Characterization of D₂ Dopamine Receptors in Dopamine‐Resistant Prolactin‐Secreting Rat Pituitary Tumors 7315a and MtTW15

Abstract

We have investigated the structure of D2 receptors present in two prolactin-secreting, dopamine-resistant, transplantable rat pituitary tumors, 7315a and MtTW15. These receptors specifically bind with high affinity the dopamine antagonist [3H]spiroperidol when membrane bound or solubilized by [3-(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate 10 mM and are pharmacologically characterized as D2 type. Target-size analysis by radiation inactivation indicated a molecular mass of approximately 100,000 and 200,000 daltons for receptors present respectively in 7315a and MtTW15 tumors either membrane bound or solubilized. The minimal size of the D2 binding site was evaluated at 94,000 daltons by photoaffinity labeling with [125I]azido-N-(p-aminophenethyl)-spiperone followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A guanine nucleotide had no effect on the displacing potency of the agonist N-propylnorapomorphine evaluated with membrane-bound or solubilized receptors obtained from either tumor. These results suggest the absence or inactivation of a guanine nucleotide binding protein in the receptorial complex of these tumors. Thus, our data indicate that a structural anomaly is present in the D2 receptorial complex of these prolactin-secreting rat pituitary tumors, which may be responsible for their resistance to the inhibitory effects of dopamine.

Structural Differences Between Dopamine D2 Receptors Present in a Rat Pituitary Adenoma and in Transplantable Rat Pituitary Tumors 7315a and MtTW15

“…The RIS values computed from the radiation inactivation curves are summarized in Table 1. In order to compare the RIS values obtained in the presence and in the absence of NPA, previously published RIS of D2 receptors obtained in the absence of NPA (Bouvier et al, 1986(Bouvier et al, , 1987 were included in Table 1.…”

“…More recently (Bouvier et al, 1987), we also characterized the D2 receptors present in DA-resistant and PRL-secreting transplantable pituitary tumors 7 3 15a and MtTW15 (Cronin et al, 1981(Cronin et al, , 1982, which are resistant to the inhibitory effects of DA, although bearing high-affinity D2 binding sites (Cronin et al, 1983). Our results show that, although sharing with normal and adenomatous receptors a 94-kDa ligand binding subunit, as visualized by photoaffinity labeling, tumoral D2 receptors have some distinct structural anomalies that may be responsible for tumoral resistance to DA.…”

“…Pituitary adenomas were induced as previously described by Bouvier et al (1986) MtTW I5 tumors, respectively, as previously described (Cronin et al, 1981(Cronin et al, , 1982Bouvier et al, 1987), to transplant these DA-resistant pituitary tumors.…”

“…The radiation inactivation size (RIS) of the D2 receptor complexes in a stabilized state was evaluated either by using intact or solubilized NPA-pretreated membranes and [3H]spiperone as ligand, or by using intact membranes and [3H]NPA as ligand. The RIS was determined by irradiating the receptors, before performing the binding assays, in a Gammacell 220, as previously described by Beauregard and Potier (1982) and Bouvier et al (1986Bouvier et al ( , 1987, and by using the empirical equation of Kepner and Macey (1968) and linear regression analysis of the radiation-induced ligand binding decay. The error in molecular size determination using RIS is lower than 15% , and appropriate controls have been included, such as determination of the RIS of the reference enzymes catalase and acetylcholinesterase.…”

“…Binding assays with [3H]spiperone were performed, as previously described by Bouvier et al (1986Bouvier et al ( , 1987, in the presence or in the absence of M (+)-butaclamol. Binding assays with [3H]NPA were done using the same methodology as with [3H]spiperone, except for the duration (30 min) and the temperature (22°C) of incubation.…”