Use of N-ethylmaleimide to directly determine the proportion of beta-adrenergic receptors exhibiting high affinity for agonists on human mononuclear leukocyte membranes.
Following incubation with the beta-adrenergic agonist isoproterenol (ISO) and the alkylating agent N-ethylmaleimide (NEM), the number of beta-adrenergic receptors measured on human mononuclear leukocyte (MNL) membranes decreased. Pretreatment induced an approximately 40% loss of measurable receptors. The proportion of receptors lost was that which formed the ternary complex of agonist-receptor-Gs protein (H-R-Gs) and exhibited high affinity for agonists. In comparison studies, the loss of receptors was found to be similar to the proportion of receptors that form the high affinity state of the receptor (%RH) for agonist in agonist competition binding studies. Acute exposure to agonist induced a similar reduction in ISO/NEM "sensitive" receptors and the %RH. These experiments indicate that pretreatment of MNL membranes with ISO/NEM is a powerful tool with which to directly determine the percentage of receptors able to form an agonist high affinity state.
Rigby
J W
JW
Vanderbilt University School of Medicine, Department of Pharmacology, Nashville, TN 37232.
Wood
A J
AJ
D001667:Binding, Competitive / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000187:drug effects*; D010869:Pindolol / Q000031:analogs & derivatives; D011869:Radioligand Assay; D011943:Receptors, Adrenergic, beta / Q000032:analysis* / Q000187:drug effects
D011943:Receptors, Adrenergic, beta; C029247:3-iodopindolol; D010869:Pindolol; D007545:Isoproterenol; D005033:Ethylmaleimide