Impaired nitric oxide production of the myenteric plexus in colitis detected by a new bioimaging system.
Direct measurement of the release of nitric oxide (NO) from the myenteric plexus has been extremely difficult to date, due to the lack of suitable methodologies. We have developed a new bioimaging system to visualize the nitrergic neurons of the myenteric plexus and investigated whether NO production is impaired in dextran sulfate sodium (DSS)-induced colitis. Longitudinal muscle layers with the myenteric plexus intact were obtained from the rat colon and were incubated with 4,5-diaminofluorescein-2-diacetate (DAF-2DA) (7 microm) for 30 min. Illumination at 450-490 nm revealed the fluorescence in the myenteric plexus. Confocal laser microscopy and three-dimensional reconstruction techniques were used to quantify the changes in the amount of NO production by the myenteric plexus. Fluorescent double-labeled immunostaining for nNOS was performed to confirm the colocalization of nNOS in 4,5-diaminofluorescein (DAF-2)-positive cells. DAF-2 fluorescence was abolished by pretreatment with N(G)-nitro-l-arginine methyl ester (l-NAME; a nonselective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a selective neuronal NOS inhibitor), and omega-conotoxin GVIA (an N-type Ca(2+) channel blocker), but not by nifedipine (an l-type Ca(2+) channel blocker). Fluorescent double-labeled immunostaining showed that DAF-2-positive cells colocalized with nNOS-positive cells. Oral administration of 5% DSS for 7 days induced distal colitis and the number of DAF-2-positive neurons were significantly reduced to 55 +/- 17% of control. DAF-2 offers a sensitive indicator for visualizing production of NO with high spatial resolution. This new system may contribute to the study of the pathophysiological role of the nitrergic pathway in the gastrointestinal tract.
Kono
Toru
T
Department of Surgery II, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan. kono@asahikawa-med.ac.jp
Chisato
Naoyuki
N
Ebisawa
Yoshiaki
Y
Asama
Toshiyuki
T
Sugawara
Mutsubu
M
Ayabe
Tokiyoshi
T
Kohgo
Yutaka
Y
Kasai
Shinichi
S
Yoneda
Masashi
M
Takahashi
Toku
T
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003092:Colitis / Q000139:chemically induced / Q000378:metabolism*; D016264:Dextran Sulfate; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D007093:Imidazoles / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009197:Myenteric Plexus / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D017207:Rats, Sprague-Dawley; D020866:omega-Conotoxin GVIA / Q000494:pharmacology
C111205:4,5-diaminofluorescein; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007202:Indicators and Reagents; D009569:Nitric Oxide; C029899:imidazole; D016264:Dextran Sulfate; D020866:omega-Conotoxin GVIA; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester