Comparison of inhibitors of superoxide generation in vascular smooth muscle cells

Background and purpose: We compared the dose-dependent reductions in cellular superoxide anion (O-2(-)) by catalytic agents: superoxide dismutase (SOD), polyethylene glycol (PEG)-SOD and the nitroxide 4-hydroxy-2,2,6,6,-tetramethylpiperidine- 1-oxyl (tempol) with uncharacterized antioxidants: 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinate iron (III)(Fe-TTPS), (-)-cis-3,3', 4', 5,7-pentahydroxyflavane (2R, 3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro1-( 2H)-benzopyran-3,5,7-triol (-epicatechin), 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) and N-acetyl-L-cysteine (NAC) with the spin trap nitroblue tetrazolium (NBT) and with the vitamins or their analogues: ascorbate, alpha-tocopherol and 6-hydroxy-2,5,7,8-tetramethylkroman-2-carboxy acid (trolox). Experimental approach: O-2(-) was generated in primary cultures of angiotensin II-stimulated preglomerular vascular smooth muscle cells from spontaneously hypertensive rats and detected by lucigenin-enhanced chemiluminescence. Key results: SOD, PEG-SOD, NAC and tempol produced a similar maximum inhibition of O-2(-) of 80-90%. -Epicatechin, NBT, ebselen and Fe-TTPS were significantly (P < 0.0125) less effective (50-70%), whereas trolox, alpha-tocopherol and ascorbate had little action even over 24 h of incubation (< 31%). Effectiveness in disrupted and intact cells was similar for the permeable agents, PEG-SOD and tempol, but was enhanced for SOD. Generation of O-2(-) was increased by NAC and NBT at low concentrations but reduced at high concentrations. Conclusions and implications: Maximum effectiveness against cellular production of O-2(-) requires cell membrane permeability and catalytic action as exemplified by PEG-SOD or tempol. NAC and NBT have biphasic effects on O-2(-) production. Vitamins C and E or analogues have low efficacy. British Journal of Pharmacology (2009) 157, 935-943; doi:10.1111/j.1476-5381.2009.00259.x; published online 18 May 2009