p-[125I]iodoclonidine, a novel radiolabeled agonist for studying central alpha 2-adrenergic receptors.
Unlabeled p-iodoclonidine was efficacious in attenuating forskolin-stimulated cAMP accumulation in SK-N-SH neuroblastoma cells. Maximal attenuation was 76 +/- 3%, with an EC50 of 347 +/- 60 nM. Comparable values of epinephrine were 72 +/- 3% and 122 +/- 22 nM. Responses to both agonists were abolished by 10 microM phentolamine. Therefore, p-iodoclonidine is an agonist in a cell culture model system of the neuronal alpha 2-adrenergic receptor. p-[125I]Iodoclonidine binding to membranes were measured using various regions of the rat brain. The agonist labeled a single population of sites present on cerebral cortical membranes, which was saturable (Bmax = 230 fmol/mg of protein) and possessed high affinity for the ligand (Kd = 0.6 nM). Binding was largely specific (93% at 0.6 nM). A variety of alpha 2-adrenergic agonists and antagonists were shown to compete for the binding of the radioligand. The binding of p-[125I]iodoclonidine was much less sensitive to agents that interact with alpha 1-adrenergic, serotonergic, and dopaminergic receptors. Approximately 65% of the binding was sensitive to guanine nucleotides. Association kinetics using 0.4 nM radioligand were biphasic (37% associate rapidly, with kobs = 0.96 min-1, with the remainder binding more slowly, with kobs = 0.031 min-1) and reached a plateau by 90 min at 25 degrees. Dissociation kinetics were also biphasic, with 30% of the binding dissociating rapidly (k1 = 0.32 min-1) and the remainder dissociating 50-fold more slowly (k2 = 0.006 min-1). Agonist binding is, therefore, uniquely complex and probably reflects the conformational changes that accompany receptor activation.
Baron
B M
BM
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Siegel
B W
BW
D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D003000:Clonidine / Q000031:analogs & derivatives* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000032:analysis / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000316:Adrenergic alpha-Agonists; D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D011942:Receptors, Adrenergic, alpha; C053113:4-iodoclonidine; D000242:Cyclic AMP; D008274:Magnesium; D003000:Clonidine