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Biomedical subjects

O D Uchitel

Publications and source records attributed to O D Uchitel.

At least 37 records · Page 2Linked to original sources
PubMed1996

Calcium channels involved in synaptic transmission at the mature and regenerating mouse neuromuscular junction.

1. The involvement of the different types of voltage-dependent calcium channels (VDCCs) in synaptic transmission at the mature and newly formed mammalian neuromuscular junction was studied by evaluating the effects of L-, P/Q- and N-type VDCC antagonists on transmitter release in normal and reinnervating levator auris preparations of adult mice. 2. Nerve-evoked transmitter release was blocked by omega-agatoxin IVA (omega-AgaIVA), a P/Q-type VDCC blocker, both in normal and reinnervating endplates (100 nM omega-AgaIVA caused > 90% inhibition). The N-type VDCC antagonist omega-conotoxin GVIA (omega-CgTX; 1 and 5 microM), as occurs in normal preparations, did not significantly affect this type of release during reinnervation. Nitrendipine (1-10 microM), an L-type VDCC blocker, strongly antagonized release in reinnervating muscles (approximately 40-69% blockade) and lacked any effect in normal preparations. 3. In reinnervating muscles, spontaneous release was not dependent on Ca2+ entry through either P- or L-type VDCCs. Neither 100 nM omega-AgaIVA nor 10 microM nitrendipine affected the miniature endplate potential (MEPP) frequency or amplitude. 4. At the newly formed endplates, K(+)-evoked release was dependent on Ca2+ entry through VDCCs of the P-type family (100 nM omega-AgaIVA reduced approximately 70% of the K(+)-evoked MEPP frequency). L-type VDCCs were found not to participate in this type of release (10 microM nitrendipine lacked any effect). 5. In reinnervating muscles, the L-type VDCC blocker, nitrendipine (10 microM), provoked a significant increase (approximately 25%) in the latency of the evoked endplate potential (EPP). This drug also caused an increase (approximately 0.3 ms) in the latency of the presynaptic currents. The P/Q- and Ny-type VDCC blockers did not affect the latency of the EPP. 6. These results show that at newly formed mouse neuromuscular junctions, as occurs in mature preparations, VDCCs of the P-type family play a prominent role in evoked transmitter release whereas N-type channels are not involved in this process. In addition, signal conduction and transmitter release become highly sensitive to nitrendipine during reinnervation. This suggests that L-type VDCCs may play a role in synaptic transmission at the immature mammalian neuromuscular junction.

Animals↗
PubMed1996

Uptake of immunoglobulin G from amyotrophic lateral sclerosis patients by motor nerve terminals in mice.

Clinical and experimental evidence support an autoimmune etiopathogenesis for amyotrophic lateral sclerosis (ALS). We have shown that local application of ALS-IgG onto nerve terminals induces dysfunction in transmission at the neuromuscular junction. It has been established that IgG and other circulating serum proteins can be taken up by motor nerve terminals, being immunolocalized in the soma where they accumulate following retrograde axonal transport. In the present study, we investigated the presence of human ALS and control IgG in the soma of mouse motoneurons. IgG was applied onto motor nerve terminals of mice by subcutaneous injections on the left levator auris longus muscle which is innervated by a branch of the facial nerve. After several injections, sections of the brainstem containing the facial nuclei were immunoprocessed to detect human IgG. For all IgG tested, motoneuron labeling was significantly more intense in the facial nucleus ipsilateral to the site of injection. In ALS-IgG-treated animals, ipsilateral labeling was significantly stronger than that found on the ipsilateral side of control IgG-treated animals. Our results are compatible with the concept that motoneurons preferentially take up, transport and/or accumulate ALS-IgG. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease.

Adult↗
PubMed1996

Calcium channel blockers and transmitter release at the normal human neuromuscular junction.

Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and omega-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or omega-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.

Action Potentials↗
PubMed1995

Effects of Ca2+ channel blockers on transmitter release and presynaptic currents at the frog neuromuscular junction.

1. The effects of the calcium channel blockers, funnel-web spider toxin (FTX), omega-agatoxin IVA (omega-Aga IVA) and omega-conotoxin GVIA (omega-CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50 = 0.02 microliter ml-1) and omega-CgTX (1 microM) but was not affected by omega-Aga IVA (0.5 microM). When FTX (0.1 microliter ml-1) was assayed on spontaneous release either in normal Ringer solution or in low Ca(2+)-high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by approximately 2-fold in both conditions. 3. Presynaptic calcium currents (ICa), measured by the perineurial technique in the presence of 10 mM tetraethylammonium chloride (TEA) and 200 microM BaCl2 to block K+ currents, were blocked by omega-CgTX (5 microM), partially blocked by FTX (1 microliter ml-1) and not affected by omega-Aga IVA (0.5 microM). 4. The presynaptic calcium-activated potassium current (IK(Ca)) measured by the perineurial technique in the presence of 0.5 microM 3,4-aminopyridine (DAP) to block voltage-dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 microM). This current was also blocked by omega-CgTX (5 microM) and by CdCl2 (200 microM) but was not affected by FTX (1 microliter ml-1). The blockade by omega-CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two omega-CgTX-sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a omega-CgTX- and FTX-sensitive population.

Animals↗
PubMed1995

Different calcium channels mediate transmitter release evoked by transient or sustained depolarization at mammalian sympathetic ganglia.

We have compared the effect of calcium channel blockers on the potassium-evoked release of tritium-labeled acetylcholine and on preganglionic spike-evoked synaptic transmission in the rat superior cervical ganglion. Transmitter release at the nerve terminals is mediated by the influx of calcium through voltage-gated calcium channels. While four types of voltage-gated calcium channels (T, L, N and P) have been identified in neurons, it is not clear which may actually be involved in excitation-secretion coupling. Release of tritiated acetylcholine evoked by sustained depolarization in high (40 mM) extracellular potassium decreased markedly in the absence of calcium or the presence of cadmium. High potassium-evoked release was substantially inhibited by the P-type channel blockers, purified from funnel-web spider toxin, and omega-agatoxin-IVA, and by the N-type channel blocker omega-conotoxin-GVIA, but was unaffected by the L-type channel blocker nitrendipine. In contrast, postganglionic compound action potentials synaptically triggered by preganglionic stimulation were strongly blocked by funnel-web spider toxin and slightly blocked by a high concentration of omega-agatoxin-IVA, but were unaffected by either omega-conotoxin-GVIA, nitrendipine or a low concentration of omega-agatoxin-IVA. Thus, at the superior cervical ganglion, funnel-web spider toxin-sensitive calcium channels play a dominant role in transmitter release evoked by transient, spike-mediated depolarization, but other types of voltage-gated calcium channels in addition to the funnel-web spider toxin-sensitive channel mediate the transmitter release that is evoked by sustained high potassium depolarization.

Acetylcholine↗
PubMed1995

Pharmacological characterization of the voltage-dependent Ca2+ channels present in synaptosomes from rat and chicken central nervous system.

The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (omega-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and omega-agatoxin IVA]. K(+)-induced Ca2+ uptake by chicken synaptosomes was blocked by omega-conotoxin GVIA (IC50 = 250 nM). This toxin at 5 microM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and omega-agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 microliter/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and omega-agatoxin IVA affected Ca2+ uptake, FTX (3 microliters/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with omega-agatoxin IVA saturation was not reached even at 5 microM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 microliter/ml) and different concentrations of omega-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and omega-conotoxin GVIA was never greater than the one observed with omega-conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by omega-conotoxin MVIID (1 microM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 microM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P-type Ca2+ channel blockers, thus indicating that P-type channels are predominant in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Transmitter release and presynaptic Ca2+ currents blocked by the spider toxin omega-Aga-IVA.

Mammalian neuromuscular transmission is resistant to L and N type calcium channel blockers but very sensitive to a low molecular weight funnel web spider venom toxin, FTX, which selectively blocks P type calcium channels. To further characterize the calcium channels involved in neuromuscular transmission we studied the effect of omega Agatoxin (omega-Aga-IVA) a polypeptide P type channel blocker from the same spider venom. We show that omega-Aga-IVA is a potent and irreversible inhibitor of the presynaptic Ca2+ currents and of acetylcholine release induced by electrical stimulation or by K+ depolarization. This provides further evidences that transmitter release at the mammalian neuromuscular junction is mediated by P type Ca2+ channels.

Acetylcholine↗
PubMed1993

Newly recognized congenital myasthenic syndrome associated with high conductance and fast closure of the acetylcholine receptor channel.

We describe here a new congenital myasthenic syndrome associated with a kinetic abnormality of the acetylcholine receptor (AChR) channel. The propositus had poor suck and cry after birth. Subsequently, she had intermittent ocular symptoms and fatigued abnormally on exertion. At age 9 years, significant weakness was detected only in the frontalis, levator palpebrae, and neck flexor muscles. Electromyography showed no decrement in limb muscles but single-fiber examination of the facial muscles was consistent with a neuromuscular transmission defect. The ocular symptoms responded partially to pyridostigmine, but the abnormal fatigability did not. Tests for anti-AChR antibodies were negative. A younger sister had elements of the same disease. An intercostal muscle specimen was obtained from the propositus at age 9 years for endplate studies. The quantal content of the endplate potential was normal. Miniature endplate currents were abnormally large and their decay time constant was abnormally short. AChR channel properties were studied by analysis of acetylcholine-induced current noise. The mean single-channel conductance was increased 1.7-fold and the mean channel open time was 30% shorter than normal. The number of AChR per endplate was normal. Electron microscopy of most endplates showed no abnormality, but a few were degenerating or simplified. The channel abnormality may stem from a point mutation in an AChR subunit affecting a single amino acid residue lining the pore of the AChR channel. The mechanism by which the physiological abnormality produces clinical symptoms is not known, but possible explanations are considered.

Adult↗
PubMed1993

Potassium channels from normal and denervated mouse skeletal muscle fibers.

The properties of singles K+ channels in normal and denervated muscles were compared using the "patch-clamp" technique. Single channels were recorded from vesicles obtained by stretching bundles of normal and denervated extensor digitorium longus (EDL) muscles. The most frequently observed channel in normal muscles was a high conductance (266 pS) Ca++ activated K+ channel. Although channel density, as estimated by patch recording, showed a significant decrease in denervated muscles, no differences were found in conductance and gating properties. Another voltage-dependent K+ channel (81 pS) was only recorded from normal muscles, but never from denervated ones. In addition, a 35 pS conductance was recorded from both normal and denervated fibers. This channel displayed neither voltage dependence nor sensitivity to tetraethylammonium (TEA). In contrast, another TEA-insensitive (16 pS) channel was recorded only from denervated muscles. We conclude that denervation induces significant changes in the distribution and expression of K+ channels in mammalian skeletal muscles.

Animals↗
PubMed1992

P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.

Action Potentials↗
PubMed1992

Long-term neuromuscular dysfunction produced by passive transfer of amyotrophic lateral sclerosis immunoglobulins.

We investigated the role of the immune system in the pathogenesis of amyotrophic lateral sclerosis (ALS) by studying the long-term consequences of ALS immunoglobulin (Ig) application on the levator auris muscle of the mouse. We applied Ig from seven ALS patients, four disease controls, and a pool of normal Ig (6 mg of Ig in 2 weeks) by subcutaneous injection; removed the muscles 4 to 12 weeks after the beginning of treatment; and recorded both spontaneous and evoked release of transmitter. None of the control Ig induced changes in transmitter, whereas five of seven ALS Ig induced a significant increase in the rate of spontaneous release, and all ALS Ig produced significant changes in the quantal content of evoked release. In muscles treated with one of the ALS Igs, synaptic activity was completely absent. Cholinesterase and silver staining demonstrated intact neuromuscular junctions in the control Ig-treated muscles and also in many areas of ALS Ig-treated muscles. Axonal degeneration and denervation were present in most muscles treated with ALS Ig. There was complete denervation when no synaptic activity could be recorded. Thus, ALS Ig appears to lead to long-lasting effects at the neuromuscular junction, and such effects may be an early stage in the immune-mediated pathogenesis of ALS.

Adult↗
PubMed1991

Effect of omega-conotoxin GVIA on neurotransmitter release at the mouse neuromuscular junction.

The effect of omega-conotoxin GVIA (omega-CgTx) was studied on spontaneous, K(+)-induced and electrically evoked neurotransmitter release at the neuromuscular junction of mouse diaphragm. omega-CgTx decreased the frequency and amplitude of basal and K(+)-induced miniature end plate potentials. This effect was abolished by raising the extracellular Ca2+ concentration. omega-CgTx had no effect on the quantal content of the electrically evoked release in this preparation.

Animals↗
PubMed1990

Ca2+ role on the effect of phorbol esters on the spontaneous quantal release of neurotransmitter at the mouse neuromuscular junction.

The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release process was studied in presence of different extracellular Ca2+ concentrations, in the mouse phrenic-diaphragm preparation. Hemidiaphragms were incubated for 2 h at room temperature in the presence or absence of TPA. TPA increased the basal frequency of miniature end plate potentials (mepp's) in a dose-dependent manner, resulting in a maximal increase of 280% at a concentration of 0.5 microM. An inverse relationship between extracellular Ca2+ concentration and TPA effect was observed: at high extracellular concentrations of Ca2+ the action of TPA decreased significatively, while at low Ca2+ concentrations the effect of TPA was remarkably augmented. The highest effect of TPA was obtained when tested in a calcium-free medium. TPA also increased mepp frequency stimulated by 10 mM K+. As at basal conditions, the effect of TPA was higher at lower concentrations of extracellular calcium. The results suggest that the effect of stimulation of PKC on neurotransmitter release at the mice neuromuscular junction is not exerted at the level of calcium influx to the nerve terminal. Moreover the action of calcium and TPA seems to be superimposed. The effect of K+ on neurotransmitter release could be explained not only by depolarization of the nerve terminal but by increasing the pool of activable PKC.

Animals↗
PubMed1990

Ca-dependent slow action potentials in neuromuscular diseases.

Slow Ca-dependent action potentials were studied in skeletal muscle fibers from different Neuromuscular Diseases (NMD). Biopsies were obtained from: 3 myopathies [Fascioscapulohumeral Dystrophy (FSH) and Polymyositis (PM)], 6 patients with other diseases (CD) [Amyotrophic Lateral Sclerosis (ALS), Central Core Disease, Mitochondrial Myopathy, Polyneuritis (PN), von Eulenberg's Paramyotonia], and 8 normal control muscles. Experiments were carried out in muscle fibers under current-clamp conditions. Membrane currents other than Ca ones were abolished or greatly diminished. Muscle fibers produced any of 3 types of responses, when stimulated by depolarizing pulses: fully developed Ca-action potentials (CaAP), abortive non-regenerative Ca responses (NrR), or only capacitive passive responses (WR). The 3 types of responses were not dependent on the basal conditions of the fibers. The frequency of observation of CaAPs was significantly higher in myopathic disease. In myopathies, 46% of the muscle fibers had CaAPs, while only 22% of fibers from CD and 15% of the fibers from normal muscles showed CaAPs. No differences were observed in the resting constants as well as in the CaAPs parameters between normal and diseased muscle fibers.

Action Potentials↗
PubMed1989

Electrical properties of normal, denervated and organ-cultured slow fibres of toad cruralis muscles.

Two types of fibres were characterized in the cruralis muscle of the toad using electrophysiological techniques: the slow and the fast fibres. Five to ten slow fibres were easily identified on the inner face of this muscle. The cruralis slow fibres developed the ability to produce action potentials 40 days after the sciatic nerve was transected at the hip level, while the slow fibres of the pyriformis muscle showed a latent period of 17 days after the same surgical treatment. However, when in addition to this procedure the nerve was transected at the point where it enters the muscle but without damaging the slow fibres, the latency was about 20 days. The slow fibres of the cruralis muscle maintained in organ culture developed the ability to produce action potentials in 24 days. During the winter the slow fibres of in vivo denervated cruralis and pyriformis muscles did not develop the ability to produce regenerative responses. More-over organ-cultured cruralis muscles taken from winter toads showed this same inability. These results further support the idea that the excitability of slow fibres is under the control of a neural factor rather than of activity. The seasonal dependence points to the fact that the metabolic state of the muscle is of crucial importance in determining the development of excitability of slow fibres.

Action Potentials↗
PubMed1988

Ca-dependent slow action potentials in human skeletal muscle.

Slow Ca-action potentials (CaAP) were studied in normal human skeletal muscle fibers obtained during surgery (fibers with both ends cut). Control studies also were carried out with intact as well as cut rat skeletal muscle fibers. Experiments were performed in hypertonic Cl-free saline with 10 or 84 mM Ca and K-channel blockers; muscles were preincubated in a saline containing Cs and tetraethylammonium. A current-clamp technique with two intracellular microelectrodes was used. In human muscle, 14.5% of the fibers showed fully developed CaAPs, 21% displayed nonregenerative Ca responses, and 64.5% showed only passive responses; CaAPs were never observed in 10 mM Ca. In rat muscle, nearly 90% of the fibers showed CaAPs, which were not affected by the cut-end condition. Human and rat muscle fibers had similar membrane potential and conductance in the resting state. In human muscle (22-32 degrees C, 84 mM Ca), the threshold and peak potential during a CaAP were +26 +/- 6 mV and +70 +/- 3 mV, respectively, and the duration measured at threshold level was 1.7 +/- 0.5 sec. In rat muscle, the duration was four times longer. During a CaAP, membrane conductance was assumed to be a leak conductance in parallel with a Ca and a K conductance. In human muscle (22-32 degrees C, 84 mM Ca, 40 micron fiber diameter), values were 0.4 +/- 0.1 microS, 1.1 +/- 0.7 microS, and 0.9 +/- 0.4 microS, respectively. Rat muscle (22-24 degrees C, 84 mM Ca) showed leak and K conductances similar to those found in human fibers. Ca-conductance in rat muscle was double the values obtained in human muscle fibers.

Action Potentials↗
PubMed1988

Immunoglobulins from amyotrophic lateral sclerosis patients enhance spontaneous transmitter release from motor-nerve terminals.

Amyotrophic lateral sclerosis (ALS) is an incapacitating neuromuscular disease of unknown etiology. Although laboratory evidence is lacking, circumstantial evidence supports the importance of immune factors in the pathogenesis of ALS. In the present study immunoglobulins from 4 of 8 ALS patients induced a significant increase in spontaneous quantal transmitter release as monitored by miniature end-plate potential (MEPP) frequency in mouse phrenic nerve-diaphragm preparations at 23 degrees C, whereas immunoglobulins from normal individuals and from patients with other neuromuscular diseases had no effect. At 32 degrees C neither normal nor disease control immunoglobulins influenced MEPP frequency, but 8 of 11 ALS immunoglobulin samples produced a significant increase in spontaneous quantal transmitter release. The enhancing effect could be prevented by 10 mM Mg2+. No effects were noted on MEPP amplitude and muscle resting potential. These findings suggest that the presynaptic terminal of the motor neuron may be an early target and that immunological factors may play an important role in the disease process.

Amyotrophic Lateral Sclerosis↗