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Excitatory Amino Acids (NMDA) Alter Nitric Oxide Synthetase (NOS) Activity in the Isolated Salt Perfused Rat Lung

Necrotizing enterocolitis (NEC) affects 10% of premature neonates. Although several potentially contributing mediators have been identified, the exact mechanism of disease pathogenesis is unknown. Increased epithelial apoptosis have been proposed to contribute to the epithelial demage in NEC. The goal of our studies was to identify mediators of epithelial apoptosis and the mechanism(s) of their action. IEC-6 rat small intestinal epithelial cells were subjected to treatment with inflammatory mediators known to induce NEC (PAF; 0.1-10μM, LPS; 10μg/ml) or a chemical NO donor (DETA; 31.125μM-1mM), and/or inhibitors of nitric oxide synthase(NOS) (LNAME; 1mM, AMT; 20nM) for 24hrs. Apoptosis was assessed using fluorescence TUNEL staining and fluorescence digital imaging microscopy or by quantifying histone-bound DNA fragments with ELISA. All effects on the rate of apoptosis were normalized to control values and were expressed as (percent control±S.E.M.; n>3). Treatment with either PAF, LPS or DETA significantly enhanced apoptosis in IEC-6 cells in a time- and concentration-dependent manner. Maximal effects were observed with 10μM PAF (292±26%), 10μg/ml LPS(326±39%) or with 250μM DETA (498±64%). The induction of apoptosis by DETA was due to NO release, since freshly isolated red blood cells, i.e. NO scavengers, completely obliterated the apoptosis-inducing effect of DETA, up to 1mM DETA concentration. Complete inhibition of all NOS activity with 1mM LNAME potently induced apoptosis (269±53%). In the presence of 1mM LNAME, low concentrations of DETA, i.e., 30-70μM DETA, were protective against LNAME-induced apoptosis, while high concentrations of DETA, i.e., 100-300μM, stimulated apoptosis. These data indicate that inflammatory mediators that induce NEC in vivo, elicit apotposis in cultured intestinal epithelial cells and that NO plays a complex role in the regulation of apoptosis in these cells. High concentrations of NO are pro-apoptotic, while low concentrations of NO are necessary to protect IEC-6 cells from apoptosis. Evaluating the exact relationship between PAF, LPS and NO in the regulation of epithelial apoptosis will contribute to our understanding of the cellular and molecular mechanisms that underlie the development of NEC.

Santiago Lamas (Associate Editor)We incorporate a new section in Redox Biology, the Editors'forum. The main objective is to attract short contributions relatedto previously published articles that may help to clarify concepts,open controversies or constructively criticize conclusions from aspecific article. The contribution will show a link to the relatedarticle and may be accompanied by the appropriate response ofthe authors to the commentary on their article.The article by Drs. Cortese-Krott and Kelm [1] promoted anextensive analysis by Dr. Tsikas about the optimal methodology tocharacterize eNOS activity in red blood cells and its relevancefor the concepts put forward by Cortese-Krott and Kelm. Theauthors were given the opportunity to respond to this commen-tary and their answer is shown below the commentary. Indeedthis is an important question as the jury appears to be still outconcerning the existence of a NOS isoform in human red bloodcells. Dr. Tsikas makes a strong case on the validity of the gaschromatography-mass spectrometry (GC-MS) NOS activity assayfor a large set of samples, including erythrocytes, and delves intothe complex chemistry involved. Drs. Cortese-Krott and Kelmlucidly respond to Dr. Tsikas with equally powerful arguments.We are proud to host this illuminating discussion on a complexchemical topic and we invite all readers to follow this example andsubmit commentaries that may contribute to enhance progress inthe understanding of challenging notions in the field of redoxbiology.http://dx.doi.org/10.1016/j.redox.2015.01.018