D2 dopamine-receptor-mediated inhibition of proliferation of rat lactotropes in culture is accompanied by changes in cell shape.

Dopaminergic agonists are effective in vivo in inhibiting lactotrope proliferation and prolactin (PRL)-secreting pituitary tumors. The purpose of the present study was to demonstrate in vitro actions of dopaminergic agents on proliferation and cell shape of rat lactotropes. Anterior pituitary cells cultured with serum-free, chemically defined medium were treated with dopaminergic agents and were labeled with 5-bromo-2'-deoxyuridine (BrdU) for 3 h before the end of culture. BrdU-labeling indices indicative of the proliferation rate of lactotropes were determined by double immunofluorescence staining for BrdU and PRL. Treatment with dopamine for 21 h decreased BrdU-labeling indices of lactotropes in a dose-dependent manner with a nadir at 3 x 10(-7) M. The inhibitory action of 10(-5) M dopamine appeared 15 h after the initiation of treatment and became pronounced with time up to 33 h. The dopamine action was mimicked by treatment with the D2 receptor agonist bromocriptine at concentrations over 10(-9) M. Phase-contrast microscopy revealed that the flat polygonal cell shape of cultured lactotropes had changed to a round refractive cell shape after treatment with dopamine or bromocriptine, and that these changes in cell shape exactly paralleled those in the BrdU-labeling index. The changes in cell shape of lactotropes were accompanied by changes in subcellular distribution of actin filaments. Pretreatment with 10(-7) M eticlopride, a D2 receptor antagonist, blocked the dopamine- or bromocriptine-induced changes in both BrdU-labeling index and cell shape. These results suggest that (1) the in vitro experimental system established in the present study is a good model for studying the mechanism of the antiproliferative action of dopamine and (2) D2-receptor-mediated inhibition of proliferation of lactotropes in serum-free culture is closely related to changes in actin organization and cell shape.

Estradiol-induced diurnal changes in lactotroph proliferation and their hypothalamic regulation in ovariectomized rats.

Pentobarbital anesthesia during the proestrous afternoon delays proliferation of lactotrophs of the anterior pituitary from estrus to diestrus 1 in cycling female rats. We determined whether estradiol treatment induced diurnal changes in rates of lactotroph proliferation in ovariectomized rats, and if so, examined whether hypothalamic neural activity was involved in the occurrence of the estradiol-induced diurnal changes. Dispersed anterior pituitary cells were obtained from ovariectomized rats bearing estradiol implants that had been treated with 5-bromo-2'-deoxyuridine (BrdU) 3 h before decapitation. BrdU-labeling indices representative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for BrdU and PRL. After treatment of ovariectomized rats with estradiol on day 0, BrdU-labeling indices of lactotrophs as determined by injecting BrdU at 1000 h increased markedly with time peaking on days 4-7. Levels of BrdU-labeling indices at 1000 h on day 4 were 2.8-fold higher than those at 2200 h on day 3 or 4 after estradiol treatment. However, levels of BrdU-labeling indices at 1000 h on day 14 were 35% lower than those at the same time on day 4 and did not differ from those at 2200 h on day 13 or 14. In addition, a difference in BrdU-labeling indices as observed between 1000 and 2200 h on day 4 in the ovariectomized rats was not detected in estradiol-treated orchidectomized male rats. Serial determinations of BrdU-labeling indices throughout day revealed that the difference in BrdU-labeling indices between 1000 and 2200 h on day 4 in the ovariectomized rats reflected estradiol-induced diurnal changes that were characterized by a peak between 0700-0900 h and a nadir between 1900-2200 h. Pentobarbital injected at 0900 or 2100 h on day 3 decreased slightly high levels of BrdU-labeling indices at 0800 h on day 4. However, pentobarbital injection at 1345 h on day 3, which was effective in blocking estradiolinduced surges of LH and PRL secretion, suppressed markedly the high levels at 0800 h on day 4. In these pentobarbital-blocked rats, the diurnal changes in BrdU-labeling indices whose peak would normally have occurred at 0700-0900 h on day 4 were delayed not by the time corresponding to the duration of pentobarbital anesthesia but exactly by 24 h. These results suggest that 1) hypothalamic and sexually dependent diurnal changes in lactotroph proliferation can be induced by short-term estradiol treatment in ovariectomized rats as well as in cycling rats, and 2) estradiol treatment for 14 days rather prevents the diurnal changes in lactotroph proliferation.

Pentobarbital anesthesia during the proestrous afternoon blocks lactotroph proliferation occurring on estrus in female rats.

Estradiol stimulates the synthesis and secretion of PRL and lactotroph proliferation, and its long-term administration induces PRL-secreting pituitary tumors. We examined changes in the number of proliferating lactotrophs throughout the estrous cycle in female rats and the involvement of the brain in the regulation of the lactotroph proliferation by anesthetizing with pentobarbital. Female rats revealing regular 4-day estrous cycles were injected ip with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) 3 h before decapitation to label DNA-replicating cells. Dispersed anterior pituitary cells obtained from these rats were stained for PRL and BrdU with a double labeling immunofluorescence technique. The rate of lactotroph proliferation was represented by the BrdU-labeling index (BrdU-labeled lactotrophs as a percentage of total lactotrophs counted). Lactotrophs from rats injected with BrdU at 1000 h showed a high BrdU-labeling index of 2.6% on estrus, whereas they showed almost undetectable levels of the BrdU-labeling index during the other stages of the estrous cycle. The anterior pituitary cells other than lactotrophs were scarcely labeled during any stages. The BrdU-labeling index began to rise by the midnight between proestrus and estrus, peaked between 0800 and 1200 h, and returned to undetectable levels by the midnight between estrus and diestrus I. Pentobarbital (35 mg/kg, i.p.) injected at 1345 h on proestrus, which was effective in blocking ovulation on estrus, eliminated completely the increase in the BrdU-labeling index as determined by BrdU injection at 1000 h on estrus. In contrast, the high BrdU-labeling index on estrus was partly suppressed to a level of 1.4% by pentobarbital injection at 0900 or 2100 h on proestrus. The rats injected with pentobarbital at 1345 h on proestrus did not show any increases in the BrdU-labeling index even after BrdU injection was delayed from 1000 to 1400 h on estrus. However, a high BrdU-labeling index of 3.7% was obtained in these animals when BrdU was injected at 1000 h on diestrus I. We conclude that 1) lactotrophs of cycling female rats proliferate selectively on the day of estrus; and 2) the proliferation of lactotrophs on estrus is not due to a direct action on the anterior pituitary of estradiol secreted from the ovaries but triggered by neural events occurring during the proestrous afternoon, which are closely related with the regulation of preovulatory surges of gonadotropin and PRL secretion.

Somatotropes and thyrotropes in the rat anterior pituitary gland cosecrete substance P: analysis by the sandwich cell immunoblot assay.

Substance P (SP) which is synthesized and secreted by anterior pituitary cells has been suggested to alter pituitary functions as a local modulator. We determined which cell type(s) of rat anterior pituitary gland secretes SP. A new technique, named the sandwich cell immunoblot assay (CIBA), was developed to identify two protein substances that are secreted simultaneously from the same cells. Monodispersed anterior pituitary cells were sandwiched with pairs of transfer membranes and incubated to absorb secretory substances on the membranes. Small reference points for identifying the location of cell blots were simultaneously put on the pairs of transfer membranes after the incubation. The validity of the sandwich CIBA was revealed by the following: (1) cell blots were detected at the same locations on the pairs of transfer membranes that were immunostained with the same antisera against the anterior pituitary hormones, and (2) the areas of these cell blots detected at the same locations on different membranes correlated well. When one of the pair of transfer membranes was immunostained for SP and the remaining immunostained for one of the anterior pituitary hormones, it was found that 78 and 27% of SP-immunoreactive cell blots showed also GH and TSH immunoreactivity, respectively. None of the SP-immunoreactive cell blots showed immunoreactivity for PRL, ACTH, LH or FSH. These results suggest that the sandwich CIBA is useful to identify two substances cosecreted from a cell and that a subpopulation of rat somatotropes or thyrotropes cosecretes SP.