Elli Kohen

MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES

Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (δIf) due to microinjection of glucose‐6‐phosphate (G6P) into EL2 ascites cells. Above 510 nm, δIF is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δIF level. The difference between δIf and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire δIF can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component.

Cell-to-cell Communication in Rat Pancreatic Islet Monolayer Cultures is Modulated by Agents Affecting Islet-Cell Secretory Activity

Single islet cells in monolayer cultures of neonatal rat pancreas were microinjected with the fluorescent dye Lucifer Yellow CH and the cultures were observed by combined phase contrast and fluorescent microscopy. The dye spread from an injected cell directly into neighboring islet cells, and successive microinjections of dye into different cells defined territories comprised of 2–6 communicating cells. The number of communicating cells could be modulated by addition to the cultures of different agents known to affect islet cell secretory activity. Cell communication was significantly increased by a high (16.7 mM) glucose concentration, by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM), and by the calcium ionophore, A23187. The effect of A23187 was transient and dose-dependent. Somatostatin (1 μg/ml) significantly inhibited cell communication. These results demonstrate that cell-to-cell communication may participate in the regulation of islet cell secretory activity.

METABOLISM OF REDUCED PYRIDINE NUCLEOTIDES IN ASCITES CELL NUCLEI.

Summary1. The conditions are described under which the fluorescence due to reduced pyridine nucleotides can be studied separately at nuclear and cytoplasmic sites of glass-grown ascites cells, by the use of a flow chamber in the microfluorimeter ofChance andLegallais.2. The addition of glucose to ascites cells leads to a reduction of pyridine nucleotides within the nucleus, thus providing evidence for the participation of nuclear pyridine nucleotides in cellular metabolism.3. Although generally nuclear and cytoplasmic pyridine nucleotides parallel each other in their response to different metabolic conditions, there are few instances (e.g., Amytal) where they do not show such parallelism. This is discussed with regard to the problem of reoxidation of nuclear reduced pyridine nucleotides.

A microspectrofluorometric approach for the study of Benzo(a)pyrene and Dibenzo(a, h)anthracene metabolization in single living cells

SummaryThe use of a microspectrofluorometer in conjunction with the microelectrophoretic intracellular injection of glycolytic intermediates, for the study of carcinogen metabolization (i.e. Benzo(a)pyrene (BP) and Dibenzo(a, h)anthracene) by single living EL2 ascites cells, has allowed the detection of fluorescence attributable to the metabolites of BP and DBA.The results obtained are in agreement with those yielded by more conventional methods: i.e. in vivo assay and in vitro reconstitution of intracellular environment. Thus, it is observed that NADPH is more active in the metabolization of BP. Furthermore, in the case of BP the fluorescence attributable to a BP metabolite, exhibits a strong similarity to 3-OH benzo(a) pyrene.

Penetration and localization of furocoumarins in single living cells studied by microspectrofluorometry

The microspectrofluorometric technique has been used to study the penetration and the localization of psoralen, 4,5,8-trimethylpsoralen and 4-aminomethyltrioxsalen in single living L-cells. The concentration of the different compounds inside the cell reached a plateau in 2 min with psoralen and aminomethyltrioxsalen and in 20 min with trioxsalen. Washing of the cells with culture medium produced only a partial removal of the three furocoumarins, distributed apparently in equivalent amount in the nucleus and cytoplasm.

Quantitative Aspects of Rapid Microfluorometry for the Study of Enzyme Reactions and Transport Mechanisms in Single Living Cells

Fluorescent molecular probes are ideally suited not only for kinetic studies of enzyme reactions in localized cell structures (i.e., mitochondria, nucleus, cytoplasm) (Chance 1970) but also for observations on transport mechanisms across intracellular membranes (Kohen, Siebert et al. 1971). Among intracellular fluorochromes, reduced pyridine nucleotides with their blue fluorescence (Sund 1968) provide a direct probe into the microenvironment of intracellular compartments (Kohen 1964) through the analysis of NAD-(NADP) reduction reoxidation transients (Kohen et al. 1970a; 1971a;1971b; 1972) resulting from rapid microelectrophoretic additions (Kohen et al. 1970a) of metabolites (i.e.,glucose-6-phosphate) to the cell cytoplasm or nucleus. The maximization of the signal-to-noise ratio in microfluorometric determinations can best be achieved through the use of an optimum choice of the fluorescence excitation source, optical filtering system and fluorescence detection system (Kohen et al. 1970a; 1971a; 1971b; 1972; Chance and Legallais 1959; Kaufman et al. 1971; Ploem 1971; Zatzick 1970; Jones et al. 1971) in addition to the properties of the fluorochrome perse (i.e. for reduced pyridine nucleotides high quantum yield when bound to proteins (Estabrook 1962),universal distribution in animal and plant cells, specific functions in various cell compartments (Kohen 1964) or metabolic pathways, association with close to 100 dehydrogenases (Sund 1968) etc.).

Rapid two-channel microfluorimetry: A method for comparative study of metabolism, phase differences or transport in the cytoplasm and nucleus

Abstract Two-channel microfluorimetry, based on electronic chopping in conjunction with microelectrophoretic addition of metabolites (e.g. glucose 6-phosphate, 6-phosphogluconate) or dyes (fluorescein) to single living cells (ascites, L), can be used for simultaneous two-site observations (i.e. nucleus vs cytoplasm, or intracellular transport). Despite considerable metabolic parallelism between nucleus and cytoplasm (the more so for the hexose monophosphate shunt), nuclear Embden-Meyerhof enzymes may be in a different configuration (higher NAD+ reduction rate, higher Hill coefficients, also a certain difficulty in the reoxidation of nuclear NADH). In most cells delays due to distance (4–5 ms per 1 μm) or the nuclear membrane (approx. 35–50 ms) seem to mater less than metabolic lags. Nuclear and cytoplasmic regions were found to be either metabolically in phase or out-of-phase (lags up to 500 ms), apparently depending upon functional state. Generally, regardless of the site of microelectrophoretic addition, transients in the mitochondria-free cytoplasm started, on average, 200 ms later than in the nucleus, but started up to 300 ms earlier in the mitochondria-rich cytoplasmic regions. Thus, hitherto unknown patterns of metabolic regulation in localized cell regions are open to exploration.

Microspectrofluorometric study of Benzo(a)pyrene metabolization in Benzo(a)pyrene-grown single living cells

SummaryA microspectrofluorometric approach in conjunction with microelectrophoretic injection of glucose-6-P (G6P), was used to study the effect of cultivation in a benzo(a)pyrene (BP)-containing medium on the metabolization of BP, by EL2 ascites cells.The fluorescence increase which follows the microinjection of G6P to single living cells. was studied in EL2 cells grown for 24-hours, 48-hours, 10 days and 57 days in a BP-containing medium, as well as EL2 cells grown for 16 days in presence of BP and placed then for 24 or 48 hours in a BP-free medium. The effects on the induction of Aryl Hydrocarbon Hydroxylase were observed for cells so treated, and it seems that for the latter the requirement of intracellular O2 for the metabolization of BP, has been verified. Furthermore, another important factor has emerged: the intracellular BP concentration. The various spectra are discussed in function of these considerations.

The influence of succinate on pyridine nucleotide reduction in glass-grown ascites cells during glycolysis

Summary1. The changes in fluorescence due to reduced pyridine nucleotides were studied by microfluorimetry in the mitochondrial and extramitochondrial regions in glass-grown ascites cells during glycolysis, and in the presence of various substrates, using the flow chamber technique, which allows addition at will of drug and substrate.2. At anaerobiosis in a drug and substrate free medium, maximum fluorescence was localized in the mitochondria. Aerobiosis resulted in a considerable decrease of fluorescence.3. Upon addition of glucose aerobic cells showed a diffuse fluorescence extending to the extramitochondrial space. In cells pretreated with 20 mM sodium succinate the response to glucose was considerably more pronounced, and the cells exhibited a uniform and very strong blue fluorescence. However when treatment with succinate followed administration of glucose, the fluorescence was more comparable to that obtained with glucose alone. The succinate effect was not blocked by malonate and could not be simulated by fumarate. There was a slight decrease of the succinate effect with dicumarol, but this was not very reproducible.4. Alternative interpretations for the succinate effect (in cells subsequently treated with glucose) are a physicochemical one which affects intracellular structure and permeability to glucose or a metabolic one which involves transfer of ATP from the mitochondria to the extramitochondrial space, where it stimulates glycolysis via the hexokinase reaction. The absence of response to inhibitors may be due to a paradoxical stimulation of glycolysis by damaging agents which can upset intracellular control mechanisms.

The effect of intracellular oxygen on the metabolization of Benzo(a)pyrene and Benzo(k)Fluoranthene. A microspectrofluorometric analysis in single living cells

SummaryThe fluorescence increase which accompanies the injection of glycolytic intermediates to Benzo(a)pyrene (BP) and Benzo(k) Fluoranthene-B(k)F treated EL2 ascites cancer cells, under aerobic and anaerobic conditions, has been studied in a microspectrofluorometer. In the carcinogen-treated cells the altered fluorescence increase pattern (in reference to control cells) which is observed at aerobiosis and attributed to BP or B(k)F metabolization, is not any more observable at anaerobiosis, in which case the fluorescence increase of the carcinogen-treated cells resembles that of the controls. This difference in behavior is discussed and a comparison is initiated between the response to injection in cells treated with BP (compound with K region) or B(k)F (compound without K region).