Anne-Catherine Dazy

Effects of maitotoxin on calcium entry and phosphoinositidebreakdown in the rabbit ciliated tracheal epithelium

Summary— Maitotoxin induces a concentration‐dependent 45Ca uptake in primary cultures of rabbit tracheal epithelial cells. This response is insensitive to the calcium channel antagonists nifedipine, diltiazem and verapamil up to 20 μM. However, verapamil at 200 μM completely prevents 45Ca uptake. Measurements of indo‐1 fluorescence show that MTX induces a very sustained (≥ 2 h) [Ca]i rise, which is completely inhibited by 200 μM of verapamil. Genistein (110 μM) (an inhibitor of tyrosine kinases) also strongly inhibits it. The inhibitory effect of 50 μM miconazole (an inhibitor of cytochrome P450) is only partial. Okadaic acid (inhibitor of protein‐phosphatases) primarily delays the response to the toxin without decreasing its magnitude. MTX induces the formation of (1,4,5) inositol trisphosphate (IP3). The MTX response curve is biphasic. Stimulation is transient (5 10 min) and is not inhibited by chelation of intracellular Cai with BAPTA, nor by verapamil (200 μM) or U73122 (10 μM) (an inhibitor of activation of PLCPI through a trimeric G protein). Results suggest that MTX independently activates a calcium transport process (which might imply phosphorylation on tyrosine residues) and a PLC not linked to a trimeric G protein.

Morphogenesis, bioelectrical polarity and intracellular streaming in a giant cell, Acetabularia mediterranea: Studies on their recovery after prolonged dark period

Abstract Acetabularia cells placed in the dark enter dormancy. They stop growing, lose their electrical polarity and their cytoplasmic streaming ceases; all three, after 2–3 weeks. When re-illuminated with continuous light, electrical polarity and cytoplasm movements recover in a few minutes, then decrease again and become firmly established only after 2–4 h. They both show a rhythm on the order of hours (period > 12 h). Morphogenesis starts during the second day. If the dark treatments last more than 6–8 weeks, then the cytoplasm regresses gradually along the stalk and after 4 months, the living part of the cell is limited to the rhizoids. Such hibernating algae grow very fast when in continuous light. However, the recovery of the electrical polarity and cellular streaming is slower than after the shorter dark periods.

RNA migration in Acetabularia mediterranea: Effects of cytochalasin B, cycloheximide and prolonged dark periods

Abstract The migration of newly synthesized RNA outside the nucleus and its distribution along the cell of Acetabularia as a function of time has been studied by means of pulse-chase experiments followed by autoradiography, trichloroacetic acid (TCA) precipitation or ribosomal RNA extraction. During the first 4–6 days of chase, the newly synthesized nuclear RNA appears in the cytoplasm following a baso-apical exit gradient. After 4–6 days, the labelled RNA becomes uniformly distributed throughout the cell. rRNA migration is inhibited by cytochalasin B suggesting the involvement of actin microfilaments in the process. Prolonged darkness prevents RNA transport and light induces its recovery. This may be interpreted as the result of ionic modifications. A short-lived protein could also be involved in the regulatory process since cycloheximide (CH) inhibits the recovery of RNA migration after dark treatment.

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro.

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca2+ (Fura2) and Na+ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o- after exposure to H2O2 or *OH (H2O2 + Fe2+). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H2O2 or *OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl-. Cai also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Nai on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, *OH inhibited the Na+ current (a measure of Na(+)-K(+)-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na+/K(+)-pump may contribute to the sustained elevation of Nai. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.

Differential effects of maitotoxin on calcium entry and ciliary beating in the rabbit ciliated tracheal epithelium

Summary— The marine toxin maitotoxin (MTX) induces stimulation of ciliary beating in primary cultures of rabbit tracheal epithelial cells. The response is time‐ and concentration‐dependent. External calcium is an absolute requirement, although at a very low concentration (50 μM for maximal effect). Pretreatment of the cells with MTX induces an early (5 min) and sustained (≥ 24 h) homologous desensitization. The response to MTX is strongly inhibited by trifluoperazin (an inhibitor of Ca‐calmodulin‐dependent enzymes) and by chelation of [Ca]i with BAPTA. However, the magnitude and kinetics of [Ca]i rise elicited by MTX do not correlate with those of the ciliary beat frequency (CBF) increase: the CBF increase is transient (with a peak at 5–10 min) while the [Ca]i rise is sustained; the CBF increase occurs at concentrations of MTX which are without an effect on [Ca]i; the CBF increase is not inhibited by 200 μM verapamil, genistein or okadaic acid, which inhibit the MTX‐induced [Ca]i rise. The CBF increase is strongly inhibited by antagonists of arachidonic acid metabolism, mepacrine and nordiguaiaretic acid. However, MTX does not stimulate cAMP synthesis. These results suggest that calcium is not the only factor involved in the biological effects of MTX and even suggest that MTX may primarily stimulate phospholipid breakdown in the cell membrane.

Measurement of the epithelial barrier integrity in tracheal cell cultures exposed to irritant compounds.

A simple method has been developed to measure epithelial barrier alteration, in order to evaluate the irritant effect of inhaled compounds on the respiratory tract. In vitro primary cell cultures from rabbit tracheal epithelium were grown on permeable filters and used to study the effect of two pollutants-acrolein and parathion-on epithelial barrier integrity. The transepithelial potential difference and [(14)C]mannitol permeability were measured. Acrolein specifically injured the tight junctions and therefore increased transepithelial permeability. Parathion did not produce any alteration of the epithelial barrier but the results suggest alteration of ionic transport.

The effects of blue and red light on Acetabularia mediterranea after long exposure to darkness

After Acetabularia mediterranea cells were kept in darkness for 2–8 weeks, all the cellular processes were arrested and the algae did not grow. In particular, the transcellular electrical potential (VAB) decreased to almost zero and cytoplasmic streaming was arrested. Upon illumination with continous blue light (BL), the first events were (as with white light (WL)), immediate increase in VAB and movements of water, followed, after a lag period of 1–3 min, by transient recovery of cytoplasmic streaming which lasted about 16 min. After 10 min (earlier than in WL), the frequency of the spontaneous action potentials increased much more than in WL. Then, after 1.5–4 hr during which VAB often decreased to zero while the cytoplasmic movements stopped, both activities resumed with diurnal oscillations. BL stimulated (as WL) rRNA synthesis, migration of rRNA from nucleus towards apex and cell growth. Upon illumination with red light (RL), VAB also increased, but water movements were much less pronounced than in BL. The transient streaming phase was shorter. The spontaneous action potentials increased in frequency much later (several hr) and much less than in BL or WL. VAB did not decreased at any time and was maintained at particularly high values. Cytoplasmic streaming resumed, but showed very attenuated or no rhythm. rRNA synthesis and migration remained low. Cell growth did not resume during the experiments.

Cadmium interactions with ATPase activity in the euryhaline alga Dunaliella bioculata

To further study the toxicity of cadmium in the euryhaline alga, Dunaliella bioculata, ATPase activity and Cd2+ interactions were investigated in this species.Ultracytochemical studies showed the presence of ATPase reaction after incubation with Ca2+ and Mg2+, on different cell structures, the cytoplasm, the nucleoplasm, the axoneme and the membrane of the flagellae. In the cytoplasm, the localization of the lead precipates suggests that they are associated with the endoplasmic reticulum.The in vitro measurement of enzyme activity in crude cell extracts obtained by a partial solubilization of deflagellated algae with Triton X100, revealed a high Mg2+ dependent pyrophosphatase activity, a weak Mg2+-ATPase and a Ca2+-ATPase (Km = 0.12 mM) which was little sensitive to vanadate. In these extracts, a Ca2+ dependent ATPase was detected at the level of a double band by a non-denaturing electrophoresis. The same activity was found in the supernatant of sonicated cells in the absence of detergent, which suggests that this ATPase could be a cytosolic enzyme.In plasma membrane fractions, vanadate-sensitive ATPase activity was measured. This reaction was activated either by Mg2+ at relatively low concentrations (Km = 150µm) or by Ca2 +, but required unusually high concentrations of this ion, 50–100 mM.The inhibitory effects of Cd2+ on Ca2+ ATPase activity in cell extracts were compared with those of other cations. The range of toxicity was: Zn2+ > Cd2+ > Cu2+ > La3+ > Co2+. For Cd2+, the IC50 was 42 µM. The nature of inhibition, though, mixed was for the most part competitive, since the competitive constant value (Ki = 7 µM) was lower than the non-competitive constant value (K′i = 35 µM).In plasma membrane fractions, ATPase activity showed a high sensitivity to the heavy metal. It was non-competitively inhibited by cadmium in a narrow range of micromolar concentrations.

The effects of blue and red light on the transcellular electric potential, cytoplasmic streaming and rRNA transport in Acetabularia acetabulum

The main characteristics of the giant cell Acetabularia acetabulum (= A. mediterranea) are its elongated shape and its pronounced polarity: the differentiated rhizoids of the base contain the nucleus but growth, enhanced metabolic activities (e.g., photosynthesis, protein synthesis) and morphogenesis occur at the opposite end of the cell, the apex. The cytoplasm streams slowly and irregularly (up to 15 μm· S–1), parallel to the axis and in both directions, carrying with it the chloroplasts and other organelles (see Puiseux-Dao, 1979; Koop & Kiermayer, 1980a). The growth and morphogenesis of Acetabularia are associated with a transcellular current (Novak & Bentrup, 1972; Goodwin & Pateromichelakis, 1979). This current is a result of the hyperpolarization of the apex (5–15 mV) as compared with the base; it is accompanied by spontaneous action potentials (Gradmann, 1976) occurring each 10 to 60 min, depending on the individual alga. The amplitudes of both electrical activities oscillate (Novak & Sironval, 1976; Dazy, et al 1980; Broda & Schweiger, 1981).