ABSTRACT

The activity of ornithine decarboxylase (ODC) in the rat anterior pituitary gland varies during the oestrous cycle, with a rise in activity seen at pro-oestrus. This enzyme, which is rate-limiting for the synthesis of the polyamines, can be specifically and irreversibly blocked by α-difluoromethylornithine (DFMO). A previous study showed that when this drug was administered to rats in vivo on the afternoon of pro-oestrus, it suppressed the normal surge in plasma prolactin levels that occurred later that day. The effect of DFMO was associated with reduced levels of putrescine in the anterior pituitary gland, suggesting that ODC activity in the lactotroph might be involved in the prolactin surge. We have examined the effects of DFMO on the secretion of prolactin from anterior pituitary cells, isolated either from male rats or from females at different stages of the oestrous cycle. The drug was found to reduce prolactin secretion stimulated by thyrotrophin-releasing hormone (TRH), but only in cells isolated from pro-oestrous animals and only for 2 days after cell isolation. Basal secretion was unaffected by DFMO. The results imply that ODC is important for TRH-stimulated prolactin secretion at pro-oestrus, and it is specific for pro-oestrus. The prolactin surge could therefore be influenced by this ODC-dependent effect of TRH. The pro-oestrous-specific response to TRH may be a consequence of the increased ODC activity seen at this time. Alternatively, the increased ODC activity could be a consequence of coupling to TRH receptors, which are known to increase in number at pro-oestrus.

Journal of Endocrinology (1993) 137, 133–139

ABSTRACT

The effects of histamine H₁- and H₂-receptor antagonists on the pituitary-thyroid axis were studied in normal and thyroxine (T₄)-treated rats. Acute administration (120 min before the test) of the H₂ antagonist cimetidine induced a significant (P<0·01) increase in the TSH response to TRH, whereas treatment with histamine (30 min before the test) or with the H₁-receptor blocker diphenhydramine (120 min before the test) was without effect. Treatment with cimetidine or ranitidine (another H₂-receptor antagonist) for 5 days induced a marked decrease in basal plasma TSH concentrations (P<0·01), with no changes in pituitary concentrations of TSH. Plasma prolactin concentrations were similarly decreased by cimetidine (P<0·01), though not by ranitidine. Neither antihistaminic altered pituitary prolactin concentrations. Despite decreasing basal concentrations of plasma TSH, cimetidine augmented the response to TRH above baseline values (P<0·01) in control rats as well as in animals with T₄-induced suppression of plasma TSH. Administration of cimetidine or ranitidine for 5 days was followed by a reduced concentration of plasma T₄ and tri-iodothyronine (T₃) (P<0·05 and P<0·01 respectively), perhaps as a result of the declining plasma TSH levels. These results provide the first evidence for the reduction of plasma TSH concentrations by H₂-receptor blockers, and may indicate that histamine can physiologically regulate TSH and prolactin secretion through H₂ receptors in the anterior pituitary. The results, however, do not disprove a central action of histamine, since the decreased plasma TSH concentrations in the presence of low plasma T₄ and T₃ concentrations, and also the decrease in plasma prolactin concentrations induced by cimetidine, suggest reduced hypothalamic stimulation.

J. Endocr. (1986) 111, 175–180

Abstract

1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)₂D₃ on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH₃) cells. Compared with vehicle-treated cells, 1,25-(OH)₂D₃ (10 nmol/l) increased specific [³H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose–response experiments, specific [³H]MeTRH binding increased with 1,25-(OH)₂D₃ concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0·5 nmol 1,25-(OH)₂D₃/l. The vitamin D metabolite, 25-OH D₃, increased [³H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)₂D₃. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)₂D₃/l for 48 h increased the maximum binding from 67·4 ± 8·8 fmol/mg protein in vehicle-treated cells to 96·7 ± 12·4 fmol/mg protein in treated cells. There was no difference in apparent K _d (1·08 ± 0·10 nmol/l for 1,25-(OH)₂D₃-treated and 0·97 ± 0·11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)₂D₃/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 μg/ml, 6 h) abrogated the 1,25-(OH)₂D₃-induced increase in [³H]MeTRH binding. Cortisol also increased [³H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)₂D₃. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)₂D₃. We conclude that the active vitamin D metabolite, 1,25-(OH)₂D₃, caused a time- and dose-dependent increase in [³H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding.

Journal of Endocrinology (1995) 147, 397–404

Abstract

It is clear that dopamine (DA) at high concentrations (>100 nmol/l) inhibits the release of prolactin (PRL). Paradoxically, this monoamine at low concentrations (<10 nmol/l) has also been shown to augment PRL secretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein subtypes that recruit opposing intracellular signaling pathways within lactotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity of G_iα3 and G_sα in primary cultures of rat pituitaries and subsequently tested the ability of these cultures to respond to high and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobulins (IgGs; 50 μg/ml) purified from preimmune serum (PII) or IgGs directed against the C-terminal portion of G_iα3 or G_sα. After immunoneutralization of these G proteins, cells were challenged with 10 or 1000 nmol Da/l and the relative amount of PRL released was assessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nmol DA/l inhibited (61·4 ±7·6% of basal values; mean ± s.e.m.) while 10 nmol DA/l augmented (120·0 ± 7·0%) PRL release in five separate experiments. Treatment of cells with anti-G_iα3 attenuated the inhibitory effect of high dose DA (87·3 ± 14·5%). However, elimination of G_iα3 activity did not significantly alter the PRL stimulatory effect of 10 nmol DA/l (121·0 ± 5·2%). Interestingly, immunoneutralization of G_sα resulted in a reciprocal shift in the activity of the lower dose of DA from stimulatory to inhibitory (69·7 ± 7·3%) while combined treatment of anti-G_iα3 and anti-G_sα abrogated the responsiveness of pituitary cell cultures to either DA treatment (1000 nmol/l, 70·7 ± 12·5% and 10 nmol/l, 87·5 ± 21·4%). These data reveal that ligand-activated DA receptors can interact with both G_iα3 and G_sα. Elimination of the stimulatory component (G_sα) favors the DA receptor activation of the inhibitory pathway (G_iα3) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary cells with 100 nmol thyrotropin-releasing hormone (TRH)/1 as a positive control for secretory integrity. As anticipated, TRH stimulated PRL release to 188·0±31·0% of basal values under control conditions. Unexpectedly, immunoneutralization of G_sα completely blocked the ability of TRH to induce PRL release (101·8 ± 12·0% This neutralizing effect was specific to G_sα in that blockade of G_iα3 activity had no significant effect on TRH-stimulated PRL release (166·2 ± 13·1%). These data are the first to support a direct role of G_sα in TRH signal transduction within PRL-secreting cells.

Journal of Endocrinology (1996) 148, 447–455

ABSTRACT

Prolactin responses to pharmacological agents were used to characterize the defect in prolactin regulation which occurs after administration of high doses of oestrogen to rats.

Animals with chronically implanted venous cannulae were injected with 2 mg oestradiol benzoate in oil and 2–3 days later prolactin concentrations were measured after injections of saline, thyrotrophin-releasing hormone (TRH), fenfluramine, apomorphine and butaclamol. The responses were compared with those in oil-injected animals.

Hyperprolactinaemia in oestrogen-treated animals was unresponsive to apomorphine, but was even more sensitive to dopamine receptor blockade than controls. These results suggest that the lactotrophs in oestrogen-treated animals are already maximally suppressed by endogenous dopamine, though ineffectively.

Although there was an increased prolactin response to TRH in oestrogen-treated animals, there was an impaired response to fenfluramine, indicating suppressed serotonergic prolactin-releasing factor mechanisms.

Maximal endogenous dopaminergic activity and suppressed prolactin-releasing factor mechanisms are appropriate hypothalamic responses to hyperprolactinaemia. The operation of these responses in the earliest stages of the development of pituitary hyperplasia indicates that oestrogen induces a disturbance of prolactin regulation in the lactotroph, independent of hypothalamic control.

J. Endocr. (1985) 104, 447–452

SUMMARY

The effects of two dopamine agonists (apomorphine and bromocriptine) and a dopamine antagonist (pimozide) on cold- or thyrotrophin releasing hormone (TRH)-induced TSH secretion were studied in normal male rats. Apomorphine given in various doses (0·5–10 mg/kg body wt) 10 min before exposure to cold significantly depressed TSH secretion. Large doses of bromocriptine (5–10 mg/kg body wt) given 1 h before exposure to cold, also blocked this response whereas a smaller dose (2·5 mg/kg body wt) given 30 min, 1, 3 or 6 h before cold exposure or repeated doses (0·1–2·5 mg/kg body wt) for 3 days did not modify cold-induced TSH secretion. Pimozide given in various doses (0·25–2·5 mg/kg body wt) 1 h before exposure to cold did not alter the cold response, but 2·5 mg/kg reversed the inhibition caused by apomorphine or bromocriptine. None of these drugs affected TRH-induced TSH secretion. These results suggest that there are no dopaminergic receptors on the pituitary thyrotrophs, but that dopamine might be an inhibitory transmitter in the brain involved in the regulation of TSH secretion in the rat.

Pituitary enlargement and hyperprolactinaemia were induced in male rats by a single subcutaneous injection of 2 mg oestradiol benzoate in oil. Two months after treatment, when oestrogen levels were normal, serial blood samples for determination of plasma concentrations of prolactin were obtained from undisturbed animals through an indwelling right atrial cannula which had been implanted 7–10 days before.

Basal concentrations of prolactin were obtained in treated and control rats, and responses of prolactin to intravenous injections of thyrotrophin releasing hormone (TRH), apomorphine, butaclamol and fenfluramine were measured.

Sustained hyperprolactinaemia with pituitary hyperplasia was achieved in only 20% of animals. Responses of prolactin to TRH were the same in control, oestrogen-treated hyperprolactinaemic and non-hyperprolactinaemic rats, indicating normal pituitary responsiveness to one prolactin releasing factor.

Complete suppression of prolactin concentrations by apomorphine occurred in hyperprolactinaemic animals, whereas no suppression could be demonstrated in animals with normal (low) basal prolactin levels, indicating good responsiveness of hyperplastic pituitary glands to dopamine inhibition.

Dopamine receptor blockade by butaclamol resulted in a vigorous prolactin response in animals with sustained hyperprolactinaemia, indicating that dopaminergic prolactin inhibitory mechanisms remain qualitatively intact, but the response was quantitatively less than in controls, suggesting insufficient hypothalamic release of dopamine.

Responses of prolactin to certain doses of fenfluramine were completely abolished in hyperprolactinaemic animals, indicating diminished sensitivity of serotoninergic prolactin releasing factor mechanisms.

Prolactin releasing factor unresponsiveness and relative insufficiency of dopaminergic activity could be regarded as physiologically appropriate responses to chronic hyperprolactinaemia. Thus oestrogen-induced chronic hyperprolactinaemia appears to be entirely of pituitary origin.

Abstract

The reduced thyroid activity during short-term starvation is associated with a lowered hypothalamic synthesis and secretion of TRH. However, little is known about the cause of the reduced thyroid function during prolonged malnutrition. We have therefore studied the effects of food reduction to one-third of normal (FR33) on the hypothalamus-pituitary-thyroid axis of male and female Wistar rats. After 3 weeks body weights of FR33 rats were almost 50% lower than those of controls. In both sexes, FR33 caused marked increases in serum corticosterone, and decreases in serum TSH, thyroxine (T₄), free T₄, tri-iodothyronine (T₃) and free T₃. While the free T₃ fraction (FFT₃) in serum decreased, the free T₄ fraction (FFT₄) tended to increase. Electrophoretic analysis indicated that decreased FFT₃ was correlated with an increased thyroxine-binding globulin, while the increase in FFT₄ seemed due to a decreased thyroxine-binding prealbumin binding capacity. Total RNA and proTRH mRNA in the hypothalamus were not affected by FR33. Median eminence and posterior pituitary TRH content tended to increase in FR33 rats, suggesting that hypothalamic TRH release is reduced in FR33 rats. Anterior pituitary TSH content was decreased by FR33 in both sexes, but pituitary TSHβ mRNA and TRH receptor status were not affected except for increased pituitary TSHβ mRNA in female FR33 rats. Although FR33 had no effect on pituitary weight, pituitary RNA and membrane protein content in FR33 rats were 50–70% lower than values in controls.

In conclusion, prolonged food reduction suppresses the pituitary-thyroid axis in rats. In contrast to short-term food deprivation, the mechanism whereby serum TSH is suppressed does not appear to involve decreases in proTRH gene expression, but may include effects on pituitary mRNA translation. Our results further support the hypothesis that TSH release may be lowered by increased corticosterone secretion, although the mechanism of this effect may differ between acute starvation and prolonged food reduction.

Journal of Endocrinology (1996) 150, 169–178

Thyroid hormones exert a range of developmental and physiological actions in all vertebrates. Serum concentrations of L-thyroxine (T4) and 3,5,3 -L-triiodothyronine (T3) are maintained by a negative feedback loop involving T3-inhibition of hypothalamic thyrotrophin releasing hormone (TRH) and pituitary thyroid stimulating hormone (TSH) secretion, and by tissue specific and hormone-regulated expression of the three iodothyronine deiodinase enzymes that activate or metabolise thyroid hormones. T3 actions are mediated by two T3-receptors, TRalpha and TRbeta, which act as hormone-inducible transcription factors. The TRalpha (NR1A1) and TRbeta (NR1A2) genes encode mRNAs that are alternatively spliced to generate 9 mRNA isoforms (TRalpha1, alpha2, alpha3, Deltaalpha1, Deltaalpha2, beta1, beta2, beta3 and Deltabeta3), of which four (TRalpha1, alpha2, beta1 and beta2) are known to be expressed at the protein level in vivo. The numerous TR mRNAs are expressed widely in tissue- and developmental stage-specific patterns, although it is important to note that levels of mRNA expression may not correlate with receptor protein concentrations in individual tissues. The TRalpha2, alpha3, Deltaalpha1 and Deltaalpha2 transcripts encode proteins that fail to bind T3 in vitro. These non-binding isoforms, in addition to TRDeltabeta3 which does bind hormone, may act as dominant negative antagonists of the true T3-binding receptors in vitro, but their physiological functions and those of the TRbeta3 isoform have not been determined. In order to obtain a new understanding of the complexities of T3 action in vivo and the role of TRs during development, many mouse models of disrupted or augmented thyroid hormone signalling have been generated. The aim of this review is to provide a picture of the physiological actions of thyroid hormones by considering the phenotypes of these genetically modified mice.