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.