Cahide 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.

PERMEATION OF LYSOSOMAL MEMBRANES IN THE COURSE OF PHOTOSENSITIZATION WITH METHYLENE BLUE AND HEMATOPORPHYRIN: STUDY BY CELLULAR MICROSPECTROFLUOROMETRY

Abstract— The photodynamically‐induced liberation of lysosomal enzymes using ß‐galactosidase as marker for the lysosomal enzymes has been studied by microspectrofluorometry on mouse L cells. Similar studies have been carried out using N‐acetyl‐ß‐D‐glucosaminidase as marker for the lysosomal enzymes of human fibroblasts. The high sensitivity of the fluorescence detection makes it possible to use 4‐methylumbelliferyl substrates for the enzymes contained in a single cell. Methylene blue and hematoporphyrin readily incorporate into both cells and upon excitation, sensitize lysosomal membrane damages, leading to enzyme release accompanying strong morphological changes.

PHOTOSENSITIZATION BY PORPHYRINS DELIVERED TO L CELL FIBROBLASTS BY HUMAN SERUM LOW DENSITY LIPOPROTEINS. A MICROSPECTROFLUOROMETRIC STUDY

Human serum LDL were used as vehicles to deliver protoporphyrin and hematoporphyrin dimer to L cell mouse fibroblasts. Topographic analysis by microspectrofluorometry on single living cells shows that after digestion of LDL, protoporphyrin is localized in cytoplasmic areas. Protoporphyrin and hematoporphyrin dimer are readily bleached by 420 ± 60 nm radiations at the high fluence rate used. Complex bleaching kinetics are observed. Spectral studies using the same technique demonstrate that an intense fluorescent emission (λmax= 450 nm) is produced immediately after the onset of irradiation with 365 ± 2 nm or 420 ± 60 nm radiations using LDL loaded with protoporphyrin or Photofrin II. These fluorescent products have been previously identified as lipofuscin‐like pigments formed by reaction of lipid photoperoxides with amino groups. The permeation of lysosomal membranes is also induced after delivery of the porphyrins by LDL. This permeation can be strongly inhibited not only by the lysosomal inhibitors chloroquine and monensin but also by a‐tocopherol. On the other hand, neither α‐tocopherol nor chloroquine or monensin inhibit the lipofuscin‐like pigment formation.

Imaging of cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level

The success of biopharmaceuticals relies on the ability to have reliable probes to interpret their mechanisms of action in situ at the intracellular level in terms of cell organelles and microcompartments. One of the most effective probes is the endogenous coenzyme NAD(P)H and its fluorescence transients obtained by the microinjection or perfusion of metabolic intermediates and modifiers, in the presence of drugs and inhibitors. The approach in fluorescence microtopography and microspectrofluorimetry is based on the premise that natural cell fluorescence (autofluorescence) holds a decisively greater potential in unravelling intracellular physiopathological processes than extrinsic fluorescence or artificial pseudocolouring. The mounting as a detector of a cooled charge‐coupled device camera or alternatively of a non‐cooled camera in conjunction with an image intensifier or an investigator (i.e. frame scan accumulator) to enhance sensitivity makes possible the detection of the low‐quantum‐yield NAD(P)H fluorescence at a level comparable to images previously obtained with high‐quantum‐yield fluorochromes. The modulation of mitochondrial autofluorescence by rotenone, carbonyl cyanide p‐ trifluoromethoxyphenylhydrazone and oligomycin, and of cytoplasmic and nuclear autofluorescence by glucose and iodacetamide in CV‐1 kidney epithelial cells, Ehrlich‐Lettre hypotetraploid CCL77 cells and Saccharomyces cerevisiae, provides examples of the usefulness of fluorescence imaging in the study of biopharmaceuticals. The method goes beyond NAD(P)H to the multiplicity of extrinsic and intrinsic probes already available or in development.

A microspectrofluorometric study of porphyrin-photosensitized single living cells. II: Metabolic alterations

The transient change in the NAD(P)H NAD(P) equilibrium following microinjection of mitochondrial (malate) or extra‐mitochondrial (6‐phosphogluconate) substrates into single living cells is strongly modified a few seconds after the onset of 365 nm irradiation in the presence of hematoporphyrin. There is no major difference in the time frame for the alterations of the Krebs cycle and pentose pathway. In view of the complex interrelationships between mitochondrial and extramitochondrial pathways, there is a reasonable chance that effects on both pathways are not unrelated‐ However there is no definite evidence for a direct correlation between the photoeffects on these two pathways.

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells: Relationship to cellular quiescence and senescence in culture

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.

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.).

MULTISITE TOPOGRAPHIC MICROFLUOROMETRY OF INTRACELLULAR AND EXOGENOUS FLUOROCHROMES

Abstract. Microspectro fluorometry combined with microinjection of metabolites allows the monitoring of pathways regulating the energy metabolism, biosynthetic or metabolizing activity of the intact living cell. The topographic scan of ˜ 100–150 adjacent regions within a cell or the spectral scan of fluorescence from a single cell region is completed within 60 ms. The in situ activity of intracellular enzymes and substrate concentration vs rate relationships are derived from the kinetic analysis of transients (e.g. NAD(P) ⇄ NAD(P)H) elicited by sequential injections with increasing doses of substrate (e.g. glucose‐6‐P, 6‐phosphogluconate). A method of approximation is used to predict the non‐linear behavior of whole biochemical systems (e.g. the sum of reactions involved in NAD(P) reduction‐reoxidation transients). Approximation to power law kinetics is validated by prediction of system behavior over a 10–100 fold variation in the input concentration of substrate.

Rapid microfluorimetry of enzyme reactions in single living cells

Abstract An optimized photon counting technique allows the microfluorimetric study of NAD + (or NADP + ) reduction-reoxidation transients in single living cells with a time resolution in the range of 1/50-1/100 sec. The transients resulting from the micro-electrophoretic addition of metabolites ( e.g. Glc-6- P or Glc-1- P ) can be analyzed in terms of early parameters ( e.g. initial lag, rise half time or full rise time) and overall parameters (time of rise and half decay, amplitude, reoxidation time). Both the initial lag and rise half time are considerably longer with Glc-1- P than with Glc-6- P , possibly due to control at the phosphoglucomutase or compartmentation of glycolytic phosphate esters. While glycolytic NAD + (or NADP + ) reduction proceeds adequately in aerobic EL2 and EAT ascites cells (although ΔNADH/Δ t is higher at anaerobiosis), it is critically dependent upon anaerobiosis in L and astrocytoma cells. Thus by rapid microfluorimetry it is possible to resolve the rising phase or other segments of the fluorescence transients into components each corresponding to a particular step in the sequence of intracellular events or control states.

Direct connection between myelinosomes, endoplasmic reticulum and nuclear envelope in mouse hepatocytes grown with the amphiphilic drug, quinacrine

Mouse hepatocytes grown in 4 microM quinacrine had numerous myelinosomes which were directly connected to expanded cisternae of the rough endoplasmic reticulum (RER). The cisternae of the RER either subtended the electron transparent space of the myelinosome, expanded to form the outer membrane of the myelinosome or penetrated into it. Material of low electron density was frequently seen within the area where the cisternae penetrated into the electron transparent space of the myelinosome. Myelinosomes were also associated with the nuclear envelope in a pattern similar to that of the RER. Quinacrine appears to bind with the phospholipids of the membranes of the endoplasmic reticulum and nuclear envelope and this drug-lipid complex is then moved into myelinosomes effectively removing the drug from the cell.