Alteration of the Myometrial Plasma Membrane Cholesterol Content with .beta.-Cyclodextrin Modulates the Binding Affinity of the Oxytocin Receptor

Abstract

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)

Engineering Mycobacterium smegmatis for testosterone production

“…An additional carbon source was added in some cases. AD was incorporated into the medium as a solution with randomly methylated β‐cyclodextrin (1:10.3, molar ratio) (Klein et al ., 1995). …”

Association of Helicobacter pylori Vacuolating Toxin (VacA) with Lipid Rafts

“…Control experiments were performed using methyl-␤-cyclodextrin-cholesterol complex (27 mg of cholesterol per gram methyl-␤-cyclodextrin), prepared as described (53,54). The cholesterol content of cells was measured using either thin layer chromatography (48) or an Infinity cholesterol reagent kit (Sigma).…”

Interaction of Serotonin1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

“…Native membranes were treated with MβCD for depleting cholesterol as described earlier (Klein et al, 1995) with some modifications. Briefly, hippocampal membranes (containing 2 mg of total protein) were incubated in a total volume of 1 ml of 50 mM Tris buffer (pH 7.4) containing 40 mM MβCD by shaking for 1 h at room temperature (23 • C).…”