Analysis of metabolic transients in intact cells by rapid microfluorimetry

Abstract

The analysis of NAD+ (or NADP+) reduction-reoxidation transients in single living cells by rapid microfluorimetry provides a mean to screen the activity of various intracellular enzymes, the interplay of regulating cofactors as well as the influence of structural compartmentalization or membrane barriers. Examples of possible applications refer to the analysis of transient parameters, the pattern of enzymatic pathways in relation to cell growth, effects of cofactors or metabolic “preloading”, etc. Through the incorporation of a nitrogen chamber the method has been extended to cells (e. g. L cells, human astrocytoma) which require anaerobiosis for glycolytic reduction of NAD+. When glucose-6-phosphate is replaced by glucose-1-phosphate the lag which precedes NAD+ reduction is prolonged from 100–200 msec up to 500–1000 msec. This can be shortened in presence of glucose-1,6-phosphate (a coenzyme for phosphoglucomutase). Differences in the flux pattern of the forward reaction at the phosphoglucomutase are found in a pleiomorphic population of L cells: e. g. glucose-1-phosphate more easily channeled towards the Embden-Meyerhof sequence in the larger non-dividing individuals. Preincubation with glycerol or xylitol leads to a prolongation of all parameters in the fluorescence transients, while cyclic AMP and ethionine lead to the opposite. The pattern of fluorescence transients makes possible a differentiation between reversible and irreversible inhibitors of LDH. Thus, by rapid microfluorimetry it is possible to resolve the early and later phases of fluorescence transients into components corresponding to characteristic steps in the sequence of intracellular events or control states.