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A Lombet

Publications and source records attributed to A Lombet.

53 records · Page 3Linked to original sources
PubMed1983

Electrophysiological characterization, solubilization and purification of the Tityus gamma toxin receptor associated with the gating component of the Na+ channel from rat brain.

Electrophysiological studies with neuroblastoma cells have shown that toxin gamma from the venom of the scorpion Tityus serrulatus is a new toxin specific for the gating system of the Na+ channel. The procedure which solubilizes the tetrodotoxin receptor from rat brain also solubilizes the Tityus gamma toxin receptor. Binding experiments on the solubilized receptor with a radioiodinated derivative of Tityus gamma toxin have shown: (i) that the TiTx gamma-receptor complex is very stable with a dissociation constant of 8.6 X 10(-12) M and a very slow dissociation (T 1/2 = 15 h); (ii) that the toxin recognizes a class of sites with a 1:1 stoichiometry with those for tetrodotoxin (Bmax = 1.3 pmol/mg protein). The radioiodinated Tityus gamma-receptor complex has been substantially purified by ion-exchange chromatography, lectin affinity chromatography and sucrose gradient sedimentation. A ratio of one Tityus gamma toxin binding site per tetrodotoxin binding site was found throughout the purification. The purified material exhibited a sedimentation coefficient of 10.4S and had an apparent mol. wt. of 270 000 on SDS-gel electrophoresis. No other polypeptide chains were demonstrated to be associated with this large protein in the Tityus gamma receptor. The main conclusion is that the tetrodotoxin binding site associated with the selectivity filter of the Na+ channel and the Tityus gamma toxin binding site associated with the gating component are probably carried by the same polypeptide chain.

Action Potentials↗
PubMed1983

Differences in the properties of Na+ channels in muscle surface and T-tubular membranes revealed by tetrodotoxin derivatives.

The effect of ethylenediamine derivatives of tetrodotoxin (enTTXI and enTTXII) on frog skeletal muscle was studied both electrophysiologically and biochemically. Electrophysiological experiments with one of these molecules (enTTXI) showed that the concentrations needed to block the early phase of the inward sodium current (K0.5 = 7 nM) are much lower than those needed to block the late phase of inward current or muscle contraction (K0.5 = 40 nM). Conversely, tubular Na+ channels are more sensitive to enTTXII than are surface Na+ channels. Toxin binding to isolated muscle membranes was studied using 3H-enTTXI and 3H-enTTXII. The first derivative (3H-enTTXI) has a higher affinity Kd = 8 nM) for Na+ channels in the surface membrane than for Na+ channels in the T-tubular membrane (Kd greater than 20 nM). In contrast 3H-enTTXII has a higher affinity for the tubular Na+ channel (Kd = 0.2 nM) than for the receptor in surface membranes (Kd = 4nM). We conclude that Na+ channels in muscle surface and T-tubular membranes have different toxin-binding properties, which must reflect a difference in molecular structure.

Animals↗
PubMed1983

Purification of binding protein for Tityus gamma toxin identified with the gating component of the voltage-sensitive Na+ channel.

The gating component associated with the voltage-sensitive Na+ channel from electroplax membranes of Electrophorus electricus has been purified by using toxin gamma from the venom of the scorpion Tityus serrulatus serrulatus. The toxin-binding site was efficiently solubilized with Lubrol PX, resulting in an extract of high initial specific activity. Purification was achieved by adsorption of the toxin-binding component to DEAE-Sephadex A-25 followed by desorption at high ionic strength and chromatography on either wheat germ agglutinin-Ultrogel or Sepharose 6B. Maximal final specific activities were at least 42% of the specific activity expected for a pure toxin-binding component. The purified material exhibited a Stokes radius of 85 A, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated a single polypeptide component of Mr 270,000. Furthermore, tetrodotoxin binding activity and Tityus gamma toxin binding activity copurified, suggesting that the selectivity filter and the gating component of the Na+ channel are carried by the same polypeptide chain.

Animals↗
PubMed1982

Normal serum and lipoprotein-deficient serum give different expressions of excitability, corresponding to different stages of differentiation, in chicken cardiac cells in culture.

Monolayers of cardiac cells from 11-day-old chicken hearts have different properties when maintained in fetal calf serum or in a lipoprotein-deficient serum (LPDS). Cells in fetal calf serum have a resting potential near -60 mV; the rate of rise of the action potential is low (less than 10 V/sec); the action potential and the contraction are essentially unaffected by tetrodotoxin (TTX); and the beating properties are unaffected by muscarinic agents. Cells in LPDS have a resting potential near -75 mV, and a fast rise of the action potential (approximately equal to 100 V/sec) that is drastically decreased by TTX with a parallel abolition of contraction, and the beat is blocked by very low concentrations of muscarinic agonists. Cells that are physiologically fully responsive to TTX and to muscarinic agents have receptors that remain stable 24 hr after protein synthesis is blocked, whereas cells that are physiologically unresponsive to TTX and muscarinic agents have receptors that are rapidly degraded with half-lives between 9 hr (TTX receptor) and 14 hr (muscarinic receptor). Differences in the physiological and biochemical properties are accompanied by changes in the cholesterol contents of the cell membranes. The properties of cardiac cells cultured in normal serum are similar to those found for cells of chicken hearts in the very early embryonic stage, whereas those of cardiac cells cultured in LPDS correspond to the late embryonic stage.

Animals↗
PubMed1982

Na+ channels with binding sites of high and low affinity for tetrodotoxin in different excitable and non-excitable cells.

The properties of interaction of tetrodotoxin with its receptor site on the voltage-sensitive Na+ channel were analysed in two ways: (a) by titrating Na+ channels with a tetrodotoxin derivative, [3H]ethylenediamine-tetrodotoxin; (b) by studying the physiological properties of interaction of the toxin with its receptor from 22Na flux measurements. Using a variety of cell types in culture, three different kinds of situations were observed. 1. Cells like N1E 115 neuroblastoma, CCl 39 fibroblasts, embryonic chick cardiomyocytes and chick skeletal myotubes only have one family of Na+ channels with high-affinity binding sites (in the nanomolar range) for tetrodotoxin. These Na+ channels are the same ones as those that are activated by the alkaloid and polypeptide toxins that accelerate 22Na+ influx. 2. C9 cells have Na+ channels with low-affinity binding sites for tetrodotoxin. These Na+ channels are also the ones that are activated by alkaloid and polypeptide toxins (the median inhibitory concentration for tetrodotoxin inhibition of 22Na+ influx through these Na+ channels in 300 nM). 3. Rat myotubes that have differentiated in culture in the absence of neuronal influence have both high-affinity binding sites (in the nanomolar range) detected with the tritiated tetrodotoxin derivative and low-affinity binding sites (on the micromolar range) detected by 22Na+ flux experiments. Only low-affinity binding sites correspond to Na+ channels that can be activated with alkaloid and polypeptide toxins.

Animals↗
PubMed1982

Sodium channel and sodium pump in normal and pathological muscles from patients with myotonic muscular dystrophy and lower motor neuron impairment.

TWO SODIUM TRANSPORT SYSTEMS HAVE BEEN ANALYZED IN THIS WORK: the voltage-sensitive sodium channel and the (Na(+), K(+)) ATPase pump. The sodium channel has been studied using a tritiated derivative of tetrodotoxin; the sodium pump has been studied using tritiated ouabain. Properties of interaction of tritiated tetrodotoxin and of tritiated ouabain with their respective receptors were observed in normal human skeletal muscle and in muscles of patients with myotonic muscular dystrophy and with lower motor neuron impairment. Levels of sodium pump and of sodium channels were measured at different stages of membrane purification. Microsomal fractions of normal human muscle have maximal binding capacities for tetrodotoxin of 230 fmol/mg of protein and of 7.4 pmol/mg of protein for ouabain. Dissociation constant for the complexes formed by the tetrodotoxin derivative and by ouabain with their respective receptors were 0.52 nM and 0.55 muM, respectively. In muscles from patients with myotonic muscular dystrophy, the maximal binding capacity for tetrodotoxin, i.e., the number of Na(+) channels was found to be very similar to that found for normal muscle. The maximal binding capacity for ouabain, i.e., the number of Na(+) pumps was three- to sixfold lower than in normal muscle. Dissociation constants for the complexes formed with the tetrodotoxin derivative and with ouabain were the same as for normal muscle. In muscles from patients with lower motor nerve impairment, the maximal binding capacities for tetrodotoxin and for ouabain were twice as high as in normal muscle. Again, dissociation constants for the complexes formed with the tetrodotoxin derivative and with ouabain were nearly unchanged as compared with normal muscle. These results suggest that sodium transport systems involved in the generation of action potentials and/or in the regulation of the resting potential are altered both in myotonic muscular dystrophy and in lower motor neuron impairment.

Adult↗
PubMed1981

Synthesis and properties of new photoactivable derivatives of tetrodotoxin.

The Pfitzner-Moffatt oxidation procedure has been used to prepare two new photoactivable derivatives of tetrodotoxin that have been synthesized with high specific radioactivities (17.5 Ci/mmol and 30 Ci/mmol). They specifically bind to axonal membranes with affinities of 5.2-14.2 nM. They dissociate from their membrane complex with half-lives of 10.8 and 20 min. In the dark, these compounds give a reversible block of the sodium channels. After ultraviolet irradiation, they induce an irreversible blockade of the nerve channels.

Animals↗
PubMed1981

Differentiation of the fast Na+ channel in embryonic heart cells: interaction of the channel with neurotoxins.

The sensitivity of embryonic cardiac cells to tetrodotoxin (TTX) increases with age. At the early embryonic stage, the maximum upstroke velocity is not affected by the presence of TTX. In the course of both in ovo and in vitro development, this velocity reaches an adult-like value of 90-120 V/sec, which is decreased in the presence of TTX to 5-10 V/sec. The differentiation of the Na+ channel has been followed by using three types of specific toxins: (i) TTX or a tritiated derivative of it, (ii) a polypeptide toxin extracted from sea anemone, and (iii) the alkaloidic toxins veratridine and batrachotoxin. Electrophysiological, including voltage-clamp experiments, and biochemical studies have shown (i) that the TTX receptor and the fast Na+ channel machinery exist even when action potentials are insensitive to TTX--the channel is then in a nonfunctional or silent form that is revealed (or chemically activated) by both the alkaloids and the polypeptide toxin--and (ii) that the total number of Na+ channels increases during development by a factor of 4 or 5. In monolayers of cardiac cells insensitive to TTX in which all Na+ channels are in a nonfunctional form, the rate of degradation of the TTX receptor follows first-order kinetics with a half-time of 9 hr. In aggregates fully sensitive to TTX, the number of TTX receptors remains perfectly stable 24 hr after blockade of protein synthesis.

Action Potentials↗
PubMed1980

Synthesis of new, highly radioactive tetrodotoxin derivatives and their binding properties to the sodium channel.

The Pfitzner-Moffatt oxidation procedure has been used to prepare five derivatives of tetrodotoxin by covalent attachment of the oxidized toxin to lysine, glycine, beta-alanine or ethylenediamine. These derivates reach specific radioactivities between 5 and 45 Ci/mmol. The equilibrium properties of binding of these tetrodotoxin derivatives to membrane-embedded or solubilized sodium channels from crustacean nerves have been analysed and compared to the binding properties of tritiated tetrodotoxin and saxitoxin to the same biological systems. All these highly radiolabelled derivatives associate to the tetrodotoxin binding component with properties similar to those of tetrodotoxin itself. The synthetic route described in this paper may be used to prepare other types of tetrodotoxin derivatives such as fluorescent derivatives for example.

Animals↗
PubMed1979

Synthesis and mode of action on axonal membranes of photoactivable derivatives of tetrodotoxin.

Two photoactivable derivatives of tetrodotoxin have been synthesized. Electrophysiological experiments on crab giant axons and competitive binding with [3H]-tetrodotoxin for the tetrodotoxin receptor indicate that they are only 4.5 to 7.5 times less active than tetrodotoxin itself. These compounds give a reversible block of the sodium channel in the dark but after ultraviolet irradiation they provoke an irreversible blockade of the channel.

Animals↗
PubMedDate not supplied

Proapoptotic effects of antiestrogens, progestins and androgen in breast cancer cells.

The promoting action of E2 in breast cancer cells has been, until now, mainly linked to its action on prolifieration. Because of the importance of an increase in apoptosis in breast cancer prevention, we have studied the possible effects of various antiestrogens, progestins and an androgen on its occurrence in three hormone-dependent breast cancer cell lines. The antiestrogens were, a triphenylethylene derivative, 4 hydroxytamoxifen (4OHTAM) and two steroidal antiestrogens, IC1182780 and RU58668. The progestins were Org2058, a pregnane derivative, tibolone (OrgOD14), a normethyltestosterone derivative and OrgOM38 (the delta4 isomer of OrgOD14) and the androgen dihydrotestosterone (DHT). Apoptosis was studied in MCF-7, ZR75-1 and T47-D cells using morphological approaches and flow cytometry. The antiestrogens, the progestins and DHT were proapoptotic but to different potencies according to the cell line studied. Indeed, the 'pure' steroidal antiestrogens were more efficient than 4OHTam in increasing apoptosis. We have also studied the level of expression of some of the proteins involved in the regulation of apoptosis. Bcl-2 and bcxL, two antiapoptotic members of the bcl-2 family proteins, were inhibited by the progestins and the antiestrogens. In contrast, the proapoptotic proteins, bax and bak seemed to be constitutively expressed. Thus, since the ratio of proapoptotic and antiapoptotic proteins determines apoptosis or cell survival, the hormone effects are operating by modulating the antiapoptotic regulators of the balance. These data demonstrate that antiestrogens, progestins, and androgens can promote apoptosis in breast cancer cells, an effect which could be of importance in the therapeutic prevention of breast cancer.

Apoptosis↗