Membrane-Bound and solubilized brain 5HT3 receptors: Improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H-Gr65630C, 3H-BRl43694, and 3H-LY278584
✍ Scribed by Keith Miller; Ellen Weisberg; Paul W. Fletcher; DR. Milt Teitler
- Book ID
- 104600429
- Publisher
- John Wiley and Sons
- Year
- 1992
- Tongue
- English
- Weight
- 866 KB
- Volume
- 11
- Category
- Article
- ISSN
- 0887-4476
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✦ Synopsis
Bovine area postrema tissue was used as a convenient source of tissue for studies of brain 5HT3 receptors. 5HT3 receptor density was determined to be 97 5 5 fmoVmg and 124 k 10 fmoVmg of protein using the commercially available 5HT3 radioligands, 3H-GR65630 and 3H-BRL43694, respectively. The equilibrium dissociation constants (K,) for ,H-GR65630 and 3H-BRL43694 were 0.5 * 0.1 nM and 1.7 * 0.3 nM, respectively. The affinities of a series of drugs for the 5HT3 receptor using the two radioligands were essentially identical; the K, values and order of affinities of agonists and antagonists were very similar to data published in studies on radiolabeling of 5HT3 receptors in rat brain. 3H-LY278584 also labeled 5HT3 receptors in bovine area postrema homogenates with a K,, of 3.1 f 0.1 nM and a B,,, of 84 ? 6 fmol/mg. In rat cortical homogenates, 3H-LY278584 produced the most reliable specific signal of 72%, with a KD of 2.6 * 0.3 nM and a B, , value of 10.5 ? 1 fmoVmg. At 1 1lM,~H-GR65630 or 3H-BRL43694 specific binding represented 28 and 50% of total radioligand binding, respectively. These data in bovine and rat brain tissues indicate that bovine area postrema can be used with 3H-GR65630, 3H-BRL43694, or 3H-LY278584 for drug screening or molecular investigations of the brain 5HT3 receptor: only 3H-LY278584 can be used for studies on the regulation andor the molecular properties of 5HT3 receptors in rat cortical homogenates. 5HT, receptors were solubilized from bovine area postrema and characterized using 3H-GR65630. Gel filtration chromatography revealed a specific binding peak with a MW of approximately 600 kDa. A 10-fold enrichment of the binding activity was achieved using wheat germ agglutinin followed by application of N-acetyl-glucosamine to elute the glycosylated proteins, indicating the receptor is a glycosylated protein.
Serotonin, Brain stem, Ion channel