Elsevier

Biochemical Pharmacology

Volume 42, Issue 3, 15 July 1991, Pages 569-584
Biochemical Pharmacology

Binding of [3H]-p-aminoclonidine to α2adrenoceptor states plus a non-adrenergic site on human platelet plasma membranes

https://doi.org/10.1016/0006-2952(91)90320-5Get rights and content

Abstract

Characterization of the binding of [3H]p-aminoclonidine ([3h]PAC) to purified plasma membranes from human platelets has revealed multiple binding sites. [3h]PAC identified site-1 in the picomolar affinity range (site-1 KD estimates ranged from 13 to 94 pM). Site-1 displayed a rank order of competition by various compounds for [3H]PAC, indicative of an α2-adrenoceptor, and was sensitive to 0.1 mM GTP. [3H]PAC also identified a second site with nanomolar affinity (site-2 KD estimates ranged from 0.7 to 1.7nM). In the presence of 0.1 mM GTP, site-2 was not diminished significantly. Also in contrast to site-1, site-2 displayed low affinity for yohimbine (YOH), (−)-epinephrine and (−)-norepinephrine (NE). Therefore, site-2 could not be an active α2-adrenoceptor; instead it had properties similar to a previously reported imidazoline-preferring binding site. A third site (site-3) bound [3h]PAC with a KD for site-3 of 26.6 ± 10.0 nM (SD). Site-3 had a rank order of competition by various compounds for 5nM [3H]yohimbine ([3H]YOH) binding which was indicative of an α2-adrenoceptor. (−)-NE competed for 5 nM [3H]YOH binding at two sites: site-1 Ki = 32 pM, site-3 Ki = 239 nM. Treatment with 0.1 mM GTP completely removed site-1 and transferred the competitive binding of (−)-NE to low affinity (Ki = 437 nM). Thus, site-3 appears to be a free α2-adrenoceptor. Bmax estimates for untreated membranes, derived from simultaneous multi-experiment curve-fitting analyses, were site-1 = 36 ± 29 fmol/mg plasma membrane protein, site-2 = 95 ± 34 fmol/mg and site-3 = 154 ± 35 fmol/mg. We are the first to report a site for [3H]PAC binding on platelets (site-2) with properties uncharacteristic of an adrenoceptor. This observation appears to be due to our use of purified plasma membrane and low ionic strength buffer. These studies relate to reports of increased binding of [3H]PAC to platelets from depressed patients.

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