Biomedical subjects

J Marsal

Publications and source records attributed to J Marsal.

At least 37 records · Page 2Linked to original sources

PubMed1996

Niflumic acid-induced increase in potassium currents in frog motor nerve terminals: effects on transmitter release.

The actions of the nonsteroidal antiinflammatory drug niflumic acid were studied on frog neuromuscular preparations by conventional electrophysiological techniques. Niflumic acid reduced the amplitude and increased the latency of endplate potentials in a concentration-dependent manner. Neuromuscular junctions pretreated with niflumic acid (0.05-0.5 mM) showed much less depression than control when they were stimulated with trains of impulses. Inhibition of acetylcholine release was reverted by raising the extracellular Ca(2+) concentration but not by simply washing out the preparations with niflumic acid-free solutions. Pretreatment with indomethacin (0.1 mM), another nonsteroidal antiinflammatory drug, did not affect the niflumic acid-induced inhibition of evoked responses. Niflumic acid (0.1 mM) did not change the amplitude of miniature endplate potentials and had a dual action on the frequency of miniatures: it decreased their frequency at 0.1 mM whereas it produced an enormous increase in the rate of spontaneous discharge at 0.5 mM. Niflumic acid (0.1 - 1 mM) reversibly increased the amplitude and affected the kinetics of presynaptic voltage-activated K+ current and Ca(2+)-activated K(+) current in a concentration-dependent manner. Niflumic acid (0.1 - 1 mM) irreversibly decreased the amplitude and reversibly affected the kinetics of the nodal Na(+) current. Indomethacin (0.1 mM) had no effect on presynaptic currents. In conclusion, niflumic acid reduces acetylcholine release by increasing presynaptic K+ currents. This may shorten the depolarizing phase of the presynaptic action potential and may reduce the entry of Ca(2+) with each impulse.

Animals↗

PubMed1996

Differential distribution of syntaxin isoforms 1A and 1B in the rat central nervous system.

Syntaxin 1 binds to several proteins of the synaptic terminal and is a central component in the pathway of protein-protein interactions that underlies docking and fusion of synaptic vesicles. Molecular studies revealed the occurrence of two isoforms, syntaxin 1A and syntaxin 1B, which coexpress in neural tissues. However, they display differential expression patterns in endocrine cell types. We generated isoform-specific antibodies that were used in Western blotting and immunocytochemical studies. First, we confirmed the sole presence of syntaxin 1A in endocrine pituitary cells. Second, we found distinctive immunolabelling patterns of each isoform in the rat olfactory system, hippocampus, striatum, thalamus and spinal cord. In addition, the principal white matter commissures displayed distinct immunoreactivity for each isoform. This report shows, for the first time, major differences between the distributions of syntaxin 1A and syntaxin 1B isoforms in the rat central nervous system.

Animals↗

PubMed1996

Expression of synaptosomal-associated protein SNAP-25 in endocrine anterior pituitary cells.

A growing body of evidence indicates that the fundamental molecular mechanism of exocytosis in the secretory pathway may be structurally similar in all eukaryotic cells. The synaptosomal-associated protein of 25 kDa (SNAP-25) is a plasma membrane protein involved in regulated exocytosis in neurons. In order to compare exocytotic components in neurons and endocrine cells, we have analyzed the expression of SNAP-25 in the rat anterior pituitary. Western blotting analysis documented the presence of SNAP-25 in anterior pituitary homogenates and cultured anterior pituitary cells. In addition to SNAP-25, other neuronal proteins involved in exocytosis (syntaxin, VAMP/synaptobrevin and Rab3A) were also detected in the anterior pituitary. The specific expression of SNAP-25 mRNA in anterior pituitary cells was also corroborated by Northern analysis. SNAP-25 immunoreactivity was located at the plasma membrane of endocrine anterior pituitary cells. Characteristically, patches of fine punctate deposits exhibited intense SNAP-25 immunoreactivity. Double-labeling immunocytochemistry revealed that SNAP-25 was mainly associated with gonadotroph cell populations. Furthermore, we demonstrate that in the anterior pituitary, SNAP-25 is selectively cleaved by clostridial neurotoxins. In conclusion, our results establish the presence of SNAP-25 in secretory anterior pituitary cells and suggest a potential role of this protein in the secretion of adenohypophysial hormone.

Animals↗

PubMed1995

Tetanus toxin inhibits spontaneous quantal release and cleaves VAMP/synaptobrevin.

Tetanus toxin decreased the frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocytosis and peptidase activity on synaptobrevin, is discussed.

Acetylcholine↗

PubMed1995

Botulinum toxin type A inhibits Ca(2+)-dependent transport of acetylcholine in reconstituted giant liposomes made from presynaptic membranes from cholinergic nerve terminals.

Giant liposomes were made from a mixture of asolectin phospholipid vesicles and presynaptic plasma membranes isolated from Torpedo cholinergic nerve endings. Acetylcholine filled giant liposomes were able to release neurotransmitter upon stimulation by the Ca2+ ionophore A23187 and Ca2+. Botulinum neurotoxin type A inhibited this Ca(2+)-dependent acetylcholine transport. Additionally, Botulinum toxin type A decreased membrane fluidity of liposomes. These results suggest that Botulinum toxin can interact directly with components of the presynaptic plasma membrane and inhibit acetylcholine translocation. Furthermore, since the reconstituted liposomes do not have synaptic vesicle components, the observed effects may account for the action of Botulinum toxin on the non-quantal release of acetylcholine from motor nerve terminals.

Acetylcholine↗

PubMed1995

Cloning characterization and expression of the cDNA encoding a neuron-specific alpha-tubulin isoform highly represented in the electric lobe of Torpedo marmorata.

A cDNA (alpha T6) encoding an alpha-tubulin from Torpedo marmorata (Tm) was isolated and sequenced. The deduced 451-amino-acid (aa) sequence codes for an alpha-tubulin of 50,161 Da. The aa sequence of alpha T6 of Tm showed a 70-99.6% identity to the other alpha-tubulins previously described. Moreover, the alpha T6 aa sequence was 95-99.6% identical to neural-specific tubulins of mouse, rat, human and siberian salmon. The corresponding mRNA is highly represented in the giant motoneurons of the electric lobe. All neuronal populations of the Tm brain exhibit variable levels of alpha T6 expression, with the highest levels in the long-axon-projecting neurons. These results suggest that this alpha-tubulin isoform may play an important role in the maintenance and/or remodeling of the neuronal cytoskeleton.

Amino Acid Sequence↗

PubMed1995

Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.

Acetylcholine↗

PubMed1995

Characterization of a rabbit serum raised against a botulinum toxin type A binding protein from presynaptic plasma membranes from Torpedo electric organ.

Botulinum neurotoxin type A blocks acetylcholine release from the peripheral nervous system. We have previously described a putative botulinum neurotoxin type A receptor of presynaptic plasma membranes from Torpedo. The electric organ of Torpedo, which is largely enriched in cholinergic nerve endings, is homologous to the neuromuscular junction, allowing us to isolate large scale of presynaptic components. In order to characterize this protein we have raised a polyclonal antibody (a-P140) against this receptor. The antiserum a-P140 recognizes a 140,000 mol. wt band in non-reducing conditions and an 80,000 band in reducing conditions. The immunohistochemistry assay reveals the P140 protein on the ventral face of the electrocytes where the nerve terminals are localized. Moreover, a-P140 antiserum recognizes the P140-BoNT/A complex after binding and cross-linking experiments. In addition, we have immunoprecipitated an in vitro translated product which is closely coincident in mol. wt to the 80,000 band of the receptor.

Animals↗

PubMed1995

Tetanus toxin mechanism of action in Torpedo electromotor system: a study on different steps in the intoxication process.

The mechanism of action of tetanus toxin was characterized in the electromotor system of Torpedo marmorata either at peripheral and central nervous system. The consecutive steps of the intoxication pathway were observed: (i) [125I]tetanus toxin specifically bound to neuronal plasma membranes isolated both from electric organ and electric lobe of Torpedo, exhibiting one and two binding sites respectively; (ii) [125I]tetanus toxin was internalized into nerve terminals and retrogradely transported to the electric lobe after its injection in the electric organ; (iii) finally, intracellular effect of tetanus toxin was studied either at electric organ and electric lobe membrane fractions. In both preparations tetanus toxin cleaved synaptobrevin, as detected by immunoblotting methods. In conclusion, our findings exhibit the presence of two different populations of acceptors for tetanus toxin in central and peripheral nervous system and show that synaptobrevin cleavage may account for intracellular toxicity in Torpedo.

Animals↗

PubMed1994

Transmission at the squid giant synapse was blocked by tetanus toxin by affecting synaptobrevin, a vesicle-bound protein.

1. The effect of whole tetanus toxin (TeTX) and of its light chain (TeTX L-chain) on transmitter release was determined by presynaptic pressure-injection in the squid giant synapse. 2. The results indicate that whole TeTX does not modify transmission while the L-chain blocks transmission within 20-30 min. This block does not involve changes in the sodium or potassium conductances responsible for spike generation or the voltage-dependent presynaptic calcium current responsible for transmitter release. 3. Western blotting of protein fractions from the squid optic lobe demonstrated the presence of a protein which reacted with specific antibodies against mammalian synaptobrevin, a vesicular protein. In addition, this protein was enzymatically cleaved by the L-chain component of the toxin in a similar fashion to its mammalian counterpart. 4. These results demonstrate that TeTX L-chain toxin acts directly on a squid synaptobrevin and prevents synaptic release probably by interfering with the docking-fusion synaptic vesicles at the active zone.

Animals↗

PubMed1994

Nerve growth factor and basic fibroblast growth factor protect cholinergic neurons against quinolinic acid excitotoxicity in rat neostriatum.

In the present work we have characterized a possible mechanism leading to the early survival of neostriatal cholinergic neurons after quinolinic acid injection. Different doses of quinolinic acid were injected in rat neostriatum and two different parameters were analysed 7 days after the lesion: choline acetyltransferase (ChAT) activity and nerve growth factor (NGF) levels. We have observed that ChAT activity decreased (until 68 nmol quinolinic acid) and NGF levels increased (until 34 nmol quinolinic acid) in a dose-dependent manner. In order to characterize the time-course of the lesion on NGF levels and ChAT activity, and the possible protective effect of NGF and basic fibroblast growth factor (bFGF) on cholinergic neurons, we have used the quinolinic acid dose (68 nmol) at which the first decrease of ChAT activity was observed. ChAT activity and NGF levels showed different patterns of response to quinolinic acid injection, since the maximal effect was reached at 1 day for ChAT activity and at 2 days for NGF levels. NGF or bFGF simultaneously injected with quinolinic acid (68 nmol) completely prevented the decrease in ChAT activity in a dose-dependent manner but NGF was more effective than bFGF. Furthermore, differences observed in ChAT activity after NGF but not bFGF treatment were correlated with changes in the number of ChAT immunoreactive cells. Finally, we have also observed that, although bFGF alone was not able to modify NGF levels, bFGF simultaneously injected with quinolinic acid produced an increase of NGF levels higher than that observed after quinolinic acid injection alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Zinc ions block rectifier potassium channels and calcium activated potassium channels at the frog motor nerve endings.

The effect of Zn2+ on presynaptic currents was investigated on frog cutaneous pectoris nerve-muscle preparations. Nerve terminal spikes were recorded with extracellular electrodes placed in the perineurial sheaths of motor nerves. Zinc ions reversibly suppressed the component of the waveform associated with K+ currents--unmasking an inward current at the terminal--and induced repetitive firing when were applied to preparations perfused with calcium containing solutions. In experiments in which delayed rectifier channels were blocked by 3,4-diaminopyridine, Zn2+ caused a prolonged and reversible inward current associated with a slight decrease in the peak calcium current generated by 3,4-diaminopyridine. Zinc ions abolished the plateau calcium current produced by the simultaneous action of 3,4-diaminopyridine and tetraethylammonium. Finally, in all the preparations in which the exposure to Zn2+ was prolonged it was observed a dramatic and irreversible reduction of the presynaptic currents. These results suggest that Zn2+ has, at least, four different effects on presynaptic currents: (1) blockade of delayed rectifier potassium currents, (2) blockade of calcium-activated potassium currents, (3) blockade of calcium currents and (4) a delayed and irreversible disruption of all ionic conductances of the terminal.

4-Aminopyridine↗

PubMed1994

Tacrine-induced increase in the release of spontaneous high quantal content events in Torpedo electric organ.

1. The anticholinesterases, tacrine (100 microM) and physostigmine (60 microM) had different effects on the amplitude distribution and kinetics of miniature endplate currents (m.e.p.cs) recorded extracellularly from the electric organ of Torpedo marmorata. 2. Tacrine increased the ratio of giant miniatures (larger than 4 mV of amplitude) to more than 20% of recorded spontaneous events. In the presence of physostigmine such events represented only 4%. 3. Both tacrine and physostigmine increased the rise time and the decay phase of normal-sized m.e.p.cs when compared to control conditions. Both effects were significantly greater for tacrine. 4. We have tested the specificity of the tacrine effect on ectoenzyme activities associated with plasma membranes of these pure cholinergic nerve endings. Tacrine does not act unspecifically on every ectoenzyme, because it is not able to block the ectoapyrase activity even at a concentration 100 fold greater than that required to inhibit 94% of AChE. 5. We conclude that the differential effects of tacrine and physostigmine can be explained in terms of undetermined presynaptic actions of tacrine, while comparable effects of the two compounds can be explained through a shared anticholinesterase activity.

Acetylcholine↗

PubMed1993

High resolution labeling of cholinergic nerve terminals using a specific fully active biotinylated botulinum neurotoxin type A.

We report here on the synthesis and characterization of a fully active biotinylated derivative of the botulinum neurotoxin type A. Different ratios of biotin: botulinum toxin were tested to optimize derivatizing conditions and a ratio of 35:1 was selected for further experiments. The average number of biotin groups per toxin molecule was estimated to be 7.8, occurring at both heavy and light chains, and almost all externally located and easily accessible to recognition by streptavidin. The modified toxin retained its toxicity and its ability to interact with biological membranes. Apart from its suitability for detection in Western blots and in microtiter well plates, biotinylated botulinum toxin proved to be adequate for morphological labeling studies at both light and electron microscopy. Peroxidase histochemistry in cryostat sections of intoxicated rat hemidiaphragm muscles showed a distinct labeling of end-plates. Electron microscopy studies were performed on the electric organ of Torpedo marmorata using colloidal gold-conjugated streptavidin for detection. After intoxication of electric organ fragments with the modified toxin, gold labels were found associated with the presynaptic plasma membrane of nerve terminals and with the membrane of synaptic vesicles. Moreover, the distribution of biotinylated botulinum toxin binding sites over the membrane of synaptosomes isolated from the electric organ of Torpedo and their relationship with intramembrane particles were analyzed using the replica-staining label-fracture technique. It was found that the toxin is never associated with intramembrane particles.

Animals↗

PubMed1993

Selective resistance of tachykinin-responsive cholinergic neurons in the quinolinic acid lesioned neostriatum.

We have studied changes on endogenous acetylcholine (ACh) release evoked by different agents from rat neostriatal slices after quinolinic acid (QA) injections. QA lesions induced a biphasic decrease on ACh release evoked by 1 microM glutamate and 50 mM KCl. ACh release evoked by selective tachykinin agonists was only significantly decreased by 250 nmol QA. These results suggest the presence of different functional cholinergic cell populations, with tachykinin-responsive cholinergic neurons selectively spared.

Acetylcholine↗

PubMed1993

Nerve growth factor and its receptor are differentially modified by chronic naltrexone treatment during rat brain development.

In order to examine the relationship between the action of opioid neurotransmitters and growth factors in the regulation of brain development, we have studied the long-term effect of the opiate antagonist naltrexone (NTX) on the content of nerve growth factor (NGF) in cortex, hippocampus, septum and neostriatum, and on NGF receptor (NGFRs) levels in cortical membranes. 50 mg/kg NTX treatment induced a decrease in the number of 125I-NGF high-affinity binding sites, without detectable changes in NGF levels. However, low doses of NTX (1 mg/kg) produced no differences in 125I-NGF binding sites, but induced a decrease in NGF levels in hippocampus, septum and neostriatum. These results suggest that NGF and NGFRs could be involved in the trophic effects of opioids during brain development.

Animals↗

PubMed1993

Control of tachykinin-evoked acetylcholine release from rat striatal slices by dopaminergic neurons.

The regulation of tachykinin-evoked acetylcholine release by the dopaminergic system in the neostriatum was examined. We studied the effect of selective and potent tachykinin agonists for each subtype of receptor ([Sar9,Met(O2)11]-Substance P for NK1; [Nle10]-Neurokinin A4-10 for NK2; and senktide for NK3) on endogenous acetylcholine release from rat striatal slices where the dopaminergic system was modified either by 6-hydroxydopamine lesion or by dopamine receptor antagonists. Unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway induced a decrease in senktide-evoked acetylcholine release and an increase in the effect of [Nle10]-Neurokinin A4-10. The same results were obtained after chronic haloperidol treatment, whereas SCH-23390 or clozapine treatment had no effect on tachykinin-evoked acetylcholine release, suggesting an involvement of D2 receptors. 6-hydroxydopamine lesion induced a diminution in the density of NK3 receptor, which could be related to the reduction in senktide-evoked acetylcholine release.

Acetylcholine↗

PubMed1993

Calcium channel antagonist omega-conotoxin binds to intramembrane particles of isolated nerve terminals.

Voltage-sensitive calcium channels play a key role in evoked neurotransmitter release and their distribution in presynaptic membranes can be critical for fast signalling at chemical synapses. Using a biotinylated derivative of the neuronal calcium channel antagonist, omega-conotoxin, and a combination of colloidal gold labeling and freeze-fracture techniques, we have labeled calcium channels present at the membrane of nerve terminals isolated from the electric organ of Torpedo marmorata. The biotinylated blocker exerts an inhibitory action on the high potassium-evoked release of adenosine triphosphate as the native toxin does and its interaction with biological membranes is specific as shown in displacement experiments. This study shows that an antagonist specific for voltage-activated calcium channels binds to intramembrane particles in presynaptic membranes, reinforcing the idea that these particles, concentrated at neurotransmitter release sites, effectively represent calcium channels.

Adenosine Triphosphate↗