Quantification of thyrotropin-releasing hormone by liquid chromatography-electrospray mass spectrometry.

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography-mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA.

In this paper we report the cloning of the chicken preprothyrotropin-releasing hormone (TRH) cDNA and the study of its hypothalamic distribution. Chicken pre-proTRH contains five exact copies of the TRH progenitor sequence (Glu-His-Pro-Gly) of which only four are flanked by pairs of basic amino acids. In addition, the amino acid sequence contains three sequences that resemble the TRH progenitor sequence but seem to have lost their TRH-coding function during vertebrate evolution. The amino acid sequence homology of preproTRH between different species is very low. Nevertheless, when the tertiary structures are compared using hydrophobicity plots, the resemblance between chicken and rat prepro-TRH is striking. The cloning results also showed that the chicken preproTRH sequence includes neither a rat peptide spacer 4 (Ps4) nor a Ps5 connecting peptide. Comparison of the cDNA sequence with the chicken genome database revealed the presence of two introns, one in the 5' untranslated region, and another downstream from the translation start site. This means that the gene structure of chicken preproTRH resembles the gene stucture of this precursor in mammals. Based on the cDNA sequence, digoxigenin-labelled probes were produced to study the distribution of preproTRH in the chicken brain. By means of in situ hybridization, preproTRH mRNA was detected in the chicken paraventricular nucleus (PVN) and in the lateral hypothalamus (LHy).

Excitatory effects of thyrotropin-releasing hormone in the thalamus.

The activity of the thalamus is state dependent. During slow-wave sleep, rhythmic burst firing is prominent, whereas during waking or rapid eye movement sleep, tonic, single-spike activity dominates. These state-dependent changes result from the actions of modulatory neurotransmitters. In the present study, we investigated the functional and cellular effects of the neuropeptide thyrotropin-releasing hormone (TRH) on the spontaneously active ferret geniculate slice. This peptide and its receptors are prominently expressed in the thalamic network, yet the role of thalamic TRH remains obscure. Bath application of TRH resulted in a transient cessation of both spindle waves and the epileptiform slow oscillation induced by application of bicuculline. With intracellular recordings, TRH application to the GABAergic neurons of the perigeniculate (PGN) or thalamocortical cells in the lateral geniculate nucleus resulted in depolarization and increased membrane resistance. In perigeniculate neurons, this effect reversed near the reversal potential for K+, suggesting that it is mediated by a decrease in K+ conductance. In thalamocortical cells, the TRH-induced depolarization was of sufficient amplitude to block the generation of rebound Ca2+ spikes, whereas the even larger direct depolarization of PGN neurons transformed these cells from the burst to tonic, single-spike mode of action potential generation. Furthermore, application of TRH prominently enhanced the afterdepolarization that follows rebound Ca2+ spikes, suggesting that this transmitter may also enhance Ca2+-activated nonspecific currents. These data suggest a novel role for TRH in the brain as an intrinsic regulator of thalamocortical network activity and provide a potential mechanism for the wake-promoting and anti-epileptic effects of this peptide.

Developmental expression of prolactin releasing peptide in the rat brain: localization of messenger ribonucleic acid and immunoreactive neurons.

Prolactin releasing peptide (PrRP) was recently identified as the stimulator of prolactin release from the anterior pituitary. PrRP mRNA is expressed in the medulla oblongata and the hypothalamus in the rat brain. The fibers containing PrRP are widely distributed in the brain, therefore, it was postulated that PrRP may act as a neurotransmitter or neuromodulator as well as an endocrine substance. To clarify the developmental changes in the expression of PrRP during brain development, we examined PrRP in rat fetuses and neonates using in situ hybridization and immunohistochemistry. The PrRP mRNA was expressed in the nucleus of the solitary tract (NTS) at embryonic day 18 (E18) and in the ventral and lateral reticular nucleus (VLRN) of the caudal medulla oblongata at E20. The PrRP mRNA in the hypothalamus was first expressed at postnatal day 13 (P13). Reverse transcription-polymerase chain reaction analysis (RT-PCR) for PrRP revealed that PCR product, a 268 bp band, was detected from either E18 in the medulla or P13 in the hypothalamus. Immunodetection with monoclonal antibody against prepro-PrRP revealed intensive staining of cells in the NTS at E18, in the VLRN at E20 or in the dorsomedial hypothalamus at P13. Immunohistochemistry using monoclonal antibody against mature PrRP at P6 showed PrRP fibers to be distributed in the paraventricular hypothalamic nucleus, periventricular hypothalamic nucleus, medial preoptic area, basolateral amygdaloid nucleus, dorsomedial hypothalamus, ventromedial hypothalamus, periventricular nucleus of the thalamus and bed nucleus of the stria terminalis as previously shown in the adult rat. PrRP fibers were also found in the optic chiasm, dorsal endopiriform nucleus, cingulum, intermediate reticular nucleus, and caudal ventrolateral reticular nucleus at P6 and P9. However, PrRP fibers were never found in the above regions in the adult animal. These findings suggest that PrRP fibers originating in the medulla oblongata have been widely distributed in the rat brain during the early postnatal day and PrRP may play various roles in the brain development.

Physiological and pathophysiological aspects of thyrotropin-releasing hormone gene expression in the human hypothalamus.

Although the tripeptide thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone to be isolated and characterized, only very few data were available on the central component of the hypothalamus-pituitary-thyroid (HPT) axis in the human brain until recently. We used immunocytochemistry to describe, for the first time, the distribution of TRH-containing cells and fibers in the human hypothalamus. Brain material was obtained with a short postmortem delay followed by fixation in paraformaldehyde, glutaraldehyde, and picric acid. Many TRH-containing cells were present in the paraventricular nucleus (PVN), especially in its dorsocaudal part. Some TRH cells were found in the suprachiasmatic nucleus (SCN), which is the circadian clock of the brain, and in the sexually dimorphic nucleus (SDN), which is in agreement with earlier observations in the rat hypothalamus. Dense TRH-containing fiber networks were present not only in the median eminence but also in a number of other hypothalamic areas, suggesting a physiological function of TRH as a neuromodulator or neurotransmitter in the human brain, in addition to its neuroendocrine role in pituitary secretion of thyroid-stimulating hormone (TSH). As a next step, we developed a technique for TRH mRNA in situ hybridization using a [35S] CTP-labeled TRH cRNA antisense probe in formalin-fixed paraffin-embedded sections. Numerous heavily labeled TRH mRNA-containing neurons were detected in the caudal part of the PVN, while some cells were present in the SCN and in the perifornical area. These results demonstrated the value of in situ hybridization for elucidating the chemoarchitecture of the human hypothalamus in routinely fixed autopsy tissue and enabled us to perform quantitative studies. As part of the neuroendocrine response to disease, serum concentrations of thyroid hormone decrease without giving rise to elevated concentrations of TSH, suggesting altered feedback control at the level of the hypothalamus and/or pituitary. In order to establish whether decreased activity of TRH cells in the PVN contributes to the persistence of low TSH levels in nonthyroidal illness (NTI), hypothalamic TRH gene expression was investigated in patients whose plasma concentrations of thyroid hormones had been measured just before death. Quantitative in situ hybridization showed a positive correlation of total TRH mRNA in the PVN and serum concentrations of TSH and triiodothyronine (T3) less than 24 hours before death, supporting our hypothesis. Current experiments aim at elucidating the mechanism by which hypothalamic thyroid hormone feedback control in TRH cells of patients with NTI is changed.

Pre- and posthatch developmental changes in hypothalamic thyrotropin-releasing hormone and somatostatin concentrations and in circulating growth hormone and thyrotropin levels in the chicken.

Thyrotropin-releasing hormone (TRH) and somatostatin (SRIH) concentrations were determined by RIA during both embryonic development and posthatch growth of the chicken. Both TRH and SRIH were already detectable in hypothalami of 14-day-old embryos (E14). Towards the end of incubation, hypothalamic TRH levels increased progressively, followed by a further increase in newly hatched fowl. SRIH concentrations remained stable from E14 to E17 and doubled between E17 and E18 to a concentration which was observed up to hatching. Plasma GH levels remained low during embryonic development, ending in a steep increase at hatching. Plasma TSH levels on the other hand decreased during the last week of the incubation. During growth, TRH concentrations further increased, whereas SRIH concentrations fell progressively towards those of adult animals. Plasma TSH levels increased threefold up to adulthood; the rise in plasma GH levels during growth was followed by a drop in adults. In conclusion, the present report shows that important changes occur in the hypothalamic TRH and SRIH concentration during both embryonic development and posthatch growth of the chicken. Since TRH and SRIH control GH and TSH release in the chicken, the hypothalamic data are compared with plasma GH and TSH fluctuations.

Método analítico para la determinación de protirelina por cromatografía líquida de alta resolución / Analytical methods for the determination of protirelin by high pressure liquid chromatography

Se desarrolló un método analítico para el control de la calidad y estudio de estabilidad de protirelina inyectable. Se realizó el análisis por cromatografía líquida de alta resolución con la utilización de un sistema isocrático de buffer fosfato-metanol (85:15) como fase móvil y una columna Lichrosorb RP-18 de 250 x 4 µm y detección ultravioleta, a una longitud de onda de 210 nm. El método analítico resultó ser lineal, preciso, exacto y específico en el rango de concentraciones estudiadas(AU)

Método analítico para la determinación de protirelina por cromatografía líquida de alta resolución / Analytical methods for the determination of protirelin by high pressure liquid chromatography

Se desarrolló un método analítico para el control de la calidad y estudio de estabilidad de protirelina inyectable. Se realizó el análisis por cromatografía líquida de alta resolución con la utilización de un sistema isocrático de buffer fosfato-metanol (85:15) como fase móvil y una columna Lichrosorb RP-18 de 250 x 4 µm y detección ultravioleta, a una longitud de onda de 210 nm. El método analítico resultó ser lineal, preciso, exacto y específico en el rango de concentraciones estudiadas

Urinary excretion of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in rats.

TRH-like immunoreactivity (TRH-LI) was estimated in methanolic extracts of rat tissues and blood by RIA using antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2, or antiserum 8880, which is specific for TRH (pGlu-His-ProNH2). TRH-LI (determined with antiserum 4319) and TRH (determined with antiserum 8880) contents were 8 and 8 ng/g in brain, 216 and 222 ng/g in hypothalamus, 6.5 and 6 ng/g in pancreas, 163 and 116 ng/g in male pituitary, 105 and 77 ng/g in female pituitary, 1 and 0.1 ng/g in salivary gland, 61 and 42 ng/g in thyroid, 12 and 3 ng/g in adrenal, 3 and 0.3 ng/g in prostate, and 11 and 0.8 ng/g in ovary respectively. Blood TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 31 and 18 pg/ml in male rats, and 23 and 10 pg/ml in female rats respectively. Unextracted serum obtained from blood kept for at least 1 h at room temperature no longer contained authentic TRH but still contained TRH-LI (males 20.3 +/- 3.1, females 15.9 +/- 3.0 pg/ml; means +/- S.E.M.). Isocratic reverse-phase HPLC showed that TRH-LI in serum is largely pGlu-Glu-ProNH2 (< EEP-NH2), a peptide previously found in prostate and anterior pituitary. In urine, TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 3.21 +/- 0.35 and 0.32 +/- 0.04 ng/ml in male rats and 3.75 +/- 0.22 and 0.37 +/- 0.04 ng/ml in female rats respectively (means +/- S.E.M.). Anion-exchange chromatography on QAE-Sephadex showed that urine of normally fed rats contains both basic/neutral TRH-LI (b/n TRH-LI) and acidic TRH-LI (aTRH-LI) in a ratio of approximately 40:60, and further analysis by HPLC indicated that aTRH-LI represents < EEP-NH2. Analysis of food extracts and urine from fasted rats demonstrated that b/n TRH-LI is derived from food particles spilled by the rats during urine collection, while aTRH-LI is endogenously produced. While urinary aTRH-LI levels were higher in female than in male rats (2.99 +/- 0.41 vs 2.04 +/- 0.20 ng/ml), the daily urinary excretion was similar in both sexes (females 15.6 +/- 1.4, males 19.5 +/- 2.0 ng/day). Intravenously injected < EEP-NH2 disappeared from serum with a half-life of approximately 1 h, and was recovered unchanged and quantitatively in urine. In contrast, when < EEP-NH2 was administered with food, only approximately 0.5% was recovered in urine. The urinary clearance rate of serum TRH-LI amounted to 0.52 +/- 0.10 ml/min in males and 0.34 +/- 0.05 ml/min in females. In view of the presence of < EEP-NH2 in the anterior pituitary gland, and the regulation of its content in parallel with gonadotrophins, we examined the possibility that serum < EEP-NH2 is of pituitary origin and correlates with gonadotrophin secretion. However, treatments that alter pituitary < EEP-NH2 content and gonadotrophin release had no effect on serum TRH-LI or urinary aTRH-LI. In conclusion, the TRH-like peptide < EEP-NH2 is present in rat serum and is excreted into the urine. Moreover, < EEP-NH2 in serum and urine is not derived from rat food and is probably not of pituitary origin.

Distribution of thyrotropin-releasing hormone (TRH)-containing cells and fibers in the human hypothalamus.

In the present study, we describe for the first time the distribution of thyrotropin-releasing hormone (TRH)-containing cells and fibers in the human hypothalamus using brain material obtained with a short postmortem delay. Following fixation in paraformaldehyde, glutaraldehyde and picric acid, excellent staining was obtained with two different TRH antisera. Many TRH-containing neurons were present in the paraventricular nucleus (PVN), especially in the dorsocaudal part of this nucleus. They were mostly parvicellular, but a few magnocellular TRH-positive neurons were observed as well. The PVN also contained a dense network of TRH fibers. The supraoptic nucleus (SON) did not show any TRH immunoreactivity, excluding the possibility of cross-reactivity of the antiserum with neurohypophysial hormones or their precursors. In addition, TRH cells were found in the suprachiasmatic nucleus (SCN), which is the circadian clock of the brain, in the sexually dimorphic nucleus (SDN) and dorsomedially of the SON. We observed small number of TRH cells throughout the hypothalamic gray in all subjects studied. A high density of TRH-containing fibers was seen not only in the median eminence but also in other hypothalamic areas, e.g., in the ventromedial nucleus (VM) and in the perifornical area. The results generally agree with earlier data in the rat, with the exception of the absence of TRH cells in the SON. The large number of sites of TRH-containing fiber terminations on neurons suggests important physiological functions of this neuropeptide as a neurotransmitter or neuromodulator in the human brain, in addition to its role as a neurohormone in pituitary secretion of thyroid-stimulating hormone (TSH).

Different effects of continuous infusion of interleukin-1 and interleukin-6 on the hypothalamic-hypophysial-thyroid axis.

The cytokines interleukin-1 (IL-1) and IL-6 are thought to be important mediators in the suppression of thyroid function during nonthyroidal illness. In this study we compared the effects of IL-1 and IL-6 infusion on the hypothalamus-pituitary-thyroid axis in rats. Cytokines were administered by continuous ip infusion of 4 micrograms IL-1 alpha/day for 1, 2, or 7 days or of 15 micrograms IL-6/day for 7 days. Body weight and temperature, food and water intake, and plasma TSH, T4, free T4 (FT4), T3, and corticosterone levels were measured daily, and hypothalamic pro-TRH messenger RNA (mRNA) and hypophysial TSH beta mRNA were determined after termination of the experiments. Compared with saline-treated controls, infusion of IL-1, but not of IL-6, produced a transient decrease in food and water intake, a transient increase in body temperature, and a prolonged decrease in body weight. Both cytokines caused transient decreases in plasma TSH and T4, which were greater and more prolonged with IL-1 than with IL-6, whereas they effected similar transient increases in the plasma FT4 fraction. Infusion with IL-1, but not IL-6, also induced transient decreases in plasma FT4 and T3 and a transient increase in plasma corticosterone. Hypothalamic pro-TRH mRNA was significantly decreased (-73%) after 7 days, but not after 1 or 2 days, of IL-1 infusion and was unaffected by IL-6 infusion. Hypophysial TSH beta mRNA was significantly decreased after 2 (-62%) and 7 (-62%) days, but not after 1 day, of IL-1 infusion and was unaffected by IL-6 infusion. These results are in agreement with previous findings that IL-1, more so than IL-6, directly inhibits thyroid hormone production. They also indicate that IL-1 and IL-6 both decrease plasma T4 binding. Furthermore, both cytokines induce an acute and dramatic decrease in plasma TSH before (IL-1) or even without (IL-6) a decrease in hypothalamic pro-TRH mRNA or hypophysial TSH beta mRNA, suggesting that the acute decrease in TSH secretion is not caused by decreased pro-TRH and TSH beta gene expression. The TSH-suppressive effect of IL-6, either administered as such or induced by IL-1 infusion, may be due to a direct effect on the thyrotroph, whereas additional effects of IL-1 may involve changes in the hypothalamic release of somatostatin or TRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of hypophysectomy on TRH and its related peptides concentrations in various rat organs.

The effects of hypophysectomy on thyrotropin releasing hormone (TRH), TRH-glycine (TRH-Gly) and pre-pro-TRH (178-199) concentrations in the rat hypothalamus, cerebrum, cerebellum, brain stem, stomach and retina were studied 7 days after the operation. The hypophysectomized rats were administered a single i.p. injection of T4 (500 micrograms/kg), T3 (100 micrograms/kg) or bovine TSH (1.25 IU/kg), and 5 rats of each subgroup were decapitated at 4 hours later. After hypophysectomy, TRH-Gly and pre-pro-TRH (178-199) concentrations in the hypothalamus increased significantly and TRH concentrations decreased after hypophysectomy. After the injection of T4, T3, TRH or TSH TRH-Gly and pre-pro-TRH (178-199) concentration in the hypothalamus of hypophysectomized rats decreased significantly, while that of TRH significantly increased. No changes in TRH, TRH-Gly and pre-pro-TRH (178-199) concentration in other tissues were observed after hypophysectomy or hormone treatment. The findings suggest that hypophysectomy stimulated TRH synthesis and release in the hypothalamus, that TRH, TSH, T3 and T4 regulate hypothalamic TRH levels, and that pro-TRH synthesis in the tissues except the hypothalamus may not be regulated by thyroid hormone.

The evaluation of hypothalamic somatostatin tone using pyridostigmine and thyrotropin releasing hormone in patients with acromegaly.

To indirectly evaluate the hypothalamic somatostatin (SS) tone in patients with acromegaly, the effects of pyridostigmine (PD), a cholinesterase inhibitor which can inhibit hypothalamic SS secretion, on TRH-induced TSH secretion and the effects of SMS 201-995 on TSH or GH secretion were studied in acromegalic patients (31-69 yr, n = 10), normal young (21-24 yr, n = 7) and normal old male subjects (62-71 yr, n = 7). After pretreatment with PD (60 mg po, -30 min), normal young subjects showed significantly enhanced TSH responses to TRH (500 micrograms i.v., 0 min) compared to single administration of TRH, whereas normal old and acromegalic patients did not show such enhancement. Plasma TSH response to a single administration of TRH in acromegalic patients was significantly lower than that of normal young and old subjects. Although normal young and old subjects showed significantly enhanced GH responses to GHRH (100 micrograms i.v. at 0 min) after the pretreatment with PD (60 mg, -30 min), no such enhancement was observed in acromegalic patients. In contrast, the decrement in plasma TSH after SMS 201-995 administration was similar between normal subjects (5 young 5 old) and 7 acromegalic patients. Further, the maximal plasma GH decrement after administration was significantly greater in acromegalic patients than in the 5 normal young and 5 old subjects p < 0.01). In conclusion, hypothalamic SS tone does not appear to be elevated in acromegalic patients compared to normal young and probably old subjects.

A cryptic peptide from the preprothyrotropin-releasing hormone precursor stimulates thyrotropin gene expression.

The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSH beta gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSH beta-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from that of TRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.

Effects of dexamethasone on TRH, TRH-glycine and pre-pro-TRH (178-199) levels in various rat organs.

The effect of dexamethasone administration on the concentration of thyrotropin releasing hormone (TRH) and pre-pro-TRH connecting peptides TRH-glycine (TRH-Gly), and pre-pro-TRH (178-199) in various rat organs was studied. Three groups of 35 rats each were injected dexamethasone (Group A: 25 micrograms/100 g; Group B: 500 micrograms/100 g) or saline (Group C: control). The subgroups of 7 rats each were decapitated at 1, 2, 3, 4 and 24 h after the injection and the levels of TRH-Gly, pre-pro-TRH (178-199) and TRH in the hypothalamus, cerebrum, cerebellum and brain stem, stomach, retina were estimated by specific radioimmunoassays. The level of TRH-Gly and pre-pro-TRH (178-199) in the hypothalamus decreased significantly in groups A and B at 1-4 hours after the injection, and then returned to pretreatment levels at 24 h after the injection. In contrast, TRH levels in the hypothalamus increased significantly in groups A and B at 1-4 h after the injection. The TRH-Gly, pre-pro-TRH (178-199) and TRH levels in other organs showed no changes after dexamethasone injection. From these findings it is concluded that dexamethasone inhibits the synthesis and secretion of TRH and the maturation of pro-TRH in the hypothalamus, while its effects on other organs is different from the hypothalamus.

Identification of the thyrotropin-releasing hormone-prohormone and its posttranslational processing in a transfected AtT20 tumoral cell line.

By using an AtT20 cell line transfected with complementary DNA for preproTRH, we have identified the proTRH polyeptide precursor [26 kilodaltons (kDa)] and shown that this molecule gives rise to the proTRH derived sequences as determined by pulse-chase and trypsinization studies. The predicted proTRH precursor composed of 231 amino acids with 5 copies of a TRH progenitor sequence (Gln-His-Pro-Gly) and 7 other cryptic peptides was demonstrated by: 1) Western Blot analysis of an AtT20 cell extract with anti-pCC10 antibodies (an antibody that recognizes the intact prohormone as well as some intermediate products of processing); 2) Immunoprecipitation of the radiolabelled 26 kDa protein with anti-pCC10 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis; 3) Gel filtration chromatography of the radiolabeled 26 kDa extracted from SDS-PAGE. 4) RIA with anti-pCC10 antiserum against peptides extracted from adult rat hypothalamus and olfactory lobe after SDS-PAGE. 5) Trypsinization of the proTRH precursor which generated the proTRH cryptic peptides preproTRH25-50 (pYE27) and preproTRH53-74 (pFT22). These moieties were also produced during trypsinization of intermediate products of processing. By means of pulse-chase studies, the 26 kDa polypeptide was shown to be the biosynthetic precursor to all the proTRH derived cryptic peptides. Cleavage at two positions in the center of the molecule (Lys107-Arg108 and Lys152-Arg153) generated two moieties of 16.5 and 15 kDa. The 15 kDa N-terminal fragment is later cleaved to a 6 kDa peptide that includes the proTRH derived peptides, pYE27, pFT22, and pEH24. The carboxy-terminal 16.5 kDa fragment of the prohormone is processed to a 9.6 kDa fragment which contains the proTRH derived peptide pST10 (preproTRH160-169) and a fragment of 5.4 kDa that may be the C-terminal peptide preproTRH208-255 recognized by antisera pAC12 and pYE17. In further processing, the 9.6 kDa molecule is cleaved to produce a 5.4 kDa peptide from either sequences 115-169 or 160-199.

Failure of bilateral paraventricular nuclear lesions to cause hypothalamic hypothyroidism in fetal sheep.

Concentrations of T4 indicative of a hyperthyroid state are present in fetal sheep plasma between 100 days gestational age (dGA) and parturition. Fetal pituitary stalk section studies indicate that, as in adults, these high fetal plasma T4 concentrations during pregnancy are controlled by the hypothalamus. We compared peripheral plasma T4 concentrations in fetal sheep with bilateral hypothalamic paraventricular nuclear (PVN) lesions (lesion group; n = 5) to fetal sheep with sham-PVN lesions (sham group; n = 4) at 131 and 146 dGA in the lesion group or at term (mean +/- SEM, 146 +/- 0.9 dGA) in the sham group. Bilateral hypothalamic PVN lesions or sham lesions were placed at 118-122 dGA. Baseline blood samples were taken between 1100-1500 h at 131 dGA in both groups, at term in the sham group, and at 146 dGA in the lesion group. In control sheep, TRH cells were found in the PVN and in a number of extra-PVN sites, and the median eminence received abundant TRH axons. In the lesion group, complete destruction of the PVN bilaterally was confirmed by histology. Extra-PVN TRH neurons remained intact in the lesioned sheep, and axons to the median eminence were reduced, but not eliminated. T4 concentrations in fetal plasma were not different in the lesion group and the sham group at 131 dGA (81 +/- 7 vs. 92 +/- 19 ng/ml) or at term (112 +/- 35 vs. 79 +/- 15 ng/ml), respectively. In contrast, fetal plasma concentrations of cortisol, which were not different in lesion and sham group fetuses at 131 dGA (1.7 +/- 0.3 vs. 1.0 +/- 0.2 ng/ml, respectively), were greatly reduced (P < 0.05) at 146 dGA in the lesion group compared to those in the sham group at term (2.0 +/- 0.5 vs. 58.8 +/- 11.5 ng/ml). We conclude that unlike in adult rats, the ovine fetal PVN is not required to maintain normal plasma T4 concentrations. The many TRH-positive cells that lie outside of the PVN in the fetal sheep appear to enable PVN-lesioned fetuses to remain euthyroid fetuses.

Effects of dexamethasone on TRH and TRH precursor peptide (Lys-Arg-Gln-His-Pro-Gly-Arg-Arg) levels in various rat organs.

The effect of an acute dexamethasone administration on thyrotropin-releasing hormone (TRH) and TRH precursor peptide (Lys-Arg-Gln-His-Pro-Gly-Arg-Arg) (p-8) levels in various rat organs has been studied. Rats were injected i.p. with 25 micrograms of dexamethasone/100 g body weight (group A), 500 micrograms of dexamethasone/100 g body weight (group B) or saline (group C). The rats were serially decapitated after the injection. TRH and p-8 levels in the hypothalamus, cerebrum, cerebellum and brain stem, stomach and eye and plasma TRH and thyrotropin (TSH) levels were measured by individual radioimmunoassays. P-8 levels in the hypothalamus decreased significantly in both group A and B at 1-4 hours after the injection, and then returned to pretreated levels at 24 hours after the injection. TRH levels in the hypothalamus increased significantly in both group A and group B at 1-4 hours after dexamethasone injection. No changes in p-8 and TRH levels were observed in other organs. In group A, plasma TRH levels tended to decrease at 1-2 hours, then to increase at 3 hours. In group B, plasma TRH levels decreased 1-4 hours after the dexamethasone injection, then increased at 24 hours. The plasma TSH levels decreased significantly at 1-4 hours in group A and group B, returned to pretreatment levels at 24 hours in group A, and increased significantly in group B at 24 hours after dexamethasone injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive thyroliberin (TRH) precursor forms in human hypothalamus and anterior pituitary tissues.

Immunoreactive (IR) proTRH forms were characterized in human hypothalamic tissue with two antisera raised against a hepta- and a decapeptide containing the TRH progenitor sequence (-Gln-His-Pro-Gly-). A similar study was performed in human normal and adenomatous anterior pituitaries, tissues in which TRH synthesis has been previously suggested. IR-proTRH was found in all the samples ranging from 42-775 fmol/mg proteins. Size exclusion chromatography identified a major 25-35 kDa form and a minor 4-8 kDa form. The existence of the major form was confirmed by immunoblotting. The results suggest that both human hypothalamic and normal or adenomatous anterior pituitary tissues synthesize similar IR-proTRH forms.

The regional distribution of thyrotropin releasing hormone, leu-enkephalin, met-enkephalin, substance P, somatostatin and cholecystokinin in the rat brain and pituitary.

There was no apparent difference in the regional distribution of neuropeptides in the brain of male and female rats. The highest levels of immunoreactive leu-enkephalin, TRH, substance P and somatostatin were found in the hypothalamus, while the striatum and the cerebral cortex had the highest concentrations of met-enkephalin and cholecystokinin respectively. The lowest concentrations of these were found in the cerebellum. Enkephalins (cerebral cortex), substance P (cerebral cortex and brain stem), and somatostatin (brain stem and striatum) showed higher level in the female while enkephalin and substance P contents in the anterior pituitary were higher in the male.