Author
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Saari, Jack |
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BODE, A - UNIVERSITY OF OREGON |
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Submitted to: Experimental Biology
Publication Type: Abstract Only Publication Acceptance Date: 4/17/1999 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: A prior study has shown that nitric oxide (NO) production is elevated in hearts of copper-deficient (CuD) rats. We further examined the effect of copper deficiency on the nitric oxide pathway in rat hearts by measuring protein levels of the inducible form of NO synthase (iNOS) by Western blot analysis. In a 2-way design, male weanling rats were provided a Cu-adequate (CuA, 6.4 mg Cu/kg) or a CuD diet (0.4 mg Cu/kg) and given daily i.p. injections of aminoguanidine (50 mg/kg/day), an inhibitor of NOS activity, or saline vehicle for 5 weeks. Their hearts were excised and homogenized on ice in buffer containing 25 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 0.32 M sucrose, 2 mM EGTA, 50 ug/ml leupeptin, 50 ug/ml aprotinin and 0.1 mM spermine. Insoluble material was removed by boiling (5 min) and centrifugation (5 min). Supernates were fractionated by electrophoresis on 12% polyacrylamide gels. Western immunoblotting was performed using mouse monoclonal antibody (1:1000) that recognizes iNOS. A goat anti-mouse IgG antibody conjugated to horseradish peroxidase (1:2000) was used to form protein-antibody complexes that were detectable by chemiluminescence. We found that relative amounts of iNOS protein were increased in hearts of CuD rats by 3 fold relative to those observed in hearts of CuA rats. Further, while aminoguanidine caused marginal elevation of iNOS protein in hearts of CuA rats, it elevated iNOS protein by 8 fold in hearts of CuD rats. From these data we speculate that a negative feedback mechanism, whereby NO modulates iNOS expression, is amplified by elevated amounts of reactive oxygen species in CuD hearts. |
