Biomedical subjects

Y Landry

Publications and source records attributed to Y Landry.

At least 55 records · Page 3Linked to original sources

PubMed1991

The use of the potential-sensitive fluorescent probe bisoxonol in mast cells.

The regulation of the plasma membrane potential of rat peritoneal mast cells at the resting state and during activation was investigated using bisoxonol as a potential-sensitive fluorescent dye. Fluorescence microphotography showed that this negatively charged probe was not only present in the plasma membrane, but was also distributed in the cytoplasm. The intracellular localization of bisoxonol was confirmed by conducting experiments which showed that bisoxonol fluorescence was not enhanced in ATP-permeabilized mast cells. Rotenone (10(-7) M) and oligomycin (10(-6) M) did not change the fluorescence of bisoxonol showing, therefore, mitochondrial depolarization was not recorded with bisoxonol and suggesting that bisoxonol may represent a useful probe to study plasma membrane potential changes in the absence of exocytosis. We showed that, in non-stimulated mast cells, the blockade of the sodium pump enhanced the fluorescence of bisoxonol as did gramicidin a non selective ionophore used to fully depolarize the cells. High concentration of potassium (30 mM) as well as different ionic channel blockers did not significantly change the fluorescence intensity of bisoxonol, suggesting that ionic channel permeabilities were not involved in maintaining the resting plasma membrane potential of mast cells. Mast cells stimulated by compound 48/80 completely lost the fluorescence, shown by fluorescence microphotography, suggesting that exocytotic phenomena might induce a dye redistribution which is not only due to changes in the plasma membrane potential. In mast cells pretreated with pertussis toxin, which blocks mast cell-exocytosis, compound 48/80 induced a delayed (2 min) decrease of bisoxonol fluorescence which was shown to be dependent on the activity of the sodium pump. Considering that bisoxonol is a useful potential-sensitive probe in exocytosis-deprived mast cells, our results suggest that the sodium pump is mainly involved in the changes of plasma membrane potential of mast cells.

Adenosine Triphosphate↗

PubMed1991

Evidence for the interaction of mast cell-degranulating peptide with pertussis toxin-sensitive G proteins in mast cells.

K(+)-channel blocker properties have been reported for mast cell-degranulating peptide (MCD) in the central nervous system, but its action mechanism in mast cells remains unknown. We studied the effect of MCD on the membrane potential of rat peritoneal mast cells using the fluorescent probe bis-oxonol. Unexpectedly, MCD induced a decrease in bis-oxonol fluorescence, in a rapid and then a slower phase, suggesting hyperpolarization of mast cells. Other K(+)-channel blockers, tetraethylammonium and 4-aminopyridine, did not significantly modify the bis-oxonol fluorescence and did not alter the effect of MCD. The late phase of bis-oxonol fluorescence decrease was inhibited by ouabain and by potassium deprivation, whereas histamine release was not affected. The first phase of putative hyperpolarization induced by MCD coincided with histamine release and with the generation of inositol polyphosphates. Prior treatment of the cells with pertussis toxin inhibited these effects of MCD. MCD stimulated the GTPase activity of purified G proteins (G0/Gi) in a concentration-dependent manner. These results indicate that the effect of MCD on mast cells is unrelated to K+ channels but that it is relevant to the activation of pertussis toxin-sensitive G proteins leading to the activation of phospholipase C. A direct interaction of MCD with G proteins is proposed, which, unlike mastoparan, does not require positive cooperativity.

Amino Acid Sequence↗

PubMed1991

Evidence for two high-affinity bradykinin binding sites in the guinea-pig lung.

We examined the binding of [3H]bradykinin ([3H]BK) to the guinea-pig lung which was saturable. Scatchard analysis indicated the existence of two binding sites, one with a high affinity (KD = 15 pM) and one with a low one (KD = 570 pM) with maximum number of binding sites of 12 and 45 fmol/mg protein, respectively. Kinetic studies confirmed the presence of these two types of binding sites and their affinity ranges. Neither the B1 agonist des-Arg9-BK, nor the B1 antagonist des-Arg9-[Leu8]BK displaced [3H]BK, demonstrating the absence of B1 receptors in the guinea-pig lung. Current B2 antagonists fully displaced the [3H]BK binding. Their potencies differed slightly according to the concentration of [3H]BK, suggesting a specificity of current B2 antagonists for the lower affinity site as opposed to the higher one. Altogether, these results do not allow us to confirm the occurrence of putative B3 receptors in guinea-pig lung.

Animals↗

PubMed1991

G-proteins as targets for non-immunological histamine releasers.

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction.

Animals↗

PubMed1991

Molecular basis for cellular effects of naturally occurring polyamines.

The naturally occurring polyamines, putrescine, spermidine and spermine, and the analogue cadaverine, induce a dose-dependent histamine release from rat peritoneal mast cells. Spermine was the most active among these polycationic metabolites, followed by spermidine and putrescine. The histamine release was inhibited by a 2 h pretreatment of the cells with pertussis toxin (100 ng/ml), demonstrating the involvement of a pertussis toxin-sensitive GTP-binding regulatory protein during the exocytotic process. Experiments performed with purified Go/Gi proteins reconstituted into phospholipid vesicles showed a direct stimulation of GTPase activity by the polyamines. This direct stimulation of G proteins and the consequent activation of the coupled effectors may represent a new mechanism of action for natural polyamines controlling receptor-dependent processes.

Animals↗

PubMed1991

The role of the sodium pump in the plasma membrane potential changes during mast cell activation.

The role of the sodium pump in the plasma membrane potential changes induced by compound 48/80 and by antigenic challenge has been investigated using a fluorescent potential sensitive probe, bis-oxonol. Compound 48/80 induced a fast decrease of the fluorescence of bis-oxonol followed by a delayed decrease. The antigenic stimulation induced only a delayed decrease of fluorescence. Zinc gluconate inhibited the first decrease but did not alter the second one. The delayed decrease was inhibited by ouabain or by the absence of potassium. These results suggest that compound 48/80 induced mast cell secretion via a zinc-sensitive mechanism followed by activation of the sodium pump. The changes in the plasma membrane potential during the antigenic stimulation are due to the activation of the sodium pump but occur after the secretion process.

Animals↗

PubMed1991

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.

Animals↗

PubMed1991

Muscarinic receptor subtypes in guinea pig airways.

The muscarinic receptors present in the guinea pig tracheobronchial tree were characterized using ligand-binding studies and functional approaches. The binding constants of four selective antagonists, pirenzepine,[11-([2-[(diethylamino) methyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-pyrido(2,3) (1,4) benzodiazepine-6-on] (AF-DX 116), methoctramine, and 4-diphenylacetoxy-Nmethylpiperidine methobromide(4-DAMP) were examined. Heterogeneity in the airway muscarinic receptors population was revealed by competitive binding experiments against [N-methyl3H]scopolamine with the M2 muscarinic antagonists AF-DX 116 and methoctramine. In guinea pig lung and trachea, AF-DX 116 and methoctramine recognized 86-88% and 50-60% of total receptors with high affinity, respectively. These receptors exhibit binding constants for these two compounds similar to those of the M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors. In lung and trachea, we found no evidence for a high-affinity [N-methyl-3H]pirenzepine binding sites. In functional studies, pirenzepine, methoctramine, and 4-DAMP inhibited the methacholine-induced contraction of lung parenchymal, main bronchial, and tracheal strips with affinities characteristic of smooth muscle M3 receptors. These results are consistent with the presence of M2 and M3 receptors in guinea pig airways. Throughout the airways, the muscarinic receptors mediating smooth muscle contraction are of the M3 subtype.

Animals↗

PubMed1991

[Modulation of respiratory smooth muscle reactivity by epithelial factors].

The epithelium of the pulmonary airways plays a complex role in the modulation of bronchial smooth muscle reactivity thanks to the metabolic properties and the mobilisation of contractant and relaxant factors which may issue directly from the epithelial cells or surrounding cells. The expression of these epithelial factors varies along the length of the tracheo-bronchial tree and with the species of animal being considered. Neuronal cells and inflammatory cells may equally intervene and interfere with relaxant and contractant factors depending on the epithelium. But the greatest difficulty in characterising these epithelial factors residues in the functional antagonism between relaxant and contractant factors when they are jointly expressed. Amongst factors depending on the respiratory epithelium prostaglandin E2 would be one of the relaxing factors and Calcitonin Gene-Related Peptide (CGRP), one of the contracting factors.

Animals↗

PubMed1990

Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80.

The neuropeptide substance P and the polyamine compound 48/80, both known to activate mast cell secretory processes, increased the rate of GTP S binding to G-proteins purified from calf brain (Go/Gi mixture). The GTPase activity of G-proteins was also increased by substance P and compound 48/80 in a dose-dependent and Mg2+-dependent way. These effects were similar to those of the wasp venom peptide mastoparan, another histamine releaser of rat peritoneal and human skin mast cells. This suggests that the secretory property of compound 48/80 and substance P is not due to a receptor-mediated process but, like mastoparan, results from a direct activation of G-proteins.

Animals↗

PubMed1990

Selective implication of thromboxane A2 and PAF-acether in two guinea pig anaphylactic models.

In an IgE and an IgG model of anaphylaxis in the guinea pig, we investigated the role of prostanoids and PAF-acether in the tracheal response in vitro to immunochallenge. Indomethacin (10(-6) M) potentiated the antigen-induced contraction in both models suggesting the synthesis of relaxant prostaglandins during the anaphylactic phenomenon. UK-38,485 (10(-5) M), a thromboxane (TxA2) synthetase inhibitor, did not modify the tracheal response in the IgE model. In the IgG model, this drug reduced the response of the tracheal strips to antigen. Ro 19-3704 (10(-6) M) and BN 52021 (10(-5) M), two potent PAF antagonists, reduced antigen-induced contraction of the tracheal strips in the IgE model. These two drugs did not modify the contractile response in the IgG model. These results indicate that PAF-acether and TxA2 play a role in the IgE and IgG model of anaphylaxis, respectively.

Anaphylaxis↗

PubMed1990

Evidence for the involvement of calcitonin gene-related peptide in the epithelium-dependent contraction of guinea-pig trachea in response to capsaicin.

Capsaicin (10(-9) to 10(-5) M) contracted guinea-pig tracheal strips. Epithelium-containing tracheal strips developed a maximum active tension which was significantly higher than that observed in epithelium-free strips. Anti-CGRP (calcitonin gene-related peptide) serum blocked the epithelium-dependent potentiation of the capsaicin-induced contraction in the intact tracheal strips, without affecting the response of the epithelium-free strips. This result suggests the occurrence of an epithelium-dependent release of CGRP. This same serum markedly reduced the contraction induced by exogenous rat CGRP in both intact and epithelium-free tracheal strips. In epithelium-free tracheal strips, capsaicin-induced contraction was abolished by spantide (10(-6) and 10(-5) M), a substance P antagonist, but, in intact tracheal strips, spantide did not abolish the capsaicin-induced contraction, showing that both CGRP and substance P release are directly induced by capsaicin. Moreover, the contractile responses to rat CGRP of intact tracheal strips from guinea pig suggest that CGRP itself might be able to release a contracting factor from the airway epithelium. Therefore, CGRP originating from the airway epithelium may play a major role in the control of airway smooth muscle tone.

Animals↗

PubMed1990

Role of phospholipase A2 and G-proteins in the IgE-dependent activation of mast cells and macrophages.

The effect of para-bromophenacyl bromide (a selective inhibitor of phospholipase A2) and pertussis toxin has been investigated on IgE-dependent histamine release and on IgE-dependent macrophage-mediated cytotoxicity. Para-bromophenacyl bromide inhibited dose-dependently IgE-dependent stimulation of mast cells and macrophages (IC50's of 5.0 X 10(-7) M and 2.5 X 10(-7) M, respectively). In contrast, pertussis toxin only inhibited the IgE-dependent stimulation of macrophages, whereas the IgE-dependent activation of mast cells was not affected. These results suggest that the transducing mechanisms following the activation of the high affinity receptor for IgE (Fc epsilon RI on mast cells) as well as the low affinity receptor for IgE (Fc epsilon RII on macrophages) induce the activation of phospholipase A2. Fc epsilon RII might be coupled to a pertussis toxin sensitive G-protein.

Acetophenones↗

PubMed1990

A pertussis toxin-sensitive G protein is required to induce histamine release from rat peritoneal mast cells by bradykinin.

Bradykinin, kallidin (Lys-bradykinin) and [Thi 5,8, D-Phe7]-bradykinin, a functional B2 antagonist, induce histamine release from rat peritoneal mast cells. The histamine release is dependent upon added calcium when mast cells are placed in calcium-free medium 30 min before being triggered with the kinins. Histamine release was dose-dependently inhibited by pertussis toxin (1-100 ng/ml) and by benzalkonium chloride (0.1-3 micrograms/ml). The efficiency of ionophore A23187 on histamine release was affected neither by pertussis toxin nor by benzalkonium chloride. The parallel response of rat peritoneal mast cells to kinins and to substance P suggest that these peptides have the same mechanisms of action i.e. activation of a pertussis toxin-sensitive G protein and of phospholipase C defining a peptidergic triggering pathway of mast cells.

Animals↗

PubMed1990

The sensitivity to Zn2+ discriminates between typical and atypical mast cells.

The effects of zinc gluconate have been studied on rat peritoneal mast cells and rat basophilic leukemia cells (RBL 2H3) stimulated by various secretagogues. The IC50's of zinc gluconate on peritoneal cells were (microM): 1.6, 1.9, 5.4 and 18 for ionophore A23187, phorbol 12-myristate 13-acetate, substance P and immunoglobulin E-antigen, respectively. Higher concentrations of zinc gluconate were required to inhibit histamine secretion from RBL 2H3 cells, i.e. 12 microM (ionophore A23187) and 140 microM (immunoglobulin E-antigen). Zinc gluconate (10(-4) to 10(-3) M) also inhibited the IgE-dependent contraction of guinea pig trachea but was unable to affect that induced by exogenous histamine. These results suggest that zinc gluconate acts intracellularly and is selective of "typical" or "connective tissue" mast cells.

Animals↗

PubMed1990

Human M1-, M2- and M3-muscarinic cholinergic receptors: binding characteristics of agonists and antagonists.

The muscarinic acetylcholine receptors were identified in membrane preparations from human tissues by the specific binding of 1-[benzilic-4,4'-3H] quinuclidinyl benzilate. Saturation binding isotherms of this radioligand yielded a total amount of receptors of 435 +/- 208, 159 +/- 65 and 913 +/- 89 fmol/mg protein, respectively, in the hippocampus, pons and submandibular gland. Non linear least squares analysis of competition binding studies with the antagonists pirenzepine and AF-DX 116 indicates that the majority of receptors are of the M1-type in the hippocampus (83%, high affinity for pirenzepine, intermediate affinity for AF-DX 116), the M2-type in the pons (low affinity for pirenzepine and high affinity for AF-DX 116), and the M3-type in the submandibular gland (low affinity for pirenzepine and AF-DX 116). Competition binding parameters of the agonists carbachol, arecoline, oxotremorine, pilocarpine and MCN-A-343 were compared for M1, M2 and M3 receptors in the human hippocampus, pons and submandibular gland. GTP caused a shift to the right and a steepening of the shallow agonist competition curves in the 3 tissues but did not affect the initially steep ones. This effect is explained by a GTP-mediated conversion of high- to low-agonist affinity sites. The extent of the nucleotide shift was much greater for M2 receptors as compared with M1 and M3 receptors. The GTP effect was impaired by the sulphydryl reagent N-ethylmaleimide, probably due to alkylation of GTP-binding proteins. Moreover, the reagent provoked also an increase of the agonist affinity for the uncoupled muscarinic receptors. For all agonists, this increase was more pronounced for the M2 receptors than for the M1 and M3 receptors. These findings suggest structural differences between the agonist binding sites of M1 and M3 receptors versus the M2 receptors.

Binding, Competitive↗

PubMed1990

Interaction of substance P, compound 48/80 and mastoparan with the alpha-subunit C-terminus of G protein.

Pretreatment of purified calf brain G proteins with activated pertussis toxin or antibodies raised against the C-terminus of their alpha subunits prevented the increase in GTPase activity induced by substance P, compound 48/80 and mastoparan. These results suggest that these mast cell secretagogues activate G proteins directly via an interaction with the C-terminus of alpha subunits of G proteins by mimicking the agonist-liganded receptors.

Amino Acid Sequence↗

PubMed1990

G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides.

The neuropeptide substance P, the venom peptide mastoparan and the synthetic polyamine compound 48/80 activate rat peritoneal mast cells, leading to rapid histamine release by exocytosis. Although these effects are inhibited by pertussis toxin and involve a transient increase in IP3, no selective membrane receptors have been identified. However, it has recently been shown that these compounds activate G proteins in vitro. Here Yves Landry and colleagues discuss the proposal that direct activation of G protein is the physiological mechanism of action of substance P on rat peritoneal mast cells, this mechanism being mimicked by mastoparan and 48/80, and possibly by other cationic amphiphilic peptides such as kinins. These compounds might be of help in defining the interaction between membrane receptors and G proteins.

Amino Acid Sequence↗