Dopamine D2Receptors in the Anterior Pituitary: A Single Population Without Reciprocal Antagonist/Agonist States

Abstract

Although dopamine agonists can recognize 2 states of the D, dopamine receptor in the anterior pituitary .**GRAPHIC**. and .**GRAPHIC**. antagonists such as [3H]spiperone could recognize these 2 sites with different affinities was examined. Using up to 30 concentrations of [3H]spiperone, only a single population of binding sites (porcine anterior pituitary homogenates) with a dissociation constant (Kd) of 130 pM could be detected. When specific [3H]spiperone binding was defined by a low concentration of (+)-butaclamol (100 nM), the apparent density was low. When defined by a high concentration of (+)-butaclamol (10 .mu.M), nonspecific sites became detectable, thus revealing 2 apparent populations of sites for [3H]spiperone, only 1 of which was specific for dopamine, NaCl reduced the Kd of the single population of specific D2 sites to 64 pM. Guanine nucleotide by itself had no effect on the Kd, but enhanced the density by 25%. Since the density-enhancement could be eliminated by extensive washing of membranes and could be restored by preincubation with dopamine, the nucleotide-induced elevation of D2 density appeared to be a result of the release of tightly bound endogenous dopamine. Thus, monovalent cations and guanine nucleotides have separate regulatory effects on the anterior pituitary D2 receptor that modulate antagonist-receptor interactions. Several maneuvers were used to test whether [3H]spiperone could differentiate between the 2 agonist-detected subpopulations of sites. Twentyfold different concentrations of [3H]-spiperone (47 pM and 1000 pM) labeled identical proportions of receptors in the .**GRAPHIC**. and .**GRAPHIC**. states as detected by the agonist 6,7-dihydroxyaminotetralin (ADTN), suggesting that spiperone labeled equal proportions of .**GRAPHIC**. and .**GRAPHIC**. sites without differential affinity for them. Competition of spiperone for .**GRAPHIC**. sites selectively labeled by the agonist [3H]n-propylnorapomorphine (NPA) had a virtually identical Kd for spiperone as did the total D2 receptor population as determined by direct binding studies (75 pM vs. 64 pM). [3H]Spiperone bound to a uniform population of .**GRAPHIC**. sites induced by preincubation with guanine nucleotide with identical affinity as to the total D2 population. These data do not support a reciprocal model for the D2 receptor (i.e., antagonist having low affinity for .**GRAPHIC**. and high affinity for .**GRAPHIC**. in a manner reciprocal to agonists). Evidently spiperone recognizes the .**GRAPHIC**. and .**GRAPHIC**. states of the receptor with equal affinity and no evidence is provided in support for reciprocal modulation of antagonist/agonist affinities.