Nitric oxide scavenging by the cobalamin precursor cobinamide.
Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B12) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NO-induced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations > or =50 microM with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra- and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin.
Broderick
Kate E
KE
Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 92093-0652, USA.
Singh
Veena
V
Zhuang
Shunhui
S
Kambo
Amanpreet
A
Chen
Jeffrey C
JC
Sharma
Vijay S
VS
Pilz
Renate B
RB
Boss
Gerry R
GR
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D004330:Drosophila; D008317:Malpighian Tubules / Q000502:physiology; D008840:Microfilament Proteins / Q000378:metabolism; D015394:Molecular Structure; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D014805:Vitamin B 12 / Q000737:chemistry* / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D003038:Cobamides; D008840:Microfilament Proteins; D009566:Nitrates; D020030:Nitric Oxide Donors; D009573:Nitrites; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; C017142:cobinamide; D009569:Nitric Oxide; D000001:Calcimycin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D014805:Vitamin B 12