Extramitochondrial Dehydrogenase Research Articles

The differential effects of dimethylnitrosamine and ethionine on mitochondrial and extramitochondrial dehydrogenases in single intact cells

In studying the bioenergetics of living cells, the microfluorometric analysis of coenzyme (NAD(P)H) responses to microinjected respiratory and glycolytic substrates enables, in principle, a search for qualitative/quantitative differences in normal versus carcinogen-treated (short-term, long-term) and malignant cells. Responses are compared in L-cells, same adapted to hypertonic media (i.e. L255, L355) and highly malignant rhabdomyosarcoma (CCL 136) cells. The largest responses to respiratory substrate (malate, isocitrate) and the lowest responses to glycolytic substrate (glucose-6- P) are in the L255, 355 cells which exhibit structural rearrangement and dense packing of mitochondria possibly due to high energy requirement for ion pumping. The converse is observed in the CCl 136 where there is no lack of these organelles, but they could be functionally deficient, as suggested by a predominant response to glucose-6- P compared to malate. In the control L-cell, the malate and glucose-6- P responses are relatively well balanced. Upon addition of dimethylnitrosamine to L-cells, there is an initial acceleration in the rate of glucose-6- P-induced NAD(P) reduction (? NADPH requirement for dimethylnitrosamine metabolization), followed by an upsurge of the malate response. In L355 cells, addition of the carcinogens dimethylnitrosamine or ethionine is followed by a strong reductive response to malate, and minimal response to glucose-6- P. The dramatic intensification of the NAD(P)H response to malate in L355 cells pretreated with an ATP trap (ethionine) or an uncoupler (dinitrophenol) strongly points to a requirement for ATP depletion. Weaker enhancement of NAD(P)H response (preferentially after glucose-6- P) is observed in the CCL 136 upon treatment with ethionine. The findings indicate the need for further study on differences in respiratory/glycolytic pathways and efficiency of ATP cycle in malignant cells exhibiting graded differences of structural/functional specialization.

Pyridine nucleotides in lung and liver of hypoxic rats

Adult male albino rats of Wistar strain in the weight range of 170–200 g were exposed to acute hypobaric hypoxia of 25,000 ft at 32°C for 90, 180, 270 and 360 min. respectively. The extramitochondrial dehydrogenases generating NADPH and the mitochondrial dehydrogenases generating NADH were studied in lung and liver. The oxidized and the reduced forms of these two nucleotides were determined at 90 min. and 360 min. of hypoxia. Hypoxia caused an initial decline in the activities of all the dehydrogenases studied. The activities of mitochondrial and pentose phosphate dehydrogenases of liver were high at 360 min. of hypoxia whereas the activities of extramitochondrial dehydrogenases of lung were generally low at all levels of hypoxic exposure. The NADPH levels of hypoxic rat lung were correspondingly low. However, the activities of liver mitochondrial dehydrogenases were elevated at 360 min. of hypoxia in spite of NADH accumulation in hypoxic rat liver. A decreased NAD/NADH ratio was observed in lung at 90 min. and 360 min. of hypoxia.

Ruby laser micro-irradiation of single tissue culture cells vitally stained with Janus green B: II. Effects on dehydrogenase activities

The effects of ruby laser irradiation on living cells have been studied histochemically in terms of seven intra- and extramitochondrial dehydrogenases. Single KB cells were stained with non-toxic concentrations of Janus green B, known for its mitochondrial specificity, then subjected to localized microirradiation of the cytoplasm. Beams of several different intensities were employed. The findings were very similar for all enzymes tested. Cells with light, and the majority (84–98 per cent) of those with moderate, primary laser damage had normal dehydrogenase activities although the latter had been shown by electron microscopy to have severely damaged mitochondria. After heavy primary damage, which was typically lethal within 2 hr after irradiation, the majority of the cells (67–94 per cent) exhibited a decrease or a complete absence of dehydrogenase activities. In such cells, ultrastructural changes of virtually all cellular organelles were observed. Most of the cells (79–85 per cent) exhibited cell membrane damage.