Biomedical subjects

N Morel

Publications and source records attributed to N Morel.

At least 37 records · Page 2Linked to original sources

PubMed2001

Lacidipine prevents endothelial dysfunction in salt-loaded stroke-prone hypertensive rats.

Endothelium-dependent vasorelaxation is defective in hypertensive rats, especially in conduit arteries. In the stroke-prone spontaneously hypertensive rat, impaired endothelium-dependent vasorelaxation appears to contribute to the pathogenesis of stroke independent of blood pressure. Because treatment with lacidipine, a long-acting calcium channel blocker, protects against stroke and cardiovascular remodeling in this model, we investigated the effect of this treatment on endothelium-dependent vasorelaxation in the aorta. Stroke-prone rats were exposed to a salt-rich diet (1% NaCl in drinking water) with or without lacidipine (1 mg. kg(-1). d(-1)) for 6 weeks. A high-sodium diet (1) increased systolic blood pressure, aortic weight, and wall thickness and plasma renin activity (P<0.05); (2) markedly reduced nitric oxide (NO)-mediated, endothelium-dependent relaxation of aortic rings to acetylcholine and the sensitivity to the relaxing effect of S-nitroso-N-acetylpenicillamine, an NO donor (P<0.001); and (3) induced an elevation of preproendothelin-1 mRNA levels in aortic tissue (P<0.01) without affecting endothelial NO synthase mRNA levels. Lacidipine treatment prevented the salt-dependent functional and structural alterations of the aorta, including the overexpression of the preproendothelin-1 gene, and increased endothelial NO synthase mRNA levels in aortic tissue (P<0.01). In conclusion, lacidipine protects stroke-prone hypertensive rats against the impairment of endothelium-dependent vasorelaxation evoked by a salt-rich diet, and this effect may contribute to its beneficial effect against end-organ damage and stroke.

Animals↗

PubMed2000

Role of mediatophore in connection with proteins of the active zone in synaptic transmission.

Mediatophore is a protein purified from Torpedo electric organ synaptosomes, which translocates acetylcholine (ACh) upon calcium action after reconstitution in artificial membranes. After expression in transfected cells, it endows these cells with a calcium-dependent release mechanism displaying clear quantal properties. The role of mediatophore in synaptic transmission is discussed in relation to the ultrastructural organization of the active zone and the cytosolic high calcium microdomains that transiently appear after presynaptic membrane depolarization.

Acetylcholine↗

PubMed2000

Comparative expression of homologous proteins. A novel mode of transcriptional regulation by the coding sequence folding compatibility of chimeras.

Recombinant acetylcholinesterases (AChE) are produced at systematically different levels, depending on the enzyme species. To identify the cause of this difference, we designed expression vectors that differed only by the central region of the coding sequence, encoding Torpedo, rat, and Bungarus AChEs and two reciprocal rat/Bungarus and Bungarus/rat chimeras. We found that folding is a limiting factor in the case of Torpedo AChE and the chimeras, for which only a limited fraction of the synthesized polypeptides becomes active and is secreted. In contrast, the fact that rat AChE is less well produced than Bungarus AChE reflects the levels of their respective mRNAs, which seem to be controlled by their transcription rates. A similar difference was observed in the coding and noncoding orientations; it seems to depend on multiple cis-elements. Using CAT constructs, we found that a DNA fragment from the Bungarus AChE gene stimulates expression of the reporter protein, whereas a homologous fragment from the rat AChE gene had no influence. This stimulating effect appears different from that of classical enhancers, although its mechanism remains unknown. In any case, the present results demonstrate that the coding region contributes to control the level of gene expression.

Acetylcholinesterase↗

PubMed2000

Action of a NO donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery.

The aim of the present study was to investigate the actions of NO donors in ratsuperior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (Vm), cytosolic calcium concentration ([Ca2+]cyt) and accumulation of inositol phosphates. In unstimulated arteries, SNAP (S-nitroso-N-acetylpenicillamine, 10 microM) hyperpolarised Vm by 3.0 +/- 0.5 mV (n = 9). In KCl-stimulated arteries, SNAP relaxed contraction without changing Vm and [Ca2+]cyt. In noradrenaline-stimulated arteries, SNAP relaxed tension, repolarised Vm and decreased [Ca2+]cyt with the same potency. Responses to SNAP were unaffected by the following K+ channel blockers: glibenclamide, 4-aminopyridine, apamin and charybdotoxin, and by increasing the KCl concentration to 25 mM. In SNAP-pretreated arteries, the production of inositol phosphates and the contraction stimulated by noradrenaline were inhibited similarly. The guanylate cyclase inhibitor ODQ abolished the increase in cyclic GMP content evoked by SNAP and inhibited the effects of SNAP on contraction, Vm and accumulation of inositol phosphates in noradrenaline-stimulated artery. These results indicate that, in rat superior mesenteric arteries activated by noradrenaline, inhibition of production of inositol phosphates is responsible for the effects of the NO donor SNAP on membrane potential, [Ca2+]cyt and contraction through a cyclic GMP-dependent mechanism.

Aluminum Compounds↗

PubMed2000

Changes of the potassium currents in rat aortic smooth muscle cells during postnatal development.

This study was designed to investigate the role and regulation of arterial K+ channels during postnatal development. Rat thoracic aortic segments were suspended for isometric tension and resting membrane potential (RMP) recording. Contraction in response to 4-aminopyridine (4-AP) was similar in 4-, 8- and 12-week-old rats but was higher in 1-day-old rats. Contraction in response to tetraethylammonium (TEA) increased after 4 weeks. TEA increased the contractions evoked by noradrenaline in the aorta from 8- and 12-week-old rats but not from 1-day- and 4-week-old rats. RMP did not change during development. Patch-clamp studies of freshly isolated smooth muscle cells from the same aortas bathed in Ca2+-free medium showed a voltage-dependent K+ current (IK) sensitive to 4-AP. This current remained stable at all ages whereas the density of the total IK, recorded in the presence of Ca2+, showed a twofold increase between 4 and 8 weeks. This current was highly sensitive to TEA and charybdotoxin. The binding site density of 125I-labelled charybdotoxin was threefold higher in the membranes of aortas from 12-week-old compared to 4-week-old rats. These results indicate that changes in K+ channel distribution occur in the rat aorta during postnatal development. These are related to an increase in the expression of charybdotoxin-sensitive Ca2+-activated K+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1999

The polymorphism of acetylcholinesterase: post-translational processing, quaternary associations and localization.

The molecular forms of acetylcholinesterase (AChE) correspond to various quaternary structures and modes of anchoring of the enzyme. In vertebrates, these molecules are generated from a single gene: the catalytic domain may be associated with several types of C-terminal peptides, that define distinct types of catalytic subunits (AChE(S), AChE(H), AChE(T)) and determine their post-translational maturation. AChE(S) generates soluble monomers, in the venom of Elapid snakes. AChE(H) generates GPI-anchored dimers, in Torpedo muscles and on mammalian blood cells. AChE(T) is the only type of catalytic subunit that exists in all vertebrate cholinesterases; it produces the major forms in adult brain and muscle. AChE(T) generates multiple structures, ranging from monomers and dimers to collagen-tailed and hydrophobic-tailed forms, in which catalytic tetramers are associated with anchoring proteins that attach them to the basal lamina or to cell membranes. In the collagen-tailed forms, AChE(T) subunits are associated with a specific collagen, ColQ, which is encoded by a single gene in mammals. ColQ contains a short peptidic motif, the proline-rich attachment domain (PRAD), that triggers the formation of AChE(T) tetramers, from monomers and dimers. The critical feature of this motif is the presence of a string of prolines, and in fact synthetic polyproline shows a similar capacity to organize AChE(T) tetramers. Although the COLQ gene produces multiple transcripts, it does not generate the hydrophobic tail. P, which anchors AChE in mammalian brain membranes. The coordinated expression of AChE(T) subunits and anchoring proteins determines the pattern of molecular forms and therefore the localization and functionality of the enzyme.

Acetylcholinesterase↗

PubMed1999

Effect of nitro-L-arginine on electrical and mechanical responses to acetylcholine in the superior mesenteric artery from stroke-prone hypertensive rat.

1. High salt diet is known to aggravate the vascular pathology in spontaneously hypertensive stroke-prone rats (SHR-SP). The aim of the present study was to assess the involvement of endothelial dysfunction in this effect. Contractile tension and membrane potential were simultaneously recorded in superior mesenteric artery rings of untreated and NaCl-loaded (1% NaCl in the drinking water) SHR-SP and normotensive Wistar Kyoto rats (WKY). 2. In unstimulated artery, hyperpolarization evoked by acetylcholine was not different in WKY and in NaCl-loaded WKY; it was reduced in SHR-SP and further reduced in NaCl-loaded SHR-SP. Hyperpolarization was unaffected by N(omega)-nitro-L-arginine (L-NA) but was abolished in high-KCl solution. 3. In noradrenaline-stimulated artery, ACh-evoked hyperpolarization and relaxation were not different in WKY and in SHR-SP. NaCl-treatment did not affect the responses to ACh in WKY but decreased maximum relaxation in SHR-SP from 93+/-2% to 72+/-7% of the contraction. In WKY, in NaCl-loaded WKY and in SHR-SP, L-NA similarly shifted the concentration-relaxation curve to ACh to the right and depressed its maximum but L-NA did not affect the hyperpolarization to ACh. In NaCl-loaded SHR-SP, L-NA blunted the effects of ACh on membrane potential and on contraction. 4. The NO donor SNAP abolished the depolarization and the contraction evoked by noradrenaline with the same potency in WKY and in untreated SHR-SP but was more potent in NaCl-loaded SHR-SP. 5. In KCl-contracted arteries the relaxations to ACh were not different in WKY and SHR-SP but NaCl-loaded SHR-SP were more sensitive to ACh. 6. The results showed that NaCl-rich diet markedly reduced the L-NA-resistant responses to ACh and increased the sensitivity to NO in SHR-SP.

Acetylcholine↗

PubMed1999

Functional reduction and associated cellular rearrangement in SHRSP rat basilar arteries are affected by salt load and calcium antagonist treatment.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a strain with high incidence of cerebrovascular accidents increased by salt-rich diet and decreased by calcium-antagonist treatment. In the SHRSP rat basilar artery the authors have previously shown reduced contractility and altered structure including regions of smooth muscle cell (SMC) disorganization. The aims of this study have been to analyze (1) the morphology of these abnormal regions, (2) the structural modifications responsible for the reduced function, and (3) the effect of salt and calcium-antagonist treatment on vascular structure and function. Wistar Kyoto and SHRSP rats, untreated or treated from week 8 through 14 with 1% NaCl or 1% NaCl + 1 mg x kg(-1) x d(-1) lacidipine, were used. Function was studied with wire myography. Structure was analyzed in fixed intact arteries with confocal microscopy. Basilar arteries from SHRSP rat showed (1) reduced contractility, (2) discrete foci of SMC disarray with altered proportion of adventitia to SMC, and (3) decreased SMC and increased adventitial cell number. Arteries from salt-loaded SHRSP rats showed a higher degree of SMC disarray and further reduction in contractility. Lacidipine treatment of salt-loaded rats significantly improved structure and function. These data suggest that vascular remodeling can provide an explanation for the observed reduction in vascular contractility of SHRSP rat basilar arteries and might show light on the effects of salt load and calcium-channel blockers in life span and the incidence of cerebrovascular accidents in SHRSP rats.

Animals↗

PubMed1999

Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase.

Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.

Acetylcholinesterase↗

PubMed1999

VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much resemble homotypic fusion.

Animals↗

PubMed1998

A therapeutic dosage of amlodipine prevents vascular hyporeactivity induced in rats by lipopolysaccharide.

The aim of this work was to investigate whether treatment with the 1,4-dihydropyridine Ca2+ antagonist amlodipine could affect the vascular hyporesponsiveness induced by cytokines. Endotoxemia was induced by Salmonella typhosa lipopolysaccharide (LPS) injection (4 mg kg(-1), i.p.). In endothelium-denuded rings of thoracic aorta from untreated rats, contractile response to noradrenaline was decreased after LPS injection, this effect was partially overcome by the addition of N(omega)-nitro-L-arginine (L-NNA, 100 microM) into the bathing solution. In amlodipine-pretreated rats (15 mg kg(-1) day(-1), orally, for one week), the effect of LPS was lower than in untreated ones and it was completely reversed by L-NNA. The relaxation of the noradrenaline-induced tone evoked by L-arginine (10 microM) in aortae of LPS-injected rats was reduced in amlodipine-pretreated rats. Amlodipine-treatment reduced both the LPS-induced Ca2+-independent NOS activity in homogenates of heart and the expression of iNOS mRNA in aortae of LPS-injected rats. However, the vascular hyporeactivity induced by exposing aortae to interleukin-1beta in vitro was not influenced by amlodipine (10 nM). Amlodipine (10 microM) also did not affect the production of nitrite in primary aortic smooth muscle cell culture challenged by LPS although nitrite production in macrophage culture challenged with LPS was significantly inhibited. The results show that rat pretreatment with amlodipine prevented the decrease of vascular responsiveness induced by LPS, an effect that may be at least partly related to reduction of in vivo NOS induction. The weak effect of amlodipine on the in vitro NOS induction indicates that the protective action in endotoxemia did not result from a short term interaction with L-type Ca2+ channels in vascular smooth muscle. Alternative mechanisms are discussed.

Amlodipine↗

PubMed1998

Increased reactivity to 5-hydroxytryptamine of portal veins from mice infected with Schistosoma mansoni.

In chronic severe infection with Schistosoma mansoni, portal hypertension accompanied by anatomical changes of the portal vasculature can develop as a consequence of granulomatous response to eggs. Mice infected unisexually with male worms were used in the present study in order to investigate a direct effect of worms on the reactivity of their host portal vein. A higher reactivity in the presence of 5-hydroxytryptamine (5-HT), but not in the presence of KCl 100 mM solution, was observed in portal vein from infected mice compared to healthy mice. It was characterized by an increase in the maximal contraction and sensitivity to 5-HT. Blockade of NO-synthase with N omega-nitro-L-arginine methyl ester (L-NAME) induced a small increase in 5-HT potency in the portal vein from non-infected mice, but did not change the amplitude of the contractions. In portal veins from infected mice, preincubation with L-NAME did not affect the reactivity to 5-HT. Histological analysis indicated endothelial damage, subendothelial fibrous plaques, and focal areas of inflammatory infiltrates in the adventitial layer. As a conclusion, these results show that unisexual infection of mice with male S. mansoni increased the reactivity of the portal vein to 5-HT which seems to be only partially related to an alteration in the endothelial production of nitric oxide.

Animals↗

PubMed1998

The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel.

Animals↗

PubMed1998

Vacuolar H+-ATPase domains are transported separately in axons and assemble in Torpedo nerve endings.

Torpedo electric organ synaptosomes possess a typical vacuolar H+-ATPase (V-ATPase), inhibited by concanamycin A and insensitive to vanadate, made of the association of a catalytic soluble sector V1 to a membrane domain V0. In the electric nerves, the 57-kDa subunit B of the V1 sector was transported to the nerve endings by the slow axonal flow and did not accumulate upstream from an axonal block. In contrast, a 500% accumulation of the 15-kDa subunit c of the V0 membrane domain was observed, demonstrating that this subunit is conveyed by the fast axonal anterograde transport. After velocity sedimentation of solubilized nerve proteins, the 57- and 15-kDa subunits were recovered in different complexes corresponding, respectively, to the V1 and V0 domains. No fully assembled V-ATPase was detected. It is concluded that V1 and V0 domains of V-ATPase are transported separately in axons, at different rates, and that they only associate once arrived in nerve endings to form the active V-ATPase.

Animals↗

PubMed1998

Prevention of salt-dependent cardiac remodeling and enhanced gene expression in stroke-prone hypertensive rats by the long-acting calcium channel blocker lacidipine.

OBJECTIVE: To analyze the effect of the long-acting calcium channel blocker lacidipine on cardiovascular remodeling induced by salt loading in a genetic model of hypertension. DESIGN: We examined the influence of threshold doses of lacidipine, with little blood-pressure lowering effect, on cardiac weight and gene expression in stroke-prone spontaneously hypertensive rats (SHRSP). METHODS: SHRSPs (8-week-old) were randomly allocated to four groups: control, salt-loaded SHRSP and salt-loaded SHRSP treated with lacidipine at 0.3 and 1 mg/kg per day. Systolic blood pressure was measured by the tail-cuff method. At the end of 6 weeks of treatment, ventricles were collected and weighed. Ventricular messenger RNA was extracted and subjected to Northern blot analysis. RESULTS: Lacidipine (0.3 mg/kg per day) not only prevented the salt-dependent cardiac hypertrophy and the slight increase in systolic blood pressure induced by salt, but also prevented, largely or completely, salt-dependent increases in ventricular levels of several gene products: skeletal and cardiac alpha-actin, beta-myosin heavy chain (beta-MHC), type I collagen, long-lasting (L)-type calcium channel and preproendothelin-1. At a higher dose of 1 mg/kg per day, lacidipine further decreased systolic blood pressure below the level of control SHRSP, completely prevented salt-dependent overexpression of the beta-MHC gene and markedly attenuated salt-dependent overexpression of the transforming growth factor-beta1 gene. CONCLUSIONS: Lacidipine prevents the cardiac remodeling and enhanced gene expression induced by salt loading in SHRSP at doses that only minimally affect the high systolic blood pressure.

Animals↗

PubMed1998

Portal veins of mice infected with Schistosoma mansoni exhibit an increased reactivity to 5-hydroxytryptamine.

In chronic severe infection with Schistosoma mansoni, portal hypertension and related vascular alterations usually develop as a consequence of granulomatous response to eggs. In order to investigate a putative direct effect of worms on the reactivity of their host portal vein, mice infected only with male worms were used in the present study. An higher reactivity to 5-hydroxytryptamine (5-HT) characterized by an increase in the maximal contraction and sensitivity was observed in portal vein from infected mice compared to healthy mice. Blockade of NO-synthase with l-NAME induced a small increase in 5-HT potency in portal vein from non-infected mice without changing the amplitude of the contractions, whereas it did not alter the reactivity of veins from infected mice. The present results show that unisexual infection of mice with male S. mansoni increased the reactivity of the portal vein to 5-HT which seems to be partially related to an alteration in the nitric oxide release by endothelium.

Animals↗

PubMed1997

Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris.

In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETDelta, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEDelta, reduced to the catalytic domain). We show that AChEH and AChET subunits are processed into the same molecular forms as in vivo or in transfected mammalian cells, but that lytic processes converting amphiphilic forms into non-amphiphilic derivatives appear to be more active in yeast. The production of glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion of monomers) demonstrates that P. pastoris can correctly process a mammalian C-terminal GPI-addition signal. Truncated rat and Bungarus AChE molecules, which exclusively generated non-amphiphilic monomers, were released more efficiently and thus produced more AChE activity. In the hope of increasing the production of AChE, we replaced the endogenous signal peptide by yeast prepeptides, with or without a propeptide. We found that the presence of a propeptide, which does not exist in AChE, does not prevent the proper folding of the enzyme, and that it may either increase or decrease the yield of secreted AChE, depending on the signal peptide. Surprisingly, the highest yield was obtained with the endogenous signal peptide. For all combinations, the yield was 2-3 times higher for Bungarus than for rat AChE, probably reflecting differences in the folding efficiency or stability of the polypeptides. The Michaelis constant (Km), the constant of inhibition by excess substrate (Kss) and the catalytic constant (kcat) values of the recombinant AChEs obtained both in P. pastoris and in COS cells, were essentially identical with those of the corresponding natural enzymes, and the Ki values of active-site and peripheral-site inhibitors (edrophonium, decamethonium, propidium) were similar.

Acetylcholinesterase↗

PubMed1997

Association of syntaxin with SNAP-25 and VAMP (synaptobrevin) during axonal transport.

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP-25, and a synaptic vesicle membrane protein, VAMP/synaptobrevin, form stable protein complexes that are involved in the docking and fusion of synaptic vesicles at the presynaptic membrane. These protein complexes have also been described in a homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ. In the present study, we performed similar experiments combining velocity sedimentation and immunoprecipitation on control or ligated electric nerves and found it was possible to distinguish syntaxin that is in the axonal plasma membrane from syntaxin that is transported by the fast anterograde axonal flow. Although syntaxin that is resident in axonal membranes is associated with SNAP-25 but not with VAMP, syntaxin that accumulates proximally to a ligature is associated with both SNAP-25 and VAMP in a stoichiometry very similar to that of the nerve terminal complex. In control nerves, lower amounts of syntaxin form a complex with VAMP, in proportion to syntaxin, which is conveyed by the axonal flow. Because added VAMP was unable to associate with syntaxin in solubilized control nerves and because neither solubilization by SDS nor dilution to the nanomolar range of syntaxin and VAMP concentrations before solubilization change the stoiechiometry between the immunoprecipitated proteins, this complex appears to be both formed prior to solubilization and stable thereafter. Hence, heterotrimeric complexes containing syntaxin, SNAP-25, and VAMP are already formed during fast anterograde axonal transport, before reaching the nerve endings.

Animals↗