In situ detection of constitutive superoxide anion production in granules of mast cells.

3,3'-Diaminobenzidine, in the presence of manganese and cobalt ions, was applied for the detection of superoxide anions in unfixed cryostat sections of rat oesophagus, trachea, skin and intact mesenterium. In all connective tissues, a blue final reaction product was found in a granular form in mast cells. The amount of final reaction product formed after incubation with diaminobenzidine and cobalt ions was increased by the addition of manganese ions. Electron microscopical analysis revealed that the electron-dense final reaction product was exclusively present in the granules of mast cells and on elastin fibres. It was found that the constitutive spontaneous formation of final reaction product in mast cells was enzymatic and dependent on the presence of oxygen in the medium. Of all the enzyme inhibitors and free radical scavengers tested, only azide strongly reduced the amount of final reaction product. It was concluded that the reaction was partly caused by peroxidase activity, but that superoxide anions are also constitutively and spontaneously produced in mast cell granules. The exact enzymatic source could not be established. Whether this property of mast cell granules plays an antimicrobial role in connective tissues can only be speculated.