title: 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)
authors.affiliation: Max-Planck-Institut für Biophysik, Frankfurt, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Klein
authors.forename: G
authors.initials: G
authors.lastname: Gimpl
authors.forename: F
authors.initials: F
authors.lastname: Fahrenholz
mesh_terms: D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D002784:Cholesterol / Q000031:analogs & derivatives / Q000032:analysis / Q000502:physiology*; D002793:Cholic Acids; D003505:Cyclodextrins / Q000378:metabolism / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006168:Guinea Pigs; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009215:Myometrium / Q000187:drug effects / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism; D011247:Pregnancy; D018045:Receptors, Oxytocin / Q000378:metabolism*; D012995:Solubility; D013256:Steroids / Q000737:chemistry / Q000378:metabolism; D014668:Vasotocin / Q000031:analogs & derivatives / Q000494:pharmacology; D047392:beta-Cyclodextrins
chemical_list: D002793:Cholic Acids; D003505:Cyclodextrins; D003902:Detergents; D008563:Membrane Lipids; D018045:Receptors, Oxytocin; D013256:Steroids; D047392:beta-Cyclodextrins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010121:Oxytocin; C048531:chapso; D002784:Cholesterol; D019204:GTP-Binding Proteins; C031215:betadex; D014668:Vasotocin