title: p-[125I]iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor.
abstract: The binding properties of p-[125I]iodoclonidine [( 125I]PIC) to human platelet membranes and the functional characteristics of PIC are reported. [125I]PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific [125I]PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. [125I]PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist [3H] bromoxidine (UK14,304), which represented approximately 40% of the sites bound by the antagonist [3H]yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of [125I]PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for [125I]PIC binding was stereoselective. Competition for [3H]bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for [3H]yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. [125I]PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM. [125I]PIC should prove useful in binding assays involving tissues with a low receptor density or in small tissue samples and in studies of cloned and expressed alpha 2-AR.
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
authors.forename: M A
authors.initials: MA
authors.lastname: Gerhardt
authors.forename: S M
authors.initials: SM
authors.lastname: Wade
authors.forename: R R
authors.initials: RR
authors.lastname: Neubig
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000316:Adrenergic alpha-Agonists / Q000493:pharmacokinetics*; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003000:Clonidine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D015016:Yohimbine / Q000493:pharmacokinetics
chemical_list: D000316:Adrenergic alpha-Agonists; D007457:Iodine Radioisotopes; D011942:Receptors, Adrenergic, alpha; C053113:4-iodoclonidine; D015016:Yohimbine; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D003000:Clonidine