Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.
BACKGROUND
Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC.


METHODS
Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy.


RESULTS
APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection.


CONCLUSIONS
The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.
Amour
Julien
J
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Brzezinska
Anna K
AK
Weihrauch
Dorothee
D
Billstrom
Amie R
AR
Zielonka
Jacek
J
Krolikowski
John G
JG
Bienengraeber
Martin W
MW
Warltier
David C
DC
Pratt
Philip F
PF
Kersten
Judy R
JR
D000777:Anesthetics / Q000494:pharmacology*; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D001794:Blood Pressure / Q000187:drug effects; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D042783:Endothelial Cells / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D006339:Heart Rate / Q000187:drug effects; D047468:Immunoprecipitation; D019157:Ischemic Preconditioning, Myocardial; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D049449:Luminescence; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D009203:Myocardial Infarction / Q000473:pathology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000502:physiology*; D010126:Ozone / Q000737:chemistry; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects
D000777:Anesthetics; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018942:Macrolides; D009569:Nitric Oxide; D010126:Ozone; D052250:Nitric Oxide Synthase Type III; C035359:monorden; D019331:NG-Nitroarginine Methyl Ester; C001277:geldanamycin