• Corpus ID: 42797350

EndothelinA Receptor Subtype Mediates Endothelin-Induced Contractility in Left Ventricular Cardiomyocytes Isolated from Rabbit Myocardium 1

@inproceedings{Kelso2000EndothelinARS,
  title={EndothelinA Receptor Subtype Mediates Endothelin-Induced Contractility in Left Ventricular Cardiomyocytes Isolated from Rabbit Myocardium 1},
  author={Elizabeth J. Kelso and Barbara J. McDermott and Bernard Silke and James Paul Spiers},
  year={2000}
}

• E. Kelso, B. McDermott, +1 author J. Spiers
• Published 2000
• Biology

Endothelin (ET)-1 is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca in the myocardium. The object of this study was to examine 1) the influence of ETA and ETB receptor subtypes, and 2) the role of the phospholipase C (PLC) pathway in mediating ET-1-induced contraction. Left ventricular cardiomyocytes were isolated from the hearts of New Zealand White rabbits (2–2.5 kg) by the use of Langendorff perfusion with collagenase. Cardiomyocyte function was examined during unloaded, electrically stimulated (0.5 Hz) contractions with a video-edge detection system. ET-1 increased cell shortening with greater potency than ET-3: mean EC50 values were 1.1 3 10 211 and 2.6 3 10 M, respectively. With the same order of potency, ET-1 and ET-3 increased (P , .05) velocity of cell shortening. The ETA receptor-selective antagonist ABT-627 shifted the ET-1-induced cell shortening response curve to the right with a pA2 value of 10.3. The ETB receptor-selective antagonist A-192621 (10-10 M) did not alter the concentration-response of ET-1. Moreover, the ETB receptor-selective agonist sarafotoxin 6c did not have any effect on cell shortening over the concentration range of 10 to 10 M. ET-1 in the presence of the PLC inhibitor U-73122 did not alter the contractile amplitude. However, ET-1 in the presence of the protein kinase C inhibitor bisindolylmalemide increased cell shortening. These findings indicate that 1) the ETA receptor subtype, and not the ETB receptor subtype, mediates the positive inotropic effect of ET-1, and 2) the response of ET-1 is mediated by a PLC pathway, but not through protein kinase C, in ventricular cardiomyocytes isolated from rabbit myocardium. Two distinct subtypes of endothelin (ET) receptors, ETA and ETB, have been distinguished pharmacologically by the different potencies displayed by ET isopeptides; ET-1 has a higher affinity than ET-3 for the ETA receptor subtype, whereas the isopeptides have almost equal affinity for the ETB receptor subtype (Warner, 1994). Both receptor subtypes are widely distributed in cardiovascular tissues; indeed, both ETA and ETB receptor subtypes have been characterized in human atrial and ventricular myocardium (Molenaar et al., 1993). An additional receptor subtype (ETC), which binds preferentially to ET-3, has been identified in bovine endothelial cells and human anterior pituitary cells, but it also may exist in the atria where receptor-binding assays have exposed high-affinity receptors that are selective for ET-3 (Masaki et al., 1994). ET-1 is more potent than ET-3 as an inotropic agent, whereas both peptides are equipotent chronotropic agents (Moravec et al., 1989; Ishikawa et al., 1991), suggesting that the inotropic response is likely to be mediated, at least in part, by the ETA receptor subtype, whereas the chronotropic response is more likely to be mediated by the ETB receptor subtype. However, all three isopeptides from the ET family, namely, ET-1, ET-2, and ET-3, were reported to produce similar positive inotropic effects in rabbit papillary muscle, suggesting that the contractile effect may be mediated by the ETB receptor subtype (Takanashi and Endoh, 1991; Molenaar et al., 1993). The use of ET receptor-selective antagonists as pharmacological tools to study functional responses has exposed increased complexity of interactions. For example, based on sensitivity to BQ-123, ET-1 has been reported to produce a positive chronotropic effect through the ETB receptor subtype and a negative chronotropic effect through Received for publication February 25, 2000. 1 This study was supported by Wellcome Trust project Grant M/95/3141. ABBREVIATIONS: ET, endothelin; ETA, endothelin receptor subtype A; ETB, endothelin receptor subtype B; BQ-123, cyclo[D-Trp-D-Asp-Pro-DVal-Leu-]; Sfx, sarafotoxin 6c; PLC, phospholipase C; ABT-627, 2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)amino carbonyl methyl)-pyrrolidine-3-carboxylic acid; A-192621, 2-(4-propoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(2,5-ethylphenyl)amino carbonyl methyl)-pyrrolidine-3-carboxylic acid; BIM, bisindolylmalemide I; U-73122, 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; PKC, protein kinase C; SR, sarcoplasmic reticulum. 0022-3565/00/2943-1047$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 2645/846869 JPET 294:1047–1052, 2000 Printed in U.S.A. 1047 at A PE T Jornals on July 7, 2017 jpet.asjournals.org D ow nladed from the ETA receptor subtype (Ono et al., 1994), and exemplifies the modulatory role of the peptide in cardiac regulation. There is a distinct lack of clarity regarding the influence of ET receptor subtype(s) that mediate the contractile response in the myocardium. The positive inotropic effects of ET isopeptides in mammalian myocardium tend to vary extensively and depend on the preparation used and the species investigated. Although rabbit myocardium has a high density of binding sites for ET, density does not appear to reflect wholly the magnitude of functional response. For example, ET-3 has no cardiotonic effect in ventricle of rat and a potent effect in ventricle of rabbit, although the density of binding sites for I-ET-3 is much higher in ventricle of rat (Ishikawa et al., 1991) than rabbit (Kasai et al., 1994). The influence of specific ET receptor subtypes on contractile parameters has not previously been examined with isolated ventricular cardiomyocytes. Isolated ventricular cells are devoid of endogenous neuronal, humoral, or endothelialderived effects that are likely to influence myocardial contractile function. Using such a single population of cells also may avert any disparity caused by myocardial tissue preparations, which have additional cell types that also potentially contain high densities of ET receptors (Ishikawa et al., 1991). In this study, ventricular cardiomyocytes were isolated from myocardium of rabbit, a species that, in contrast to the rat, responds markedly to interventions that increase intracellular Ca (Li et al., 1991). Cumulative concentration-response relationships were assessed in single cells with the agonists ET-1 (ETA/B receptor nonselective), ET-3, and sarafotoxin 6c (Sfx) (ETB receptor selective). Receptor-selective antagonists were chosen that had both high potency and selectivity. Both ABT-627 and A-192621 (Abbott Laboratories, Chicago, IL) are reported to have 1400-fold selectivity for the ETA and ETB receptor subtypes, respectively (Douglas et al., 1994). The influence of the phospholipase C (PLC) pathway also was investigated with selective signaling transduction inhibitors. Experimental Procedures Isolation of Ventricular Cardiomyocytes. Ventricular cardiomyocytes were obtained after enzymatic dissociation. New Zealand White male rabbits (2.5–3 kg) were anaesthetized with sodium pentobarbitone (50 mg/kg i.v.) after heparinization (400 I.U./kg i.v.). The chest was opened and the heart quickly removed and cannulated through the ascending aorta, on a modified Langendorff perfusion apparatus (Kelso et al., 1995). Blood was flushed from the coronary vasculature with a Ca-free modified Krebs-Ringer buffer containing 110 mM NaCl, 2.6 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.2 mM KH2PO4 and 11 mM glucose (pH 7.4; 37°C), which had just previously been aerated with 95% O2, 5% CO2. The perfusate was subsequently supplemented with 0.12% (w/v) collagenase and recirculated for approximately 15 min while maintained at 37°C and continuously aerated with 95% O2, 5% CO2. After enzymatic digestion, the hearts were cut at the atrioventricular junction, sliced vertically toward the apex, and chopped into cubes of 0.7 mm with a mechanical tissue chopper (McIlwain, Mickle Laboratory Engineering Co. Ltd., Surrey, UK). The minced tissue was placed in the collagenase-containing perfusate that had been supplemented with 0.2% (w/v) BSA, and the mixture triturated with a 10-ml serological pipette for approximately 5 min. The dispersed cells were filtered through a nylon mesh gauze of pore size 200 mm and washed twice. Ca was restored by means of centrifugation at 25g twice, and the cells were resuspended in modified Krebs-Ringer solutions containing 250 and 500 mM CaCl2, respectively. Finally, the cells were layered onto a solution of 4% (w/v) BSA containing 1 mM CaCl2, and left to settle by gravity, at 37°C. After approximately 5 min, the supernatant was aspirated and the resulting cell material resuspended at a density of 1 to 2 mg of protein/ml in a storage medium (M199 with Earle’s salts, containing 5 mM creatine, 5 mM taurine, 2 mM carnitine, 100 I.U./ml streptomycin, 100 mg/ml penicillin, pH 7.4) at 37°C. Suspensions of cardiomyocytes were .70% viable as estimated by their elongated rod-shaped morphology. Contractile Measurement. An aliquot of the cell suspension was placed in a transparent recording chamber (;150 ml) mounted on a heated microscope stage (37°C; Zeiss, Jena, Germany), and allowed to settle for 10 min before being bathed with an aerated (100% O2) Krebs-Henseleit solution of the following composition: 125 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2 mM CaCl2, 10 mM HEPES, and 11 mM glucose (pH 7.4). Cells were field stimulated (Grass stimulator) at 0.5 Hz with biphasic pulses of 0.5-ms duration at 60 V via Ag/AgCl2 wires embedded in the wall of the recording chamber. The cells were visualized at 12803 magnification and cell shortening was monitored with a video-edge detection system (VED 40; Crescent Electronics, Sandy, UT). The resulting signal was digitized (Digidata 1200; Axon Instruments, Foster City, CA) and recorded on computer for subsequent analysis with WCP software (Dr. J. Dempster, Department of Pharmacology, University of Strathclyde, Strathclyde, Scotland). This system has a time resolution of 16.7 ms and a spatial resolutio