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Use of [ methyl- 3H]yohimbine as a radioligand for alpha-2 adrenoreceptors on intact platelets. Comparison with dihydroergocryptine
Journal article   Peer reviewed

Use of [ methyl- 3H]yohimbine as a radioligand for alpha-2 adrenoreceptors on intact platelets. Comparison with dihydroergocryptine

Donald E Macfarlane, Betty L Wright and David C Stump
Thrombosis research, Vol.24(1), pp.31-43
1981
DOI: 10.1016/0049-3848(81)90029-3
PMID: 6120583

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Abstract

The binding of the non-selective α-adrenergic receptor antagonist [ 3H]dihydroergocryptine and the selective α 2-adrenergic receptor antagonist [ methyl- 3H]yohimbine to intact blood platelets was investigated. Loosely bound dihydroergocryptine dissociated from washed platelets diluted with plasma in a temperature dependent fashion, and at 37°C this was substantially complete in 30 seconds. The remaining radioactivity was resolvable into a non-specific component equivalent to about 10μl supernatant per 10 8 platelets and a saturable component that was suppressed by yohimbine amounting to about 400 sites per platelet. The binding to these sites was slow, and did not equilibrate in 20 minutes. Addition of phentolamine, yohimbine or non-radioactive dihydroergocryptine at this time resulted in the prompt dissociation of about one third of the specifically bound dihydroergocryptine, and a further one third dissociated over the next hour. In contrast, the non-specific binding of yohimbine to intact platelets after plasma dilution amounted to the equivalent of 0.5 − 1.5 μl supernatant per 10 8 platelets. Specific binding and dissociation was exponential with K 1 = 5.25 × 10 5M −1 sec −1, K −1 = 3.3 × 10 −3sec −1. The derived dissociation constant was 6.3 nM, and the rate of dissociation was not materially affected by the presence or absence of non-radioactive yohimbine or epinephrine. Equilibrium binding revealed 200–350 non-interactive sites per platelet with dissociation constant 2–4 nM. Binding was completely suppressed by agents which interact with the α 2-receptor with the following rank of potency: epinephrine < clonidine < ρ-aminoclonidine < yohimbinic acid < phentolamine < yohimbone. Dihydroergocryptine and dihydroergotamine also completely suppressed binding. Prazosin, an α 1-adrenergic receptor antagonist suppressed binding only at concentrations above about 10μM. It is concluded that dihydroergocryptine appears to bind to more than one site, one of which has such a slow rate of dissociation that equilibrium with low concentrations of dihydroergocryptine will not be achieved for several hours. However, yohimbine bound with the characteristics of a bimolecular reaction with an α 2-receptor site, and this reagent is expected to be a useful radioligand for the α 2-adrenergic receptor on intact platelets.
 radioligand binding yohimbine Alpha receptors human platelets dihydroergocryptine

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