[Method of measurement of cadmium influx by fura-2 titration in MDCK cell evidenced. Fluorescence video-microscopy study]. / Méthode de mesure des flux entrants de cadmium par titration du fura-2, à l'échelle d'une cellule MDCK. Etude par vidéo-microscopie de fluorescence.

Cadmium influx rate in mammal kidney cells (MDCK) is analyzed using an original method based on fura-2 titration. The method relies on the high affinity of the fluorophore for the metal. It follows that the excitation spectrum shift of fura-2 can be linearly correlated to the influx rate of cadmium. Fluorescence digital imaging microscopy allows the study at single cell and intra-cellular organite levels. Results show that the cadmium uptake seems to be carrier dependent. Metal fluxes are potential independent, with a temperature effect caracterized by a Q10 of 2.3 +/- 0.2. No effect of verapamil is noticed; however, cadmium transport is inhibited by external calcium. Apparent dissociation constant for the cadmium uptake is estimated at 4.5 10(-5) M at 20 degrees C. An additional passive transmembrane diffusion process is also evidenced.

Herbicide isoproturon-specific binding in the freshwater macrophyte Elodea densa--a single-cell fluorescence study.

The experimental approach to the effects of the phenylurea herbicide Isoproturon (IPU), on the photosynthesis activity of leaf cells of the freshwater macrophyte Elodea densa was based on the fast induction kinetics of the PSII chlorophyll a in vivo fluorescence. FI/FP ratios determined on the induction curves exhibited a noticeable effect at 5 micrograms IPU.liter-1. They appeared to be the most reliable parameter for the quantitative evaluation of the dissociation constant of the complex "IPU/D1 protein" (Kd = 1.2 x 10(-7) M) and of the concentration of free IPU either at the thylakoid level or in the surrounding medium of the leaf epithelial cells. Total IPU bioaccumulation at the whole plant level as a function of [IPU] in the medium indicated that the D1 protein represented the main binding site for IPU in this aquatic plant species.

Multidrug resistance bypass in cells exposed to doxorubicin-loaded nanospheres. Absence of endocytosis.

Multidrug resistance bypass has been achieved in vitro with polyalkylcyanoacrylate nanospheres loaded with doxorubicin as previously shown by Cuvier et al. Fluorescence-imaging experiments were performed to determine if the endocytosis of the particles by the cells could be responsible for this activity. The results obtained with three lines of sensitive and resistant cells (K562, MCF7, and C6) demonstrate that the particles were not internalized by the cells, nor were they adsorbed onto the cell surface. In contrast, these nanospheres were very efficiently endocytosed by phagocytic cells such as macrophages. No correlation was observed between the fate of the particles, endocytosed or not, and their cytotoxic activity. It was concluded that no endocytosis step was involved in the mechanism of the multidrug resistance bypass obtained by associating a drug to polymeric particles.

Spatial and temporal distribution of [Ca2+]i in normal human myotubes. A fura-2 imaging study.

The spatio-temporal distribution of intracellular, free calcium ions, [Ca2+]i, induced in human myotubes by electrical stimulation typically showed a relatively large increase of [Ca2+]i in the vicinity of the plasmalemma. The similarity of this distribution, with that observed after the application of caffeine, and the lack of any effect of lanthanum, strongly suggest that the main source of Ca2+ participating in the electrically induced transient is the sarcoplasmic reticulum. Aneurally cultured human myotubes therefore display a 'skeletal muscle type' coupling between membrane depolarization and calcium release. However, the relatively slow time course of the electrically induced transients compared to rat and mouse myotubes, together with the inability of Ca2+ released from the sarcoplasmic reticulum to activate the contractile machinery, implies that aneurally cultured human myotubes achieve only a limited degree of differentiation. The relevance this may have to an apparent delay between the electrically induced rise in intranuclear Ca2+ relative to cytosolic Ca2+ remains to be determined but, at this stage of differentiation, there appears to be some form of barrier to free diffusion between the two cellular compartments.

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes.

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca2+, 37 degrees C, pH 7.4), 10%-20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca2+]i) of these cells was 106 +/- 4 nM versus 77 +/- 3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity approximately 50 microns/s, maximal Ca amplitude = 195 +/- 11 nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca2+]i increase induced by K+ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca2+]i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).

Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca2+ in rat myotubes.

Rat myotubes have a resting [Ca2+]i of about 82 nM. Myotubes 3-5 days old (quiescent myotubes) display electrically induced and spontaneous transients in the intracellular concentration of free Ca2+ ions ([Ca2+]i) uncoupled to any detectable contraction. By contrast, 1- to 2-day-old myotubes are insensitive to electrical stimuli and, after 6 days in culture, stimulated myotubes always show [Ca2+]i transients and twitch contractions. The spatial distribution of [Ca2+]i variations in quiescent myotubes is heterogeneous, local increases in [Ca2+]i being mainly observed near the periphery of the cell. The small effect of different external Ca2+ concentrations and of Cd2+ on the amplitude of the [Ca2+]i oscillation indicates that the main source of Ca2+ may be the sarcoplasmic reticulum. This conclusion is supported by the close similarity between electrically induced and caffeine-induced [Ca2+]i maps. These findings suggest that, at an early stage of myotube ontogenesis, a part of the excitation/contraction coupling, as membrane ionic channels, voltage sensors and Ca2+ release and reuptake mechanisms, is functional but, apparently, still uncoupled to the contractile machinery.

Effect of local anaesthetics on mitochondrial membrane potential in living cells.

Using the laser dye rhodamine 123, we demonstrated that local anaesthetics can reach mitochondria in cell culture and reversibly decrease, or even collapse, their transmembrane potential. This effect is highly dependent on the lipid-solubility of the local anaesthetic and can be facilitated by the presence of a lipophilic anion.

[Is muscular acetylcholinesterase activity correlated with the intracellular concentration of free calcium?]. / L'activité de l'acétylcholinestérase musculaire est-elle corrélée à la concentration du Ca2+ intracellulaire libre?

The role of the calcium ion Ca2+ as an agent of intracellular control in a variety of physiological processes is well established. In vertebrate skeletal muscle fibers, Ca2+ is involved in muscle contraction, modulation of membrane permeability and regulation of metabolic activity. Recently it was suggested that ion fluxes through membranes regulate the level of two cholinergic macromolecules, the acetylcholine receptor and the A12 form of acetylcholinesterase (AChE), the presumed synaptic form of the enzyme. Muscle cells paralysed by veratridine, which maintains the Na+ channel in the open state, showed an increase in total AChE and in the levels of the A12 form. The effect of veratridine on AChE was blocked in the presence of agents that block Ca2+ permeability suggesting that Ca2+ is involved in this effect. To understand whether the level of muscle AChE is related in some way to the level of free intracellular Ca2+, we analysed the variations of Ca2+ levels in rat muscle cells treated by agents which modify the ionic permeabilities. This level was determined by spectrofluorimetry using the fluorescent Ca2+ indicator: Quin 2. However no correlation between these parameters was observed in our experimental conditions.

Reduction of membrane-bound dopamine beta-hydroxylase from the cytoplasmic surface of the chromaffin-granule membrane.

Resealed bovine chromaffin-granule 'ghosts' were used for assaying the membrane-bound form of dopamine beta-hydroxylase. Hydroxylation of the substrate tyramine is dependent on its accumulation within the 'ghosts', where the active site of the enzyme is located. Free tyramine in the medium is at a low concentration, low ionic strength and a relatively high pH (7.0), so that even in the presence of a reducing agent (co-reductant) the unaccumulated amine is hydroxylated at a negligible rate. 'Ghosts' contain an endogenous co-reductant, which is shown to be catecholamine remaining in the membrane itself after granule lysis. Catecholamine that is free in solution in the medium or in the interior of the 'ghosts' is not effective as co-reductant, nor is ascorbate, in contrast with the situation with soluble dopamine beta-hydroxylase. Ferrocyanide is an active co-reductant, however, giving a hydroxylation rate approximately equal to the tyramine accumulation rate: it does not enter the 'ghosts', nor does the enzyme appear to utilize ferrocyanide sealed inside the 'ghosts'. A mechanism must therefore exist for transferring electrons across the membrane from the cytoplasmic surface to the matrix surface. NADH is not an electron donor for the enzyme, nor is cytochrome b-561 involved.

Chemical studies on yeast hexokinase. Specific modification of a single tyrosyl residue with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

1. Of the 15 tyrosyl residues/subunit of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase) only one residue is specifically modified at pH 8.0 with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. 2. The acylation of this single tyrosyl residue leads to the loss of the enzyme activities (hexokinase and ATPase) by a first-order process, which can be fully reversed by treatment with hydroxylamine. 3. ATP does not protect the enzyme against chemical modification and inactivation; however, glucose exerts a noticeable though indirect protection effect against chemical modification and inactivation. 4. The chemically modified enzyme, purified by column chromatography, has 14% of the activity of the native enzyme, but the Km for ATP-Mg or glucose remains unchanged as does the pH optimum of activity. Results of conformational studies (ultracentrifugation, fluorescence, thermostability and chemical reactivity of the sulfhydryl groups) indicate that the decrease of enzyme activity due to the modification of the tyrosyl residue is related to a localized perturbation of the enzyme active-center region.