Ginette Lagacé

G protein modulation by estrogens

Levels of various G protein subunits were assayed by immunoblot and densitometry, using specific antibodies, in anterior pituitaries and striata of female rats exposed to physiological or pharmacological modifications of ovarian hormone levels and, for comparison, in the same tissues of coeval male rats. Treatment of ovariectomized rats with 17 beta-estradiol 10 micrograms/rat/day for 5, 10 or 20 days induced a time-dependent rise in plasma prolactin (PRL) levels. While no change in G protein levels was observed in the striatum, estrogen treatment induced a significant reduction of all pituitary G protein levels except those of alpha i1, which remained unchanged, and of alpha s42, which increased in a time-dependent manner. A highly significant correlation was observed between pituitary alpha s42 values and plasma PRL levels. During the estrous cycle, pituitary values of alpha o, alpha i3 and alpha s47 were generally lower than those of ovariectomized rats, suggesting the existence of tonic inhibitory influence of circulating ovarian hormones. Pituitary levels of alpha o, alpha i1 and alpha s42 also showed a significant modulation during the various phases of the estrous cycle, and those of alpha o, alpha i3, alpha s47 and beta were significantly lower in female than in male rats. No significant effects of estrous cycle hormone variations or sex differences were observed in the values of striatum G proteins. In conclusion, these data clearly indicate that ovarian hormones, and particularly estrogens, have a significant and specific effect on pituitary G protein levels which may modulate the secretion of pituitary hormones such as PRL.

G proteins in normal rat pituitaries and in prolactin-secreting rat pituitary tumors

It is still undetermined which GTP-binding (G) protein is involved in the regulation of prolactin (PRL) release and through which effector. This study shows that, when compared to normal pituitary tissue, the levels of alpha o protein were very low in dopamine (DA)-resistant, PRL-secreting pituitary tumors 7315a and MtTW15, while alpha o mRNA was present in the two tumors. In the MtTW15 tumor alpha i1, alpha i2 and alpha i3 levels were decreased while those of alpha s42 and alpha s47 were increased, and in the 7315a tumor alpha i2, alpha i3 and beta levels were decreased and those of alpha s47 increased. In an estrone-induced, DA-sensitive prolactinoma the levels of alpha i3 were greatly reduced. DA was unable to inhibit basal PRL release by 7315a and MtTW15 and basal cAMP accumulation by adenomatous and MtTW15 cells. Vasoactive intestinal peptide (VIP) increased both cAMP accumulation and PRL release by all cell preparations which could be suppressed by DA with adenomatous and 7315a but not with MtTW15 cells. These and previously published results provide circumstantial evidence that alpha o, alpha i1 and alpha i3 are all involved in the transduction of the DA inhibitory message while alpha s47 transduces cAMP activating messages and alpha s42 is responsible for the constitutive activation of L-type Ca2+ channels, adenylate cyclase and baseline PRL release.

Regulation of Basal and stimulated prolactin release in prolactin-secreting rat pituitary tumors*.

To better understand the mechanisms of the inhibitory effects of dopamine on pituitary prolactin release, we have utilized an estrone‐induced, benign and dopamine‐sensitive rat pituitary adenoma and two malignant, transplantable and dopamine‐resistant rat pituitary tumors, 7315a and MITW15. Enzymatically dispersed and Percoll purified cells obtained from the three tissues were incubated for 30 min in media with or without Na+ and in the presence or the absence of dopamine and/or various prolactin releasers for evaluating the secretion of prolactin under baseline and experimental conditions. In some experiments, the cells were pretreated for 16 h with pertussis toxin to evaluate the eventual presence and role of pertussis toxin‐sensitive G proteins. Dopamine inhibited baseline prolactin release by adenomatous lactotrophs in a Na+‐dependent manner, but was totally inactive with 7315a and MtTW15 cells. The Ca2+ channel agonist BAY K 8644 stimulated prolactin release with all three preparations and its effects were enhanced by a Na+‐free medium. Dopamine antagonized the stimulatory effects of BAY K 8644 with adenomatous and 7315a cells only, even in the absence of Na+. Pertussis toxin pretreatment significantly increased baseline prolactin release by adenomatous and MtTW15 cells and abolished dopamine inhibition of adenomatous lactotrophs baseline hormone release. BAY K 8644, TRH and vasoactive intestinal peptide, stimulated prolactin release by adenomatous cells and this effect was antagonized by dopamine in a pertussis toxin‐sensitive manner. All prolactin releasers, except TRH, were effective also with 7315a cells, and its actions were not blocked by pertussin toxin. The stimulatory effects of BAY K 8644 and vasoactive intestinal peptide on 7315a cells were enhanced by pertussis toxin pretreatment. The results obtained with an almost pure preparation of adenomatous lactotrophs confirm the existence of a dual mechanism of dopamine inhibitory action on prolactin release: 1) a Na+‐dependent action exerted on baseline, and 2) a Na+‐independent action exerted on stimulated prolactin release. They also indicate that both actions are exerted through pertussis toxin‐sensitive G proteins. Furthermore, our results show the presence in transplantable pituitary tumors 7315a and MtTW15 of multiple and diverse anomalies in the regulation of prolactin release probably due, at least partly, to anomalies of one or more G proteins and/or neurotransmitter receptors.

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

Abstract: We have investigated the structure of dopamine (DA) D2 receptors present in an estrone‐induced, prolactin (PRL)‐secreting, DA‐sensitive adenoma and in two PRL‐secreting and DA‐insensitive transplantable tumors 7315, and MtTW15, in order to identify better the anomalies present in DA‐resistant lactotrophs. D2 receptors were found in both a high‐ and a low‐affinity state in adenomatous lactotrophs as shown by displacement studies with the agonist N‐propylnorapomorphine (NPA), but only in the low‐affinity state in the two DA‐resistant tumors. Treatment with the alkylating agent N‐ethylmaleimide induced a disappearance of the high‐affinity state of the D2 receptor in the adenoma and a reduction in receptor concentration, but did not have any effect on the affinity of receptors present in DA‐resistant tumors. Moreover, target size analysis and radiation inactivation studies of D2 receptors, using membranes preincubated with NPA and [3H]spiperone as ligand or using [3H]NPA as ligand on membranes preparations, have shown the presence of distinct structural differences between adenomatous and tumoral D2 receptors and between the two tumoral receptors themselves; these results suggest that the normal functional unit of the D2 receptor is a dimer associated with a guanine nucleotide‐binding protein (G protein) subunit and that tumoral D2 receptors may exist in various polymeric forms unassociated with G proteins. The anomalies found to be present in tumoral D2 receptor complexes may be responsible for the insensitivity of these tumors to dopaminergic agonists’inhibitory activity on PRL release and tumor growth.

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 MtTWIS. These receptors specifically bind with high affinity the do‐pamine 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 MtTWIS 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‐polyacryl‐amide gel electrophoresis. A guanine nucleotide had no effect on the displacing potency of the agonist N‐propyl‐norapomorphine 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.