Free calcium and calpain I activity.

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

[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.

Movements of fluorescent probes in the mechanism of cell fusion induced by poly(ethylene glycol).

It has been claimed that purified poly(ethylene glycol) (PEG) is able only to aggregate cells and not to fuse them. In our hands, purified PEG 6000 (recrystallized/dialysed) induces both aggregation and fusion of human erythrocytes, and the mechanism of fusion by the purified polymer has been investigated with fluorescent probes. No movement of a carbocyanine probe or of octadecyl rhodamine B chloride from labelled to unlabelled cells occurred in the absence of PEG or with cells treated with concanavalin A, protamine or spermine. With 40% PEG, however, both probes immediately started to diffuse into the membranes of unlabelled cells. This indicates that continuity between the phospholipid bilayer membranes of adjacent erythrocytes (i.e. membrane fusion) is established within seconds in concentrated solutions of the polymer, and precedes the cell fusion event that is induced by purified PEG. These observations are consistent with the idea that micro-regions of shared phospholipid bilayer may be formed in the membranes of cells when they are forced together as a consequence of the dehydrating action of PEG. Intact erythrocytes were cytoplasmically labelled with 6-carboxyfluorescein to avoid the possibility that loading the cells with a cytoplasmic marker by hypotonic haemolysis might modify their response to PEG. Unlike the lipid probes, carboxyfluorescein did not diffuse from labelled to unlabelled cells in the presence of 40% PEG, and there was little diffusion on subsequent dilution of the polymer solution to 13%. However, after the PEG solution had been replaced by an isotonic buffer, a rapid transfer of the cytoplasmic fluorophore to unlabelled cells often occurred. This is considered to be more consistent with the osmotic rupture of a membranous barrier, such as a shared bilayer, between the labelled and unlabelled cells than with the return of cytoplasmic viscosity to normal when the PEG is removed. Possible reasons are discussed for the reported inability of purified PEG to fuse fibroblasts with hypotonically loaded human erythrocytes.

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration.

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.

[Relation between the axonal membrane microfluidity and excitability]. / Relations entre la microfluidité des membranes axonales et l'excitabilité.

The olfactory nerve of the garfish, the rabbit vagus nerve and the sciatic nerve of the frog labelled with pyrene or triethylammonium-butyl-pyrene show during the action potential a transient decrease in the Ie/Im ratio which suggest a small transient decrease in nerve membrane fluidity.

Modifications of membrane cholesterol content affects electrical properties and prolactin release of cultured pituitary cells.

Treatment of cloned pituitary cells (GH3/B6) with cholesterol-enriched liposomes, which presumably increases membrane cholesterol content, affects the passive and active electrophysiological properties and stimulates the release of prolactin (PRL).

The relationship between the membrane potential of neurosecretory nerve endings, as measured by a voltage-sensitive dye, and the release of neurohypophysial hormones.

The membrane potential of isolated rat neurohypophyses and isolated neurosecretosomes (neurosecretory nerve endings) was monitored with the voltage sensitive fluorescent probe diS-C3-(5). K ions, in contrast to Na or Cl ions, give rise to large changes of the fluorescent signal. The fluorescent response is linearly related to log[K+]0 at values higher than 10 mM, whereas at lower [K+]0 the permeability of the membrane for Na ions has to be taken into account. Veratridine increases the fluorescent signal only in the presence of external sodium; this effect is blocked by tetrodotoxin. After prolonged K-induced depolarisation, addition of veratridine to the medium gives a further change in fluorescence of diS-C3-(5) associated with release of vasopressin. Vasopressin release from isolated neurohypophyses started to increase significantly only above 25 mk [K+]0, while the depolarization of the membrane was linearly related to log[K+]0. The results are consistent with the view that neurosecretory nerve endings have voltage-dependent calcium channels that regulate the amount of hormone released during depolarisation.

Electrophysiological modifications induced by the fluorescent probe, pyrene, on Myxicola giant axons.

Current- and voltage-clamp experiments on Myxicola giant axons labelled with pyrene showed decreased Na+ and K+ conductances. The low-frequency membrane capacity and the gating charge transfer were slightly reduced. It may be inferred that pyrene is incorporated in some hydrophobic membrane domains close to the ionic channels.

[Release of hydrogen peroxide by peritoneal macrophages from mice infested with Trypanosoma musculi]. / Libération de peroxyde d'hydrogène par les macrophages péritoneaux de Souris infestées par Trypanosoma musculi.

Peritoneal macrophages from Mice infested with Trypanosoma musculi release large amounts of hydrogen peroxide. In Mice infested with T. musculi, increase in hydrogen peroxide release induced by P.M.A. is well correlated with the course of parasitemia, suggesting acute control mechanisms.

Temperature dependence of the fluorescence of pyrene labeled crab nerve membranes.

A method, using albumin-pyrene complexes, has been developed for labeling, in a controlled manner, crab leg nerves whose excitability was preserved. The excimer-to-monomer fluorescence intensity ratio of pyrene, embedded in nerve membrane lipids and in their crude lipid extracts, is a fluidity parameter which displayed the following features with temperatures. a--a temperature-dependent increase of fluidity b--three breaks (6 degrees, 19 degrees and 37 degrees C) in the physiological medium c--In Ca++-depleted sea water, the 37 degrees characteristic temperature vanished. These breaks may reflect some lateral phase separations of the lipid components of nerve membranes. The calcium dependent temperature break may involve a segregation of acidic phospholipids while the other two breaks (6 degrees and 19 degrees C) may be due to neutral lipids phase separation. The relationship of these findings to nerve function is discussed.