Abstract
In a previous study (Hardy et al. (1994) J. Biol. Chem. 269, 18535-18540), we observed that the manganese-based superoxide dismutase mimetic Mn(II)-dichloro(1,4,7,10,13-pentaazacyclopentadecane) (MnPAM) inhibited neutrophil-mediated cell injury in vitro. We have extended these studies with the low molecular weight superoxide dismutase mimic to evaluate the role of superoxide in neutrophil-mediated tissue injury in vivo. In a dose-dependent manner, MnPAM inhibited colonic tissue injury and neutrophil accumulation into the colonic tissue induced by the intracolonic instillation of dilute aqueous acetic acid in mice. Tissue injury was assessed by visual and histological analysis. Neutrophil infiltration was determined by tissue myeloperoxidase activity and confirmed by histological analysis. Two novel Mn(II) dichloro complexes of the carbon-substituted macrocycles 2-methyl-1,4,7,10,13-pentaazacyclopentadecane (MnMAM) and 2-(2-methylpropyl)-1,4,7,10,13-pentaazacyclopentadecane (MnBAM) effectively catalyzed the dismutation of superoxide with catalytic rate constants (kcat) of 3. 31 x 10(7) M-1 s-1 and 1.91 x 10(7) M-1 s-1, respectively, as determined by stopped-flow kinetic analysis at pH 8.1 and 21 degrees C. The superoxide dismutase mimetics MnMAM and MnBAM also attenuated dilute aqueous acetic acid-induced tissue injury and neutrophil infiltration into colonic tissue; however, two Mn(II) complexes that had little or no detectable SOD activity (kcat </= 0.1 x 10(7) M-1 s-1), specifically the Mn(II) dichloro complexes of 1,4,7,10,13-pentaazacyclohexadecane and 1,4,7,11,14-pentaazacycloheptadecane, failed to inhibit the colonic tissue injury or infiltration of neutrophils in mice treated intracolonically with dilute aqueous acetic acid. These results are consistent with a proinflammatory role for superoxide in the mediation of neutrophil infiltration in vivo.
Highlights
Considerable experimental data exist to suggest that inflamed tissue and tissue subjected to ischemia-reperfusion are under oxidative stress induced by polymorphonuclear leukocytes [1,2,3,4,5]
We have utilized the technique of stopped-flow kinetic analysis to directly monitor the decay of superoxide as a means to quantitate the ability of a putative superoxide dismutase (SOD) mimetic to catalyze the dismutation of superoxide [21, 25]
The purpose of this study was to utilize synthetic, low molecular weight SOD mimetics and structurally related complexes with little or no detectable SOD activity as molecular probes to evaluate the importance of superoxide in tissue injury and neutrophil infiltration in a model of inflammation in vivo
Summary
Considerable experimental data exist to suggest that inflamed tissue and tissue subjected to ischemia-reperfusion are under oxidative stress induced by polymorphonuclear leukocytes [1,2,3,4,5]. Since the SOD enzymes are not membrane-permeable, the enzyme is limited in regard to its effectiveness as a probe for elucidating the role of intracellular superoxide in neutrophilmediated tissue injury. There has been considerable interest in the design and synthesis of membrane-permeable, low molecular weight mimetics of the enzyme SOD (14 –19), which could be utilized to probe the role of superoxide in neutrophil-mediated tissue injury as well as in other physiologic and pathologic processes. Of critical importance to this study was the accurate measurement of the ability of the SOD mimetics to catalyze the dismutation of superoxide, so that a protective effect could be correlated to the SOD activity of the mimetics. We have discovered that a Mn(II) dichloro complex of 1,4,7,10,13-pentaazacyclopentadecane (MnPAM) effectively catalyzes the dismutation of superoxide [26]. Structure-activity relationship studies with two novel, carbon-substituted Mn(II)based SOD mimetics and two Mn(II)-based complexes with little or no detectable SOD activity indicate the importance of superoxide in the mediation of neutrophil infiltration in vivo
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