Biomedical subjects

F Eusebi

Publications and source records attributed to F Eusebi.

At least 91 records · Page 5Linked to original sources

PubMed1987

Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture.

The expression and the physiological properties of acetylcholine receptors (AChRs) of mononucleated myogenic cells, isolated from either embryonic or adult muscle of the mouse, have been investigated using the gigaohm seal patch-clamp technique in combination with immunocytochemistry (with an anti-myosin antibody) and alpha-bungarotoxin binding techniques. Undifferentiated (myosin-negative) embryonic myoblasts, grown either in mass culture or under clonal conditions, were found to be unresponsive to ACh and did not bind alpha-bungarotoxin. On the contrary, undifferentiated satellite cells (from adult muscle) exhibited channels activated by ACh and alpha-bungarotoxin binding sites similar to those observed in differentiated (myosin-positive) embryonic myoblasts and myotubes. Two classes of ACh-activated channels with different opening frequencies were identified. The major class of channels had a conductance of about 42 pS and mean open time of 3.1-8.2 msec. The minor class of channels had smaller conductance (about 17 pS) and similar open time. During differentiation, the conductance of the two channels did not change significantly, while channel lifetime became shorter in myotubes derived from satellite cells but not in myotubes derived from embryonic myoblasts. The relative proportion of small over large channels was significantly larger in embryonic than in adult myogenic cells.

Animals↗

PubMed1987

Single acetylcholine-activated channels in cultured rhabdomyoblasts.

Acetylcholine receptor (AChR) was found to be present on the cell surface of the human rhabdomyoblast (RD) cell line. Two classes of ACh-activated channels have been observed, one with a large conductance and long duration and the other with smaller conductance and short duration, similar to those of human myotubes. RD membrane exhibited a specific binding to the alpha-bungarotoxin indicating the presence of nicotinic AChRs. These results support the hypothesis that rhabdomyosarcomas derive from myogenic precursors.

Bungarotoxins↗

PubMed1987

Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes.

1. Single-channel currents induced by acetylcholine (ACh) were recorded from unstriated and non-innervated embryonic chick myotubes using the cell-attached patch-clamp technique. 2. ACh applied to the non-patched membrane decreased both channel opening probability and conductance. These ACh-induced effects occurred also when the non-patched membrane was exposed to nominally Ca2+-free extracellular medium, but were absent when it was treated with curare. 3. ACh-induced membrane current recorded under whole-cell patch-clamp conditions decreased in amplitude and time course when myotubes were intracellularly loaded with guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), but not with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or cyclic adenosine-5'-monophosphate (cyclic AMP). Internal perfusion of GTP gamma S affected the ACh-induced openings in a similar manner to the non-patch ACh application. 4. These results suggest that ACh, in addition to its direct effect, acts indirectly on the nicotinic receptor channels by delivering an intracellular messenger and through the activation of a putative G protein.

Acetylcholine↗

PubMed1986

Cyclic AMP regulates the life time of acetylcholine-activated channels in cultured myotubes.

'Giga-seal' patch-clamp recording was performed in embryonic chick myotubes at day 3 to 4 of culture. Myotubes were exposed to agents that enhance the concentration of cytosolic cyclic AMP (cAMPi) and their action on acetylcholine- (ACh) activated channels was investigated. While the conductance and the closed time was unaffected by forsokolin, cholera toxin, dibutyryl cyclic AMP and 8-bromo-cyclic AMP, these agents lengthened the ACh-activated channel life time with efficacy that paralleled with their capability to increase the cAMPi.

Acetylcholine↗

PubMed1986

Liposome-delivered phosphatidylcholine enhances the acetylcholine sensitivity of dystrophic mouse myotubes.

Myotubes were obtained in vitro from satellite cells of normal and dystrophic C57BL/6J/dydy mice. The acetylcholine sensitivity (mV/nC) of dystrophic myotubes determined with conventional electrophysiological techniques, was lower than that of normal myotubes. Incubation of dystrophic myotubes with liposomes containing phosphatidylcholine (a lipid present in higher amounts in normal adult muscle) significantly increased their acetylcholine sensitivity.

Acetylcholine↗

PubMed1986

Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction.

Spontaneous transmitter release was studied at frog neuromuscular junctions exposed to the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PrkC). TPA at concentrations between 10(-7) and 10(-6) M induced a dose-dependent increase in miniature end-plate potential frequency. This frequency increase was enhanced by raising [Ca]o, diminished by lowering the temperature of the bath and virtually abolished in Ca2+ -free Ringer. The TPA effect was only poorly reversible after washing. TPA was ineffective in increasing spontaneous release of transmitter at phosphatidylcholine-pretreated neuromuscular junctions. It is suggested that PrkC might play a role in neuromuscular transmission processes.

Animals↗

PubMed1986

Postsynaptic effects of the phorbol ester TPA on frog end-plates.

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PKc), were examined on the frog neuromuscular junction. The depolarization elicited by iontophoretically applied acetylcholine (ACh) was reversibly decreased by 20-60% when muscle fibres were exposed to 1-5 X 10(-7) M TPA. Liposome-delivered phosphatidylcholine (100 micrograms/ml) prevented this effect. A similar decrease in ACh-sensitivity was produced by diacylglycerol (diolein), a physiological activator of PKc, but in this case the decrease was only partially reversible. In TPA-Ringer, the peak size of miniature end-plate potentials exhibited a small decrease; miniature end-plate currents were reduced in size and their decay time constant became longer and relatively independent of membrane potential. The possibility that these TPA-induced actions are mediated by activation of PKc is discussed.

Acetylcholine↗

PubMed1986

Effect of phorbol esters and liposome-delivered phospholipids on the differentiation program of normal and dystrophic satellite cells.

Satellite cells, isolated from hind limb of normal C57BL/6J mice, differentiate in culture in the presence of concentrations of phorbol esters which inhibit differentiation of embryonic myoblasts. However, if phosphatidylserine containing liposomes were added to the culture medium together with TPA, differentiation of satellite cells was reversibly inhibited. Under these conditions, the withdrawal of these cells from the cell cycle still occurred as in untreated cells. Phosphatidylserine liposomes alone or liposomes containing phosphatidylcholine (either alone or in combination with TPA) had no effect on satellite cell differentiation. In the case of satellite cells from dystrophic C57BL/6J/dydy mice, TPA addition (0.1 microM) to the culture medium partially (about 70%) inhibited morphological and biochemical differentiation. This effect could be prevented by preincubating dystrophic satellite cells with liposomes containing phosphatidylcholine but not other phospholipids. These data indicate that it is possible to change the sensitivity to TPA of satellite cells by modifying the phospholipid composition of their plasma membrane. Possible relationships of these phenomena with activation of protein kinase C or phosphatidylinositol breakdown have been investigated. The results obtained are discussed with regard to possible modulation of the intracellular response to agonist binding.

Animals↗

PubMed1986

Putative second messengers affect cell coupling in the seminiferous tubules.

Junctional transfer of ions in monolayer primary cultures of Sertoli cells and in intact seminiferous tubules from 20 day-old rats has been investigated by using electrophysiological techniques. The electrotonic coupling of both intact tubule and monolayer culture was inhibited by the presence of dibutyryl cyclic AMP (10(-4) M) in the medium. In contrast, 1-oleoyl-2-acetylglycerol (10(-5) M) and 12-O-tetradecanoyl-phorbol-13-acetate (10(-7) M) decreased the junctional resistivity and increased the extent of the coupling in immature tubules. The significance of this modulation of the cell coupling in the seminiferous epithelium is discussed.

Animals↗

PubMed1986

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro.

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.

Acetylcholinesterase↗

PubMed1985

Acetylcholine stimulates phosphatidylinositol turnover at nicotinic receptors of cultured myotubes.

Acetylcholine treatment of [3H]inositol pre-labelled cultured chick embryo myotubes results in the stimulation of phosphatidylinositol breakdown, as shown by the measurement of inositol-1-phosphate accumulating in the presence of lithium. The described effect is dependent on agonist concentration and incubation time, and is inhibited by tubocurarine and alpha-bungarotoxin. The activation of phosphatidylinositol breakdown by acetylcholine at extrajunctional nicotinic receptors is likely to be involved in the modulation of the functional activity of the receptor.

Acetylcholine↗

PubMed1985

Calcium entry induced by acetylcholine action on snail neurons.

A study was made of excitatory and inhibitory responses elicited by acetylcholine (ACh) in neurons of the snail Eobania vermiculata. At resting potential, ACh evoked a depolarizing inward current in some neurons (D-cells) and a hyperpolarizing current in others (H-cells). The currents elicited by ACh were nonlinearly dependent on membrane potential. After either D- or H-cells were equilibrated in chloride-free isotonic calcium, ACh evoked a depolarizing inward current which reversed sign at about -55 mV. These results suggest that ACh causes an influx of Ca2+ in both types of neurons.

Acetylcholine↗

PubMed1985

Cell-to-cell communication in cultured Sertoli cells.

Junctional transfer of ions in monolayer primary cultures of Sertoli cells has been studied by using conventional techniques. Cells were extensively electrically coupled. Membrane voltage displacements caused by the injected current pulses were measured at variable interelectrode distance. Data analyses were based on a thin-sheet model for current flow (Jongsma and van Rijn, 1972) and yielded a generalized space constant of 248 microns and an internal resistivity of 1.7 kOhm.cm in standard medium. Electrical coupling was inhibited by A23187 Ca2+ ionophore (5 microM).

Animals↗

PubMed1985

Response of a neuronal membrane to applied sinusoidal currents.

The membrane model of Connor and Stevens was used to calculate the response of a neuron to an injected sinusoidal current. This stimulating current simulates the effects of cell exposure to incident time-harmonic electromagnetic fields. The theoretical results obtained with this model were compared with intracellular recordings carried out during the injection of a sinusoidal transmembrane current through a single microelectrode. Experimental and theoretical data were substantially in agreement for current amplitudes that can be induced by actual exposure conditions.

Animals↗

PubMed1985

Increased endocytosis of acetylcholine receptors by dystrophic mouse myotubes in vitro.

Multinucleated myotubes, grown in vitro from satellite cells of dystrophic mice (C57BL/6J/dydy) exhibit a reduced sensitivity to ACh. This reduction correlates with a reduced density of 125I-alpha-bungarotoxin (125I-BTX) binding sites on the surface of dystrophic myotubes. Denervated adult muscle fibers from dystrophic mice respond to Ach similarly to denervated normal muscle fibers. Furthermore, cultured dystrophic myotubes, treated with a brain extract which induces AChR clusterization, still show an impaired response to ACh and reduced 125I-BTX binding. Thus AChR function appears altered in dystrophic muscle cells in culture while it appears normal in dystrophic adult muscle, regardless of whether the receptors are dispersed on the membrane or clustered at the junctional site. Metabolic studies on the reduced AChR level in dystrophic myotubes revealed a dramatically reduced half-life (2 vs 10 hr) while the rate of synthesis was unchanged. An increased rate of internalization of AChR was observed in dystrophic myotubes with a corresponding relative increase of the "hidden AChR pool," which could be partially reduced by agents which disrupt the cytoskeleton. No structural alterations could be detected on the AChR molecule as its sedimentation coefficient and subunit composition appeared identical between normal and dystrophic myotubes. Thus the increased turnover of AChR in dystrophic myotubes either reflects subtle alterations of the molecule or a more generalized increase of endocytosis in this form of myopathy.

Animals↗

PubMed1985

gamma-Amino butyric-N-acid sensitivity of mouse and human oocytes.

gamma-Aminobutyric acid (GABA)-sensitivity was studied in mouse and human oocytes using electrophysiological techniques. Isolated mouse oocytes at the germinal vesicle (GV) or metaphase II stage, and human oocytes at the GV stage or following resumption of meiosis in culture, were sensitive to GABA. The transmitter usually hyperpolarized the membrane, with a concomitant decrease followed by an increase in membrane conductance, at threshold concentrations as low as 10(-10) M. GABA response was not evoked in Cl-free medium. Bicuculline (10(-5)-10(-6) M) reversibly blocked GABA (10(-9)-10(-4) M) responses. In contrast mouse fertilized eggs were insensitive to GABA at concentrations of 10(-5) M or lower. A possible biological role of the neurotransmitter GABA is discussed.

Animals↗

PubMed1985

Cell-to-cell electrical coupling in seminiferous cords of mouse fetal testis.

Experiments performed on mouse fetal testes in vitro using electrophysiological techniques and intracellular injections of the fluorescent tracer disodium fluorescein showed that junctional transfer occurs between cells inside the seminiferous cords. This suggests a possible metabolic cooperation between testis cells at an early stage of gonad development.

Animals↗

PubMed1985

Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes.

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.

Acetylcholine↗