Regulation of the expression of soluble guanylyl cyclase by reactive oxygen species.
OBJECTIVE
Superoxide anions produced during vascular disease scavenge nitric oxide (NO), thereby reducing its biological activity. The aim of the present study was to investigate whether reactive oxygen species (ROS) have a direct effect on soluble guanylyl cyclase (sGC) subunit levels and function and to ascertain the mechanism(s) involved.


METHODS
Rat aortic smooth muscle cells (RASM) or freshly isolated vessels were exposed to reactive oxygen species (ROS)-generating agents and sGC subunit expression was determined at the mRNA and/or protein level. cGMP accumulation was also determined in RASM exposed to ROS.


RESULTS
Incubation of smooth muscle cells with H(2)O(2), xanthine/xanthine oxidase (X/XO) or menadione sodium bisulphite (MSB) significantly decreased protein levels of alpha1 and beta1 subunits of sGC and reduced SNP-induced cGMP formation. Similarly, sGC expression was reduced in freshly isolated vessels exposed to ROS-generating agents. The ROS-triggered inhibition of alpha1 and beta1 levels was not blocked by proteasome inhibitors, suggesting that decreased sGC protein was not due to protein degradation through this pathway. Real time RT-PCR analysis demonstrated a 68% reduction in steady state mRNA levels for the alpha1 subunit following exposure to H(2)O(2). In addition, alpha1 promoter-driven luciferase activity in RASM decreased by 60% after H(2)O(2) treatment.


CONCLUSIONS
We conclude that oxidative stress triggers a decrease in sGC expression and activity that results from reduced sGC steady state mRNA levels. Altered sGC expression is expected to contribute to the changes in vascular tone and remodeling observed in diseases associated with ROS overproduction.
Gerassimou
C
C
Department of Critical Care and Pulmonary Services, G.P. Livanos-M. Simou Laboratories, University of Athens, Evangelismos Hospital, Athens, Greece.
Kotanidou
A
A
Zhou
Z
Z
Simoes
D C M
DC
Simoes
D D C
DD
Roussos
C
C
Papapetropoulos
A
A
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D046509:Tissue Culture Techniques; D014158:Transcription, Genetic; D024483:Vitamin K 3 / Q000494:pharmacology; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
D020030:Nitric Oxide Donors; D021122:Protein Subunits; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D018160:Receptors, Cytoplasmic and Nuclear; D009599:Nitroprusside; D019820:Xanthine; D024483:Vitamin K 3; D006861:Hydrogen Peroxide; D014969:Xanthine Oxidase; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP