Characterization of (-)-[3H]dihydroalprenolol binding to intact and broken cell preparations of human peripheral blood lymphocytes.
In this study we compared characteristics of (-)-[3H]dihydroalprenolol ([3H]DHA) binding sites in crude membrane preparations of human peripheral blood lymphocytes with those of intact, viable cells. A valid determination of specific beta-adrenergic receptor binding in both preparations was obtained by defining non-specific [3H]DHA binding with 10(-6) M l- or dl-propranolol or 10(-3) M l-isoproterenol. Higher concentrations of propranolol were used in prior reports on lymphocyte membranes. We showed that these concentrations may inhibit non-specific binding, causing non-saturability and inhomogeneity of the estimated 'specific' binding. In the intact cell preparations, inclusion of 10(-4) M phentolamine was necessary to reduce the high degree of non-specific binding. By contrast, phentolamine (10(-4) M) showed no effect on the [3H]DHA binding to membrane preparations. At 37 degrees C the [3H]DHA binding to beta-adrenergic receptor sites in both intact and broken cell preparations was rapid and reversible. The sites were stereoselective, as l-propranolol was about two orders of magnitude more potent to inhibit [3H]DHA binding than was the d-isomer. In both preparations, agonists competed for specific binding with a rank order of potency isoproterenol greater than epinephrine greater than norepinephrine, which indicated a beta 2-type of adrenergic receptor. The specific [3H]DHA binding was saturable and Scatchard analysis revealed comparable numbers of homogeneous, non-cooperative binding sites (approximately 1250 receptors/cell in the membrane preparations and 1700 receptors/cell in the intact cells). In spite of these similarities the membrane sites showed a lower affinity for the antagonists [3H]DHA and propranolol than did the intact cell sites, whereas their affinity for the agonists was increased. These differences indicate that the membrane system might be less suited to provide physiologically significant information about the beta-adrenergic receptor system.
D000526:Alprenolol / Q000031:analogs & derivatives*; D002462:Cell Membrane / Q000378:metabolism; D004082:Dihydroalprenolol / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D010646:Phentolamine / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D011941:Receptors, Adrenergic / Q000032:analysis*; D011943:Receptors, Adrenergic, beta / Q000032:analysis* / Q000187:drug effects; D014316:Tritium
D006150:Guanine Nucleotides; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D014316:Tritium; D004082:Dihydroalprenolol; D000526:Alprenolol; D011433:Propranolol; D010646:Phentolamine