When unfixed cardiac muscle from the rat or hamster was incubated in substrate-free media containing cerous chloride buffered to pH 7.5 with Tris-maleate-sucrose, electron-dense reaction bodies were formed in the sarcoplasm and, to a greater extent, inside and budding from mitochondria. Fixation in glutaraldehyde prevented the reaction product appearing within mitochondria, but had no effect on either the bodies associated with outer mitochondrial membranes or those apparently free in the sarcoplasm or outside the cells. No reaction product was observed in unfixed liver, kidney or skeletal muscle.
The electron-dense reaction product within cardiocytes was absent in fixed tissues treated beforehand with acetone and in fresh tissues preincubated with the D-aminoacid oxidase inhibitor kojic acid. It was substantially reduced by preincubation in Tiron (a superoxide scavenger), diethyldithiocarbamic acid (an inhibitor of copper-containing enzymes such as diamine oxidase and superoxide dismutase) or EDTA, and totally if any of these compounds was included in the incubation medium. However,
p-chloromercuribenzenesulphonate, a non-penetrating and non-specific inhibitor of NAD (P) H oxidoreductases, and atebrine limited the amount of reaction product formed in all sites by much less. Chlorpromazine inhibited the formation of reaction product in the sarcoplasm but gave rise to a coagulated product within mitochondria. With Clorgyline, in contrast, no product was deposited in mitochondria and large amorphous masses of reaction product appeared in the sarcoplasm.
None of the following, when included in the incubation medium, had any significant effect: 3-amino-1, 2, 4-triazole, catalase, dicoumarol, glutathione peroxidase, mannitol, methanol, Pargyline,
o-phenanthroline, potassium cyanide, rotenone, sodium azide, sodium benzoate, sodium pyruvate, superoxide dismutase and thiourea.
It is concluded that the electron-dense product arises from a reaction of cerium ions with either a lipid peroxide or endogenous hydrogen peroxide generated by a metal-containing thiol enzyme, possibly an oxidised form of monoamine oxidase A with the properties of a diamine oxidase. The significance of the cerium reaction in the ageing of cells is discussed briefly.
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