Characterisation of [3H]lysergic acid diethylamide binding to a 5-hydroxytryptamine receptor on human platelet membranes

doi:10.1016/0014-2999(84)90515-6

European Journal of Pharmacology

Volume 97, Issues 1–2, 13 January 1984, Pages 87-93

https://doi.org/10.1016/0014-2999(84)90515-6 Get rights and content

Abstract

Specific binding of [³H]lysergic acid diethylamide (LSD) to human platelet membranes, as defined by 300 nM spiperone, was saturable over the concentration range of 0.25–2.5 nM [³H]LSD. At 0.5 nM [³H]LSD the half-time for association at 37°C was 56 min, half-time for dissociation was 173 min, and the kinetically derived affinity was 0.24 nM. In 19 control subjects equilibrium binding studies gave an affinity of 0.53±0.02 nM (mean ± S.E.M.) and capacity of 57.1 ± 5.6 fmol/mg protein (mean ± S.E.M.).The inhibition profile was consistent with that of a 5-hydroxytryptamine (5-HT) receptor. There was a significant correlation between the inhibition of [³H]LSD binding and the inhibition of 5-HT-induced shape change, but not inhibition of active platelet uptake of 5-HT. There was also a significant correlation between the inhibition of [³H]LSD binding to human platelet membranes and human frontal cortex. Platelet [³H]LSD binding may therefore be a useful model for study of peripheral and central 5-HT receptors in man.

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Citation Excerpt :
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Analysis of an extensive database of human platelet 5-HT_2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT_2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. B_max declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K_D during the half-life of B_max. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg $protein ml$ (at 0.2 nM of ligand). Above 0.1 mg $protein ml$ , percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 $mg ml$ , well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.
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In the present study, the effects of the thiol oxidising agent diamide upon the properties of rat brain β₁-adrenergic and human platelet serotonin_2Areceptor recognition sites have been investigated using [³H](−)CGP-12177 (in the presence of ICI-118551) and [³H]LSD as ligands. (−)Isoprenaline inhibited [³H](−)CGP-12177 binding with n_Hvalues of 0.87, 0.67, and 0.56 for added Mg²⁺concentrations of 0, 2.5, and 25 mM, respectively. Pretreatment with diamide increased the n_Hto above unity for the inhibition of the binding by (−)isoprenaline, without a concomitant effect on the inhibition of the binding by (−)propranolol. In contrast, diamide reduced the affinity of human platelet serotonin_2A-receptors for antagonists, but did not consistently induce n_Hvalues above unity for the inhibition of antagonist binding by serotonin. These results suggest that cooperative interactions reported for cardiac muscarinic receptors may also occur for β₁-adrenergic receptors in the rat brain.

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Characterisation of [3H]lysergic acid diethylamide binding to a 5-hydroxytryptamine receptor on human platelet membranes

Abstract

Brain Res.

European J. Pharmacol.

Life Sci.

J. Biol. Chem.

European J. Pharmacol.

Characterisation of [³H]lysergic acid diethylamide binding to a 5-hydroxytryptamine receptor on human platelet membranes