Antiestrogen binding sites in brain and pituitary of ovariectomized rats

In addition to interacting with estrogen binding sites in a number of tissues, antiestrogens have recently been shown to interact with a separate, estrogen-non-compatible, antiestrogen-specific binding site (AEBS). In order to better understand possible mechanisms by which the antiestrogens may effect behavioral and physiological changes, we have examined AEBS in several areas of the brain and pituitary in adult, ovariectomized rats. Single point binding assays with 2 nM [3H]tamoxifen (TAM) in the presence of saturating amounts (1 microM) E and +/- 1 microM TAM indicated the existence of specific binding to AEBS throughout the brain and pituitary. In most areas of the brain (cortex, cerebellum, amygdala, area postrema/nucleus of the solitary tract region) as well as pituitary. Scatchard analyses revealed the presence of a single AEBS with a dissociation constant (Kd = 1-4 x 10(-9) M) similar to that previously reported for other tissues. However, in both hypothalamus and preoptic area, an additional, higher affinity site (Kd = 6-9 x 10(-11) M) was found. Competitive inhibition studies revealed that there was little competition by the potent estrogen agonist, diethylstilbesterol, for AEBS binding. Antiestrogens competed in the following order: tamoxifen greater than or equal to nafoxidine much greater than keoxifene. Additional competitive inhibition studies were run using neurotransmitter antagonists. The phenothiazines, chlorpromazine and fluphenazine, bind to both D1 and D2 dopamine receptors and effectively compete with [3H]TAM for binding at the AEBS. Other pharmacological substances, including specific antagonists of the D2 sites, as well as antagonists of the norepinephrine, opiate, histamine, GABA and acetylcholine systems, were ineffective competitors for [3H]TAM binding.