Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta.
BACKGROUND
Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well.


RESULTS
We therefore studied the effects of 3-day NTG treatment of rats and rabbits on activity and expression of the immediate NO target soluble guanylyl cyclase (sGC) and on the cGMP-activated protein kinase I (cGK-I). Tolerance was induced either by chronic NTG infusion via osmotic minipumps (rats) or by NTG patches (rabbits). Western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and Northern blot analysis revealed significant and comparable increases in the expression of sGC alpha(1) and beta(1) subunit protein and mRNA. Studies with the oxidative fluorescent dye hydroethidine revealed an increase in superoxide in the endothelium and smooth muscle. Stimulation with NADH increased superoxide signals in both layers. Although cGK-I expression in response to low-dose NTG was not changed, a strong reduction in vasodilator-stimulated phosphoprotein (VASP) serine239 phosphorylation (specific substrate of cGK-I) was observed in tolerant tissue from rats and rabbits. Concomitant in vivo and in vitro treatment with vitamin C improved tolerance, reduced oxidative stress, and improved P-VASP.


CONCLUSIONS
We therefore conclude that increased expression of sGC in the setting of tolerance reflects a chronic inhibition rather than an induction of the sGC-cGK-I pathway and may be mediated at least in part by increased vascular superoxide.
Mülsch
A
A
University Hospital Eppendorf, Division of Cardiology, Hamburg, Germany.
Oelze
M
M
Klöss
S
S
Mollnau
H
H
Töpfer
A
A
Smolenski
A
A
Walter
U
U
Stasch
J P
JP
Warnholtz
A
A
Hink
U
U
Meinertz
T
T
Münzel
T
T
D000279:Administration, Cutaneous; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta / Q000187:drug effects* / Q000201:enzymology; D001205:Ascorbic Acid / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004361:Drug Tolerance; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006162:Guanylate Cyclase / Q000378:metabolism*; D015918:Infusion Pumps, Implantable; D007262:Infusions, Intravenous; D008297:Male; D008840:Microfilament Proteins; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D005996:Nitroglycerin / Q000008:administration & dosage / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D015290:Second Messenger Systems; D013481:Superoxides / Q000378:metabolism; D014665:Vasodilator Agents / Q000008:administration & dosage / Q000494:pharmacology*; D000098662:Vasodilator-Stimulated Phosphoprotein
D000975:Antioxidants; D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D014665:Vasodilator Agents; D000098662:Vasodilator-Stimulated Phosphoprotein; D013481:Superoxides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D005996:Nitroglycerin; D006152:Cyclic GMP; D001205:Ascorbic Acid