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G Romey

Publications and source records attributed to G Romey.

At least 91 records · Page 5Linked to original sources
PubMed1984

The presence in pig brain of an endogenous equivalent of apamin, the bee venom peptide that specifically blocks Ca2+-dependent K+ channels.

An apamin-like factor has been isolated from pig brain after extraction of the tissue and purification on sulfopropyl-Sephadex C-25 and on reversed-phase high pressure liquid chromatography. The apamin-like factor has the following properties: (i) it prevents 125I-labeled apamin binding to its specific receptor site present on rat brain synaptosomes, (ii) it is active in the radioimmunoassay for apamin (i.e., it prevents 125I-labeled apamin precipitation by anti-apamin antibodies), (iii) it induces contraction of guinea pig intestinal smooth muscle previously relaxed with epinephrine, and (iv) it blocks Ca2+-dependent K+ channels responsible for the long-lasting afterpotential hyperpolarization following the action potential in rat skeletal muscle cells in culture. All these properties are those of apamin itself. The apamin-like factor is a peptide that, like apamin, is destroyed by trypsin and unaffected by chymotrypsin. These results suggest the presence in mammalian brain of a potent Ca2+-dependent K+-channel modulator.

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PubMed1984

Apamin: a specific toxin to study a class of Ca2+-dependent K+ channels.

Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30 000 daltons.

Animals↗
PubMed1983

Cells with neuronal properties in permanent cultures of quail embryo neuroretinas infected with Rous sarcoma virus.

Neuroretina cells from 7-day quail embryos infected 'in vitro' with the mutant ts NY-68 of Rous sarcoma virus, were established into permanent cultures. An initial stage of cellular proliferation was followed by a period of minimal multiplication. After recovery from this crisis, cell proliferation resumed. About 30% of the cells had binding sites for tetanus toxin and the monoclonal antibody A2B5 which seem to be specific for neurons, and an ultrastructural study suggested the presence of neurons and Müller (astroglial) cells. The specific activity of glutamic acid decarboxylase, the enzyme responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid was high (10-30 nmol CO2/h/mg of protein) and electrophysiology showed that some cells had 'active' membranes. After about 18 months in culture, approximately 20% of the cells were able to respond to electrical stimulation by producing action potentials which were inhibited by 10(-7) M tetrodotoxin. These electrophysiological properties are stable: they have been repeatedly found at regular intervals throughout a 20 months period. Furthermore, a clone in which all tested cells are excitable, has been derived from the mass culture. Quail embryo neuroretina cells with typical neuronal properties can thus be established into permanent cultures after infection with Rous sarcoma virus.

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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

A neuronal clone derived from a Rous sarcoma virus-transformed quail embryo neuroretina established culture.

Neuroretina (NR) is an evagination of the central nervous system (CNS) which is composed of photoreceptors, glial (Müller) cells and horizontal, bipolar, amacrine and ganglion neuronal cells. We describe here the usefulness of Rous sarcoma virus (RSV) in the establishment of a neuronal clone from quail embryo neuroretina. When primary cultures of chick and quail embryo neuroretina cells are transformed by RSV, neuronal markers such as ribbon synapses, choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD) specific activity are present. These RSV-transformed primary cultures can be established into permanent cell lines from which neuronal clones have been isolated. One of them, clone QNR/D, can generate tetrodotoxin(TTX)-inhibitable action potentials on electrical stimulation, has a high GAD activity and binds monoclonal antibodies raised against chick embryo neuroretina. The presence of these neuronal markers suggests that the QNR/D clone is derived from cells of the amacrine or ganglionic lineage. This is the first time that a neuronal cell clone of defined origin has been obtained from the CNS. The neuronal markers of the QNR/D clone are expressed at both the permissive and the non-permissive temperatures for transformation.

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PubMed1983

Identification and properties of phencyclidine-binding sites in nervous tissues.

[3H]Phencyclidine (PCP) binds to a single class of noninteracting binding sites in rat brain membranes with an affinity Kd of 0.25 microM and a maximal binding capacity BM of 2.4 pmol/mg of membrane protein. PCP derivatives also interact with the muscarinic and mu-opiate receptors in rat brain membranes with affinities that are one or two orders of magnitude lower than those observed for the [3H]PCP-binding sites. Activities of 25 PCP derivatives in the rotarod assay are closely correlated to affinities of these molecules for the [3H]PCP-binding sites, but not for the muscarinic or mu-opiate receptors. Monohydroxylation of PCP generally decreases the affinity of PCP for the [3H]PCP- and muscarinic-binding sites and does not change the affinity for the mu-opiate receptor. The metaphenolic derivative of PCP does not follow these general rules; the affinities of this derivative for the [3H]PCP- and mu-opiate-binding sites are 8 and 430 times higher, respectively, than those of PCP itself. Voltage-clamp experiments with N1E 115 neuroblastoma cells show that PCP is an efficient blocker of both the K+ channel (EC50 = 2.6 microM) and the Na+ channel (EC50 = 9.2 microM).

Animals↗
PubMed1982

The Ca2+-dependent slow K+ conductance in cultured rat muscle cells: characterization with apamin.

The interaction of apamin, a bee venom neurotoxin, with rat skeletal muscle cell membranes has been followed using both an electrophysiological and a biochemical approach. Voltage-clamp analyses have shown that apamin, at low concentrations, specifically blocks the Ca2+-dependent slow K+ conductance in rat myotubes and myosacs . A specific binding site for apamin in rat muscle cell membranes has been characterized with the use of a highly radiolabelled apamin derivative [( 125I]apamin). The dissociation constant for the apamin-receptor complex is 36-60 pM and the maximal binding capacity is 3.5 fmol/mg of protein. [125I]Apamin binding to rat muscle membranes is displaced by quinine and quinidine with K0.5 values of 110 microM and 200 microM, respectively.

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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

Apamin as a selective blocker of the calcium-dependent potassium channel in neuroblastoma cells: voltage-clamp and biochemical characterization of the toxin receptor.

This paper describes the interaction of apamin, a bee venom neurotoxin, with the mouse neuroblastoma cell membrane. Voltage-clamp analyses have shown that apamin at low concentrations specifically blocks the Ca2+-dependent K+ channel in differentiated neuroblastoma cells. Binding experiments with highly radiolabeled toxin indicate that the dissociation constant of the apamin-receptor complex in differentiated neuroblastoma cells is 15-22 pM and the maximal binding capacity is 12 fmol/mg of protein. The receptor is destroyed by proteases, suggesting that it is a protein. The binding capacity of neuroblastoma cells for radiolabeled apamin dramatically increases during the transition from the nondifferentiated to the differentiated state. The number of Ca2+-dependent K+ channels appears to be at most 1/5th the number of fast Na+ channels in differentiated neuroblastoma. The binding of radiolabeled apamin to its receptor is antagonized by monovalent and divalent cations. Na+ inhibition of the binding of 125I-labeled apamin is of the competitive type (Kd(Na+) = 44 mM). Guanidinium and guanidinated compounds such as amiloride or neurotensin prevent binding of 125I-labeled apamin, the best antagonist being neurotensin.

Animals↗
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.

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