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N Morel

Publications and source records attributed to N Morel.

At least 73 records · Page 4Linked to original sources
PubMed1993

Activity of dihydrothienopyridine S312 enantiomers on L-type Ca2+ channels in isolated rat aorta and cerebral microvessels.

The activity of the two enantiomers of the dihydrothienopyridine S312 was characterized in isolated rat aorta and cerebral microvessels. The interaction of S312 with 1,4-dihydropyridine and phenylalkylamine binding sites was also investigated in depolarized rat cerebral microvessels and in membranes from rat ileum. Both S-(+)-S312 and R-(-)-S312 dose dependently inhibited KCl-evoked contraction of the rat aorta, with IC50 values of 0.14 (0.13-0.16) and 2.98 (2.67-3.33) nM, respectively. When the aorta was preincubated with S-(+)-S312 in a depolarizing medium, the inhibitory effect was significantly increased, but this increased inhibition was not reversed by incubation in physiological medium. The effect of R-(-)-S312 was not affected by preincubation in a depolarizing medium. In rat cerebral microvessels, S-(+)-S312 inhibited the KCl-induced contraction and KCl-stimulated Ca2+ influx with similar potency. [3H](+)-PN 200-110 specific binding was competitively displaced by the two enantiomers in depolarized cerebral microvessels. The calculated Ki values were 0.12 nM for S-(+)-S312 and 2.4 nM for R-(-)-S312. Only 20% of [3H]D888 specific binding in rat ileum membranes was displaced by S-(+)-S312. The dissociation rate of [3H]D888 was markedly decreased by S-(+)-S312, and this allosteric interaction was significantly more marked than with nitrendipine. It is concluded that the dihydrothienopyridine S312 could interact with Ca2+ channels in a manner different to that of genuine dihydropyridines.

Animals↗
PubMed1993

The same 15 kDa proteolipid subunit is a constituent of two different proteins in Torpedo, the acetylcholine releasing protein mediatophore and the vacuolar H+ ATPase.

Using the monoclonal antibody 15K1, we have studied, at the cellular and subcellular levels, the distribution of a 15 kDa proteolipid, identified as the subunit of mediatophore, a presynaptic membrane protein able to release acetylcholine when activated by calcium. Aside from the electric lobe, the antigen distribution in the brain of Torpedo paralleled that of the synaptic vesicle antigen SV2 and did not appear to be related to that of acetylcholine and choline acetyltransferase. The 15 kDa proteolipid antigen was therefore present in all nerve endings and not restricted to cholinergic ones. At the ultrastructural level, on cholinergic nerve endings, the antigen was detected associated to synaptic vesicles and, to a lesser extent, to the presynaptic plasma membrane. Indeed, considering the high sequence homology between the mediatophore subunit (Birman et al., 1990) and the proteolipid subunit of the vacuolar type H+ ATPase, a major enzyme constituent of synaptic vesicles, this distribution was not surprising. To determine whether antibody 15K1 recognizes the vacuolar type H+ ATPase, we chose a non neuronal cell type which possesses a high content of this enzyme, the kidney proton secreting epithelial cells. Indeed, antibody 15K1 intensely labelled the apical plasma membrane of mitochondria rich epithelial cells in kidney tubules. A high density of the antigen was also found associated to intracellular membrane structures such as lysosomal multivesicular bodies, both in kidney epithelial cells and in electromotoneurons. The 15 kDa proteolipid antigen was associated with other vacuolar H+ ATPase subunits in kidney membranes which was not the case in presynaptic plasma membranes. This illustrates that the 15 kDa proteolipid antigen is a constituent of two different protein complexes, which exhibit very different functional properties.

Acetylcholine↗
PubMed1993

Effect of N,N'-dicyclohexylcarbodiimide on compartmentation and release of newly synthesized and preformed acetylcholine in Torpedo synaptosomes.

Using isolated cholinergic synaptosomes prepared from Torpedo electric organ, we studied the effects of N,N'-dicyclohexylcarbodiimide (DCCD) on acetylcholine (ACh) synthesis, compartmentation, and release after stimulation. Whereas ACh synthesis was unchanged, ACh compartmentation inside synaptosomes was affected by the presence of DCCD. In resting conditions, the uptake into the synaptic vesicle pool of newly synthesized ACh (i.e., [14C]ACh synthesized in the presence of the drug) was progressively and markedly inhibited as the duration of DCCD preincubation was increased, whereas compartmentation of endogenous ACh was unchanged in the presence of DCCD. After stimulation, the release of endogenous ACh from DCCD-treated synaptosomes was similar to that of control, in contrast to the release of [14C]ACh, which was markedly inhibited. This inhibition was observed whatever the conditions of stimulation used (gramicidin D, calcium ionophore A23187, or KCl depolarization). The study of the compartmentation of [14C]ACh during stimulation revealed a transfer of highly labeled ACh from the free to the bound ACh compartment in the presence of DCCD, suggesting the existence of several ACh subcompartments within the free and bound ACh pools. The present results are discussed in comparison with the previously reported effects of vesamicol (AH5183) on ACh compartmentation and release.

Acetylcholine↗
PubMed1992

Effect of nitric oxide on membrane potential and contraction of rat aorta.

Influence of nitric oxide (NO) on the membrane potential of rat aorta was assessed by blocking endothelial NO synthase with N omega-nitro-L-arginine (NOArg). Membrane potential was measured by two different methods: intracellular microelectrodes and [3H]tetraphenylphosphonium bromide ([3H]TPP+) uptake. Blocking of NO synthesis with NOArg (10(-4) M) depolarized the membrane by 4-6 mV. The NOArg-induced depolarization was suppressed by the NO donor SIN-1 (10(-5) M). Incubation with NOArg (10(-4) M) decreased the basal level of cGMP, and increased the basal 45Ca2+ influx as well as the sensitivity of contractile response to KCl. Results indicate that NO released by endothelial cells permanently hyperpolarizes the membrane of rat aorta smooth muscle cells and thereby may control the opening of voltage-dependent Ca2+ channels.

Amino Acid Oxidoreductases↗
PubMed1992

Effect of N,N'-dicyclohexylcarbodiimide on acetylcholine release from Torpedo synaptosomes and proteoliposomes reconstituted with the proteolipid mediatophore.

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.

Acetylcholine↗
PubMed1991

Release of acetylcholine by Xenopus oocytes injected with mRNAs from cholinergic neurons.

Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of approximately 120,000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetylcholine and were able to synthesize [14C]acetylcholine from 1-[14C]acetate. With KCl depolarization and upon treatment with a Ca2+ ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca(2+)-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentration, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. Immunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh-releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances.

Acetates↗
PubMed1991

Binding sites for 1,4-dihydropyridine Ca(2+)-channel modulators in rat intestinal smooth muscle.

The contractile response of intestinal smooth muscle to depolarization is characterized by a phasic and a tonic component which are differently sensitive to blockade by 1,4-dihydropyridines. As this difference in sensitivity could be related to different binding sites associated with distinct calcium channels, we analyzed the binding of the calciumantagonist 1,4-dihydropyridine (+)PN 200-110 [isopropyl-4-(2,1,3-benzodiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxycarbonyl-pyridine-3-carboxylate] in longitudinal smooth muscle of the rat ileum. We carried out saturation binding experiments on intact tissue exposed to physiological and depolarizing (100 mmol l-1 K+) solution, and on different membrane fractions: the total microsomal fraction, the light microsomal fraction (enriched with plasma membranes) and the mitochondrial fraction. Binding of 3H(+)PN 200-110 to the intact longitudinal smooth muscle of rat ileum appeared to be voltage-dependent, KD decreased in depolarized tissue whereas Bmax was unchanged (change in membrane potential was assessed by measuring the distribution of 3H-tetraphenylphosphonium bromide). In membrane fractions two binding sites were detected, a high-affinity site associated with plasma membrane and a low-affinity site presumably associated with mitochondria (abundant in the fractions where the cytochrome c oxidase activity was high, and undetectable in the light microsomes poor in cytochrome c oxidase activity). The KD value of the high-affinity binding in isolated membrane fractions was similar to the KD value measured in intact depolarized tissue. The low affinity binding increased at high ionic strength and did not display any stereoselectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Evidence for an association of the 15-kDa proteolipid of mediatophore with a 14-kDa polypeptide.

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.

Animals↗
PubMed1991

Detection with monoclonal antibodies of a 15-kDa proteolipid in both presynaptic plasma membranes and synaptic vesicles in Torpedo electric organ.

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.

Animals↗
PubMed1991

Characterization in rat aorta of the binding sites responsible for blockade of noradrenaline-evoked calcium entry by nisoldipine.

1. The effectiveness of the calcium antagonist, 1,4-dihydropyridine nisoldipine, as an inhibitor of contraction and 45Ca entry evoked by noradrenaline in rat aorta has been investigated and correlated with binding characteristics in intact artery. 2. Contractions evoked by noradrenaline were concentration-dependently depressed by nisoldipine (0.3-300 nM). About 60% of the response was resistant to inhibition, while KCl-induced contractions could be completely blocked. Noradrenaline-induced contractions were also less sensitive to nisoldipine inhibition than were KCl-induced contractions. 3. Preincubation of the aorta with nisoldipine in high KCl depolarizing solution increased the inhibition of the contraction evoked by a short application of noradrenaline or KCl to a similar extent. 4. The inhibition by nisoldipine of 45Ca influx evoked either by KCl depolarizing solution or by noradrenaline correlated well with the inhibition of the contractile responses. However, while KCl-stimulated 45Ca influx was totally abolished by nisoldipine (300 nM), 38% of the noradrenaline-stimulated 45Ca influx was resistant to inhibition by nisoldipine (300 nM). 5. The study of [3H]-(+)-PN 200-10 ([3H]-(+)-isradipine) binding in intact aorta showed the presence of a homogeneous population of specific binding sites. KD values were dependent on the KCl concentration in the bath while Bmax was unaffected. Binding of [3H]-(+)-isradipine was also increased in tissue exposed to noradrenaline; in the presence of 10(-5) M noradrenaline, binding parameters of [3H]-(+)-isradipine were close to the values obtained in aorta bathed in 20 mM KCl solution. 6. Displacement of [3H]-(+)-isradipine specific binding by nisoldipine was determined in segments of mesenteric artery and of aorta. The potency of nisoldipine was dependent on the incubation conditions applied to the vessel, as follows: KCl (100 mM) depolarizing solution greater than noradrenaline (10(-5) M) = KCl (25 mM) solution greater than physiological solution. The Ki value measured in aorta exposed to noradrenaline (10(-5) M) was close to the IC50 value of nisoldipine on the noradrenaline-evoked contraction. 7. The membrane potential value of rat aorta was estimated by the distribution of [3H]-tetraphenylphosphonium bromide ([3H]-TPP+), [3H]-TPP+ uptake concentration-dependently decreased when the KCl concentration in the bath was increased from 5.9 to 130 mM. Noradrenaline also concentration-dependently decreased [3H]-TPP+ uptake; the maximum effect (1-10 microns noradrenaline) was comparable in amplitude to the effect of 25 mM KCl solution. 8. It is concluded that in rat aorta, noradrenaline activates voltage-operated calcium channels that contain the specific, voltage-sensitive binding sites for calcium antagonistic dihydropyridines. The existence of a fraction of noradrenaline-stimulated '"Ca entry that is resistant to nisoldipine blockade suggests that another Ca2 + entry pathway is also opened by the agonist.

Animals↗
PubMed1991

Immunological identification of a new 14X10(3) Mr membrane-bound protein in Torpedo electric organ.

A series of monoclonal antibodies binding to different epitopes shared by a 14 x 10(3)Mr membrane-bound polypeptide has been obtained. By indirect immuno-fluorescence, it was shown that the 14 x 10(3)Mr antigen is present in various cell types in Torpedo electric organ and muscle, especially fibroblasts, capillary endothelial cells, axonal cuff cells and, to a lesser extent, Schwann cells. At the electron-microscope level, after immunogold labelling, the antigen was found associated with the external surface of the plasma membrane of these cells, with the exception of the axonal cuff cells where part of the labelling was intracellular. The possible biological role of this 14 x 10(3)Mr protein is unknown but preliminary experiments suggest that this antigen has affinity for other Torpedo electric organ membrane proteins.

Animals↗
PubMed1990

Modulation of cultured pulmonary microvessel and arterial endothelial cell barrier structure and function by serotonin.

Pulmonary microvessel endothelial cell and pulmonary artery endothelial cell monolayers in tissue culture were treated with serotonin (5-hydroxytryptamine; 5-HT) alone or in conjunction with histamine, bradykinin, the thromboxane analog U-46619, and the actin modulating agent cytochalasin B. After agent treatment, cross-sections through endothelial cell (EC) monolayers were examined by light microscopy and the percentage and widths of intercellular openings were quantitated. To correlate structural changes in the endothelial barrier with an alteration in permeability, EC monolayers cultured on micropore filters were assayed for transit of Evan's blue albumin (EBA) following treatment with vasoactive mediators. 5-HT was found to decrease the patency of endothelial junctions by up to 94%, compared to untreated monolayers, and to prevent or reverse the appearance of interendothelial gaps induced by histamine, bradykinin, U-46619, and cytochalasin B. The 5-HT effect was dose and time dependent, with a maximal increase in junctional apposition observed at a concentration of 10(-6) M for 30 min. This response was significantly blocked by the 5-HT antagonists LSD and ketanserin. The formation or reduction of interendothelial gaps by histamine, bradykinin, and U-46619 and by 5-HT, respectively, was positively correlated to changes in monolayer permeability to EBA. These results suggest that pulmonary edema caused by inflammatory mediators in part may be a consequence of transient increases in pulmonary EC junctional gaps, and that 5-HT may contribute to the homeostatic maintenance of endothelial barrier integrity.

Animals↗
PubMed1990

Acetylcholine translocating protein: mediatophore at rat neuromuscular synapses.

The neuromuscular synapses of the rat sternomastoid muscles contain a membrane protein, mediatophore, that endows artificial membranes with a calcium-dependent acetylcholine release mechanism. Mediatophore and choline acetylase had similar distributions along the muscle. Sciatic nerve membranes contain mediatophore, and a purified preparation was obtained from the nerve.

Acetylcholine↗
PubMed1990

Cerebrovascular effect of calcium antagonists.

Activity of calcium antagonists was assessed in microvessels (internal diameter 5-50 microns) isolated from rat brain. High KCl solution induced an increase of the lanthanum-resistant 45Ca influx and a marked reduction in microvessel diameter. Both responses to depolarization were inhibited by the calcium antagonists nimodipine and flunarizine. Binding studies with 3H(+)PN200-110 used as a specific ligand of Ca channels revealed the existence of voltage-dependent, stereoselective binding sites for dihydropyridine calcium antagonists.

Animals↗
PubMed1990

Modulation of the action of calcium antagonists in arteries.

This paper is a review of the experimental evidence showing that specific binding sites for dihydropyridine Ca antagonists are involved in inhibition of stimulus-dependent Ca entry into arterial cells and thereby in inhibition of the contractile response. The apparent affinity of the dihydropyridine binding site is related to the proportion of a high- and a low-affinity state which is regulated by membrane potential but could also be dependent upon other factors such as G proteins. Among Ca antagonists, a subgroup of agents exhibiting voltage dependence may be identified. Their apparent pharmacological potency is highly dependent on resting membrane potential and on the duration of the depolarizing stimulus.

Animals↗
PubMed1990

Calcium antagonists and vasoconstrictor effects in intracerebral microarterioles.

The purpose of this work was to study the contractile activity of intracerebral microarterioles and their sensitivity to the calcium antagonist nimodipine. Potassium depolarization evoked contraction and rhythmic activity that was blocked by nimodipine (IC50 0.08 nM). High concentrations of noradrenaline and prostaglandin F2 alpha were needed to elicit a contractile response. Intracerebral microarterioles were very sensitive to endothelin (ED50, 0.2 nM). The contractions evoked by concentrations of endothelin lower than or equal to ED50 were relaxed by nimodipine, which also blocked the amplified response to potassium chloride depolarization occurring with subthreshold concentrations of endothelin. We discuss these observations in relation to brain ischemia.

Animals↗