ABSTRACT

The hormone 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) has been shown to selectively enhance agonist-induced TSH release in the rat thyrotroph in vitro. The interaction of 1,25-(OH)₂D₃ with tri-iodothyronine (T₃) and cortisol was studied in primary cultures of dispersed anterior pituitary cells. TRH (1 nmol/l)-induced TSH release over 1 h was enhanced by 70% (P<0·01) following exposure to 10 nmol 1,25-(OH)₂D₃/l for 24 h. Pretreatment with T₃ (1 pmol/l–1 μmol/l) for 24 h caused a dose-dependent inhibition of TRH-induced TSH release. Net TRH-induced TSH release was inhibited by 85% at T₃ concentrations of 3 nmol/l or greater. Co-incubation with 1,25-(OH)₂D₃ resulted in enhanced TRH-induced TSH release at all T₃ concentrations tested (P<0·001). The increment of TRH-induced TSH release resulting from 1,25-(OH)₂D₃ pretreatment was equivalent in the presence or absence of maximal inhibitory T₃ concentrations. At 1 nmol T₃/1, there was a two- to threefold relative increase in 1,25-(OH)₂D₃-enhanced TRH-induced TSH release. Incubation with cortisol (100 pmol/l–100 nmol/l) had no effect on basal or TRH-induced TSH release, nor did it alter 1,25-(OH)₂D₃-enhanced TRH-induced TSH release when added 24 h before, or at the time of addition of 1,25-(OH)₂D₃. Actinomycin D and α-amanitin abolished 1,25-(OH)₂D₃-enhanced TSH secretion.

These data demonstrate that the action of 1,25-(OH)₂D₃ in the thyrotroph required new RNA transcription, and was not affected by cortisol. In the presence of T₃, the response of the thyrotroph to TRH induced by 1,25-(OH)₂D₃ was increased. We have shown that 1,25-(OH)₂D₃ has significant effects on the action of TRH and T₃ in vitro. These findings support the proposal that 1,25-(OH)₂D₃ may modulate TSH secretion in vivo.

Journal of Endocrinology (1989) 121, 451–458

Thyrotropin (TSH) is secreted not as one distinct hormone, but rather as a group of isohormones which differ in their oligosaccharide composition. Although the mechanisms regulating TSH glycosylation are not fully understood, there is strong evidence that TRH plays an important role. The aim of our study was to determine the dynamic influence of TRH on TSH microheterogeneity. Sera were obtained from euthyroid volunteers (n=20) before and 30, 60, 120, 180 and 240 min after intravenous, nasal and oral administration of TRH in three independent runs (randomized order, at a time-interval of 3 weeks between each run). TSH was immuno-concentrated and analysed by isoelectric focusing (IEF) and lentil lectin affinity chromatography. TSH immunoreactivity was measured by an automated second-generation TSH immunoassay. Overall, serum TSH concentrations reached maximal values 30 min after intravenous, 60 min after nasal and 180 min after oral TRH stimulation. IEF analysis revealed 63.3+/-3.3% of pituitary standard TSH (IRP 80/558) in the neutral pH range (8>pH>6). In contrast, 30 min after TRH stimulation 80.8+/-3.7% (P<0.001) and 60 min after TRH stimulation 44.9+/-2.2% (P<0.001) of the TSH of euthyroid probands were found in this pH range, whereas 180 min after TRH stimulation 58.4+/-2.3% (P<0.001) were detected in the acidic pH range (pH<6). This shift of TSH composition in euthyroidism after TRH stimulation was confirmed by lentil lectin analysis of TSH: core-fucose content of euthyroid TSH was 73.4+/-3.8% 30 min and 22.9+/-3.2% 120 min after TRH stimulation in contrast to basal (53.3+/-1.8%; P<0.001) and pituitary standard (IRP 80/558) TSH (63.0+/-0.9%; P<0.001). In conclusion, in euthyroidism, TRH stimulation time-dependently changes the distribution pattern of the TSH isoforms from an alkaline and neutral to a more acidic one. This corresponds to the secretion of isohormones with altered bioactivity which could influence the fine-tuning of thyroid function.

ABSTRACT

An influence of thyrotrophin-releasing hormone (TRH) on TSH heterogeneity in close association with de-novo biosynthesis was studied in rat anterior pituitary glands. Hemipituitary glands from adult male rats were incubated in Krebs–Henseleit–glucose media containing [³H]glucosamine and [¹⁴C]alanine for 3 and 6 h in the presence or absence of 10 ng TRH per ml. Fractions of TSH in the pituitary extracts were obtained using affinity chromatography coupled with an anti-rat TSH globulin. These TSH fractions were analysed by isoelectric focusing. The control pituitary glands were composed of four component peaks (isoelectric point (pI) 8·7, 7·8, 5·3 and 2·5) of [³H]glucosamine and [¹⁴C]alanine incorporated into TSH, and the amounts of radioactivity of these components were increased with the incubation time. Of these peaks, radioactive components of pI 8·7 and 7·8 coincided with the non-radioactive TSH components measured by radioimmunoassay. Addition of TRH increased incorporation of [¹⁴C]alanine into TSH in each of the components to a greater extent than that of [³H]glucosamine. In addition, new components with pI 7·2, 6·5 and 6·2, each component corresponding to each unlabelled TSH component, were demonstrated in the presence of TRH. Because addition of TRH did not change the amounts of [¹⁴C]alanine-labelled TSH in the media, the newly formed components were assumed to be connected with protein synthesis occurring in the anterior pituitary gland, which may be specific substances in response to TRH administration. These results indicate that TRH principally elicits an increase in protein synthesis in TSH at the anterior pituitary level, resulting in an alteration of TSH heterogeneity.

J. Endocr. (1984) 103, 165–171

Abstract

Pyroglutamylglutamylprolineamide, which was first discovered in mammalian prostate, differs from thyrotrophin-releasing hormone (TRH) by substitution of glutamic acid for histidine at position two of the tripeptide. Recently, the newly discovered peptide has been identified in substantial concentrations in the rat anterior pituitary gland and, in this study, we have investigated the effects of the peptide on rat anterior pituitary cells in culture. GH₃ cells were chosen to examine the possible effects of the new peptide, particularly in relation to its effects on the TRH receptor. This cell-type was deficient, in comparison with normal rat pituitary cells, in the new TRH-related peptide and appeared to be an ideal model cell in which to study the effects of pGlu-Glu-ProNH₂. TRH (0·01–100 nm) was found to stimulate the secretion of both GH and prolactin from GH₃ cells whereas pGlu-Glu-ProNH₂ had no effect within the same concentration ranges. In contrast, at micromolar concentrations pGlu-Glu-ProNH₂ exhibited intrinsic TRH-like activity causing stimulation of both GH and prolactin release from GH₃ cells. Both TRH and pGlu-Glu-ProNH₂ appeared to act through the same intracellular signalling mechanism, causing significant increases in intracellular inositol phosphate within the expected concentration ranges. However, pGlu-Glu-ProNH₂ (up to 1 mm) displaced neither [³H]TRH nor [³H]MeTRH from membrane-binding sites on GH₃ cells, suggesting that the effects of the new peptide were mediated through a second receptor. The physiological relevance of these effects of pGlu-Glu-ProNH₂ requires further investigation.

Journal of Endocrinology (1994) 142, 111–118

The effects of dopamine and thyrotrophin-releasing hormone (TRH) on prolactin release was studied in 14 intact and six pituitary stalk-sectioned (SS) female rhesus monkeys (Macaca mulatta). Baseline prolactin values were ninefold higher in SS animals (149 ± 16 ng/ml) than in intact animals (16 ± 1 ng/ml).

Prolactin release after intravenous administration of TRH in doses of 0,125,250, 500 and 1000 ng revealed that SS monkeys were more sensitive to the prolactin-releasing activity of this tripeptide than were intact animals. A significant (P < 0·05) increment in serum prolactin was observed in SS animals after injection of 125 ng TRH whereas 250 ng was required to raise prolactin levels in the circulation of intact animals significantly (P <0·05). Furthermore, at each comparable dose level of TRH, the increment in serum prolactin was distinctly greater in SS animals than in intact monkeys.

Infusion of dopamine at the rate of 10 μg/kg body weight per min significantly (P <0·05) lowered prolactin levels within 60 min in intact animals and no further decline was observed with 20 or 40 μg dopamine. Serum prolactin concentrations were not affected by saline infusion or by 5 μg dopamine. Infusion of dopamine at the rate of 10 μg/kg body wt per min also resulted in significant (P <0·01) suppression of serum prolactin in SS animals. This prolactin decrease was apparent within 40 min. Prolactin release after 500 ng TRH was less in these dopamine-treated SS monkeys than after an infusion of saline. Higher doses of dopamine (20 and 40 μg) did not cause a further decrease in basal serum prolactin concentrations, but these two dopamine treatments blocked the increase in prolactin elicited by 500 ng TRH.

The results suggest that the removal of hypothalamic influence, possibly related to the effects of dopamine, renders the pituitary gland more sensitive to the prolactin-releasing action of TRH.

Abstract

We investigated the effects of diabetes mellitus on the hypothalamo-hypophysial-thyroid axis in male (R×U) F₁ and R-Amsterdam rats, which were found to respond to streptozotocin (STZ)-induced diabetes mellitus with no or marked increases, respectively, in plasma corticosterone. Males received STZ (65 mg/kg i.v.) or vehicle, and were killed 1, 2 or 3 weeks later. At all times studied, STZ-induced diabetes mellitus resulted in reduced plasma TSH, thyroxine (T₄) and 3,5,3′-tri-iodothyronine (T₃). Since the dialyzable T₄ fraction increased after STZ, probably as a result of decreased T₄-binding prealbumin, plasma free T₄ was not altered during diabetes. In contrast, both free T₃ and its dialyzable fraction decreased during diabetes, which was associated with an increase in T₄-binding globulin. Hepatic activity of type I deiodinase decreased and T₄ UDP-glucuronyltransferase increased after STZ treatment. Thus, the lowered plasma T₃ during diabetes may be due to decreased hepatic T₄ to T₃ conversion.

Median eminence content of TRH increased after STZ, suggesting that hypothalamic TRH release is reduced during diabetes and that this is not caused by impaired synthesis or axonal transport of TRH to the median eminence. Hypothalamic proTRH mRNA did not change in diabetic (R×U) F₁ rats during the period of observation, but was lower in R-Amsterdam rats 3 weeks after STZ. Similarly, pituitary TSH and TSHβ mRNA had decreased in R-Amsterdam rats by 1 week after STZ treatment, but did not change in (R×U) F₁ rats. The difference between the responses in diabetic R-Amsterdam and (R×U) F₁ rats may be explained on the basis of plasma corticosterone levels which increased in R-Amsterdam rats only. Hypothalamic TRH content was not affected by diabetes mellitus, but the hypothalami of diabetic rats released less TRH in vitro than those of control rats. Moreover, insulin had a positive effect on TRH release in vitro.

In conclusion, the reduced hypothalamic TRH release during diabetes is probably not caused by decreases in TRH synthesis or transport to the median eminence, but seems to be due to impaired TRH release from the median eminence which may be related to the lack of insulin. Inhibition of proTRH and TSHβ gene expression in diabetic R-Amsterdam rats is not a primary event but appears to be secondary to enhanced adrenal activity in these animals during diabetes.

Journal of Endocrinology (1997) 153, 259–267

ABSTRACT

Prolactin secretion by a human pituitary tumour cell line produced in our laboratory was stimulated by TRH, vasoactive intestinal peptide (VIP) and epithelial growth factor (EGF). All raised the intracellular concentration of free calcium (Ca²⁺ _i) of cells loaded with a fluorescent quinoline Ca²⁺ indicator in suspension, but the effect of TRH was much more rapid and less prolonged than that of VIP and EGF. Both TRH and VIP also increased Ca²⁺ _i in GH₃ rat pituitary tumour cells, but in this cell line the effect of VIP was only found in attached cells grown on cover-slips. In both human and rat cell lines, the increase in Ca²⁺ _i produced by TRH was independent of extracellular calcium, whereas this was a requirement for the action of VIP and EGF. It is concluded that the prolactin secretogogues, VIP and probably EGF, increase Ca²⁺ _i through an effect on plasma membrane calcium channels and that this effect differs from that of TRH.

J. Endocr. (1987) 114, 119–123

ABSTRACT

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 μmol/l (increase over basal, 118 ±16·5 (s.e.m.) fmol TRH; P <0·001 vs basal). This effect was mimicked by the specific D₂-agonist drugs bromocriptine (0·1 μmol/l) and LY 171555 (0·1 μmol/l) (increase over basal values, 137·5±13·75 fmol and 158·6± 10·7 fmol respectively; P <0·001 vs basal), but not by the D₁-agonist SKF 38393A. The stimulatory effect of dopamine (1 μmol/l) was blocked in a stereospecific manner by the active (d) but not by the inactive (l) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D₂-antagonist domperidone (0·01 μmol/l) whereas the D₁-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0·01 μmol/l) of this D₁ -antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D₂-receptors.

J. Endocr. (1987) 115, 419–424

ABSTRACT

The effect of thyrotrophin-releasing hormone (TRH) on the circulating plasma levels of tri-iodothyronine (T₃) and thyroxine (T₄) was determined in the same group of animals (four cattle and four Murrah buffaloes) during hot dry (HD), hot humid (HH) and cold environmental conditions. Plasma T₃ and T₄ concentrations were measured during 2 h before and up to 12 h after the administration of TRH (200 μg i.v.). In the preinjection period in both cattle and buffaloes T₃ levels were significantly lower in HH conditions. No significant difference in basal (preinjection) T₃ levels was observed during HD and cold seasons in cattle. The highest T₃ levels were obtained in buffaloes during HD season with intermediate values during the cold months. Plasma T₄ levels in these animals were reversed during HD and HH months. In both cattle and buffaloes there was a biphasic response of T₃ and T₄ to TRH treatment and this varied with time and in size. The season significantly affected the T₃ response to TRH in cattle and buffaloes but the T₄ response differed in the two species. The ratio of T₄/T₃ was higher during HH condition compared with other seasons in both cattle and buffaloes. The climate significantly affected the thyroidal response to TRH.

J. Endocr. (1986) 108, 57–61

ABSTRACT

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.

J. Endocr. (1985) 105, 183–188