Leupeptin preserves cardiac nitric oxide synthase 3 during reperfusion following long-term cardioplegia.
The objective of this study was to investigate how long-term cardioplegia/reperfusion affects cardiac nitric oxide synthase 3 (NOS3). To this aim, rat hearts were mounted in a perfusion apparatus and equilibrated with a modified Krebs-Henseleit solution (KH). The hearts were then arrested by soaking them in cold St. Thomas Hospital II solution (STH) for 5, 7, and 15 h. Reperfusion was performed by low-flow cold STH delivering for 1 h followed by 15-min aerobic normothermic KH perfusion. Cardioplegia preserved the amount of NOS3 irrespective of the duration of the cardiac arrest. NOS3 content was also unaffected by reperfusion following 5 and 7 h of cardioplegia. On the contrary, reperfusion performed after 15 h of cardioplegia caused a marked reduction in the amount of NOS3 protein, in both endothelial and cardiac muscle cells, and NOS activity. The involvement of intracellular proteolysis as a cause of reduction in NOS3 cardiac level was then investigated by delivering 0.1 mmol/L of either calpain I and II inhibitors or 0.05 mmol/L leupeptin during heart reperfusion. Only the treatment with leupeptin preserved NOS3, indicating that lysosomal proteases rather then cytoplasmic calpains were mainly responsible for the cleavage of this enzyme. The observed decrease in GSH/GSSG ratio and activation of JNK in the reperfused heart suggested that proteolysis could be triggered by reactive oxygen species.
Muscari
Claudio
C
Department of Biochemistry G. Moruzzi, University of Bologna, Bologna, Italy. claudio.muscari@unibo.it
Capanni
Cristina
C
Giordano
Emanuele
E
Stefanelli
Claudio
C
Bonavita
Francesca
F
Stanic
Ivana
I
Bonafè
Francesca
F
Caldarera
Claudio Marcello
CM
Guarnieri
Carlo
C
D000818:Animals; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006324:Heart Arrest, Induced / Q000379:methods*; D007976:Leupeptins / Q000494:pharmacology*; D008297:Male; D046529:Microscopy, Electron, Transmission; D015428:Myocardial Reperfusion Injury / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000201:enzymology / Q000473:pathology / Q000648:ultrastructure; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D005978:Glutathione; C032854:leupeptin; D019803:Glutathione Disulfide