The effect of phenazine methosulphate on the histochemical demonstration of oxidative enzymes in retinal cultures

Abstract

Cultures of retinal tissue from newborn rabbits were examined after 2 weeks in vitro to ascertain the effect of the electron carrier phenazine methosulphate on the activity pattern of oxidative enzymes. The investigation was performed on formalin fixed explants with histochemical methods. The neuroectodermal cells, i.e. nerve cells and neuroglial cells and photoreceptor cells, showed higher activity of all the investigated enzymes than did the mesenchymal cells. Cytochrome oxidase activity was reduced by the addition of 10 −4 M phenazine methosulphate in the cultured cells. The electron mediator did not cause any significant change in the distribution or intensity of the activity of NADH 2 diaphorases or NADPH 2 diaphorase. The citric acid enzymes revealed an increase in activity especially in the inner segments of the photoreceptor cells and in the nerve cells. The strongest increase in activity after the addition of phenazine methosulphate to the incubation solution was obtained with lactic dehydrogenase. During assays for lactate dehydrogenase the inner segments of the photoreceptor cells, the neuroglial cells and the nerve cells were rapidly covered with formazan precipitates, and were therefore easy to observe in the explants. The soma of the photoreceptor cells showed moderate lactate dehydrogenase activity if incubated with phenazine methosulphate, but none without phenazine methosulphate. The enzymes of the hexosmonophosphate shunt, i.e. glucose-6-phosphate and 6-phosphogluconate dehydrogenases, possessed activity patterns similar to that of lactate dehydrogenase. Cellular enzyme proteins were dissolved during incubation as indicated by colouring of the incubation medium. The neuroectodermal cells all showed their own characteristic enzyme patterns. The inner segment and the soma of the photoreceptor cells displayed high activity levels of citric acid cycle and glycolytic enzymes while the nerve cells had low to moderate activity levels. The Müller neuroglial cells were characterized by their high glycolytic activity.