The influence of oestrogen administration in vivo on in vitro prolactin release. Interaction between dopamine and thyrotrophin releasing hormone.

The influence of oestrogen administered to the ovariectomized rat on the interaction between dopamine (DA) and thyrotrophin releasing hormone (TRH) on the release of radioimmunoassayable (RIA) and [3H]leucine incorporated into prolactin ([3H]PRL) was examined in vitro. Dopamine had a more marked suppressing effect on newly synthetized PRL (80%), as determined [3H]PRL, than on total PRL (50%), as determined by RIA-PRL. The administration of 5 micrograms of oestradiolbenzoate (OeB) for 7 days resulted in blocking the suppressing effect of DA when RIA-PRL was measured but not when [3H]PRL was measured. The administration of 5 micrograms of OeB enabled TRH to partially override the suppressing effect of DA and the degree of response was more marked when RIA-PRL was measured than when [3H]PRL was measured. The administration of 50 micrograms of OeB for 3 days enabled TRH to override the DA blockade of prolactin release to levels comparable to that of the control when RIA-PRL was measured but had little to no effect on [3H]PRL. The results are discussed in relation to the two storage pools of PRL in the pituitary and the data suggest that DA acts predominantly to suppress the newly synthetized, rapidly releasable pool. Oestrogen acts to block DA action on the older more stable PRL pool. The ability of TRH to override the DA blockade of PRL release depends upon the presence of oestrogen; here TRH acts predominantly on the older more stable pool of PRL. Oestrogen's action on disrupting the DA suppression of PRL release appears to be related to the time of day the hormone is administered subsequent to when the pituitary is exposed to DA in vitro.     

Effect of the interaction between pro-leu-gly-NH2 and catecholaminergic drugs on MSH release from rat pituitaries incubated in vitro

The effect of the interaction between Pro-Leu-Gly-NH2 (PLG) and catecholamines on the release of MSH by rat pituitaries glands was studied in vitro. The catecholamines dopamine (DA) and norepinephrine (NE) inhibited the release of alpha-MSH from pituitary glands incubated in vitro. The DA effect appeared at low concentrations while NE was effective in concentrations 20 times greater than DA. These effects were blocked by an alpha-adrenergic blocker agent. On the other hand, epinephrine (E) at the concentrations tested did not modify MSH release. When 50 or 500 ng/ml of DA is added to the medium containing PLG concentrations it strengthened the inhibitory effect of PLG release, whereas the action of NE and PLG was additive; at the highest tested concentration E (5,000 ng/ml) blocked the inhibition of MSH induced by PLG. It is concluded that, whereas the inhibitory release of MSH by catecholamines is mediated by an alpha-adrenergic receptor, the inhibitory effect of PLG is not exerted through a catecholaminergic receptor since its action is not prevented by any of the catecholamine receptor blockers studied. Thus, it seems that specific PLG receptors are present at the pituitary level.     

Influence of bromocriptine and oestrogen on prolactin synthesis, secretion and tumour growth in vivo in rats

The effects of diethylstilboestrol implants and bromocriptine administration on serum prolactin concentrations, prolactin messenger RNA (mRNA) and pituitary tumour weight were examined. Intact female Fischer 344 rats were implanted s.c. with 10 mg diethylstilboestrol (DES) under light anaesthesia. All animals except the control group carried the implant for 7 weeks at which time the rats were subdivided into five groups: A, control; B, DES for 7 weeks; C, DES for 7 weeks followed by withdrawal of DES for 1 week; D, DES for 7 weeks followed by withdrawal of DES and administration of bromocriptine for 1 week; E, DES for 8 weeks with concurrent administration of bromocriptine during the last week. Serum concentrations of prolactin were measured by radioimmunoassay, prolactin mRNA concentrations were measured by dot-blot hybridization and sodium dodecylsulphate-polyacrylamide gel electrophoresis of the in-vitro translated mRNA. Pituitary growth was estimated by changes in pituitary weight and assessed by light and electron microscopic examination. Treatment with DES dramatically increased serum prolactin concentrations and prolactin mRNA and induced pituitary tumour formation as shown by histological changes. Withdrawal of DES for 1 week did not lead to a decrease in pituitary tumour weight but was accompanied by a decrease in serum prolactin concentrations and prolactin mRNA from peak concentrations although they remained significantly increased above controls. Treatment with bromocriptine after DES implants were removed led to a significant reduction in pituitary tumour weight and a decrease in serum prolactin concentrations and prolactin mRNA. Histology of the pituitary tumour after the bromocriptine treatment showed pituitary cells similar to those from normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of in vivo treatment with estrogen on luteinizing hormone synthesis and release by rat pituitaries in vitro.

The influence of estrogen on uptake of [3H]glucosamine and [14C]alanine and their incorporation into LH and total protein was investigated. Ovariectomized rats were sacrificed 22 h after injection with either oil or estradiol benzoate (EB, 50 microng/rat). Quartered anterior pituitary glands were incubated for 4 h with radioactive precursors in the presence or absence of 3.6 X 10-8M synthetic gonadotropin-releasing hormone (GnRH). Labeled LH was isolated by immunoprecipitation with specific anti-LH-beta serum. Both EB and GnRH significantly elevated the amount of [3H]glucosamine-LH appearing in the medium, the tissue, and the total system (medium + tissue), but they increased the amount of [14C]alanine-LH only in the medium. There was a significant positive interaction between EB and GnRH on the amounts of [3H]glucosamine-LH and [14C]alanine-LH in the medium and of [3H]glucosamine-LH in the tissue and total system. EB enhanced [3H]glucosamine uptake and incorporation into total protein, but GnRH had little or no effect on these parameters. In time course studies rats were injected with either oil or EB at 22, 11, or 5.5 h prior to sacrifice. At all times EB significantly increased synthesis and release of [3H]-glucosamine-LH and release of total immunoreactive LH (IR-LH) by pituitaries incubated with GnRH. The amounts of labeled and IR-LH released into the medium increased linearly with time after EB injection, but the amount of labeled LH in the total system plateaued at 5.5 h after EB injection. In another study, estradiol (E2, 5 microng/rat) dissolved in 1% ethanol-saline was injected at 0.5, 1.0, 2.0, or 4 h prior to sacrifice. Incorporation of [3H]glucosamine into tissue protein and release of [3H]glucosamine-LH was stimulated within 2 h after E2 injection. However, incorporation of [3H]glucosamine into LH was not stimulated until 4 h after E2 injection. These results suggest that estrogen and GnRH regulate LH synthesis at different sites, and that the effect of estrogen is non-specific compared to that of GnRH. The synthesis of the carbohydrate moiety of LH appears to be subjected to hormonal regulation more readily than the synthesis of the polypeptide moiety.     

Role of type IV collagen in prolactin release from anterior pituitaries of male rats

We previously demonstrated that laminin, a component of basement membranes, modulates pituitary hormone secretion. In the present study, we evaluated the effect of type IV collagen, another component of this membrane, on the release of prolactin (PRL) by anterior pituitary gland from adult male rats. Hemipituitaries were incubated for 3 h with type IV collagen or antibodies against it and PRL release was studied. Rabbit IgG to type IV collagen at concentrations of 10⁻⁷−10⁻⁵ M had a significant stimulatory effect on PRL release, in comparison to normal rabbit serum IgG or medium alone used as controls. Type IV collagen induced a significant inhibitory effect on basal release of PRL at a concentration of 30 μg/mL. A slight decrease in PRL release was detected in thyrotropin-releasing hormone-stimulated hemipituitaries incubated with type IV collagen at all concentrations used. These results suggest that type IV collagen, similar to laminin-1, modulates PRL released from hemipituitaries, in vitro.     

Estrogen-induced prolactin and DNA synthesis in immature female rat pituitaries.

Estradiol is likely involved in stimulating developmental changes in the ability of the rat pituitary to secrete prolactin. To investigate the possibility that these changes involve proliferation of prolactin cells, estradiol effects on pituitary growth and prolactin synthesis were examined. Estradiol treatment of immature female rats stimulates increases in pituitary weight, [3H] thymidine incorporation, DNA content and prolactin synthesis. Treatment of rats with the DNA synthesis inhibitor, hydroxyurea, partially blocked the ability of estradiol to stimulate prolactin synthesis suggesting that at least part of the effect of estrogen is due to cell proliferation. These results suggest that estrogen-induced proliferation of prolactin cells is involved in the developmental processes of the pituitary.     

The characteristics of melanocyte stimulating hormone release from incubated pituitaries of the lizard, Anolis carolinensis

A procedure for studying melanocyte stimulating hormone (MSH) release from incubated hemipituitaries of the lizard, Anolis carolinensis, is described. Hemipituitaries incubated in vitro at 30° for 5 hr, released bioassayable MSH into the incubation medium; an initial phase of rapid release lasted .5-1 hr, followed by a lower rate of release during the remainder of the incubation. There was no evidence that MSH released from hemipituitaries and accumulated in the media influenced subsequent MSH release during the incubations. Release of MSH was dependent on the presence of Ca²⁺ in the medium. Increase of medium K⁺ to 56 mM did not alter the rate of MSH release at any stage of the incubation. The explanted lizard pars intermedia responded to low temperature in an unusual way. Tissue incubated at 15° released MSH at a lower rate than at 30°, but tissue incubated at 1–2° released MSH at a rate indistinguishable from that of tissue at 30°. MSH release at 1–2° was prevented by omitting Ca²⁺ from the medium, suggesting that MSH release in the cold was not the result of cell damage. When tissue was incubated at 1–2° during the first half-hour of incubation and allowed to rewarm to 30° there was no detectable increase in the rate of MSH release; when the same tissue was recooled to 1–2° during the fourth hour and rewarmed during the fifth, a greatly increased release of MSH resulted, indicating that during the initial period of release, but not at later times, the tissue was resistant to the effect of cooling on hormone release. Theophylline (8 mM) significantly increased MSH release at all stages of the incubation except during the first half-hour, suggesting that MSH release may be stimulated by raised intracellular cyclic AMP levels. Imidazole (10 mM), a cyclic AMP phosphodiesterase stimulator, significantly reduced MSH release by 82% during the first half-hour of incubation and again at 1–3 hr by 44%, but had no significant effect on MSH release at other times. These results may indicate that “basal” release at the earliest stage is linked to the adenyl cyclase-cyclic AMP mechanism, but it is not certain that MSH release is tightly linked to cyclic AMP production at later stages of the incubation.     

Mechanisms of release of prolactin from fowl anterior pituitary glands incubated in vitro: effects of calcium and cyclic adenosine monophosphate

Fowl anterior pituitary glands were bisected and each half was pretreated in either Medium 199 or medium containing EGTA to deplete endogenous calcium (Ca2+) stores, after which they were incubated in Medium 199, or Ca2+-free medium, containing prolactin release-stimulating agents and verapamil, a Ca2+ channel blocker. High K+ concentrations, hypothalamic extract, synthetic thyrotrophin-releasing hormone (TRH) and dibutyryl cyclic AMP (dbcAMP) all stimulated release of prolactin from control (non EGTA-treated) hemianterior pituitary glands. The effects of TRH and dbcAMP were not additive, but the response to submaximal concentrations of TRH was augmented by theophylline, a phosphodiesterase inhibitor. Reduction of Ca2+ availability with EGTA or verapamil reduced basal release of prolactin, prevented the prolactin-stimulating effects of high K+ concentrations and TRH, and markedly attenuated responses to hypothalamic extract and dbcAMP, EGTA being more effective than verapamil. Increasing the Ca2+ concentration of the medium did not augment basal or stimulated release of prolactin. These results suggest that both Ca2+ and cyclic AMP may act as intracellular mediators in the release of prolactin. Both basal and stimulated release of prolactin depend upon the presence of Ca2+. Although influx from the medium may be the major source of Ca2+, endogenous stores of Ca2+, perhaps mobilized by dbcAMP, may be able to maintain some release of prolactin. The prolactin-stimulating effects of TRH may be mediated by cyclic AMP.