Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation.
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.
Kasseckert
Sascha A
SA
Institute of Physiology, Justus Liebig University, Aulweg 129, D-35392 Giessen, Germany.
Schäfer
Claudia
C
Kluger
Angelika
A
Gligorievski
Dragan
D
Tillmann
Julia
J
Schlüter
Klaus-Dieter
KD
Noll
Thomas
T
Sauer
Heinrich
H
Piper
Hans Michael
HM
Abdallah
Yaser
Y
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
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