Altered production of nitric oxide and reactive oxygen species in rat nodose ganglion neurons during acute hypoxia.
Nitric oxide (NO) production in the sensory neurons of the rad nodose ganglion was studied by examining the distribuiotn of NO synthase (NOS) by use of NADPH diaphorase (NADPHD) histochemistry and immunohistochemistry ofr the presence of isoformes of NOS: neuronal (nNOS), endothelial (eNOS) and the inducible isoform (iNOS). Distribution and changes in NO production during acute hypoxia were studied in vital vibratome sections with the fluorescent marker for NO, diaminotriazolofluorescein (DAF-2T). Furthermore, changes in reactive oxygen species (ROS) in vibratome slices were examined utilizing 2',7'-dichlorofluorescein (DCF). By use of these histochemical methods, a positive NADPH reaction and positive immunoreactivity for eNOS were noted in all neurons observed. While for nNOS immunoreactivity, both strongly positive cells but also many negative cells are seen., no iNOS immunoreactive cells were observed. In vital vibratome slices, a dot-like distribution of fluorescence for DAF-2T, indicating production of NO, was observed in the nodose ganglion cells. Neurons exposed to hypoxia showed stronger DAF-2T fluorescence than cells exposed to normoxia, indicating an increased production of NO during hypoxia. When Ca(2+) was removed from the incubation buffer, the intensity of fluorescence for DAF-2T decreased but did not disappear completely. Using a photoconversion technique, DAF-2T was localized in the inner membrane of mitochondria in the ganglion cells by electron microscopy. The level of DCF signals for detection of ROS was higher in neurons incubated in the normoxic medium than those incubated under conditions of hypoxia. Nerve cells exposed to hypoxia followed by reoxygenation (3 min in normoxic conditions) showed higher fluorescence for DCF than those exposed to normoxia. The results of the present study demonstrate clearly that the basal production of NO in viscerosensory neurons is increased during hypoxia and is due to the isoform eNOS rather than nNOS, moreover, that ROS is augmented by reoxygenation but not during hypoxia.
Yamamoto
Yoshio
Y
Institute of Anatomy and Cell Biology, Justus-Liebig-University, Aulweg 123, 35385 Giessen, Germany. yyoshio@iwate-u.ac.jp
Henrich
Michael
M
Snipes
Robert L
RL
Kummer
Wolfgang
W
D000208:Acute Disease; D000818:Animals; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000860:Hypoxia / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009620:Nodose Ganglion / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014230:Triazoles
D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D014230:Triazoles; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase