Calcium‐handling of Endothelin A and B Receptors in Coronary Artery upon Post Ischemic Reperfusion Injury in Rats

Abstract

BackgroundIschemia‐reperfusion has been demonstrated to increase coronary artery resistance and increase the sensitivity toward the potent vasoconstrictor peptide endothelin‐1 (ET‐1). The increased sensitivity to ET‐1 is associated with an up‐regulation of vasoconstrictive ETB receptors in the vascular smooth muscle cells. This project aims to investigate any causal link between the ETB receptor‐upregulation and a shift in calcium sensitivity/handling in the vascular smooth muscle cells after experimental myocardial ischemia‐reperfusion injury.MethodsMale SD rats (~320 gram) were either subjected to experimental left anterior descending (LAD) coronary artery occlusion for 30 min followed by 24 hours of reperfusion, or sham operation. Contractile properties of artery segments distal to the ligature were measured with wire‐myography or along with simultaneous measures of the intracellular Ca2+ concentration ([Ca2+]i) using Fura‐2. The Ca2+ sensitivity of the arteries was determined during stimulation of the ETB‐R agonist Sarafotoxin 6c (S6c) followed by cumulative addition of extracellular Ca2+ (up to 2.5 mM). The experiments were subsequent repeated with ET‐1 as stimulating agent to access dual ETB‐R and ETA‐R mediated Ca2+ sensitization. Thapsigargin, 2‐APB and Felodipine were in addition used in order to identify Ca2+ handling mechanisms and their relative contribution for the ETB‐R and ETA‐R mediated contraction following ischemia‐reperfusion injury.ResultsPost ischemic reperfusion injury significantly increased vascular tone of the LAD coronary artery in response to [Ca2+]o. Prior incubation with either 125 mM of K+ or the ETB‐R antagonist BQ788 showed, that this difference in contractility was neither due to changes in membrane depolarization or a constitutive activity of the ETB‐R after ischemia and reperfusion injury. The evoked agonist‐responses of the Endothelin receptor subtypes, confirmed the enhanced ETB‐R mediated tone after ischemia‐reperfusion injury, whereas dual ETA‐R and ETB‐R mediated tone seems unaffected between surgery groups. The enhanced ETB‐R contractility develops an equal [Ca2+]i –tone relationship mainly from extracellular Ca2+ influx, through activation of voltage‐gated Ca2+ channels.ConclusionThe ETA‐R mediated contraction was unaffected between surgery groups and show equal dependence on intracellular store‐release and extracellular Ca2+ influx. The ET‐1 evoked [Ca2+]i –tone balance was leftward‐shifted opposed to the ETB‐R and suggest the ETA‐Rs to additionally stimulate downstream Ca2+ sensitivity mechanisms.Support or Funding InformationFaculty of Health and Medical Sciences, University of Copenhagen, Denmark. Lundbeck Foundation, Grant of Excellence, Denmark. R59‐AR‐5404.