GASTROENTEROLOGY 1996;111:56 – 64 Colitis and Interleukin 1b Up-regulate Inducible Nitric Oxide Synthase and Superoxide Dismutase in Rat Myenteric Neurons JOHN F. VALENTINE,* CYNTHIA L. TANNAHILL, ‡ SHARON A. STEVENOT, § JUDY E. SALLUSTIO, § HARRY S. NICK, ‡ and ERVIN Y. EAKER § *Division of Gastroenterology, Department of Medicine and ‡ Department of Biochemistry and Molecular Biology, University of Florida, Gainesville; and § Gainesville Veterans Administration Medical Center, Gainesville, Florida Background & Aims: Manganese superoxide dismutase The production of NO in the presence of excessive (MnSOD) is rapidly induced in myenteric plexus neu- reactive oxygen species or in a reduced antioxidant envi- rons (MPNs) in acute colitis and may protect cells from ronment allows the toxic effects of NO synthesis to pre- nitric oxide toxicity. Inducible nitric oxide synthase dominate. 1,6 The principal mechanism of toxicity in this (iNOS) regulation was examined in acute colitis, and scenario is through the reaction of NO with superoxide MnSOD and iNOS were examined in primary cultures to generate peroxynitrite. 1,6,7 The colon may be extremely of MPNs. Methods: Acute colitis in rats was induced susceptible to NO and oxygen free radical – mediated in- with 5% acetic acid. iNOS messenger RNA (mRNA) was jury because the colon expresses relatively low levels of analyzed by Northern analysis, and reduced nicotin- antioxidant enzymes. 8 Additionally, the inflamed colon amide adenine dinucleotide phosphate diaphorase was is exposed to high levels of oxygen free radicals 9 as a used to identify potential NO synthase activity. MnSOD result of direct release from activated neutrophils 10 and and iNOS mRNA levels were evaluated in cultured intracellular production after exposure to the cytotoxic MPNs after treatment with tumor necrosis factor a, cytokines interleukin (IL) 1 11 and tumor necrosis factor interleukin (IL) 1b, or IL-6. iNOS and MnSOD protein expression in control and IL-1b – treated neurons was (TNF ). 11,12 evaluated by immunofluorescence microscopy. Re- The superoxide dismutases (SODs) catalyze the reac- sults: iNOS mRNA was detected in the mucosal and tion 2O 2 0 / 2H / r H 2 O 2 / O 2 , thus eliminating the muscularis layers after the initiation of colitis. Reduced superoxide radical. Hydrogen peroxide is further reduced nicotinamide adenine dinucleotide phosphate diapho- to H 2 O / O 2 by catalase and glutathione peroxidase. In rase localized NO synthase activity to MPNs in controls eukaryotes, three SODs have been described. Manganese and in epithelial cells and MPNs in the inflamed colon. superoxide dismutase (MnSOD) is a nuclear-encoded mi- In MPN cultures, tumor necrosis factor a and IL-1b tochondrial matrix protein. 13 Copper-zinc superoxide treatment resulted in induction of MnSOD, but only IL- dismutase (Cu/ZnSOD) is a cytoplasmic enzyme, 14 and 1b induced iNOS. Immunolocalization confirmed that extracellular SOD is a copper containing secreted pro- the neurons were the primary source of iNOS and tein. 15 Induction of MnSOD is protective against radical- MnSOD. Conclusions: Induction of MnSOD and iNOS mediated damage as well as against TNF-a and IL-1 are coordinated and may limit NO cytotoxicity. The func- cytotoxicity, 11,12 implicating a free radical – mediated cy- tion of iNOS in gut neurons remains to be delineated. totoxicity mechanism. Regulation of MnSOD may func- tion as a protective defense against cytokine toxicity and T mitochondrial oxygen radical production. In intestinal he expanding role of nitric oxide as a neurotransmit- ter, physiological regulator, and immunomodula- epithelial cell lines, MnSOD expression is regulated by tor 1–3 includes effects in the gastrointestinal tract and the cytokines TNF-a, IL-1a, and IL-1b, as well as by liver. 4,5 Three forms of nitric oxide synthase (NOS) have bacterial endotoxin. 16 Extracellular SOD is induced by been recognized. Neuron constitutive NOS (brain NOS or type 1 NOS), inducible NOS (iNOS or type 2 NOS), Abbreviations used in this paper: Cu/ZnSOD, copper-zinc superox- and endothelial constitutive NOS (type 3 NOS). Types ide dismutase; IL, interleukin; iNOS, inducible nitric oxide synthase; 1 and 3 NOS are constitutively expressed, and their activ- MnSOD, manganese superoxide dismutase; MPN, myenteric plexus neuron; NADPH, reduced nicotinamide adenine dinucleotide phos- ity is dependent on mobilization of intracellular Ca 2/ . phate; NOS, nitric oxide synthase; SOD, superoxide dismutase; TNF, Type 2 NOS is an inducible high-output pathway with tumor necrosis factor. sustained Ca 2/ -independent activity that can last for  1996 by the American Gastroenterological Association 0016-5085/96/$3.00 days. 2 / 5e0f$$0019 06-03-96 10:37:14 gasa WBS-Gastro