Role of superoxide dismutase in in vivo and in vitro nitrate tolerance.
We assessed whether pharmacological inhibition of CuZn-superoxide dismutase (SOD) mimics the molecular mechanism of either in vitro or in vivo nitrovasodilator tolerance. In endothelium-intact aortic rings from in vivo tolerant rabbits the GTN- and acetylcholine (ACh)-induced maximal relaxation was attenuated by 36 and 23%, respectively. In vitro treatment of control rings with GTN (1 h 10 microM) similarly attenuated the vasorelaxant response to GTN, but not to ACh. Formation of superoxide radicals (*O2-) in endothelium-intact rings (lucigenin-chemiluminescence) increased 2.5 fold in in vivo tolerance, but significantly decreased in in vitro tolerance. The membrane associated NADH oxidase activity was increased 2.5 fold in homogenates of in vivo tolerant aortae, but was not changed in in vitro tolerant aorta. Conversely, SOD activity and protein expression was halved in in vivo tolerance, but SOD activity was not altered by in vitro tolerance. The *O2- scavenger tiron (10 mM) effectively restored the vasorelaxant response to GTN in in vivo tolerant aortic rings, but not the reduced response to GTN in in vitro tolerant rings. Pretreatment (1 h) of vessels with diethyldithiocarbamate (DETC; 10 mM) attenuated vasorelaxant responses to GTN and ACh, increased vascular *O2- production, and inhibited SOD activity in vessel homogenates to a similar degree as observed in in vivo tolerance. DETC-treatment of in vivo-tolerant vessels induced an additional increase in *O2- production. Increased *O2- production in in vivo nitrate tolerant aorta is associated with activation of vascular NADH oxidase and inactivation of CuZnSOD. Therefore, in vivo tolerance can be mimicked by in vitro inhibition of CuZnSOD, but not by in vitro exposure to GTN, which does not affect vascular *O2- production, NADH oxidase and CuZnSOD.
Münzel
T
T
Division of Cardiology, The University Hospital Eppendorf, Hamburg, Germany. muenzel@uke.uni-hamburg.de
Hink
U
U
Yigit
H
H
Macharzina
R
R
Harrison
D G
DG
Mülsch
A
A
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001808:Blood Vessels / Q000201:enzymology; D004050:Ditiocarb / Q000494:pharmacology; D004361:Drug Tolerance; D005260:Female; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D005996:Nitroglycerin / Q000494:pharmacology*; D011817:Rabbits; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects
D009097:Multienzyme Complexes; D013481:Superoxides; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D004050:Ditiocarb; D013482:Superoxide Dismutase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D005996:Nitroglycerin; D000109:Acetylcholine