Direct measurement of the accumulation and mitochondrial conversion of nitric oxide within Chinese hamster ovary cells using an intracellular electron paramagnetic resonance technique.
We have developed an electron paramagnetic resonance (EPR) method for the nondestructive detection and quantification of intracellular NO in real time. Based upon this technique, we have obtained evidence for the metabolism of this bioregulatory molecule by mitochondria. Line-broadening of the EPR signal of a coal derivative, fusinite, was calibrated as a function of NO concentration in aqueous solution. The methodology was validated using two compounds which release NO in a controlled and predictable manner with first-order rate constants k1 = 5.0 x 0.10(-3) s-1 and k'1 = 3.4 x 10(-4) s-1 (35 degrees C). Fusinite was internalized in Chinese hamster ovary cells (CHO) by phagocytosis, after which the cells were allowed to consume the available O2, producing an hypoxic environment. The NO released from one of the NO donors, added to the culture fluid at an initial concentration of 50 microM, was directly measured in the intracellular environment as line-broadening of the fusinite EPR signal. The linewidth diminished with time, indicating that NO was being converted to a non-paramagnetic species by the cells with an apparent zero-order rate constant of 5 x 10(8) NO molecules cell-1 min-1 (20 degrees C). Addition of cyanide to the culture medium (5 mM final concentration) inhibited this disappearance of NO. NO also was converted in the presence of isolated mitochondria in the absence of oxygen. These observations suggest that under hypoxic conditions, there exists in CHO cells a metabolic pathway for the conversion of NO to diamagnetic species, which involves interactions with mitochondria.
Clarkson
R B
RB
Illinois EPR Research Center, University of Illinois, Urbana 61801, USA.
Norby
S W
SW
Smirnov
A
A
Boyer
S
S
Vahidi
N
N
Nims
R W
RW
Wink
D A
DA
D000818:Animals; D016466:CHO Cells / Q000378:metabolism* / Q000648:ultrastructure; D002244:Carbon / Q000032:analysis; D003031:Coal / Q000032:analysis; D006224:Cricetinae; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D013696:Temperature
D003031:Coal; C091644:fusinite; D009569:Nitric Oxide; D002244:Carbon