Metabolic control and compartmentation in single living cells.

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6-phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation-reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular interconversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two-step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two-step NAD(P)H response to sequentially injected fructose 1,6-diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell-to-cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.

The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells.

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observation within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.

Multichannel analysis of intracellular control and intercellular transfer of molecules.

The metabolic regulation and exchanges within intracellular organelles or a cell cluster are studied by multichannel microfluorometry and microinjection of metabolites or tracers. The determination of structure-function relationships relies on the retrieval of cells after microfluorometry, for subsequent morphological evaluation. Rate constants of coenzyme reduction-reoxidation were deduced from a mathematical model of NAD(P) in equilibrium with NAD(P)H transients due to microinjection of metabolites into cultured cells belonging to a variety of normal or malignant lines. Nuclear and cytoplasmic sites operate synchronously or not, depending upon metabolic demand or pathological alterations. Intercellular transit times are determined for tracers and metabolites. Within cell clusters 'communicating territories' are described, which can show metabolically a multicellular integrated state. Microfluorometry in conjunction with ultrastructural and other studies can be used to develop a cybernetic model of the living cell, also yielding dynamic models of cooperative and regulatory interactions between different kinds of specialised cells within a cell cluster.

New metabolic parameters for the characterization of cells.

Microspectrofluorometric evaluation of coenzyme-linked transient changes in blue fluorescence, triggered by microinjections of metabolic intermediates, allows the definition of dynamic parameters in the characterization of cells. The observed fluorescence transients can be simulated by appropriate equations accounting for NAD(P) reduction-reoxidation, with NAD(P) as rate-limiting or not. From the above, the rate constants K1 and K2 of NAD(P) reduction and reoxidation can be determined. Other useful parameters in the metabolic evaluation of different cell lines, comprising normal and transformed fibroblasts, glia-glioma, melanoma lines, and a mouse embryo clone, can be derived from the relationship between injected dose of substrate and rise or decay rates of NAD(P) in equilibrium or formed from NAD(P)H transients. Reoxidation of NAD(P)H seems to be a useful target for such studies in view of possible impairment in malignant cells and X-irradiated cells. Cells followed by fluorometry are retrieved for subsequent ultrastructural and other analyses. Thus, the metabolic patterns associated with the operation of intracellular pathways or organelle interactions, and their aberrations can be recognized. On this basis eventually a classification of different cell lines according to structure-function should be feasible.