Biomedical subjects

F Grassi

Publications and source records attributed to F Grassi.

At least 109 records · Page 6Linked to original sources

PubMed1989

Voltage-dependent GABA-induced modulation of calcium currents in chick sensory neurons.

Externally applied gamma-aminobutyric acid (GABA) quickly and reversibly reduces by 60% voltage activated Ca2+ currents in chick dorsal root ganglion cells. This action is antagonized by depolarization, with characteristic time and voltage requirements. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) mimicks and blocks the GABA effect, respectively. A 3-state model describing the reactions involved is proposed.

Animals↗

PubMed1989

Regulation of acetylcholine receptor function by the phorbol ester TPA in rat skeletal muscle.

(1) The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of the protein kinase C (PrkC), on the function of junctional nicotinic acetylcholine receptors (nAChR) was examined on muscle fibres isolated from the M. flexor digitorum brevis of the rat. (2) In the presence of TPA the sensitivity of the whole endplates to iontophoretically applied ACh exhibited multiphasic oscillations: an early decrease followed by a delayed increase and, at the end again, a decrease to below pretreatment levels. This effect was more pronounced as the TPA concentration was increased in the range of 0.1-1 microM and was blocked by the PrkC-inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). (3) TPA (0.1-0.5 microM) shortly applied to patch-clamped fibres caused a slight decrease in nAChR-channel slope conductance without affecting the mean lifetime. In a patch the opening frequency increased over time, after an initial decrease. (4) It is concluded that specific activation of the PrkC may be of regulatory significance on nAChR function.

Animals↗

PubMed1989

Regulation of muscle acetylcholine receptor-channel function by interferon.

The effect of the antiviral agent interferon (IFN) on the function of the nicotinic acetylcholine receptor (AChR) channel, has been investigated in both mammalian cultured myotubes and adult fibres, using the single channel recording patch-clamp technique. Shortened AChR-channel lifetime, and occasionally reduced channel conductance and slowed opening frequency were seen with fibroblast IFN (IFN-beta) in the mouse myotubes, and with IFN-beta and leucocytes IFN (IFN-alpha), in the rat muscle fibres. These effects paralleled an increase in the cytosolic level of cAMP. This suggests that IFN exerts a regulatory action on AChR function. A similar regulatory action on other receptor may be responsible for some of the neurological side effects observed in patients treated with IFN.

Animals↗

PubMed1988

Effects of calcitonin gene-related peptide on synaptic acetylcholine receptor-channels in rat muscle fibres.

Cultured myotubes and freshly dissociated muscle fibres from adult rats were exposed to calcitonin gene-related peptide (CGRP) and studied by patch-clamp recording during the peptide-induced maximal accumulation of cellular cyclic AMP (cAMP). Acetylcholine receptor- (AChR-) channel properties in myotubes were not modified by the presence of CGRP (10(-7) M). The peptide, applied to the non-patched membrane, significantly increased the variance of the AChR-channel amplitude distribution at the synaptic region of muscle fibres, and three classes of AChR-channels were resolved immediately after peptide application. AChR-channels at extrasynaptic regions of fibres from denervated muscles were unaffected by CGRP. It is suggested that CGRP may regulate the synaptic AChR-channel conductance through second messenger systems.

Animals↗

PubMed1988

Microwave effects on acetylcholine-induced channels in cultured chick myotubes.

The behavior of cultured myotubes from chick embryos exposed to microwaves has been experimentally analyzed. Recordings of acetylcholine-induced currents have been obtained via patch-clamp techniques using both cell-attached (single-channel current recording) and whole-cell (total current recording) configurations. During the exposure to low-power microwaves the frequency of the ACh-activated single channel openings decreased, while the ACh-induced total current showed a faster falling phase. Channel open time and conductance were not affected by microwave irradiation. It is concluded that the exposure to microwaves increases the rate of desensitization and decreases the channel opening probability. The nonthermal origin and the molecular interaction mechanisms governing these electromagnetic-induced effects are discussed.

Acetylcholine↗

PubMed1988

Influence of protein kinase C-stimulation by a phorbol ester on neurotransmitter release at frog end-plates.

1. The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the stimulation-evoked neurotransmitter release has been investigated by measuring the quantal content (m) of end-plate potentials at frog neuromuscular junctions (Rana temporaria, M. sartorius). 2. After addition of TPA (0.1 up to 1 mumol/l) to the Ringer solution the m-values increased in a concentration-dependent manner up to more than 3 times the control values. 3. Inhibition of the activity of the protein kinase C through the inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) blocked this effect of TPA. 4. The TPA effect was much more conspicuous when the m-value was reduced by raising the extracellular Mg2+ concentration. Between the control m-values and the n-fold increase in the m-value enhanced by TPA a hyperbolic relation was observed. 5. It is concluded that protein kinase C stimulation affects predominantly the spontaneous release of neurotransmitter at the frog neuromuscular junction and only very poorly the stimulation-evoked one.

Animals↗

PubMed1988

Spontaneous channel activity induced by tumor promoter TPA in chick myotubes.

Patch-clamp recordings were used to study the activation of ion channels in the cell membrane of cultured embryonic chick myotubes treated with the specific activator of protein kinase C, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 1 x 10(-7) M). Myotubes exhibited a spontaneous channel activity when the TPA-induced dedifferentiative processes developed. This consisted in the activation of inward current channels (approximately 35 pS conductance; approximately 6 ms open time). These spontaneously active channels were insensitive to alpha-bungarotoxin, curare, atropine and tetrodotoxin and were not inhibited by the withdrawal of TPA. It is suggested that a prolonged stimulation of the protein kinase C causes a irreversible deregulation of the membrane channel function during cell dedifferentiation.

Animals↗

PubMed1988

Reduced acetylcholine-induced channel activity in dystrophic mouse myotubes.

Single channel recording patch-clamp technique was used in the mouse to compare the acetylcholine (ACh)-induced channel behaviour between normal and dystrophic myotubes. While open time and slope conductance were equivalent, ACh-induced channel opening frequency was more than 4-fold reduced in dystrophic compared to normal myotubes. In addition, the steady-state phosphorylation of the ACh receptor (AChR), tested by immunoprecipitation of 32P-labeled cells, indicated that the alpha-subunit was more heavily phosphorylated in the dystrophic myotubes. We propose that the degree of alpha-subunit phosphorylation of the AChR, which parallels the reduced AChR-channel opening probability, determines desensitization of the AChR in dystrophic myotubes.

Acetylcholine↗

PubMed1987

Anti-human tumor antibodies induced in mice and rabbits by "internal image" anti-idiotypic monoclonal immunoglobulins.

MBr1 is a murine monoclonal antibody, defining a saccharidic epitope [CaMBr1] of a human tissue-specific, tumor-associated globoside, present on the mammary carcinoma cell line MCF-7. The same epitope is shared by glycoproteins present on normal and neoplastic mammary epithelial cells, and by mucins from some ovarian cyst fluids. We have used MBr1 as the monoclonal antitumor antibody in an idiotypic sequence of immunizations in order to obtain and characterize "internal images" of the original epitope to be used as substitutes of the nominal antigen in serologic immunoassays. Two monoclonal anti-idiotypic antibodies (beta-1 and beta-2), which reacted with paratope-related idiotopes on MBr1, were obtained. The analysis of the antigenic and immunogenic properties of these molecules by both "antigen" and "antibody" competition assays provided evidence that both beta-1 and beta-2 bear "internal images" of the MBr1-defined epitope. Moreover, when injected in mice and rabbits both beta-1 and beta-2 induced anti anti-idiotypic antibodies, which mimicked MBr1 in binding MCF-7 as well as normal and neoplastic mammary gland epithelial cells. These data are discussed in terms of their possible application to the production of tumor-associated antigen substitutes and their use in serologic immunoassays.

Animals↗

PubMed1987

Acetylcholine receptor channel properties in rat myotubes exposed to forskolin.

Rat myotubes exposed to forskolin were studied by patch-clamp technique in cell-attached single channel recording configuration. Channel open time and opening frequency of the main class of acetylcholine receptor- (AChR-) channels (accounting for more than 90% of all unitary events) decreased in the presence of forskolin (20-100 microM). The forskolin-induced action on the AChR function fully developed with a delay of 30-60 minutes from the peak of cytosolic cyclic AMP (cAMPi) concentration. Both cAMP (1 mM), applied intracellularly for 10 min, and dibutyryl cAMP (0.5 mM), applied extracellularly for 90 min, did not accelerate the rate of desensitization of myotubes studied in whole-cell patch-clamp recording configuration. It was concluded that the action of forskolin on AChR-channel function of rat myotubes could be not associated with the cAMPi-dependent phosphorylation of AChR.

Animals↗

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

HIV env glycoprotein shares a cross-reacting epitope with a surface protein present on activated human monocytes and involved in antigen presentation.

A serological cross-reactivity between env gp120 glycoprotein of the human immunodeficiency virus (HIV) and a human cellular surface protein has been defined by a monoclonal antibody (M38) raised against HIV. The cellular antigen is a protein of ca. 80 kDa expressed on a small fraction of mononuclear cells in peripheral blood and in lymph nodes. The protein behaves as an activation antigen of the monocytic lineage since it is expressed by monocytes in plastic-adherent culture conditions and by interferon-gamma-treated monocytes and pro-monocytic U937 cells. The protein is involved in antigen presentation since the antibody efficiently inhibits the proliferation of responsive lymphocytes in autologous tetanus toxoid presentation assays. In the T lymphoblastoid line H9, the protein is present in very small amounts, is not induced by interferon-gamma and increases after HIV infection. Sera from lymphoadenopathy syndrome and acquired immunodeficiency syndrome (AIDS) patients fail to detect the cellular protein, although containing antibodies reacting with gp120. We propose that both viral and cellular structures recognized by the monoclonal antibody (mAb) are involved in interactions with CD4 molecules of T helper lymphocytes and that such molecular mimicry might be relevant in the pathology of HIV infection. This view is supported by the finding that BL/10T4, a CD4-specific mAb, binds to M38 neutralizing its interactions with HIV and with monocytes. mAb M38 thus behaves as the internal image of CD4. This single property would explain all its diverse binding characteristics.

Antibodies, Monoclonal↗

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

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

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↗