Nitric Oxide Production By Head Kidney Cells Of Fundulus heteroclitus Elicited By In Vitro Challenge With Vibrio parahaemolyticus   Joseph E. Burgents1, Karen G. Burnett2, and Louis E. Burnett2   1Department of Biology, The College of New Jersey, Trenton, NJ; 2Grice Marine Laboratory, University of Charleston, Charleston, SC   Peroxynitrite is among the most toxic products of the respiratory burst in vertebrate macrophages.  Presumed to function in anti-bacterial defense, the molecule results from the combination of nitric oxide (NO) with superoxide anion (O2-).  Peroxynitrite is highly labile; therefore, its production is not measured directly but instead is assumed to correlate positively with measurements of NO production.  In some reports, NO production can be induced in teleost macrophages by stimulation with LPS alone (goldfish); application of a source of gamma interferon (IFN) generally increases the kinetics and magnitude of NO production (gilthead sea bream, rainbow trout and turbot).  Despite these reports, the importance of NO production to resistance against live bacteria challenge in teleost fish remains unclear.  The immediate goal of this project was to determine whether phagocytic cells of the teleost fish, Fundulus heteroclitus, could be induced to produce NO in vitro in response to challenge with the gram-negative bacterium, Vibrio parahaemolyticus.  Mitogen-activation factor (MAF), presumed to be a rich source of IFN, was produced by stimulating head kidney cells with 10 μg/ml Con A and 5 ng/ml PMA for 3 hours.  The mitogen-activated cells were washed with HBSS and cultured for 48 hours.  The resulting supernatant was removed, stored at –20oC and used as a source of MAF.  The capacity of the culture system to support NO production was tested by allowing freshly isolated head kidney cells to adhere to 96 well microtiter plates for 3-5 hours.  The medium and non-adherent cells were replaced with medium containing 5% FCS, along with LPS (0, 5, 10, and 20 μg/ml) and MAF at dilutions of 0, 1:2, 1:4 and 1:8.  After 48 hours, supernatants were removed for quantification of NO by the Greiss reaction.  Over 96 hours, LPS stimulation alone did not induce significant production of NO.  Combinations of MAF and LPS supported NO production to a higher level than LPS, although the increase was not significant.  However, co-culture of macrophages with MAF and heat-inactivated V. parahaemolyticus at 1:1 (cells:bacteria) stimulated a significant increase in NO production.  Increasing the bacterial challenge to 1:10 did not enhance NO production.  To our knowledge, this is the first time that NO production has been measured in fish stimulated with gram-negative bacteria in vitro or in vivo, although an attenuated gram-positive bacterium, Renibacterium salmoninarum has been shown to induce in vivo production of serum NO in rainbow trout.  These experiments comprise the first steps in evaluating the importance of NO to anti-bacterial defense in Fundulus heteroclitus. (Supported by NSF-REU Grant #DBI-9876926).