PubMed Health⌕ Search

Biomedical subjects

M Molinaro

Publications and source records attributed to M Molinaro.

At least 73 records · Page 4Linked to original sources
PubMed1987

Acetylcholine may regulate its own nicotinic receptor-channel through the C-kinase system.

Acetylcholine (ACh)-activated channel properties were examined on an aneural culture of chick embryo myotubes by using patch-clamp techniques. Changes in conductance, open time and closed time were induced by the selective activator of the calcium- and phospholipid-dependent C-kinase (PKc), 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of TPA was mimicked by exogenous phospholipase C and was blocked by the PKc inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine. In addition to its gating action, ACh was shown to stimulate phosphoinositide turnover and to translocate PKc from the cytosol to the cell membrane. Both these ACh-induced effects were inhibited by curare and not substantially affected by atropine. Bath-applied ACh outside the patch-pipette in the cell-attached patch-clamp mode, had a strong effect on the ACh-activated channels in the patch membrane, in a way that resembled the action of TPA. These findings raise the possibility that ACh regulates its own nicotinic receptors through the C-kinase system.

Acetylcholine↗
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

Altered distribution of protein kinase C in dystrophic muscle cells and its modulation by liposome-delivered phospholipids.

The activity and subcellular distribution of the calcium-phospholipid dependent protein kinase (protein kinase C) were studied in normal and dystrophic muscle cells in vitro. Clonal strains of satellite cells, isolated from normal and dystrophic (C57BL/6J/dydy) mice, differentiate in vitro at a comparable level (over 80% of fusion). Differentiated myotubes were homogenized and separated into a soluble and a particulate fraction. The activity of protein kinase C was assayed in both fractions, and was found to be mainly in the cytosol of normal cells, whereas it was mainly associated to the membrane fraction of dystrophic cells. This altered distribution of the enzyme was likely consequent to alterations in the phospholipid composition of the dystrophic cell membrane, since it was possible to partially revert the situation by modifying the membranes with liposome-delivered phospholipids. Splenic lymphocytes from dystrophic mice showed an altered distribution of protein kinase C similar to that observed in muscle cells. The possible biochemical basis and the functional consequences of this altered distribution of the enzyme in the dystrophic cells are discussed.

Animals↗
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

Proliferating and quiescent cells exhibit different subcellular distribution of protein kinase C activity.

The activity of calcium, phospholipid-dependent protein kinase (PKc), which is thought to play an important role in cell proliferation, has been measured in the particulate and soluble fractions of cultured cells, under different proliferative conditions. Our results indicate that proliferating cells display higher PKc activity than quiescent cells. Furthermore, in both normal and transformed cells, PKc is preferentially associated with the particulate fraction when the cells are proliferating, while in mitotically quiescent cells the majority of the enzyme activity is found in the soluble fraction. These data suggest tha PKc activity and subcellular distribution undergo spontaneous changes according to the proliferative state of the cells.

Animals↗
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

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

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

Emergence of TPA-resistant 'satellite' cells during muscle histogenesis of human limb.

Human satellite cells, obtained by surgical biopsies of traumatized legs of healthy individuals, were grown in culture in the presence of different concentrations of the phorbol ester tetradecanoyl-phorbol 12 acetate (TPA). Satellite cells, after an initial duplicative period, fused into large multinucleated myotubes which readily synthesized myosin and acetylcholine receptor (AChR). The presence of TPA at concentrations up to 10(-7) M did not affect the differentiation pattern, while higher concentrations were toxic. Thus human satellite cells are capable of differentiating in the presence of phorbol esters which block differentiation of embryonic myoblasts [1]. We then examined the appearance of TPA-resistant cells during human muscle histogenesis, since we had observed that differentiation of human myoblasts from a 6-week-old limb was completely and reversibly inhibited by 10(-7) M TPA. Differentiation of myoblasts from 6-, 7- and 8-week-old fetuses was completely inhibited by TPA. Myoblasts from 10-week-old limbs did not form myotubes in the presence of TPA; however, immunohistochemical staining with an antimyosin antibody revealed the presence of a few mononucleated myosin-positive cells which escaped the TPA-induced block of differentiation. At 12 weeks of development, a few oligonucleated, myosin-positive myotubes developed in cultures treated with TPA, and the level of AChR expressed (measured as [125I] alpha-bungarotoxin bound) reached 20% of controls. At 14 weeks of development, about half of the cells in culture were TPA-resistant and by 16 weeks of development no major differences could be detected between control and treated cells. We conclude from these data that a population of TPA-resistant myogenic cells emerges between the 10th and 14th week of human limb development and suggest that this population represents satellite cells.

Adolescent↗
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↗
PubMed1984

Reduced acetylcholine sensitivity in dystrophic mouse myotubes in vitro.

Acetylcholine (ACh) sensitivity in cultured myotubes from normal or dystrophic mice was investigated using conventional techniques. Dystrophic multinucleate myotubes were found to be less responsive to the ACh with respect to the control preparation. Such a reduced sensitivity to ACh in dystrophic myotubes was accompanied by a reduced binding to 125I-alpha-bungarotoxin. The possible biological significance of these results on the muscle disease is discussed.

Acetylcholine↗
PubMed1984

Phosphorylation of specific polypeptides induced by 12-O-tetradecanoylphorbol-13-acetate in chick embryo fibroblasts.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces, in cultured chick embryo fibroblasts, a generalised increase of the incorporation of labelled inorganic phosphate, and stimulates the phosphorylation of at least two polypeptide bands, 26 K and 65 K. Stimulation of the phosphorylation of 26 K and 65 K occurs within minutes of the addition of TPA to the culture medium of chick embryo fibroblasts, but it can also be evidenced at later times. Removal of TPA from the culture medium causes reversion of this effect. Stimulation of the phosphorylation of 26 K is also induced by the Ca2+-ionophore A23187, but the calmodulin inhibitor trifluoperazine does not inhibit the TPA induced stimulation of polypeptide phosphorylation. Agents increasing the intracellular cAMP concentration do not stimulate the phosphorylation of 26 K and 65 K. The results obtained suggest that the phosphorylation of specific polypeptides, probably induced by TPA through a Ca2+-phospholipid dependent mechanism, may represent an early regulative event which may be relevant for the pleiotropic effect of TPA in cultured normal cells.

Animals↗