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      Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematode Haemonchus contortus Translated title: Effets de la teneur en cholestérol sur l’activité des glycoprotéines P et sur l’état physique de la membrane, et conséquences pour la résistance aux anthelminthiques chez le nématode Haemonchus contortus

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          Abstract

          Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

          Translated abstract

          Les membranes plasmiques des eucaryotes protègent les cellules contre les attaques chimiques. Les xénobiotiques, absorbés par diffusion passive, s’accumulent dans les membranes où ils sont capturés par des transporteurs, parmi lesquels les glycoprotéines P (Pgp). Chez les nématodes, les coques des œufs et les cuticules constituent des barrières de protection supplémentaires contre les xénobiotiques. On en sait peu sur le rôle de ces structures dans le transport des molécules chimiques. Les Pgp, membres de la famille des transporteurs ABC, sont présents dans les coques et les cuticules. Des changements dans l’activité de ces protéines ont également été mis en corrélation avec des altérations des lipides, tels que la teneur en cholestérol, des coques des œufs. Cependant, les mécanismes cellulaires sous-jacents à ces effets restent flous. Nous montrons ici que la diminution expérimentale de la teneur en cholestérol des coques des œufs d’ Haemonchus contortus, avec la méthyl-beta-cyclodextrine (MβCD), entraîne une augmentation de la fluidité membranaire favorisant l’activité des Pgp et une augmentation de la résistance aux anthelminthiques. Cet effet est modulé par le degré initial de résistance aux anthelminthiques des œufs. Ces résultats suggèrent que la fluidité de la coque joue un rôle majeur dans la modulation de l’activité des Pgp. Ils confirment que l’activité des Pgp est fortement influencée par le microenvironnement local, en particulier les stérols, comme observé dans certains modèles de vertébrés. Ainsi, les barrières de coques des oeufs pourraient jouer un rôle actif dans le transport des xénobiotiques.

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          Most cited references 90

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          Use of cyclodextrins for manipulating cellular cholesterol content.

          Previous studies from this laboratory have demonstrated that exposure of tissue culture cells to cyclodextrins results in rapid cholesterol depletion. In the present study, we have developed experimental systems for using solutions of cyclodextrins, either 2-hydroxypropyl beta-cyclodextrin or methylated beta-cyclodextrin, complexed with varying amounts of free cholesterol to manipulate cell cholesterol content. Cholesterol delivered via the cyclodextrin has been found to be metabolically active, as measured by the acyl-coenzyme A:cholesterol acyltransferase (ACAT)-mediated esterification of [3H]cholesterol in Fu5AH rat hepatoma cells and Chinese hamster ovary cells. The methylated beta-cyclodextrin was found to be a more efficient donor in all cell types studied, with an average cholesterol uptake of at least 100 microg cholesterol/mg protein within 6 h. By modifying the cyclodextrin:cholesterol molar ratio, it is possible to manipulate the cellular cholesterol content of cells, producing conditions ranging from net cholesterol enrichment to depletion. The use of cyclodextrins provides a convenient, precise and reproducible method for modulating the cholesterol content of tissue culture cells.
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            Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor.

            To investigate the effect of cholesterol on the oxytocin receptor function in myometrial membranes, we developed a new method to alter the membrane cholesterol content. Using a methyl-substituted beta-cyclodextrin, we were able to selectively deplete the myometrial plasma membrane of cholesterol. Vice versa, incubating cholesterol-depleted membranes with a preformed soluble cholesterol-methyl-beta-cyclodextrin complex restored the cholesterol content of the plasma membrane. Binding experiments showed that, with the removal of cholesterol from the membrane, the dissociation constant for [3H]oxytocin is enhanced 87-fold (from Kd = 1.5 nM to Kd = 131 nM), therefore shifting the oxytocin receptor from high to low affinity. Increasing the cholesterol content of the cholesterol-depleted membrane again restored the high-affinity binding (Kd = 1.2 nM). The presence of 0.1 mM GTP gamma S did not significantly change the number of high-affinity binding sites for [3H]oxytocin in native plasma membranes, in membranes depleted of cholesterol, and in plasma membranes with restored cholesterol content. The number of high-affinity binding sites for the oxytocin antagonist [3H]PrOTA was dependent in the same way on the cholesterol content as for [3H]oxytocin. Substitution of the membrane cholesterol with other steroids showed a strong dependence of the oxytocin receptor function on the structure of the cholesterol molecule. The detergent-solubilized oxytocin receptor was not saturable with [3H]oxytocin even at concentrations up to 10(-6) M of radioligand. Addition of the cholesterol-methyl-beta-cyclodextrin complex to the detergent-solubilized oxytocin receptor induced a saturation of the solubilized binding sites (Bmax = 0.98 pmol/mg) for oxytocin (Kd = 16 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
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              Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.

              Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2020
                14 January 2020
                : 27
                : ( publisher-idID: parasite/2020/01 )
                Affiliations
                [1 ] INRAE, UE-1277 Plateforme d’infectiologie expérimentale (PFIE), Centre de Recherche Val de Loire 37380 Nouzilly France
                [2 ] INRAE, Université de Tours, UMR-1282 Infectiologie et Santé Publique (ISP), Centre de Recherche Val de Loire 37380 Nouzilly France
                [3 ] INRAE, CNRS, HARAS NATIONAUX, IFCE, Université de Tours, UMR-0085 PRC Physiologie de la Reproduction et des Comportements, Centre de Recherche Val de Loire 37380 Nouzilly France
                Author notes
                [a]

                CAPF: Membre du Consortium Anti-Parasitaires et Fongiques.

                [b]

                INRAE, Laboratoire de Cytométrie en Flux, Centre de Recherche Val de Loire, 37380 Nouzilly, France.

                [c]

                Directeur de Recherche honoraire de l'UMR-1282.

                Article
                parasite190071 10.1051/parasite/2019079
                10.1051/parasite/2019079
                6959138
                31934848
                © M. Riou et al., published by EDP Sciences, 2020

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 6, Tables: 3, Equations: 1, References: 83, Pages: 13
                Categories
                Research Article

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