Localization and Characterization of Binding Sites with High Affinity for [3H]Ouabain in Cerebral Cortex of Rabbit Brain Using Quantitative Autoradiography

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

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Characterization of high affinity binding sites for vasopressin in bovine adrenal medulla

Neuropeptides ◽

10.1016/0143-4179(84)90116-1 ◽

1984 ◽

Vol 4 (5) ◽

pp. 413-420 ◽

Cited By ~ 15

Author(s):

Ferenc A. Antoni

Keyword(s):

Adrenal Medulla ◽

Binding Sites ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

Bovine Adrenal Medulla

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Solubilization and characterization of d-fenfluramine binding sites from bovine cerebral cortex

Life Sciences ◽

10.1016/0024-3205(94)00421-8 ◽

1994 ◽

Vol 54 (15) ◽

pp. 1109-1118 ◽

Cited By ~ 2

Author(s):

Valeria Gagliardini ◽

Carlo Taddei ◽

Mario Salmona ◽

Paul Pham ◽

Tiziana Mennini ◽

...

Keyword(s):

Cerebral Cortex ◽

Binding Sites

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[3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.6.1588023 ◽

1992 ◽

Vol 40 (6) ◽

pp. 771-779 ◽

Cited By ~ 13

Author(s):

A A Maki ◽

D G Baskin ◽

W L Stahl

Keyword(s):

Nervous System ◽

Rat Brain ◽

Cardiac Glycoside ◽

Binding Sites ◽

Quantitative Autoradiography ◽

Affinity Binding ◽

Ouabain Binding ◽

High Affinity Binding ◽

High Affinity ◽

Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

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