Agonist binding at alpha2-adrenoceptors of human platelets using 3H-UK-14,304: regulation by Gpp(NH)p and cations

Summary The agonist/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 K_D-value of 2.37 nmol/l in agreement with the high-affinity KD-value (K_DH = 1.60 ± 0.15 nmol/1) 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 < 1 min) of 80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 mol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of /15 of rapidly (t_/12 < 2 min) and /45 of slowly dissociating ligand (k–1 = 0.033±0.004 min^–1). Application of a sequential binding model resulted in K_D-values from this approach also in agreement with K_DH 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 ₂-adrenoceptor interaction: (–)adrenaline > clonidine > (–)noradrenaline > (–)isoprenaline and yohimbine = rauwolscine > phentolamine > prazosin >- corynanthine > 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 ₂-adrenoceptor, with high (K_DH = 1.77 ± 0.50 nmol/l) and low affinity (K_DL = 71.2 ± 11.6 nmol/l) respectively. From these experiments a fraction of 56.9%±2.1% of the total number of ₂-adrenoceptors (B_max = 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 (K_DH = 1.60±0.15 nmol/l; K_DL = 66.2±10.7 nmol/l; B_maxH = 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 (3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the ₂-adrenoceptor. Adrenoceptor antagonists had equal affinities irrespective of the receptor states labeled by the agonist or antagonist radioligand. A loss of the high-affinity binding capacity (B_maxH) of the agonist due to the presence of Gpp(NH)p was delineated from ³H-UK-14,304 saturation isotherms. An IC₅₀-value of 0.181 ± 0.007 mol/l for this Gpp(NH)p-effect was calculated. Divalent cations such as magnesium and manganese (10 mmol/l) increased specific binding of ³H-UK-14,304 by a factor of 3, without any influence on binding of the antagonist ³H-yohimbine. In contrast, sodium chloride strikingly decreased high-affinity binding of the agonist radioligand (IC₅₀ = 41.9 ± 3.7 mmol/l). Unlike Gpp(NH)p, sodium chloride (> 30 mmol/l) additionally promoted a marked decrease of the affinity of UK-14,304 at the low-affinity binding component. In contrast to the effects on agonist binding, sodium chloride concentrations of 30 to 300 mmol/l increased the binding affinity of the antagonist ³H-yohimbine about 2-fold. The sodium substitute N-methyl-D-glucamine was without effect on binding of ³H-UK-14,304 indicating that the influence of sodium chloride on binding properties was not due to changes in osmolarity. In conclusion these results suggest that ³H-UK-14,304 labels preferentially the agonist induced or stabilized high-affinity state (_2H) of the platelet ₂-adrenoceptor.Abbreviations UK 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline - Gpp(NH)p guanylyl-imidodiphosphate - EDTA ethylene dinitrilo tetraacetic acidPreliminary data were presented at the Joint Meeting of the Belgian, Dutch and German Pharmacological and Toxicological Societies at Aachen, FRG, September 23–26, 1985 (Schloos et al. 1985)This report is part of the thesis to be presented by J. Schloos in partial fulfillment of the requirements for a Doctor of Natural Science degreeSend offprint requests to J. Schloos at the above address