BINDING OF [H-3] PARA-AMINOCLONIDINE TO ALPHA-2-ADRENOCEPTOR STATES PLUS A NONADRENERGIC SITE ON HUMAN PLATELET PLASMA-MEMBRANES

被引:58
作者
PILETZ, JE
ANDORN, AC
UNNERSTALL, JR
HALARIS, A
机构
[1] CASE WESTERN RESERVE UNIV, SCH MED, METROHLTH MED CTR, DEPT NEUROSCI, CLEVELAND, OH 44106 USA
[2] CASE WESTERN RES UNIV, DEPT NEUROL, CLEVELAND, OH USA
[3] CASE WESTERN RES UNIV, DEPT PHARMACOL, CLEVELAND, OH USA
[4] CASE WESTERN RES UNIV, UNIV HOSP CLEVELAND, CTR ALZHEIMERS, CLEVELAND, OH USA
[5] VET ADM MED CTR, DEPT PSYCHIAT, ST LOUIS, MO 63125 USA
[6] ST LOUIS UNIV, ST LOUIS, MO 63103 USA
关键词
D O I
10.1016/0006-2952(91)90320-5
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
Characterization of the binding of [H-3]p-aminoclonidine ([H-3]PAC) to purified plasma membranes from human platelets has revealed multiple binding sites. [H-3]PAC identified site-1 in the picomolar affinity range (site-1 K(D) estimates ranged from 13 to 94 pM). Site-1 displayed a rank order of competition by various compounds for [H-3]PAC, indicative of an alpha-2-adrenoceptor, and was sensitive to 0.1 mM GTP. [H-3]PAC also identified a second site with nanomolar affinity (site-2 K(D) estimates ranged from 0.7 to 1.7 nM). 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 alpha-2-adrenoceptor; instead it had properties similar to a previously reported imidazoline-preferring binding site. A third site (site-3) bound [H-3]PAC with a K(D) for site-3 of 26.6 +/- 10.0 nM (SD). Site-3 had a rank order of competition by various compounds for 5 nM [H-3]yohimbine ([H-3]YOH) binding which was indicative of an alpha-2-adrenoceptor. (-)-NE competed for 5 nM [H-3]YOH binding at two sites: site-1 K(i) = 32 pM, site-3 K(i) = 239 nM. Treatment with 0.1 mM GTP completely removed site-1 and transferred the competitive binding of (-)-NE to low affinity (K(i) = 437 nM). Thus, site-3 appears to be a free alpha-2-adrenoceptor. B(max) 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 [H-3]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 [H-3]PAC to platelets from depressed patients.
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页码:569 / 584
页数:16
相关论文
共 56 条
[31]  
MOONEY JJ, 1982, MOL PHARMACOL, V21, P600
[32]   LIGAND - A VERSATILE COMPUTERIZED APPROACH FOR CHARACTERIZATION OF LIGAND-BINDING SYSTEMS [J].
MUNSON, PJ ;
RODBARD, D .
ANALYTICAL BIOCHEMISTRY, 1980, 107 (01) :220-239
[33]   LARGE-SCALE PURIFICATION OF ALPHA-2-ADRENERGIC RECEPTOR-ENRICHED MEMBRANES FROM HUMAN-PLATELETS - PERSISTENT ASSOCIATION OF GUANINE-NUCLEOTIDES WITH NON-PURIFIED MEMBRANES [J].
NEUBIG, RR ;
SZAMRAJ, O .
BIOCHIMICA ET BIOPHYSICA ACTA, 1986, 854 (01) :67-76
[34]  
NEUBIG RR, 1985, MOL PHARMACOL, V28, P475
[35]   MECHANISM OF AGONIST AND ANTAGONIST BINDING TO ALPHA-2 ADRENERGIC-RECEPTORS - EVIDENCE FOR A PRECOUPLED RECEPTOR GUANINE-NUCLEOTIDE PROTEIN COMPLEX [J].
NEUBIG, RR ;
GANTZOS, RD ;
THOMSEN, WJ .
BIOCHEMISTRY, 1988, 27 (07) :2374-2384
[36]   SIMPLIFIED METHOD OF QUANTITATING PROTEIN USING BIURET AND PHENOL REAGENTS [J].
OHNISHI, ST ;
BARR, JK .
ANALYTICAL BIOCHEMISTRY, 1978, 86 (01) :193-200
[37]   INCREASED H-3 CLONIDINE BINDING IN THE PLATELETS OF PATIENTS WITH DEPRESSIVE AND SCHIZOPHRENIC DISORDERS [J].
PANDEY, GN ;
JANICAK, PG ;
JAVAID, JI ;
DAVIS, JM .
PSYCHIATRY RESEARCH, 1989, 28 (01) :73-88
[38]   ALPHA-2-ADRENERGIC RECEPTORS IN HUMAN POLYMORPHONUCLEAR LEUKOCYTE MEMBRANES [J].
PANOSIAN, JO ;
MARINETTI, GV .
BIOCHEMICAL PHARMACOLOGY, 1983, 32 (14) :2243-2247
[39]  
PARIS H, 1989, MOL PHARMACOL, V35, P345
[40]   ALPHA-2 ADRENERGIC-RECEPTOR SUBTYPES INDICATED BY [H-3] YOHIMBINE BINDING IN HUMAN-BRAIN [J].
PETRASH, AC ;
BYLUND, DB .
LIFE SCIENCES, 1986, 38 (23) :2129-2137