Role of sulfhydryl-dependent dimerization of soluble guanylyl cyclase in relaxation of porcine coronary artery to nitric oxide.
OBJECTIVE
Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO).


RESULTS
The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine.


CONCLUSIONS
These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.
Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100191, China.
Xiaoxu
X
Zheng
Lei
L
Ying
Juan
J
Liu
Dou
D
Dou
Qiong
Q
He
Susan Wai Sum
SW
Leung
Ricky Y K
RY
Man
Paul M
PM
Vanhoutte
Yuansheng
Y
Gao
D000818:Animals; D003331:Coronary Vessels / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology*; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000502:physiology; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000502:physiology*; D000860:Hypoxia / Q000201:enzymology / Q000503:physiopathology; D066298:In Vitro Techniques; D008297:Male; D009569:Nitric Oxide / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000502:physiology*; D000071756:Soluble Guanylyl Cyclase; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D034421:Sus scrofa; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*
D009603:Nitroso Compounds; D018160:Receptors, Cytoplasmic and Nuclear; D013439:Sulfhydryl Reagents; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D004229:Dithiothreitol