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Biomedical subjects

A Lombet

Publications and source records attributed to A Lombet.

At least 37 records · Page 2Linked to original sources
PubMed1987

Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel.

Binding studies indicate that ciguatoxin and brevetoxin allosterically enhance in a very similar way the binding of [3H]batrachotoxinin A 20-alpha-benzoate to the neuronal Na+ channel protein. Moreover ciguatoxin competitively inhibits the binding of [3H]brevetoxin-3 to rat brain membranes. The affinity of ciguatoxin for the Na+ channel is at least 20-50-times higher than that of brevetoxin. These results indicate that ciguatoxin and brevetoxins act at the same binding site on the sodium channel.

Animals↗
PubMed1987

Characterization of mitochondrial-uncoupling protein in bovine fetus and newborn calf.

Development changes in the content of the mitochondrial-uncoupling protein (UCP) have been studied in adipose depots of bovine fetuses and a newborn calf as well as in adipose depots of newborn and aging lambs. The occurrence of UCP unique to brown adipose tissue (BAT) was investigated by GDP binding, photoaffinity labeling with 8-azidoadenosine 5'-triphosphate, and immunoblots using specific antibodies directed against rat UCP. A protein of 32,000 relative molecular weight was characterized in both species with properties similar to those of rodent UCP. In bovine, UCP became detectable in the perirenal adipose tissue at day -80 and its content increased until birth. Both in bovine (perirenal, subscapular, and retroperitoneal sites) and in ovine (perirenal, subscapular, retroperitoneal, and pericardiac sites), all adipose tissues except the subcutaneous adipose tissue contained at birth UCP and thus can be considered as BAT. The data indicate that the perirenal adipose depot should play in bovine and ovine a major thermogenic role at birth, whereas perirenal and pericardiac adipose tissues of lambs held under cold conditions for 45 days after birth did not show any immunoreactive UCP.

Adipose Tissue↗
PubMed1986

The voltage-dependent sodium channel is co-localized with the acetylcholine receptor at the vertebrate neuromuscular junction.

Isolated motor endplates from mouse intercostal muscles can be obtained after subcellular fractionation. On these motor endplates, localization of the nicotinic receptor and of the voltage-dependent Na+ channel coincides as demonstrated by double labeling with rhodamine alpha-bungarotoxin and a specific anti-Na+ channel monoclonal antibody. High density of Na+ channel at the motor endplate is confirmed by the enrichment in TTX binding sites as compared to the crude homogenate. In contrast isolated motor endplates are almost completely devoid of Ca2+ channel antagonist binding sites.

Animals↗
PubMed1986

[3H]-tetrodotoxin binding in neuronal and non-neuronal spinal cord cultures.

The binding of [3H]-tetrodotoxin [TTX], a sodium channel blocker, was studied in dissociated spinal cord cultures derived from fetal mice. A comparison was made between cultures that consisted of a mixture of neurons and non-neuronal background cells (N + BG) with those that were comprised of only background cells (BG). Specific binding of 1 nM [3H]-TTX was studied in 28-day-old cultures. The IC50 for TTX displacement of [3H]-TTX binding was 10 nM for (N + BG) cultures and 15 nM for (BG). The binding of [3H]-TTX to (N + BG) cultures was approximately 9-fold greater than that observed for the (BG) cultures. During development from day 6 to day 28, the binding of [3H]-TTX in (N + BG) cultures increased about 10-fold per dish or about 30% as expressed as fmol bound per mg protein. Nitrendipine did not displace [3H]-TTX in day 6 (N + BG) cultures, although previous studies indicated that TTX displaced [3H]-nitrendipine binding in developing spinal cord cultures.

Animals↗
PubMed1986

The presence of Na+ channels in myometrial smooth muscle cells is revealed by specific neurotoxins.

This paper shows the presence, in rat myometrial smooth muscles, of low affinity binding sites for tetrodotoxin with a K0.5 value of 2 microM. Electrophysiological experiments using both intact strips and single isolated myometrial cells in culture have shown that veratridine and sea anemone toxins reveal functional Na+ channels. The activity of these channels was blocked by tetrodotoxin (10 microM) or by removal of Na+ ions. Results presented here are the first direct demonstration of the existence in rat myometrium of Na+ channels of the tetrodotoxin-resistant type.

Action Potentials↗
PubMed1986

Axonal transport of Na+,K+-ATPase identified as a ouabain binding site in rat sciatic nerve.

Na+,K+-ATPase levels were measured in different segments of rat sciatic nerves by in vitro binding of [3H]ouabain. Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve, indicating an anterograde and retrograde axoplasmic transport of Na+,K+-ATPase. Accumulation of Na+,K+-ATPase at the ligature was time dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of the ligature was also visualized by autoradiographic studies in longitudinal section of sciatic nerves using [3H]ouabain.

Animals↗
PubMed1985

Axonal transport of the voltage-dependent Na+ channel protein identified by its tetrodotoxin binding site in rat sciatic nerves.

Na+ channels levels were measured in different segments of rat vagus and sciatic nerves by in vitro binding using a tritiated ethylene-diamine tetrodotoxin derivative ([3H]en-TTX). Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve indicating an anterograde and retrograde axoplasmic transport of Na+ channels. Accumulation of Na+ channels at the ligature was time-dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of a ligature was also visualized by autoradiographic studies in longitudinal sections of sciatic nerves using [3H]en-TTX.

Animals↗
PubMed1985

Biochemical characterization of plasma membrane isolated from human skeletal muscle.

Specific components of ion translocation systems were studied in excitable plasma membranes isolated from normal human muscle. Na+-K+ ATPase and ouabain-sensitive K+ phosphatase activities were 8.9 +/- 1 mumol Pi/h per mg protein and 96 +/- 9 nmol/min per mg protein, respectively. Scatchard analysis of equilibrium binding assays with [3H]ouabain showed non-linear curves consistent with high- and low-affinity sites (estimated Kd 3 nM and 0.22 microM). Two families of receptors with different affinities for a tritiated TTX derivative (estimated Kd 0.4 and 4 nM) were also identified suggesting the existence in human muscle of at least two classes of voltage-dependent Na+ channels. In addition (+)-[methyl-3H]PN200-110, a potent Ca2+ antagonist used for labeling voltage-dependent Ca2+ channels, was observed to bind to a homogeneous population of receptors in the plasma membrane (Kd = 0.2 nM).

Calcium Channel Blockers↗
PubMed1984

Autoradiographic localization of tetrodotoxin-sensitive Na+ channels in rat brain.

The localization of tetrodotoxin-sensitive Na+ channels in rat brain was studied using a tritiated derivative of tetrodotoxin. The autoradiographic distribution of a tritiated ethylenediamine derivative of tetrodotoxin [( 3H]en-TTX) binding showed a high concentration of sites in cortical layers, hippocampus, globus pallidus, substantia nigra and the molecular layer of the cerebellar cortex. Lower levels were found principally in the striatum and median forebrain bundle. The white matter was not labelled. The characteristic distribution of tetrodotoxin-sensitive Na+ channels was compared to that of nitrendipine-sensitive Ca2+ channels and to that of apamin-sensitive Ca2+-dependent K+ channels.

Animals↗
PubMed1984

Characterization, solubilization, affinity labeling and purification of the cardiac Na+ channel using Tityus toxin gamma.

Saturable, high-affinity binding of iodinated toxin gamma from Tityus serrulatus scorpion venom (TiTx gamma) to Na+ channel receptor was identified in sarcolemma membrane of chick heart. A binding capacity of 450-600 fmol/mg of protein was found similar to that of tetrodotoxin-binding component. The enrichment of these membrane-bound toxin binding sites follows that of other sarcolemma markers. Kinetic data and displacement of 125I-TiTx gamma from its binding sites by unlabeled TiTx gamma gave an equilibrium dissociation constant (Kd) of 1-3 pM. The gating component and the selectivity filter of the voltage-sensitive Na+ channel, identified as binding sites of TiTx gamma and of tetrodotoxin respectively, have been efficiently solubilized with Nonidet P-40. Purification was achieved by ion-exchange chromatography on DEAE-Sephadex A-25, affinity chromatography on wheat-germ-agglutinin-Sepharose and sucrose density gradient centrifugation. An enrichment of 1400-fold from the original detergent extract was measured for both toxin binding sites (1120-1230 pmol/mg of protein). Sodium dodecyl sulfate gel electrophoresis reveals a single large polypeptide component of Mr230000-270000. The purified material exhibits an apparent sedimentation coefficient of 8.8S. Covalent cross-linking of 125I-TiTx gamma to its membrane-embedded cardiac receptor shows that the cross-linked material, solubilized and purified by the same procedure comprises a single polypeptide chain of the same Mr of 230000-270000. Furthermore, as seen for Electrophorus electricus electroplax and rat brain, the tetrodotoxin-binding component and the TiTx gamma-binding component are carried by the same polypeptide chain. The functional Na+ channel might be an oligomer of this subunit of Mr23000-270000.

Affinity Labels↗
PubMed1984

Paranodal dysmyelination and increase in tetrodotoxin binding sites in the sciatic nerve of the motor end-plate disease (med/med) mouse during postnatal development.

Motor end-plate disease (med), in the mouse, is a hereditary neuromuscular defect, caused by a single gene mutation and characterized by a progressive muscle weakness. +Med/+med mice die 21-23 days after birth and the neurobiological abnormalities already reported are nerve terminal sprouting and swelling and neurotransmission failures. We studied +med/+med mice at preclinical (9-11 days after birth) as well as at clinically recognized stages of the disease. The nonmyelinated gaps of the nodes of Ranvier in the +med/+med sciatic nerve are found to be significantly widened in +med/+med animals compared to control littermates, even in the preclinical stage, although the nodes of Ranvier are not yet ultrastructurally mature. The maximal binding capacity for [3H]ethylene-diamine tetrodotoxin, expressed in femtomoles per milligram of protein, is significantly increased in +med/+med sciatic nerves. Thus, Na+ channels, which are known to be located mainly at the nodes of Ranvier in normal myelinated axons, are increased in number in +med/+med mice even before the disease becomes clinically established. Both the ultrastructural and biochemical developmental abnormalities of the node of Ranvier rapidly approach their maximal expression as the behavioral signs develop. Such nerve abnormalities may be closely related to the physiological impairment of nerve impulse conduction which leads to the pathophysiological expression of motor end-plate disease.

Aging↗
PubMed1984

New scorpion toxins with a very high affinity for Na+ channels. Biochemical characterization and use for the purification of Na+ channels.

Biochemical characterization of the Tityus gamma toxin receptor associated with the voltage-sensitive Na+ channel was carried out in different tissue preparations with the use of an iodinated toxin derivative. The affinity of the toxin for the receptor is high with a dissociation constant of 4 X 10(-12) M for rat synaptosomes. The density of binding sites is in the range of 0.3 to 2 pmol/mg of protein. Toxin gamma does not seem to bind to Na+ channels located on transverse-tubule membranes of skeletal muscle, but only to Na+ channels located on the sarcolemma. Both affinity labelling and radiation inactivation analysis indicate a molecular weight for the toxin receptor of 270 000 daltons. The same molecular weight is found using the tetrodotoxin. Only one single major protein component of the Na+ channel was purified from Electrophorus electroplax, rat brain membranes and chick heart membrane using the toxin gamma as a marker. The molecular weight of this component is 230 000-270 000 daltons. Reconstitution of the purified Na+ channel into planar lipid bilayers has been carried out. Two different types of electrically excitable channels with conductances of 150 and 25 pS were detected. The activity of both channels is blocked by saxitoxin.

Animals↗
PubMed1983

The Na+ channel in mammalian cardiac cells. Two kinds of tetrodotoxin receptors in rat heart membranes.

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.

Animals↗
PubMed1983

Affinity labelling of the tetrodotoxin-binding component of the Na+ channel.

Tetrodotoxin-binding sites were covalently labelled with a highly tritiated derivative of tetrodotoxin. Cross-linking experiments, using dissucinimidyl suberate, on partially purified tetrodotoxin-binding component from electroplax of Electrophorus electricus, revealed covalent labelling of a single polypeptide chain of MW 270,000.

Affinity Labels↗
PubMed1983

Complete monitoring of the purification of the plasma membrane from rabbit skeletal muscle.

A fast and reproducible purification procedure for rabbit skeletal muscle plasma membrane is described. Each step was monitored by determination of tetrodotoxin, ouabain and insulin receptors. A ouabain-sensitive K+-stimulated and a Ca2+-dependent phosphatases, probably identical to, respectively the (Na+-K+) and Ca2+-ATPases, were also evaluated. All plasma membrane receptors and the ouabain-sensitive activity accumulated in the lightest fraction separated by sucrose gradient centrifugation (peak at 18% sucrose; purification from crude homogenate, 30-fold).

Animals↗
PubMed1983

Ontogenic appearance of Na+ channels characterized as high affinity binding sites for tetrodotoxin during development of the rat nervous and skeletal muscle systems.

The appearance of the voltage-dependent Na+ channel during the fetal and post-natal development of rat brain, cerebellum and skeletal muscle has been followed using a highly radiolabelled derivative of tetrodotoxin. The number of Na+ channels is low at the fetal stage and increases drastically during post-natal development. The time-course of this increase is different in brain, cerebellum and skeletal muscle. Changes in affinity of the Na+ channel for tetrodotoxin occur during brain and cerebellum development. The results are discussed in relation with the maturation of the three types of excitable tissues.

Aging↗
PubMed1983

Molecular size of different neurotoxin receptors on the voltage-sensitive Na+ channel.

Measurements were made of the molecular sizes of two distinct receptors on the Na+ channel in rat brain synaptosomes that are specific for different neurotoxins. Radiation inactivation of the binding of radiolabeled derivatives of the toxins was consistent with Mr = 260,000 for the tetrodotoxin receptor and Mr = 266,000 for the receptor specific for two scorpion toxins, toxin II from Centruroides suffusus suffusus and toxin gamma from Tityus serrulatus serrulatus. Covalent cross-linking of the latter to its receptor similarly indicated Mr = 270,000. It seems most likely that these two distinct receptors reside on the same molecule.

Amphibian Proteins↗