Characterization of basal nitric oxide production in living cells.
Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.
López-Figueroa
M O
MO
Mental Health Research Institute, University of Michigan, Ann Arbor 48109, USA. molf@umich.edu
Caamaño
C
C
Marin
R
R
Guerra
B
B
Alonso
R
R
Morano
M I
MI
Akil
H
H
Watson
S J
SJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002461:Cell Line, Transformed; D019793:Fluorescein; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008027:Light; D009252:NADPH Dehydrogenase; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
C414689:4,5-diaminofluorescein diacetate; D007202:Indicators and Reagents; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase; D019793:Fluorescein