[³H]Cirazoline as a Tool for the Characterization of Imidazoline Sites

Recent studies have shown that cirazoline, an alpha 1-adrenoceptor agonist, has greater affinity than do other imidazoline or guanidinium compounds at imidazoline recognition sites. In this report we used [3H]cirazoline as a probe to characterize imidazoline recognition sites present in membrane homogenates of rat brain and kidney as well as pancreatic beta HIT T15 cells. Specific binding of [3H]cirazoline to these various homogenates was saturable and reversible and was resolved into two classes of high affinity binding sites. Competition inhibition studies of [3H]cirazoline binding to these different membrane preparations were performed with alkaloid, phenylethylamine, imidazoline, and guanidinium compounds. Catecholamines and non-imidazoline adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or yohimbine did not compete with [3H]cirazoline (Ki > 10 microM). Under our experimental conditions, only guanidinium and imidazoline derivatives had high affinities for [3H]cirazoline binding sites. Unlabeled cirazoline, clonidine, bromoxidine, idazoxan, and amiloride had the highest affinities with this respective rank order. These results suggest that [3H]cirazoline is a novel high affinity radioligand that specifically labels nonadrenergic imidazoline-guanidinium sites in the brain, kidney, and beta cells. Furthermore, the obtained rank order of inhibition suggests that [3H]cirazoline binding does not distinguish between I1 and I2 sites. In addition, we compared the specific binding of [3H]cirazoline with that of the alpha 2-adrenoceptor antagonist [3H]rauwolscine in chinese hamster ovary (CHO) cell lines stably expressing human alpha 2C2-, alpha 2C4-, and alpha 2C10-adrenoceptor subtypes. Using [3H]rauwolscine as a probe, each of these transfected cell lines expressed high levels for the three different alpha 2-adrenoceptor subtypes (Bmax values were between 2 and 7 pmol.mg-1 protein). In contrast, none of these cell lines displayed measurable imidazoline recognition sites. In summary, [3H]cirazoline is a novel high affinity radioligand that specifically labels imidazoline recognition sites without significant alpha- or beta-adrenoceptor binding. Furthermore, our results using alpha 2-adrenoceptor transfected cells confirm that the imidazoline recognition sites and each of the cloned alpha 2-adrenoceptor subtypes represent distinct macromolecular entities.