Biomedical subjects

C Solsona

Publications and source records attributed to C Solsona.

At least 37 records · Page 2Linked to original sources

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↗

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

Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney.

Adenine Nucleotides↗

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↗

PubMed1992

Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo.

Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane. 125I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: Kd = 0.11 +/- 0.03 nM, Bmax = 50 +/- 10 fmol.mg prot-1; presynaptic plasma membrane: Kd = 0.2 +/- 0.05 nM, Bmax = 150 +/- 15 fmol.mg prot-1) and a low affinity binding site (synaptosomes: Kd approximately 26 nM, Bmax approximately 7.5 pmol.mg prot-1; presynaptic plasma membrane: Kd approximately 30 nM, Bmax approximately 52 pmol.mg prot-1). The binding of 125I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either 125I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M(r) approximately 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.

Acetylcholine↗

PubMed1992

Omega-conotoxin differentially blocks acetylcholine and adenosine triphosphate releases from Torpedo synaptosomes.

We have examined the effect of several blockers of voltage-sensitive calcium channels on the release of acetylcholine and ATP from synaptosomes isolated from Torpedo marmorata electric organ. Depolarization of these nerve terminals with high K(+)-containing solutions resulted in a calcium-dependent release of both molecules. Cadmium ions (10(-6) to 10(-3) M) inhibited similarly both releases whereas nickel ions (10(-4) M) in the external medium did not affect either neurotransmitter or nucleotide release. Both releases were completely resistant to the effect of 1,4-dihydropyridines (antagonists nimodipine, nifedipine and agonist Bay K 8644) and of a related compound (diltiazem) at concentrations up to 10(-5) M. These drugs failed to cause any effect even when synaptosomes were submaximally depolarized during incubation. Omega-conotoxin (10(-8) to 5 x 10(-5) M) showed a differential effect on acetylcholine and ATP releases. Nucleotide release was inhibited 90% at the highest concentration tested (50 microns) while acetylcholine release was only moderately decreased (30%). EC50 values for acetylcholine and ATP were of 167 and 2 microM respectively. The results suggest the implication of different types of calcium channels in the release of these molecules.

Acetylcholine↗

PubMed1991

Effects of potassium depolarization on intracellular compartmentalization of ATP in cholinergic synaptosomes isolated from Torpedo electric organ.

It is well known that acetylcholine (ACh) and ATP are co-stored and co-released in nerve terminals of the electric organ of Torpedo. Cholinergic synaptosomes were subjected to a cycle of freezing and thawing showing that ATP is distributed in two operational pools like those described for ACh. The bound pool is resistant to freezing and thawing, and it is presumably protected by membranes. When metabolically active ATP was prelabelled with [3H]adenosine, 76% of the radioactivity was associated with the free pool of ATP. When the preparation was depolarized in a calcium containing medium, there was a decrease in the specific radioactivity of ATP in the free pool and an increase in the bound pool. These results reflect that the patterns of distribution of ACh and ATP, in this synaptosomal preparation, are similar in resting conditions and during K+ depolarization.

Adenosine Triphosphatases↗

PubMed1991

Neostriatal dopaminergic terminals prevent the GABAergic involvement in the mu- and delta-opioid inhibition of KCl-evoked endogenous acetylcholine release.

Endogenous acetylcholine (ACh) release from rat neostriatal slices was inhibited by the mu-opioid agonist [D-Ala2,Gly(ol)5]-enkephalin (DAGO) both in 6-hydroxydopamine (6-OHDA)-lesioned and non-lesioned neostriatum. However, the delta-opioid agonist [D-Pen2,D-Pen5]-enkephalin (DPDPE) could not inhibit KCl-evoked ACh release in the 6-OHDA-lesioned striatum. This result suggests that delta-opioid agonists act on dopaminergic terminals to inhibit the cholinergic neurons. In unlesioned rats, GABAA or GABAB) antagonists (bicuculline or phaclofen, respectively) prevented mu- or delta-opioid inhibition of endogenous ACh release evoked by glutamate, but not by potassium. However, in the 6-OHDA-lesioned side, DAGO inhibition of KCl-evoked ACh release was antagonized by either of the GABA antagonists. Our results suggest that the dopaminergic neurotransmission, favored by KCl, blocks the GABAergic involvement in the mu- and delta-opioid inhibition of endogenous ACh release.

Acetylcholine↗

PubMed1991

Neurokinin receptors differentially mediate endogenous acetylcholine release evoked by tachykinins in the neostriatum.

The regulation of neostriatal cholinergic function by tachykinins (TKs) has been studied by measuring endogenous ACh released from rat neostriatal slices. Septide (SEP; a highly selective substance P analog), neurokinin A (NKA), and neurokinin B (NKB) elicited endogenous ACh release in a concentration-dependent manner. The rank order in potency was the following: NKB (EC50 approximately 0.5 nM) greater than NKA (EC50 approximately 7 nM) greater than SEP (EC50 approximately 12 nM). Spantide (SPA) was less effective (39% inhibition) than [D-Arg6, D-Trp7,9, N-Methyl-Phe8]-substance P fragment 6-11 (53% inhibition) at antagonizing ACh release evoked by SEP and NKA. Smaller doses of the antagonists inhibited the effects of SEP compared to NKA, and the effects of NKB could only be antagonized by SPA. These findings suggest the involvement of the three neurokinin (NK) receptors in ACh release evoked by TKs with the following rank order: NK3 greater than NK2 greater than NK1. 6-Hydroxydopamine lesions of nigrostriatal neurons and tetrodotoxin (TTX) intoxication of striatal tissue revealed two different patterns of regulation of cholinergic function by TKs. On the one hand, SEP and NKA evoked ACh release, independently of the nigrostriatal dopaminergic system, by acting on NK1 and NK2 receptors that are probably localized on the somatodendritic field of cholinergic neurons receiving substance P terminals. On the other hand, dopaminergic terminals seem to regulate NKB neurons that modulate cholinergic neurons, because NKB-evoked ACh release decreased by 24% in the denervated striata. In addition, TTX partially blocked (50%) ACh release evoked by NKB, suggesting that NKB acts on NK3 receptors at both the nerve terminals and the somatodendritic field of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1990

The effects of TMB-8 on acetylcholine release from frog motor nerve: interactions with adenosine.

The putative intracellular calcium (Ca) antagonist TMB-8 was shown to reduce postjunctional sensitivity and quantal acetylcholine (ACh) release at low micromolar concentrations. At 10-fold higher concentrations, TMB-8 also blocked caffeine-induced Ca release (as monitored electrophysiologically by changes in ACh release) but did not impair the ability of adenosine to inhibit quantal ACh release. This last result implies that TMB-8 and adenosine exert their inhibitory actions at different steps in the depolarization-secretion coupling sequence.

Acetylcholine↗

PubMed1990

The release of adenosine at the electric organ of Torpedo. A study using a continuous chemiluminescent method.

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ of Torpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H2O2 by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction of Torpedo electric organ. The presence of alpha, beta-methylene ADP, an inhibitor of 5'-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.

Adenosine↗

PubMed1990

Opiates depress ACh and ATP release from cholinergic synaptosomes by blocking calcium uptake.

We have studied the effects of opiates on ATP and acetylcholine (ACh) release from cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. The release of ATP was inhibited by morphine and this action was reversed by naloxone. Morphine, [D-Met2-Pro5]enkephalinamide and [D-Ala2-Leu5]enkephalin also inhibited acetylcholine release. Naloxone prevented these inhibitory effects. The action of enkephalin on ACh release was less effective than that of morphine. The calcium uptake by nerve terminals of Torpedo electric organ was also inhibited by morphine, either under resting or depolarizing conditions, and this effect was reversed by naloxone. Using the quick freeze-fracture method, the structural changes induced by morphine in the presynaptic membrane were also studied. Morphine prevents the rearrangement of intramembrane particles (IMPs) at both freeze-fractured faces of the synaptosomal presynaptic membrane after depolarization. It is concluded that opiates depress the ATP and the ACh releases from cholinergic synaptosomes by inhibiting the calcium uptake by the nerve terminals and the rearrangement of the IMPs after potassium-induced depolarization. Furthermore, ACh release, but not ATP release, seems to be related with the rearrangement of IMPs in the presynaptic membrane.

Acetylcholine↗

PubMed1990

Tetanus toxin blocks potassium-induced transmitter release and rearrangement of intramembrane particles at pure cholinergic synaptosomes.

We have studied the action of tetanus toxin on the release of acetylcholine from a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. We have also studied the morphological changes induced by chemical stimulation on the presynaptic plasma membrane of poisoned synaptosomes. These changes were studied by means of freeze-fracture techniques. We found that tetanus toxin blocks the release of acetylcholine from isolated nerve terminals in a dose-dependent manner. The maximal inhibition is achieved at a concentration of 12.5 nM in 10 min. This effect is prevented by tetanus toxin antiserum. Tetanus toxin also blocks the rearrangement of intramembrane particles at plasma membrane of poisoned synaptosomes, specifically the decrease of small (less than or equal to 9.5 nm diameter) intramembranous particles at the protoplasmic hemimembrane leaflet and the increase of large (greater than 9.5 nm diameter) intramembrane particles at the external hemimembrane leaflet induced by potassium stimulation. These results suggest that intramembrane particle rearrangement could be related to acetylcholine secretion.

Acetylcholine↗

PubMed1990

Phorbol esters induce neurotransmitter release in cholinergic synaptosomes from Torpedo electric organ.

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

The action of botulinum toxin on cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. Detection of a putative toxin receptor.

Botulinum neurotoxin type A (BoNTx) inhibits the release of acetylcholine (ACh) from Torpedo electric organ synaptosomes. We have studied several biochemical and morphological aspects in order to characterize the molecular interactions of BoNTx intoxication in our preparation. 1. We are describing for the first time an electrophoretic band from cholinergic presynaptic plasma membrane (PSPM) that is recognized by 125I-BoNTx as a putative BoNTx receptor. 2. Furthermore we describe direct interaction of botulinum toxin-gold complexes with synaptic vesicles through the three-step model of the BoNTx intoxication.

Animals↗

PubMed1989

GABAergic modulation of acetylcholine release in cholinergic synaptosomes from Torpedo marmorata electric organ.

GABAA- and GABAB-receptor-specific agonists inhibit the depolarization-evoked release of acetylcholine in cholinergic synaptosomes from Torpedo electric organ. Over 60% of the release is inhibited by a 10(-4) M concentration of GABA itself. IC50s for muscimol and baclofen are 1.3 x 10(-4) and 2.2 x 10(-6) M, respectively. The effect of muscimol is totally blocked by the direct antagonist bicuculline methiodide, and also by the allosteric antagonists methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, picrotoxinin and tert-butylbicyclo-orthobenzoate; the effect of baclofen is blocked by delta-aminovalerate. Furthermore, the inhibitory action of muscimol on acetylcholine release is substantially enhanced by flunitrazepam and pentobarbital. These results suggest the existence of typical GABAA and GABAB receptors in the presynaptic nerve terminals of the Torpedo electric organ regulating the liberation of acetylcholine and therefore the discharge of the electroplaques.

Acetylcholine↗

PubMed1989

Increase in reactive cholesterol in the presynaptic membrane of depolarized Torpedo synaptosomes: blockade by botulinum toxin type A.

We have investigated the redistribution of filipin-cholesterol complexes at freeze-fractured presynaptic membrane of pure cholinergic synaptosomes isolated from Torpedo electric organ during acetylcholine release. After chemical depolarization, filipin-induced lesions increase at the presynaptic membrane. These changes do not take place when synaptosomes are stimulated in a calcium-free medium. Botulinum neurotoxin type A blocks both acetylcholine release and the rearrangement of filipin-induced lesions induced by depolarization. Since botulinum neurotoxin type A does not block either membrane depolarization or calcium entry into the nerve terminal, our results suggest that the redistribution of filipin-cholesterol complexes is linked to the acetylcholine release process.

Acetylcholine↗