Membrane-bound and solubilized brain 5HT3 receptors: improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H-GR65630, 3H-BRL43694, and 3H-LY278584.
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 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 (KD) 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 Ki 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 KD of 3.1 +/- 0.1 nM and a Bmax 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 Bmax 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 for drug screening or molecular investigations of the brain 5HT3 receptor: only 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Miller
K
K
Department of Pharmacology and Toxiology, Albany Medical Center, New York 12208.
Weisberg
E
E
Fletcher
P W
PW
Teitler
M
M
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry / Q000187:drug effects*; D002417:Cattle; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D002850:Chromatography, Gel; D017829:Granisetron; D007093:Imidazoles / Q000494:pharmacology; D007191:Indazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011985:Receptors, Serotonin / Q000187:drug effects*; D014326:Tropanes / Q000494:pharmacology
D017829:Granisetron; D007093:Imidazoles; D007191:Indazoles; D007211:Indoles; D011985:Receptors, Serotonin; D014326:Tropanes; C059068:GR 65630; C061531:LY 278584