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G Romey

Publications and source records attributed to G Romey.

At least 55 records · Page 3Linked to original sources
PubMed1992

Different types of K+ channel current are generated by different levels of a single mRNA.

A cloned human voltage-sensitive K+ channel HLK3 which is present in T-lymphocytes and in the brain was expressed in Xenopus oocytes and after permanent transfection of a human B-lymphocyte cell line (IM9). Injections of low cRNA concentrations into Xenopus oocytes led to the expression of a transient K+ current, with saturating current-voltage (I-V) relationship, which was abolished by repetitive stimulations due to a slow recovery from inactivation. This transient K+ channel current was fully inhibited by 10 nM charybdotoxin. Injection of high concentrations of the same RNA led to a non-inactivating K+ current, with linear I-V curve, which did not undergo use-dependent inactivation and was hardly sensitive to 10 nM charybdotoxin. Intermediate behaviour due to changing proportions of these two types of K+ channel expression were observed at intermediate RNA concentrations. Transient and non-inactivating K+ currents were also observed by both whole-cell and single channel patch-clamp recording from HLK3 transfected IM9 cells. The main conductance of the channel in the two different modes (inactivating and charybdotoxin-sensitive or non-inactivating and charybdotoxin-resistant) is the same (12-14 pS). Destruction of the cytoskeletal elements with cytochalasin D, colchicine or botulinum C2 toxin in oocyte experiments prevented expression of the sustained mode of the K+ channel. The results suggest that the sustained mode obtained at high RNA concentrations corresponds to channel clustering involving cytoskeletal elements. This differential functional expression of K+ channels associated with different levels of mRNA appears as a new important factor to explain the biophysical and pharmacological diversity of voltage-sensitive K+ channels.

Animals↗
PubMed1992

Adriamycin promotes neurite outgrowth in the "neurite-minus" N1A-103 mouse neuroblastoma cell line.

Adriamycin, an anticancer agent acting on topoisomerase II, promotes the arrest of cell division and neurite extension in a "neurite-minus" murine neuroblastoma cell line, N1A-103. This morphological differentiation is accompanied by a blockade in the S phase of the cell cycle, modification of the amount of peripherin, and appearance of the beta 7-tubulin isoform. Yet, adriamycin-induced N1A-103 cells fail to express other neuronal markers, such as long-lasting Ca2+ channels, synaptophysin, and the shift in the proportion of the beta'1 tubulin isoform to the beta'2 isoform, whose appearance parallels the terminal differentiation of the wild type neuroblastoma cell line N1E-115. Hence, a comparison of the behavior of these two cell lines leads to the proposal that there are two programs of neuroblastoma differentiation: one where expression is triggered by the arrest of cell division and which is observed in adriamycin-induced N1A-103 variant cells, and the other, presumably occurring further downstream, which would involve further changes in morphogenesis and acquisition of new electrophysiological properties.

Animals↗
PubMed1991

Cloning, expression, pharmacology and regulation of a delayed rectifier K+ channel in mouse heart.

Neonatal mouse cardiac poly(A)+ mRNA microinjection into Xenopus oocytes directed the expression of a delayed rectifier K+ current. A cDNA encoding this channel, called mIsK, was cloned from a neonatal mouse heart cDNA library whose properties were studied after expression of the complementary RNA in Xenopus oocytes. Among the different known K+ channel blockers, only the class III antiarrhythmic clofilium inhibited mIsK in the 10-100 microM range. The channel was completely insensitive to other antiarrhythmics such as quinine, quinidine, sotalol or amiodarone. mIsK was enhanced by increasing intracellular Ca2+ and by microinjected Ca(2+)-calmodulin dependent protein kinase II. These stimulations were reversed by the calmodulin antagonist W7. Conversely, the phorbol ester PMA, the diacylglycerol analog OAG and microinjected purified protein kinase C inhibited mIsK. This inhibitory effect could be prevented by the protein kinase C inhibitor staurosporine. These results were consistent with the presence of consensus sequences for kinase II and kinase C in the mIsK structure. Cultured newborn mouse ventricular cardiac cells exhibited a delayed rectifier K+ current which had biophysical properties similar to those of cloned mIsK and which was inhibited by clofilium and protein kinase C activators. In situ hybridization experiments revealed that mIsK mRNA was homogeneously distributed in the cardiac tissue. Neonatal mouse heart expressed the most mIsK mRNA compared with various other rat and mouse tissues. Since this K+ channel generates a current which appears to be involved in the control of both the action potential duration and the beating rate, these results suggest an important role for the mIsK channel in cardiac cell physiology and cardiac pathology.

Amino Acid Sequence↗
PubMed1991

Indolizinsulphones. A class of blockers with dual but discriminative effects on L-type Ca2+ channel activity and excitation-contraction coupling in skeletal muscle.

The alpha 1 subunit of the L-type Ca2+ channel plays a dual role in skeletal muscle. It is essential both for L-type Ca2+ channel activity and for the functioning of the voltage-sensor structure that is situated in the triads as a key element for excitation-contraction coupling. This paper shows, with mouse muscle cells in primary culture, that indolizinsulphone SR33557 which has its binding site on the alpha 1 subunit blocks both L-type Ca2+ channel activity and contraction as the more classical 1,4-dihydropyridine blockers. However, unlike other Ca2+ channel blockers, it can pharmacologically discriminate between the two different roles of the alpha 1 subunit. SR33557 inhibition of both contractile and L-type Ca2+ channel activities is very voltage dependent and increases at depolarized potentials. Complete blockade of contraction was observed at low SR33557 concentrations (K0.5 = 20 nM) and was associated with only minor L-type Ca2+ channel blockade (30%). The remaining and major part of the L-type Ca2+ channel activity (70%) was blocked at much higher SR33557 concentrations (K0.5 = 0.6 microM). The results indicate that SR33557 has a much higher affinity for the alpha 1 subunit inserted into the voltage-sensor structure. They also suggest that the voltage-sensor structure, which probably includes most of the total T-tubule alpha 1 subunit, has intrinsic (but relatively small) Ca2+ channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.

Animals↗
PubMed1990

Induction of normal ultrastructure by CGRP treatment in dysgenic myotubes.

The calcitonin gene-related peptide (CGRP) restores an apparent normal ultrastructure in mdg/mdg muscle cells in vitro, including a normal triadic organization which is known to be essential for excitation-contraction (E-C) coupling. However, neither slow L-type Ca2+ channel activity nor E-C coupling, which are absent in mdg/mdg muscle, were re-established. These observations suggest a potential role of CGRP (and also of cAMP as the intracellular messenger) in the morphological development of the muscle fiber.

Animals↗
PubMed1990

Leiurotoxin I (scyllatoxin), a peptide ligand for Ca2(+)-activated K+ channels. Chemical synthesis, radiolabeling, and receptor characterization.

Leiurotoxin I (scyllatoxin) is a 31-amino acid polypeptide from the venom of the scorpion Leiurus quinquestriatus hebraeus which has been previously isolated and sequenced by others. This paper reports (i) the total synthesis of this scorpion neurotoxin as well as some aspects of its structure-function relationships; (ii) the synthesis of the analog [Tyr2]leiurotoxin I (scyllatoxin) that has been monoiodinated at high specific radioactivity (2000 Ci/mmol) and has served for the characterization of the properties of 125I-[Tyr2]leiurotoxin I binding sites (Kd = 80 pM, molecular mass of 27 and 57 kDa for two polypeptides in the leiurotoxin I binding protein); (iii) the similarity of physiological actions between leiurotoxin I and apamin. Both toxins contract Taenia coli previously relaxed with epinephrine, both toxins block the after-hyperpolarization due to Ca2(+)-activated K+ channel activity in muscle cells in culture; (iv) the probable identity of binding sites for apamin and leiurotoxin I. In spite of a different chemical structure apamin competitively inhibits 125I-[Tyr2] leiurotoxin I binding and vice versa. Moreover, the peculiar effects of K+ on 125I-[Tyr2]leiurotoxin I binding are identical to those previously observed for 125I-apamin binding.

Amino Acid Sequence↗
PubMed1990

A genetic model for the study of abnormal nerve-muscle interactions at the level of excitation-contraction coupling: the mutation muscular dysgenesis.

Excitation-contraction in muscle fibers are coupled through a complex mechanism involving multiproteic components located at a specialized cellular site, the triadic junction. Triads in normal muscle fiber result from the apposition of sarcoplasmic reticulum citernae and T-tubule and possess strikingly organized ultrastructural elements, bridging both types of membranes, the "junctional feet". Muscular dysgenesis in the mouse is characterized by total muscle inactivity in the developing skeletal muscles due to excitation-contraction uncoupling. Triads have been found to be disorganized with no "junctional feet" and dihydropyridine (DHP) binding sites are decreased with no slow Ca2+ currents, suggesting a basic defect in the excitation-contraction coupling machinery itself. We may hypothesize that muscular dysgenesis results in a marked defect in a functional protein involved in the morphogenesis of the triad and/or directly involved in Ca2+ release for contraction.

Action Potentials↗
PubMed1989

Ontogenesis and localization of Ca2+ channels in mammalian skeletal muscle in culture and role in excitation-contraction coupling.

The mechanism of excitation-contraction (E-C) coupling in skeletal muscle is not yet well established. Cultured mouse skeletal muscle cells have been used to study the relationships between triad formation, Ca2+ channel activities, and contractions. The ontogenesis of voltage-dependent Ca2+ channels and their localization in relation to the ability of muscle to contract and the ultrastructural organization of sarcomeres and triads have been investigated by using an electrophysiological approach together with an electron microscope study. At an early stage of development, both fast (Ifast) and slow (Islow) types of Ca2+ channels are found at the surface membrane. At later stages of development, fast Ca2+ channels remain at the surface membrane, while slow Ca2+ channels migrate to the transverse-tubule membrane. The voltage dependence of fast Ca2+ channels compared to the voltage dependence of contraction clearly shows that these Ca2+ channels have no direct role in E-C coupling. Detubulation at all stages of development has confirmed that T tubules contain essential elements for E-C coupling. However, this work also shows that Ca2+ flowing through slow Ca2+ channels situated in the T-tubular system is not important for contraction. Myotubes lacking slow Ca2+ channels or having no slow Ca2+ channel transport activity (jumps to high membrane potentials, no external Ca2+, block of Islow by Co2+) still retain contraction.

Animals↗
PubMed1989

SR33557, an indolizinsulfone blocker of Ca2+ channels: identification of receptor sites and analysis of its mode of action.

SR33557 belongs to a new class of molecules (indolizinsulfones) that act on the same receptor complex that has been characterized for other classical calcium channel effectors. The main binding properties of SR33557 to rabbit skeletal muscle are as follows. (i) Unlabeled SR33557 completely inhibits the specific binding of all classes of calcium channel antagonists such as dihydropyridines [(+)-[3H]PN200-110], phenylalkylamines ([3H] verapamil), benzothiazepines (d-(cis)-[3H]diltiazem), and diphenybutylpiperidines ([3H]fluspirilene). In all these cases inhibition of binding is of a noncompetitive nature. (ii) [3H]SR33557 binds with high affinity to T tubule membranes (KD = 0.08 nM) and the maximum binding capacity (Bmax = 78 pmol/mg of protein) is the same as that found for other classes of Ca2+ channel antagonists. Photoaffinity labeling confirms that [3H]SR33557 associates with the same protein of Mr 165,000 that binds the classical calcium channel inhibitors. 45Ca2+ uptake experiments performed with the rat aortic cell line A7r5, the insulin-secreting cell line RINm5F, and the pheochromocytoma cell line PC12 demonstrate that SR33557 fully inhibits the 1,4-dihydropyridine-sensitive 45Ca2+ uptake elicited by depolarization. A very good correlation was found between inhibition of 45Ca2+ uptake and of [3H]dopamine release in PC12 cells and between inhibition of 45Ca2+ uptake and of L-type Ca2+ current in A7r5 cells under whole-cell patch-clamp conditions.

Adrenal Gland Neoplasms↗
PubMed1989

Molecular properties of potassium channels.

The paper describes the molecular pharmacology and biochemistry of three types of K+ channels, the calcium-activated potassium channels, ATP-regulated potassium channels and voltage-sensitive potassium channels.

Adenosine Triphosphate↗
PubMed1988

Targets for calcium channel blockers in mammalian skeletal muscle and their respective functions in excitation-contraction coupling.

The L-type Ca2+ channel is blocked by 1,4-dihydropyridines (DHP), by phenylalkylamines, by diphenylbutylpiperidines or by benzolactams. We first show with mouse muscle cells in culture that all these L-type Ca2+ channel blockers block contraction. However, voltage-clamp analysis associated to contraction measurements also clearly show that Ca2+ influx through L-type Ca2+ channels is not required for contraction. Therefore, there is a need for a voltage-sensor which would be responsible for the excitation-contraction (E-C) coupling. We are showing here that the voltage-sensor involved in E-C coupling and the L-type Ca2+ channel have a similar pharmacology. Some of the blockers used are more active on the voltage sensor, others on the L-type Ca2+ channel.

Action Potentials↗
PubMed1988

Vasopressin modulates the spontaneous electrical activity in aortic cells (line A7r5) by acting on three different types of ionic channels.

A7r5 smooth muscle (aorta) cells have a spontaneous electrical activity. Application of vasopressin produces a hyperpolarization accompanied by an interruption of the spontaneous activity, which is followed by a depolarization associated with a recovery of the spiking activity. Vasopressin action is produced by an action of the peptide on three different types of ionic channels. Vasopressin activates a Ca2+-sensitive K+ conductance, presumably by producing inositol 1,4,5-trisphosphate intracellularly and liberating Ca2+ from internal stores. This activation is transient (0.5-4 min) and is related to the vasopressin-induced hyperpolarization. Intracellular perfusion of inositol trisphosphate triggers by itself a transient K+ current and prevents subsequent activation by vasopressin. Vasopressin inhibits an L-type Ca2+ channel through both protein kinase C activation and a [Ca2+]i-dependent inactivation mechanism triggered by inositol trisphosphate production. The addition of the activation of a Ca2+-sensitive K+ channel and of the inhibition of a voltage-sensitive Ca2+ channel is responsible for the transient blockade of the spontaneous activity. Vasopressin also provokes the activation of an inward current (2-20 min) due to a nonselective channel able to transfer Ca2+, Na+, K+, and Cs+ across the membrane. This effect of the peptide is associated with the depolarization following the hyperpolarization phase.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

A novel high affinity class of Ca2+ channel blockers.

Benzolactams (HOE 166 and analogs) form a new class of molecules acting on the 1,4-dihydropyridine-sensitive L-type Ca2+ channels. The main binding properties of HOE 166 and analogs to rabbit skeletal muscle membranes are as follows. (i) The compounds have a specific binding site to which they associate with a high affinity (0.25 nM for HOE 166). (ii) Unlabeled HOE 166 and analogs completely inhibit 1,4-dihydropyridine binding [(+)-[3H]PN 200-110] in a competitive way. (iii) Affinity values measured for HOE 166 inhibition of (+)-[3H]PN 200-110 (K0.5 = 0.25 nM and K1 = 0.55 nM) and of [3H]HOE 166 binding (K0.5 = 0.5 nM) are in good agreement. They also fit with results from direct binding experiments with tritiated HOE 166 (Kd = 0.27 nM) and from kinetic experiments (Kd = 0.39 nM). (iv) HOE 166 completely inhibits the specific binding of other classes of Ca2+ channel antagonists such as phenylalkylamines [(-)[3H] desmethoxyverapamil], benzothiazepines (d-cis-[3H]diltiazem), diphenylbutylpiperidines ([3H]fluspirilene), and [3H]bepridil. In all these cases the binding inhibition is of a noncompetitive nature. (v) The maximum binding capacity for [3H]HOE 166 binding to transverse tubule membranes, 65 pmol/mg of protein, is the same as that found for other classes of Ca2+ channel antagonists. 45Ca2+ uptake experiments performed with the rat aortic cell line A7r5 and the insulin-secreting cell line RINm5F demonstrate that HOE 166 and analogs fully inhibit the 1,4-dihydropyridine-sensitive 45Ca2+ influx elicited by depolarization. There is a good correlation between inhibitory potencies of compounds in the HOE 166 series measured on (+)-[3H]PN 200-110 binding to A7r5 membranes and on the activity of Ca2+ channels followed by 45Ca2+ fluxes with the same cells. Structure-function relationships of HOE 166 and analogs for Ca2+ channel blockade in A7r5 and RINm5F cells were also in good correlation. Finally, voltage-clamp experiments confirmed that voltage-dependent L-type Ca2+ channels are completely blocked by 100 nM HOE 166 even at a membrane potential held at -80 mV.

Animals↗
PubMed1987

Identification in mammalian brain of an endogenous substance with Na+ channel blocking activities similar to those of tetrodotoxin.

A substance with Na+ channel blocking activities has been isolated from pig brain after extraction and purification on sulfopropyl-Sephadex C-25, reversed-phase and carboxymethyl Synchropak high pressure liquid chromatography columns. The peptidic material i) displaces [3H]ethylenediamine tetrodotoxin ([3H]en-TTX) from its binding sites on rat brain membranes, (ii) it blocks 22Na+ influx induced by veratridine and sea anemone toxin on neuroblastoma and embryonic chick heart cells in culture, (iii) it specifically decreases the height of the action potential generated in frog sciatic nerve, and (iv) it blocks the fast Na+ current in voltage-clamped neuroblastoma cells. These properties are similar to those of tetrodotoxin while the endogenous factor is a peptide that is destroyed by proteases. These results suggest the presence in pig brain of a potent Na+ channel modulation activity.

Action Potentials↗
PubMed1987

Excitation-contraction uncoupling in the developing skeletal muscle of the muscular dysgenesis mouse embryo.

The muscular dysgenesis recessive autosomal mutation is characterized by a total lack of muscular contraction and a myofibrillar non-organization. Many abnormalities involved in the excitation-contraction coupling are found in mdg/mdg myotubes: 1) the internal structural organization of the membrane coupling between the sarcoplasmic reticulum (SR) and the transverse (T)-tubule forming the triadic association is defective: the triad number is decreased in the muscle and there are a lack of periodic densities between the SR and T-tubule apposed membranes. 2) the voltage-dependent Ca2+ channel contents, identified by binding with the specific blocker PN 200-110, are decreased. The two fast (30 ms) and slow (100 ms) Ca2+ currents present in normal myotubes are absent in mdg/mdg myotubes in vitro. 3) the Ca2+-dependent K+ conductance triggering an action potential followed by a long lasting after hyperpolarization (ahp) is absent in mdg/mdg myotubes. This indicates a lack of the free intracellular Ca2+ increased by the action potential. These results suggest that: 1) the lack of differentiated triadic junctions is directly correlated with very low amounts of voltage-dependent Ca2+ channels; 2) the low amount of Ca2+ channels results directly in decreased Ca2+ currents; 3) the decreased Ca2+ currents are the consequence of the low intracellular Ca2+ concentration which is not sufficient to trigger a contraction. However, the addition of normal motoneurones to mdg/mdg myotubes in culture induces, few days later, an increase in Ca2+ currents.

Animals↗