title: Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation.
abstract: OBJECTIVE
  Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion.
  
  
  RESULTS
  Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development.
  
  
  CONCLUSIONS
  sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.
authors.affiliation: Institute of Physiology, Justus Liebig University, Aulweg 129, D-35392 Giessen, Germany.
authors.forename: Sascha A
authors.initials: SA
authors.lastname: Kasseckert
authors.forename: Claudia
authors.initials: C
authors.lastname: Schäfer
authors.forename: Angelika
authors.initials: A
authors.lastname: Kluger
authors.forename: Dragan
authors.initials: D
authors.lastname: Gligorievski
authors.forename: Julia
authors.initials: J
authors.lastname: Tillmann
authors.forename: Klaus-Dieter
authors.initials: KD
authors.lastname: Schlüter
authors.forename: Thomas
authors.initials: T
authors.lastname: Noll
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sauer
authors.forename: Hans Michael
authors.initials: HM
authors.lastname: Piper
authors.forename: Yaser
authors.initials: Y
authors.lastname: Abdallah
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004489:Edema, Cardiac / Q000378:metabolism / Q000517:prevention & control; D004721:Endoplasmic Reticulum / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D020536:Enzyme Activators / Q000494:pharmacology*; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D006706:Homeostasis; D006834:Hydrazines / Q000494:pharmacology; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects; D000071756:Soluble Guanylyl Cyclase; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000494:pharmacology; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D002227:Carbazoles; D006152:Cyclic GMP; D017869:Cyclic GMP-Dependent Protein Kinases; D020536:Enzyme Activators; D006162:Guanylate Cyclase; D006834:Hydrazines; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000071756:Soluble Guanylyl Cyclase; D013449:Sulfonamides; D062367:ortho-Aminobenzoates; D000099211:Phospholamban; C073601:KT 5823; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; C515616:5-chloro-2-(5-chlorothiophene-2-sulfonylamino)-N-(4-(morpholine-4-sulfonyl)phenyl)benzamide