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

Abstract

Bovine area postrema tissue was used as a convenient source of tissue for studies of brain 5HT₃ receptor density was determined to be 97 ± 5 fmol/mg and 124 ± 10 fmol/mg of protein using the commercially available 5HT₃ radioligands, ³H‐GR65630 and ³H‐BRL43694, respectively. The equilibrium dissociation constants (K_D) for ³H‐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_i 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_D of 3.1 ± 0.1 nM and a B_max of 84 ± 6 fmol/mg. In rat cortical homogenates, ³H‐LY278584 produced the most reliable specific signal of 72%, with a K_D of 2.6 ± 0.3 nM and a B_max value of 10.5 ± 1 fmol/mg. At 1 nM, ³H‐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 can be used for studies on the regulation and/or the molecular properties of 5HT₃ receptors in rat cortical homogenates. 5HT₃ 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. © Wiley‐Liss, Inc.