title: Agonist binding at alpha 2-adrenoceptors of human platelets using 3H-UK-14,304: regulation by Gpp(NH)p and cations.
abstract: The agonist/alpha 2-adrenoceptor interactions at human platelet membranes have been examined in radioligand binding studies with the full agonist ligand 3H-UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] and the antagonist ligand 3H-yohimbine. From association kinetics of different concentrations of 3H-UK-14,304 (0.75-8.1 nmol/l) a KD-value of 2.37 nmol/l in agreement with the high-affinity KD-value (KDH = 1.60 +/- 0.15 nmol/l) obtained from equilibrium binding studies was derived. In the presence of Gpp(NH)p about 6% of specific radioligand binding was observed in the association reaction. Addition of Gpp(NH)p at equilibrium resulted in a rapid loss (t 1/2 less than 1 min) of approximately 80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 mumol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of 1/5 of rapidly (t 1/2 less than 2 min) and 4/5 of slowly dissociating ligand (k-1 = 0.033 +/- 0.004 min-1). Application of a sequential binding model resulted in KD-values from this approach also in agreement with KDH from equilibrium binding studies. The rank order of potency for different agonists and antagonists to compete for binding with 3H-UK-14,304 indicated an alpha 2-adrenoceptor interaction: (-)adrenaline greater than or equal to clonidine greater than (-)noradrenaline greater than (-)isoprenaline and yohimbine = rauwolscine greater than phentolamine greater than prazosin greater than or equal to corynanthine greater than timolol respectively. The analysis of competition isotherms of UK-14,304 versus 3H-yohimbine (Hill-coefficient = 0.59 +/- 0.03) showed that the agonist binds to two affinity states of the alpha 2-adrenoceptor, with high (KDH = 1.77 +/- 0.50 nmol/l) and low affinity (KDL = 71.2 +/- 11.6 nmol/l) respectively. From these experiments a fraction of 56.9% +/- 2.1% of the total number of alpha 2-adrenoceptors (Bmax = 198.4 +/- 8.0 fmol/mg of protein) in the high-affinity state was calculated. Similar results were obtained from 3H-UK-14,304 saturation isotherms according to a two-state binding model (KDH = 1.60 +/- 0.15 nmol/l; KDL = 66.2 +/- 10.7 nmol/l; BmaxH = 57.6% +/- 2.3%). Adrenoceptor agonists competed for specific binding of 3H-UK-14,304 and 3H-yohimbine in a manner that suggests that the 3H-UK-14,304 (approximately 3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the alpha 2-adrenoceptor.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000959:Antihypertensive Agents; D001667:Binding, Competitive / Q000187:drug effects; D001792:Blood Platelets / Q000378:metabolism*; D000068438:Brimonidine Tartrate; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*; D015016:Yohimbine / Q000378:metabolism
chemical_list: D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D000959:Antihypertensive Agents; D002412:Cations; D008024:Ligands; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D015636:Magnesium Chloride; D015016:Yohimbine; D006165:Guanylyl Imidodiphosphate; D000068438:Brimonidine Tartrate; D006160:Guanosine Triphosphate; D008274:Magnesium