Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants.
An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.
Kukreja
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RC
Division of Cardiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Weaver
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AB
Hess
M L
ML
D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000097:blood; D005609:Free Radicals; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood*; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009195:Peroxidase / Q000097:blood; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure*; D013481:Superoxides / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002712:Chlorides; D005609:Free Radicals; D013481:Superoxides; D006861:Hydrogen Peroxide; D009195:Peroxidase; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium