ET-1 and ET-2 are similar in terms of their biologic activities, whereas ET-3 has quite different effects (8, 11, 22). Cloning of a cDNA encoding the ET receptor showed at least two distinct subtypes, ETA and ETB receptors (1, 28). It has been reported that ET-1 and ET-2 activate these receptor subtypes nonselectively, whereas ET-3 is relatively ETB receptor specific (27, 28).

Among the diverse effects of ET in various organs and tissues, it should be emphasized that ET is one of the most potent positive inotropic endogenous substances in mammalian heart (6, 12, 15, 18, 23). In addition, ETs induce a positive chronotropic response. In rat atrial preparations, ET-1 and ET-3 induced positive inotropic and chronotropic responses (11). The ET-1-induced positive inotropic response was accompanied by a prolongation of action potential duration (APD) in guinea pig left atria (6, 12). On the other hand, Kim (16) reported that all three isoforms of ETs induced a negative chronotropic response in isolated rat atrial cells. More recently, Ono and colleagues showed that ET-1 shortened the APD in isolated atrial cells (25) and transiently decreased the beating rate of isolated right atria of guinea pigs (24). In these studies the negative chronotropic effects of ETs have been attributed to the activation of muscarinic acetylcholine (ACh) receptor-operated K+ current (IK ACh) through the pertussis toxin (PTX)-sensitive GTP-binding protein. To gain greater insight into the cause of the apparent discrepancy, this study was designed to examine the effects of ETs on IK ACh in the absence and presence of muscarinic stimulation, because IK ACh is an important determinant of APD in atrial cells and there may be a tonic stimulation of muscarinic receptors in in situ heart.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Isolation

Single atrial cells of the guinea pig heart were isolated by an enzymatic dissociation method, as previously described (31). Briefly, female guinea pigs, weighing 250-350 g, were anesthetized with an intraperitoneal injection of pentobarbital sodium. Their hearts were removed, immediately mounted on a Langendorff apparatus, and retrogradely perfused with 1) normal N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Tyrode solution for 10 min, 2) nominally Ca2+-free Tyrode solution for 10 min, and then 3) Ca2+-free Tyrode solution containing 0.2 mg/ml collagenase (Wako, Osaka, Japan) for 20-30 min. After digestion, the hearts were perfused with a high-K+-low-Cl- solution [modified Kraftbrühe (KB) solution] (9, 31). Atrial tissue was cut into small pieces in the modified KB solution, and the pieces were gently agitated to isolate cells. The cell suspension was stored in a refrigerator (4°C) and used on the same day.

Solutions

The composition of the normal HEPES-Tyrode solution was (in mM) 143 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, and 5 HEPES-NaOH buffer (pH 7.4). The composition of the modified KB solution was (in mM) 70 KOH, 50 L-glutamic acid, 40 KCl, 20 taurine, 20 KH2PO4, 3 MgCl2, 10 glucose, 1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 HEPES-KOH buffer (pH 7.4). For unitary current recordings in cell-attached mode, a depolarizing bath solution containing (in mM) 140 KCl, 1.8 MgCl2, 1 EGTA, 5.5 glucose, and 5 HEPES-KOH buffer (pH 7.4) was used. The composition of the pipette solution for whole cell clamp was (in mM) 110 potassium aspartate, 20 KCl, 1 MgCl2, 5 potassium ATP, 5 potassium phosphocreatine, 10 EGTA, and 5 HEPES-KOH buffer (pH 7.4). GTP (sodium salt, 100 µM) or guanosine 5'-O-(thiotriphosphate) (GTPgamma S; lithium salt, 100 µM) was also added to the pipette solution. The free Ca2+ concentration in the pipette solution was adjusted to pCa 8.0 according to the calculation by Fabiato and Fabiato (3) with the correction of Tsien and Rink (33). For unitary current recordings, a pipette solution containing (in mM) 140 KCl, 1.8 CaCl2, and 5 HEPES-KOH buffer (pH 7.4) was used. Carbachol (1 µM) was added to the solution. In some of the experiments, 30 nM ET-1 or 30 nM ET-3 was added to the solution instead of carbachol. In these experiments, 10 µM atropine and 100 µM theophylline were also included in the pipette solution to block muscarinic and adenosine receptors.

Data Acquisition and Analysis

Whole cell voltage clamp. Whole cell membrane currents were recorded by the patch-clamp method (5). Single atrial cells were placed in a recording chamber (0.4 ml volume) attached to an inverted microscope (model IMT-2, Olympus, Tokyo, Japan) and superfused with the HEPES-Tyrode solution at a rate of 2 ml/min. The temperature of the bath solution was kept constant at 36 ± 1.0°C. Patch pipettes were made from borosilicate glass capillaries (1.5 mm OD) using a vertical microelectrode puller (model PB-7, Narishige, Tokyo, Japan). The tip resistance was 3-5 MOmega when filled with the solution described above. The electrode was connected to a patch-clamp amplifier (model CEZ-2300, Nihon Koden, Tokyo, Japan). Command pulse signals were generated by a 12-bit digital-to-analog converter controlled by pCLAMP software (Axon Instruments, Foster City, CA). Current signals were filtered at 1 kHz, digitized, and stored on the hard disk of an IBM-compatible computer with a 200-Mbyte hard disk (Compaq Prolinea 4/50, Houston, TX). A liquid junction potential between the pipette solution and the bath solution of -8 mV was corrected. After a gigaohm seal between the tip of the electrode and the cell membrane was established, the cell membrane was ruptured by negative pressure to make the whole cell configuration. The IK ACh was activated by the extracellular application of 1-10 µM carbachol or 100 µM adenosine to the GTP-loaded cells or by the intracellular loading of 100 µM GTPgamma S, a nonhydrolyzable analog of GTP, in atrial cells held at -40 mV. Effects of various concentrations of ETs on the preactivated IK ACh were examined. To calculate percent inhibition of IK ACh, the difference between the steady-state current in the solution containing 10 µM carbachol, which maximally activated IK ACh in atrial cells held at -40 mV, and the current level in the absence of any agonists was taken as 100%. In addition, to clarify the receptor subtype(s) involved in the effects of ETs, influences of selective ETA and ETB receptor antagonists BQ-485 (perhydroazepin-1-yl-L-leucyl-D-tryptophanyl-D-tryptophan) (13) and BQ-788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma -MeLeu-D-Trp(CooMe)-D-Nle-ONa] (10) were also studied. In some experiments, BQ-123 [cyclo-(D-Trp-D-Asp-L-Pro-D-Val-L-Leu)], another ETA receptor antagonist (7), was used instead of BQ-485. It was confirmed that these ET antagonists per se did not affect the whole cell currents in guinea pig atrial cells.

Whole cell current-clamp mode. Current-clamp experiments were also performed in the whole cell recording mode at 36 ± 1.0°C. External and pipette solutions were the same as those used in the whole cell voltage-clamp experiments. After the establishment of the whole cell clamp mode, the cells were stimulated by rectangular 2-ms currents through the pipette at a rate of 0.2 Hz. After a stabilization of action potential configuration, effects of various drugs on the action potential were evaluated.

Unitary current recordings. The unitary current recordings were performed in the cell-attached configuration of the patch-clamp technique. Patch pipettes were coated near their tips with silicone and heat polished. When filled with the pipette solution described above, the tip resistance was 5-8 MOmega . After a gigohm seal between the patch electrode and the cell membrane was formed in the HEPES-Tyrode solution, cells were exposed to the depolarizing external bath solution. The holding potential was clamped at -80 mV, and the inward ACh receptor-operated K+ (KACh) channel activities were recorded. The signals were obtained by the same patch-clamp amplifier at room temperature (20-25°C) and stored on a video cassette recorder (model NV-H6, Panasonic, Osaka, Japan) through a pulse code modulator (model VR-10B, Instrutech, New York, NY). Later, the data were transferred to the hard disk of the computer at a sampling rate of 10 kHz, filtered at 1.5 kHz through a digital Gaussian filter, and analyzed by pCLAMP software. Briefly, the amplitude of the single channel current was obtained by constructing an amplitude histogram or by measuring the distance of two horizontal lines set by eye at the closed and open levels. There was no significant difference in the unitary amplitude determined by the two methods. To obtain the single channel conductance, measurement of the single channel current amplitude was repeated at different test potentials in some of the experiments. Channel openings were identified by an algorithm that used amplitude information and measured with an interactive threshold for detecting events that was set at 50% of the expected amplitude. Channel activity is represented by the product NPo, i.e., the number of channels in the patch (N) times the ratio of the channel open time to the total sampled time (Po).

Drugs

ET-1 and ET-3 were obtained from the Peptide Institute (Osaka, Japan), BQ-485 from Banyu Pharmaceutical (Ibaraki, Japan), BQ-123 from Funakoshi (Tokyo, Japan), BQ-788 and sarafotoxin S6c from Novabiochem (Laufelfingen, Switzerland), PTX from Kaken Pharmaceutical (Tokyo, Japan), carbachol chloride, adenosine, glibenclamide, staurosporine, phorbol 12-myristate 13-acetate (PMA), and D-myo-inositol 1,4,5-trisphosphate (IP3) from Sigma Chemical (St. Louis, MO), atropine sulfate monohydrate and theophylline from Wako, and 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) from Seikagaku (Tokyo, Japan). ET-1, ET-3, BQ-485, BQ-123, BQ-788, and sarafotoxin S6c were dissolved in sodium phosphate buffer as a stock solution. Glibenclamide, staurosporine, PMA, and KN-62 were dissolved in dimethyl sulfoxide. PTX was dissolved in the modified KB solution with albumin (1 mg/ml). All other compounds were dissolved in distilled water.

Statistics

Values are means ± SE. Analysis by Student's t-test was performed for paired and unpaired observations. P < 0.05 was taken as significant. Half-maximal effective concentration (EC50) was obtained using a Macintosh computer (Apple Computer, Cupertino, CA) and Kaleida Graph program (Synergy Software, Reading, PA).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of ETs on the Whole Cell Outward Current

ET-1 (30 nM) induced a transient outward current in atrial cells held at -40 mV (Fig. 1A). A similar increase in the outward current was observed in eight cells exposed to 30 nM ET-1. However, ET-1 produced a transient potentiation followed by a sustained inhibition of the outward current when IK ACh was preactivated by an application of carbachol (1 µM) to the bath (Fig. 1B). ET-1 (30 nM) transiently increased the carbachol (1 µM)-induced IK ACh by 57.7 ± 3.8% of the preactivated level and then decreased it by 64.7 ± 5.8% of the preactivated level in three cells. Effects of ET-3 on IK ACh were qualitatively similar. ET-3 also induced a transient outward current in six cells (Fig. 1C) and produced a biphasic effect on the preactivated IK ACh (Fig. 1D). ET-3 (30 nM) transiently increased the carbachol (1 µM)-induced IK ACh by 12.3 ± 4.7% and decreased it by 55.0 ± 3.7% in five cells. However, the potentiating effect of ET-3 on IK ACh was weaker than that of ET-1 when effects of the same concentration (30 nM) of ETs were compared (Fig. 1, B and D). The inhibitory effects of ET-1 and ET-3 on the preactivated IK ACh were sustained, and the outward current recovered only slightly on washout of ETs. Even with the addition of ET antagonists such as BQ-485 and BQ-788, the inhibition of IK ACh could not be reversed promptly.


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  		Fig. 1.   Effects of endothelin (ET)-1 and ET-3 on membrane current of atrial cells clamped at -40 mV in absence (A and C) and presence (B and D) of 1 µM carbachol (CCh). Pipette solution contained 100 µM GTP. Horizontal bars, time scale and zero current level.

In atrial cells preincubated with PTX (5 µg/ml, 36°C, 120 min), neither 30 nM ET-1 (n = 4) nor 30 nM ET-3 (n = 4) induced the transient outward current. Recent reports have indicated that ACh can activate the ATP-sensitive K+ current in atrial cells (34) and ET-1 can inhibit the current in ventricular cells (17). Therefore, we conducted similar experiments in the presence of the ATP-sensitive K+ channel blocker glibenclamide. ET-1 (30 nM, n = 2) and ET-3 (30 nM, n = 2) still induced a transient outward current in the presence of 10 µM glibenclamide. In addition, the ETs produced a transient potentiation followed by a sustained inhibition of the outward current induced by 1 µM carbachol in glibenclamide-treated atrial cells (n = 2 each). In some experiments, ramp pulse protocol (repolarization or hyperpolarization pulse from +10 to -100 mV with a slope of -1.2 mV/ms) was used to record a quasi-steady-state current. In carbachol (1 µM)-treated cells the reversal potential of the outward current (around -80 mV) was not influenced by the addition of ET-1 or ET-3. Taken together, the current activated and inhibited by ET-1 or ET-3 would be IK ACh.

When IK ACh was maximally activated by a higher concentration (10 µM) of carbachol, only inhibitory effects of ETs on the outward current were observed (Fig. 2, A and B). ET-1 (30 nM) and ET-3 (30 nM) inhibited the IK ACh by 76.7 ± 10.3% (n = 6) and 73.7 ± 6.5% (n = 7), respectively. Although carbachol and adenosine act on different membrane receptors, i.e., M2-muscarinic ACh receptors and A1-adenosine receptors, adenosine can also induce IK ACh through the activation of PTX-sensitive GTP-binding protein in atrial cells (20). ET-1 (30 nM) elicited an almost identical inhibitory effect on the adenosine (100 µM)-induced IK ACh, which was also persisted after the washout (Fig. 2C). ET-3 also inhibited the adenosine-induced outward current. ET-1 (30 nM) and ET-3 (30 nM) inhibited the adenosine-induced IK ACh by 69.4 ± 6.7% (n = 8) and 62.0 ± 2.8% (n = 4), respectively. Intracellular loading of GTPgamma S (100 µM), a nonhydrolyzable GTP analog, gradually activated a persistent outward current, even in the absence of any agonists. Addition of 30 nM ET-1 inhibited the GTPgamma S-induced outward current by 87.5 and 90.0% without transient activation in two cells. ET-3 (30 nM) also inhibited the GTPgamma S-induced current by 88.9 and 76.9% in two cells without activation of the outward current. Because ETs commonly inhibited carbachol-, adenosine-, and GTPgamma S-induced IK ACh, it is likely that ETs inhibit IK ACh by mechanism(s) other than blocking the muscarinic receptors. Although ET-3 inhibited the preactivated IK ACh, sarafotoxin S6c (30 nM), a specific ETB receptor agonist (27), failed to inhibit the carbachol (10 µM)-induced IK ACh (Fig. 2D). Similar results were obtained in four other cells.


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  		Fig. 2.   Effects of ET-1 (A and C), ET-3 (B), and sarafotoxin S6c (D) on outward current that was almost maximally activated by 10 µM carbachol (A, B, and D) or 100 µM adenosine (C). Atrial cells were held at -40 mV, and pipette solution contained 100 µM GTP. Horizontal bars, time scale and zero current level.

Figure 3 summarizes the concentration-response relationships of the inhibitory effects of ETs on the carbachol (10 µM)-induced IK ACh in the absence and presence of ET receptor antagonists in the atrial cells held at -40 mV. This high concentration of carbachol was used to minimize the potentiating effects of ETs and quantify only their inhibitory effects on IK ACh. ET-1 and ET-3 inhibited IK ACh in a concentration-dependent fashion, and the maximal inhibition of IK ACh (~75%) was achieved with 30 nM ET-1 and 30 nM ET-3. The EC50 values of ET-1 and ET-3 for inhibiting the carbachol-induced IK ACh were 314 pM and 1.13 nM, respectively.


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  		Fig. 3.   Concentration-response relationships of inhibitory effects of ET-1 (A) and ET-3 (B) on carbachol (10 µM)-induced ACh receptor-operated K+ current in absence (control) and presence of ET receptor antagonists. Holding potential was -40 mV. Values are means ± SE of 5-12 experiments.

Pretreatment with 30 nM BQ-485, a selective ETA antagonist (13), shifted the concentration-response curve for the inhibitory effect of ET-1 to the right ~30-fold. In contrast, 30 nM BQ-788, a selective ETB antagonist (10), slightly shifted the curve to the right, but this effect was not statistically significant. The EC50 values of ET-1 were 10.1 nM in the presence of 30 nM BQ-485 and 830 pM in the presence of 30 nM BQ-788 (Fig. 3A). The inhibitory effect of ET-3 was more sensitive to BQ-485. The concentration-response curve for the inhibitory effect of ET-3 on IK ACh was shifted by 30 nM BQ-485 to the right ~900-fold. Moreover, the curve was shifted by 3 nM BQ-485 by 60-fold. In addition, 30 nM BQ-788 also shifted the concentration-response curve for the ET-3 effect to the right ~30-fold. The EC50 values of ET-3 in the presence of 3 nM BQ-485, 30 nM BQ-485, and 30 nM BQ-788 were 72.7 nM, 1.04 µM, and 28.7 nM, respectively (Fig. 3B). BQ-123 (30 nM), another selective ETA antagonist (7), also blocked the inhibitory effect of 30 nM ET-3 (n = 3, data not shown). These results suggest that ET-1 inhibits IK ACh through the activation of ETA receptors, whereas ET-3 inhibits IK ACh through the activation of BQ-485- and BQ-788-sensitive atypical ET receptors.

To elucidate the subcellular mechanism(s) by which ETs inhibit IK ACh, we evaluated influences of various compounds modulating the signal transduction system on ET-induced IK ACh inhibition. Because ETs are known to facilitate phosphatidylinositol hydrolysis (6, 15), we examined whether activation of protein kinase C (PKC) or production of IP3 was involved in the inhibitory effects of ETs on IK ACh. ET-1 (3 nM) and ET-3 (3 nM) inhibited the carbachol (10 µM)-induced IK ACh by 62.0 ± 8.0% (n = 9) and 48.7 ± 7.2% (n = 10), respectively. Pretreatment with 30 nM staurosporine, a PKC inhibitor, failed to affect the inhibition of IK ACh by these ETs (Fig. 4A). The inhibition of IK ACh by ET-1 and ET-3 in the presence of staurosporine was 57.0 ± 5.8% (n = 7) and 53.7 ± 5.5% (n = 8), respectively, not significantly different from the control condition. PMA (100 nM), a PKC activator, could not mimic the inhibitory effects of ETs in five cells (data not shown). To test the involvement of IP3 in the ET-induced IK ACh inhibition, we used a pipette solution containing a high concentration (20 µM) of IP3. We thought that the preactivation of the IP3 pathway may damp the ET-induced IK ACh inhibition if IP3 production is a prerequisite for the IK ACh inhibition. Intracellular perfusion of cells with IP3 abolished the ET-3-induced inhibition of IK ACh but not the ET-1-induced inhibition (Fig. 4B). The inhibition of IK ACh by ET-1 and ET-3 was 52.1 ± 7.6% (n = 5) and 2.4 ± 1.5% (n = 5), respectively. The ET-3-induced inhibition of IK ACh with this pipette solution was significantly smaller than that in the control condition. However, inclusion of 100 µM IP3 also failed to inhibit the ET-1-induced IK ACh inhibition (53.1 ± 7.2%, n = 3). In addition, the IK ACh density recorded with IP3-containing pipette solution (16.6 ± 3.0 pA/pF, n = 6) was not significantly different from that with normal pipette solution (19.3 ± 2.1 pA/pF, n = 6) during the activation with 10 µM carbachol. Therefore, IP3 inclusion might indirectly interfere with the inhibitory pathway on IK ACh by ET-3 but not by ET-1. IP3 is known to produce a Ca2+ release from the sarcoplasmic reticulum, and the increased Ca2+ might activate calmodulin. Accordingly, we examined the influence of KN-62, a Ca2+-calmodulin-dependent protein kinase II inhibitor (32), on the inhibitory effects of ETs on IK ACh. However, KN-62 failed to affect the inhibition of IK ACh by ETs (Fig. 4C). The inhibition of IK ACh by ET-1 and ET-3 in the presence KN-62 was 71.9 ± 6.4% (n = 6) and 40.3 ± 6.0% (n = 8), respectively. Therefore, the IP3-calmodulin pathway does not seem to be involved in the IK ACh inhibition by ET-1 or ET-3.


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  		Fig. 4.   Influence of treatment with staurosporine (A) or KN-62 (C) and pertussis toxin (PTX, D) and intracellular loading of inositol 1,4,5-trisphosphate (IP3, B) on inhibitory effects of ET-1 and ET-3 on carbachol (10 µM, A-C)- and guanosine 5'-O-(thiotriphosphate) (GTPgamma S, 100 µM, D)-induced ACh receptor-operated K+ current. Cells were held at -40 mV. Staurosporine and KN-62 were applied at least 10 min before exposure to ETs. When pipette solution containing IP3 was used, ETs were added to bath solution at least 10 min after rupture of cell membrane. PTX was added to cell suspension at 5 µg/ml at 36°C for 120 min. Horizontal bars, time scale and zero current level.

Recently, ETA receptors were shown to couple to PTX-sensitive G protein (14, 25, 35). The activation of IK ACh in response to 10 µM carbachol was completely abolished in atrial cells preincubated with PTX. Intracellular loading of 100 µM GTPgamma S was still capable of activating IK ACh through direct activation of Gi proteins. ET-1 (30 nM) and ET-3 (30 nM) inhibited the GTPgamma S-induced IK ACh, even in the PTX-treated cells (Fig. 4D), indicating that the inhibitory effects of ETs on IK ACh are not mediated by PTX-sensitive G proteins.

Effects of ETs on the Single KACh Channel Current

The unitary KACh channel current was recorded from cell-attached patches using patch pipettes containing 1 µM carbachol. In these experiments, bath solution and pipette solution contained 140 mM K+, and the pipette potential was clamped at various potentials. When pipette potential was positive, the unitary IK ACh was recorded as an inward current, which is shown as a downward deflection (Fig. 5). The current-voltage relationship for the single channel current was determined in four cells, and the mean slope conductance was 47.6 ± 1.6 pS (n = 4) and displayed inward rectification. To minimize the influence of spontaneous decrease in the channel activity due to the desensitization, ETs were added to the bath solution at least 8 min after the establishment of the cell-attached configuration. In addition, a relatively high concentration of ETs was used to shorten the time course of the effects. Application of 100 nM ET-1 or 100 nM ET-3 inhibited the channel activity through the reduction of the open probability without affecting the amplitude of unitary events (Fig. 5, A and B). Channel activity can be expressed as NPo, where N is the number of KACh channels in the patch membrane and Po is the open probability. ET-1 (100 nM) decreased NPo from 0.461 ± 0.064 to 0.259 ± 0.056 (n = 6, P < 0.05), and ET-3 (100 nM) decreased it from 0.344 ± 0.101 to 0.158 ± 0.051 (n = 5, P < 0.05; Fig. 5, C and D). Inhibitory effects of ETs were sustained, and NPo scarcely recovered after the washout. These findings suggest that ETs inhibit IK ACh via production of soluble intracellular second messenger(s).


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  		Fig. 5.   Effects of 100 nM ET-1 (A) and 100 nM ET-3 (B) on unitary ACh receptor-operated K+ current recorded in cell-attached membrane patches. Pipette solution contained 1 µM carbachol, and holding potential was -80 mV. C and D: changes in open probability (NPo) after ET-1 and ET-3, respectively. Values are means ± SE of 5-6 experiments. * P < 0.05 vs. control.

Single channel activity was also recorded from cell-attached patches with patch pipettes containing 30 nM ET-1 or 30 nM ET-3. The patch pipettes also included 10 µM atropine and 100 µM theophylline to block muscarinic and adenosine receptors. In this condition the channel openings were observed. The mean slope conductances with patch pipettes containing ET-1 and ET-3 were 46.6 ± 0.5 (n = 9) and 46.9 ± 0.4 pS (n = 7), respectively, and showed inward rectification. The channel conductance was not significantly different from that recorded with patch pipettes containing 1 µM carbachol, suggesting KACh channel activity. The NPo values of the channel with ET-1- and ET-3-containing pipettes were 0.156 ± 0.027 (n = 9) and 0.144 ± 0.034 (n = 7), respectively. These NPo values were slightly greater than the NPo of the basal spontaneous opening of KACh channels recorded using pipette solution containing atropine and theophylline (0.057 ± 0.015, n = 9), indicating KACh channel activation by ET-1 and ET-3.

Effects of ETs on Action Potential

Action potential of guinea pig atrial cells stimulated at a rate of 0.2 Hz was recorded in the whole cell current-clamp mode. The baseline characteristics of action potentials were as follows: resting membrane potential (RMP) was -73.4 ± 1.2 mV, action potential amplitude was 131.1 ± 1.6 mV, APD at 50% repolarization (APD50) was 72.9 ± 7.5 ms, and APD at 90% repolarization (APD90) was 107.1 ± 8.0 ms (n = 21). ET-1 (30 nM) shortened APD90 by 87.0 and 80.1% with a slight increase in RMP in two cells. ET-3 (30 nM) also shortened APD90 by 59.8 and 71.2% in two cells. When the APD was shortened by 1 µM carbachol, ET-1 and ET-3 transiently shortened APD further and then partly restored APD toward control (Fig. 6A). After the application of carbachol, APD90 was 16.1 ± 3.8% of the control and was prolonged to 36.3 ± 8.6% by 30 nM ET-1 (n = 8, P < 0.05) and to 29.4 ± 4.0% by 30 nM ET-3 (n = 9, P < 0.05; Fig. 6B). Carbachol (1 µM) significantly increased RMP from -74.3 ± 1.3 to -77.3 ± 1.1 mV (n = 17). Addition of 30 nM ET-1 and 30 nM ET-3 significantly decreased RMP to -73.3 ± 2.3 (n = 8) and -72.8 ± 1.7 mV (n = 9), respectively. Thus ET-1 and ET-3 functionally antagonized the muscarinic receptor-mediated action potential shortening.


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  		Fig. 6.   Effects of 30 nM ET-1 and 30 nM ET-3 on action potential of atrial cells stimulated at 0.2 Hz in presence of 1 µM carbachol. A: actual records of action potentials. B: summarized data of changes of action potential duration at 90% repolarization (APD90) after carbachol and carbachol + ET. Values are means ± SE of 8-9 experiments. * P < 0.05 vs. carbachol alone.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Since the discovery of ETs, it has been demonstrated that they produce positive inotropic and chronotropic responses in cardiac tissues of various mammalian species (6, 11, 12, 15, 18, 23). However, underlying mechanism(s) responsible for these effects of ETs have not been fully understood. Effects of ETs on the membrane current system of cardiac cells have been examined in several studies. In terms of effects of ET-1 on the Ca2+ current (ICa), inconsistent results have been reported; ET-1 decreased ICa in guinea pig ventricular cells (30, 35), whereas it increased ICa with the use of a pipette solution containing GTP in rabbit ventricular cells (21). Another study (4) showed that ET-1 enhanced the delayed rectifier K+ current through the activation of PKC in guinea pig ventricular cells. A recent report from our laboratory (17) has demonstrated that ET-1 but not ET-3 partially inhibits the ATP-sensitive K+ current in guinea pig ventricular cells. Other studies (16, 25) have shown that ET-1 activates IK ACh through the activation of PTX-sensitive GTP-binding proteins, which resulted in shortening of the action potential and a negative chronotropic response in guinea pig and rat atrial cells. However, the ET-1-induced activation of IK ACh cannot readily account for the positive inotropic and chronotropic responses observed in isolated atrial preparations (6, 11, 12, 24). In in situ heart, tonic stimulation of muscarinic receptors may exist. Therefore, it may also be important to examine the effects of ETs on the preactivated IK ACh.

In the absence of any agonist, ET-1 produced a PTX-sensitive outward current in atrial cells held at -40 mV. These observations are consistent with previous reports, in which ET-1 elicited a PTX-sensitive K+ current in rat (16) and guinea pig atrial cells (25). Although Kim (16) showed that not only ET-1 but also ET-3 activated the outward current in rat atrial cells, Ono et al. (25) reported that ET-3 failed to elicit the outward current in guinea pig atrial cells. Consistent with the former study, 30 nM ET-3 also activated the PTX-sensitive outward current in atrial cells, although the activation of the current was somewhat smaller and slower than that by ET-1 in this study. In addition, ET-1 and ET-3 activated the outward current in the presence of the ATP-sensitive K+ channel blocker glibenclamide. Furthermore, ET-3 as well as ET-1 shortened APD in single atrial cells. These results suggest that the ETs commonly activate IK ACh.

This study has demonstrated that ET-1 and ET-3 produced dual effects on IK ACh, i.e., enhancement followed by inhibition in the absence and presence of glibenclamide, when the IK ACh was preactivated by the muscarinic agonist carbachol. When the IK ACh was maximally activated by a higher concentration of carbachol, ETs produced only an inhibition of IK ACh, which was only partially reversed by the washout of the peptide. The inhibitory effects of ET-1 and ET-3 were concentration dependent, and their EC50 values for inhibiting the carbachol (10 µM)-induced IK ACh were 0.31 and 1.13 nM, respectively. The reduction of IK ACh after ETs was reflected by changes in action potential configuration. ET-1 and ET-3 partially reversed the carbachol-induced action potential shortening.

In the present study ET-1 and ET-3 inhibited the carbachol-induced IK ACh within the same order of magnitude; the EC50 for ET-1 was approximately three times smaller than that for ET-3. The concentration-response curve for the inhibitory effect of ET-1 was shifted by BQ-485 but not by BQ-788, suggesting that the ET-1 effect is mediated by ETA receptors. However, it cannot be concluded that the ET-3 effect is also mediated by ETA receptors, because ET-3 is supposed to be a relatively specific ETB agonist and to show ~100 times less affinity for ETA receptors than ET-1 (27). In addition, the concentration-response curve for the inhibitory effect of ET-3 was shifted by BQ-788 and more effectively by BQ-485. Moreover, sarafotoxin S6c, which specifically recognizes ETB receptors but not ETA receptors (29), failed to inhibit the IK ACh. Therefore, the ET-3 effect may be mediated by "atypical" ET receptors, i.e., BQ-788- and BQ-485-sensitive and S6c-insensitive receptors. Thus ET receptors mediating the ET-3 effect on IK ACh could not be readily classified into ETA or ETB subtypes. Similar atypical ET receptors have been described in rat atrial muscles (26). There may be an additional type of ET receptor that has not been discriminated on the basis of molecular biologic techniques.

According to Kim (16) and Ono et al. (25), the activation of IK ACh by ETs is mediated by the PTX-sensitive G protein. However, the inhibitory effect of ETs on IK ACh that was induced by GTPgamma S was not influenced by PTX. Because it is known that ET-1 and ET-3 produce phosphoinositide hydrolysis (6, 15), we tested the involvement of two downstream messengers, PKC and IP3, in the inhibitory effects of ET-1 and ET-3 on IK ACh. PKC is unlikely to be involved in the inhibitory effects of ETs on IK ACh, because staurosporine failed to modify the inhibitory effects of ETs and PMA could not mimic the effects of ETs.

In terms of the experiments using the pipette solution containing IP3, unexpected results were obtained. Originally, we considered that intracellular loading of excessive IP3 may damp the ET-induced IK ACh inhibition if IP3 inhibits KACh channels by acting as a ligand or by releasing Ca2+ from the sarcoplasmic reticulum. In fact, the inclusion of IP3 in the pipette solution almost abolished the inhibitory effect of ET-3 on IK ACh but not that of ET-1. However, the inhibition of the Ca2+-calmodulin pathway, which might be activated by IP3-induced Ca2+ release from the sarcoplasmic reticulum of cardiac cells (2), failed to affect the IK ACh inhibition by ET-1 and ET-3. In addition, the density of IK ACh recorded with IP3-containing pipette solution was not significantly different from that recorded with normal pipette solution. Therefore, a reasonable interpretation would be that ET-3 may inhibit IK ACh by some mechanism(s) that can be antagonized by intracellular IP3 overload. ET-1 might inhibit IK ACh by some intracellular mechanism(s) that is clearly different from those of ET-3.

Both ETs inhibited the single KACh channel activity recorded in the cell-attached mode using a carbachol-containing pipette solution. These findings suggest that both ETs appear to inhibit IK ACh via soluble second messenger(s) and not via a membrane-delimited process. However, we obtained smaller effects of ETs on the single KACh channel activity, although we employed a higher concentration (100 nM) of ETs than those used in the whole cell experiments. One possible explanation might be that access of some soluble messenger(s) to the K+ channels in the patch membrane might be limited. Further experimentation is needed to clarify the precise mechanism(s) of the IK ACh inhibition by ETs.

This study has demonstrated that ET-1 and ET-3 inhibit the preactivated IK ACh, although the receptors and mechanisms involved in the IK ACh inhibition could not be fully clarified. Previously, it was reported that ET-1 and ET-3 produce positive chronotropic and inotropic responses in guinea pig atrial preparations (6, 12, 24). It was reported that ET-1 prolonged the APD in isolated guinea pig atria (6, 12). Therefore, inhibition of IK ACh might in part contribute to the electromechanical responses to ETs in atrial preparations. However, ETs are known to elicit marked positive inotropic responses without affecting APD in a ventricular preparation in which IK ACh does not contribute to the repolarization of the action potential (15, 17). An increase in ICa and/or the Ca2+ sensitivity of cardiac myofilament resulting from the activation of the Na+/H+ exchanger (19) may be also important for the electromechanical responses to ETs.

    ACKNOWLEDGEMENTS

The authors thank M. Tamagawa and I. Sakurada for superb technical assistance and I. Sakashita for excellent secretarial services.

    FOOTNOTES

This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan.

Address for reprint requests: H. Nakaya, Dept. of Pharmacology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan.

Received 4 September 1996; accepted in final form 3 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Arai, H., S. Hori, I. Aramori, H. Ohkubo, and S. Nakanishi. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348: 730-732, 1990[Medline].

2.   Ehrlich, B. E., E. Kaftan, S. Bezprozvannaya, and I. Bezprozvanny. The pharmacology of intracellular Ca2+ release channels. Trends Pharmacol. Sci. 15: 145-149, 1994[Medline].

3.   Fabiato, A., and F. Fabiato. Calculator programs for computing the composition of solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. Paris 75: 463-505, 1979[Medline].

4.   Habuchi, Y., H. Tanaka, T. Furukawa, Y. Tsujimura, H. Takahashi, and M. Yoshimura. Endothelin enhances delayed potassium current via phospholipase C in guinea pig ventricular myocytes. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H345-H354, 1992[Abstract/Free Full Text].

5.   Hamil, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391: 85-100, 1981[Medline].

6.   Hattori, Y., H. Nakaya, J. Nishimura, and M. Kanno. A dual-component positive inotropic effect of endothelin-1 in guinea pig left atria: a role of protein kinase C. J. Pharmacol. Exp. Ther. 266: 1202-1212, 1993[Abstract/Free Full Text].

7.   Ihara, M., K. Noguchi, T. Saeki, T. Fukuroda, S. Tsuchida, S. Kimura, T. Fukami, K. Ishikawa, M. Nishikawa, M. Nisikibe, and M. Yano. Biological profiles of highly potent novel endothelin antagonists selective for ETA receptor. Life Sci. 50: 247-255, 1991.

8.   Inoue, A., M. Yanagisawa, S. Kimura, Y. Kasuya, T. Miyauchi, K. Goto, and T. Masaki. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. USA 86: 2863-2867, 1989[Abstract/Free Full Text].

9.   Isenberg, G., and U. Klöckner. Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium." Pflügers Arch. 395: 6-18, 1982[Medline].

10.   Ishikawa, K., M. Ihara, K. Noguchi, T. Mase, N. Mino, T. Saeki, T. Fukuroda, T. Fukami, S. Ozaki, T. Nagase, M. Nishikibe, and M. Yano. Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788. Proc. Natl. Acad. Sci. USA 91: 4892-4896, 1994[Abstract/Free Full Text].

11.   Ishikawa, T., L. Li, O. Shinmi, S. Kimura, M. Yanagisawa, K. Goto, and T. Masaki. Characteristics of binding of endothelin-1 and endothelin-3 to rat hearts: developmental changes in mechanical responses and receptor subtypes. Circ. Res. 69: 918-926, 1991[Abstract/Free Full Text].

12.   Ishikawa, T., M. Yanagisawa, S. Kimura, K. Goto, and T. Masaki. Positive inotropic action of novel vasoconstrictor peptide endothelin on guinea pig atria. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H970-H973, 1988[Abstract/Free Full Text].

13.   Itoh, S., T. Sasaki, K. Ide, K. Ishikawa, M. Nishikibe, and M. Yano. A novel endothelin ETA receptor antagonist, BQ-485, and its preventive effect on experimental cerebral vasospasm in dogs. Biochem. Biophys. Res. Commun. 195: 969-975, 1993[Medline].

14.   James, A. F., L. N. Xie, Y. Fujitani, S. Hayashi, and M. Horie. Inhibition of the cardiac protein kinase A-dependent chloride conductance by endothelin-1. Nature 370: 297-300, 1994[Medline].

15.   Kasai, H., M. Takanashi, C. Takasaki, and M. Endoh. Pharmacological properties of endothelin receptor subtypes mediating positive inotropic effects in rabbit heart. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2220-H2228, 1994[Abstract/Free Full Text].

16.   Kim, D. Endothelin activation of an inwardly rectifying K+ current in atrial cells. Circ. Res. 69: 250-255, 1991[Abstract/Free Full Text].

17.   Kobayashi, S., H. Nakaya, T. Takizawa, Y. Hara, S. Kimura, T. Saito, and Y. Masuda. Endothelin-1 partially inhibits ATP-sensitive K+ current in guinea pig ventricular cells. J. Cardiovasc. Pharmacol. 27: 12-19, 1996[Medline].

18.   Kohmoto, O., H. Ikenouchi, Y. Hirata, S. Momomura, T. Serizawa, and W. H. Barry. Variable effects of endothelin-1 on [Ca2+]i transients, pHi, and contraction in ventricular myocytes. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H793-H800, 1993[Abstract/Free Full Text].

19.   Krämer, B. K., T. W. Smith, and R. A. Kelly. Endothelin and increased contractility in adult rat ventricular myocytes: role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na+-H+ exchanger. Circ. Res. 68: 269-279, 1991[Abstract/Free Full Text].

20.   Kurachi, Y., T. Nakajima, and T. Sugimoto. On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. Pflügers Arch. 407: 264-274, 1986[Medline].

21.   Lauer, M. R., M. D. Gunn, and W. T. Clusin. Endothelin activates voltage-dependent Ca2+ current by a G protein-dependent mechanism in rabbit cardiac myocytes. J. Physiol. (Lond.) 448: 729-747, 1992[Abstract/Free Full Text].

22.   Maggi, C. A., S. Giuliani, R. Pataccini, P. Rovero, A. Giachetti, and A. Meli. The activity of peptides of the endothelin family in various mammalian smooth muscle preparations. Eur. J. Pharmacol. 174: 23-31, 1989[Medline].

23.   Moravec, C. S., E. E. Reynolds, R. W. Stewart, and M. Bond. Endothelin is a positive inotropic agent in human and rat heart in vitro. Biochem. Biophys. Res. Commun. 159: 14-18, 1989[Medline].

24.   Ono, K., K. Eto, A. Sakamoto, T. Masaki, K. Shibata, T. Sada, K. Hashimoto, and G. Tsujimoto. Negative chronotropic effect of endothelin 1 mediated through ETA receptors in guinea pig atria. Circ. Res. 76: 284-292, 1995[Abstract/Free Full Text].

25.   Ono, K., G. Tsujimoto, A. Sakamoto, K. Eto, T. Masaki, Y. Ozaki, and M. Satake. Endothelin-A receptor mediates cardiac inhibition by regulating calcium and potassium currents. Nature 370: 301-304, 1994[Medline].

26.   Panek, R. L., T. C. Major, G. P. Hingorani, A. M. Doherty, D. G. Taylor, and S. T. Rapundalo. Endothelin and structurally related analogs distinguish between endothelin receptor subtypes. Biochem. Biophys. Res. Commun. 183: 566-571, 1992[Medline].

27.   Sakurai, T., M. Yanagisawa, and T. Masaki. Molecular characterization of endothelin receptors. Trends Pharmacol. Sci. 13: 103-108, 1992[Medline].

28.   Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, K. Goto, and T. Masaki. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348: 732-735, 1990[Medline].

29.   Sokolovsky, M. Structure-function relationships of endothelins, sarafotoxins, and their receptor subtypes. J. Neurochem. 59: 809-821, 1992[Medline].

30.   Tohse, N., Y. Hattori, H. Nakaya, M. Endou, and M. Kanno. Inability of endothelin to increase Ca2+ current in guinea-pig heart cells. Br. J. Pharmacol. 99: 437-438, 1990[Medline].

31.   Tohse, N., H. Nakaya, and M. Kanno. alpha 1-Adrenoceptor stimulation enhances the delayed K+ current of guinea pig ventricular cells through the activation of protein kinase C. Circ. Res. 71: 1441-1446, 1992[Abstract/Free Full Text].

32.   Tokumitsu, H., T. Chijiwa, M. Hagiwara, A. Mizutani, M. Terasawa, and H. Hidaka. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 265: 4315-4320, 1990[Abstract/Free Full Text].

33.   Tsien, R. Y., and T. Rink. Neutral carrier ion-sensitive microelectrode for measurement of intracellular calcium. Biochim. Biophys. Acta 559: 623-638, 1980.

34.   Wang, Y. G., and S. L. Lipsius. Acetylcholine activates a glibenclamide-sensitive K+ current in cat atrial myocytes. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1322-H1334, 1995[Abstract/Free Full Text].

35.   Xie, L.-H., M. Horie, A. F. James, M. Watanuki, and S. Sasayama. Endothelin-1 inhibits L-type Ca currents enhanced by isoproterenol in guinea-pig ventricular myocytes. Pflügers Arch. 431: 533-539, 1996[Medline].

36.   Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Masaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988[Medline].

AJP Heart Circ Physiol 273(4):H1745-H1753
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