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

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Abstract
The agonist/α2-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 μmol/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 α2-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 α2-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 α2-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 α2-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 μmol/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 (α2H) of the platelet α2-adrenoceptor.