TRH Treatment Research Articles

ERK and RSK are necessary for TRH-induced inhibition of r-ERG potassium currents in rat pituitary GH3 cells

The transduction pathway mediating the inhibitory effect that TRH exerts on r-ERG channels has been thoroughly studied in GH3 rat pituitary cells but some elements have yet to be discovered, including those involved in a phosphorylation event(s). Using a quantitative phosphoproteomic approach we studied the changes in phosphorylation caused by treatment with 1μM TRH for 5min in GH3 cells. The activating residues of Erk2 and Erk1 undergo phosphorylation increases of 5.26 and 4.87 fold, respectively, in agreement with previous reports of ERK activation by TRH in GH3 cells. Thus, we studied the possible involvement of ERK pathway in the signal transduction from TRH receptor to r-ERG channels. The MEK inhibitor U0126 at 0.5μM caused no major blockade of the basal r-ERG current, but impaired the TRH inhibitory effect on r-ERG. Indeed, the TRH effect on r-ERG was also reduced when GH3 cells were transfected with siRNAs against either Erk1 or Erk2. Using antibodies, we found that TRH treatment also causes activating phosphorylation of Rsk. The TRH effect on r-ERG current was also impaired when cells were transfected with any of two different siRNAs mixtures against Rsk1. However, treatment of GH3 cells with 20nM EGF for 5min, which causes ERK and RSK activation, had no effect on the r-ERG currents. Therefore, we conclude that in the native GH3 cell system, ERK and RSK are involved in the pathway linking TRH receptor to r-ERG channel inhibition, but additional components must participate to cause such inhibition.

Retrospective analysis of infants designated as positive on mass-screening for congenital hypothyroidism at kagoshima university.

Mass-screening for congenital hypothyroidism has identified cases of mild hypothyroidism, transient hypothyroidism, and transient hyperthyrotropinemia as well as typical hypothyroidism. In this paper, we examine the clinical data of the cases found positive in the screening test at our hospital. From 1989 to 1999 there were 72 patients with positive screening tests who started levothyroxine sodium (l-T4; Thyradin-S) as supplement therapy. At the age of 3 to 4 yr the patients were re-evaluated to determine whether treatment should be continued. Thyroid scintigraphies were done at the same time. We divided these cases into 4 groups. Those in group 1A started l-T4 in early infancy without a TRH test because of obvious clinical evidence of hypothyroidism, and treatment was continued after re-evaluation (n=37). Those in group 1B also started treatment in early infancy without a TRH test, but treatment was discontinued after re-evaluation (n=20). Patients in group 2A started l-T4 after evaluation by a TRH test and treatment was continued after re-evaluation (n=14), while those in group 2B started treatment after a TRH test, but after re-evaluation, treatment was discontinued (n=1). In group 2A, only a low dose of l-T4 was needed, and a slightly elevated TSH and slightly decreased free T4 (FT4) were observed after the drug washout period. However, these patients had an exaggerated response to the TRH test at re-evaluation. These findings indicate that this group, forming not a small part of whole screening-positive subjects, had mild hypothyroidism. Such patients require careful follow-up and repeated evaluation to determine whether treatment should be continued.

Responses of seasonally anovulatory mares to daily administration of thyrotropin-releasing hormone and(or) gonadotropin-releasing hormone analog.

Seventeen seasonally anovulatory light horse mares were treated daily, starting January 5 (d 1), for 28 d with GnRH analog (GnRH-A; 50 ng/kg BW) and(or) thyrotropin-releasing hormone (TRH; 5 microg/kg BW) in a 2 x 2 factorial arrangement of treatments to test the hypothesis that combined treatment may stimulate follicular growth and development. Ovaries were examined via ultrasonography and jugular blood samples were collected every 3 d. Frequent blood samples were collected after treatment injections on d 1, 2, 4, 7, 11, 16, and 22; on d 29, all mares received an i.v. mixture of GnRH, TRH, sulpiride, and EP51389 (a growth hormone secretagogue) to assess pituitary responsiveness. No consistent effects (P > 0.1) of treatment were observed for plasma LH, FSH, prolactin, or thyroxine concentrations in samples collected every 3 d. The only effect on ovarian follicle numbers was a reduction in number of follicles 11 to 19 mm in diameter due to TRH treatment (P = 0.029). No mare ovulated during treatment. On the days of frequent sampling, mean LH (P = 0.0001) and FSH (P = 0.001) concentrations were higher in mares receiving GnRH-A and tended to increase from d 1 through 7. In contrast, mean prolactin (P = 0.001) and thyroid-stimulating hormone (P = 0.0001) concentrations were high in mares receiving TRH on d 1 but rapidly decreased thereafter. When mares were administered the secretagogue mixture on d 29, the LH response was greater (P = 0.0002) in mares that had previously received GnRH-A but the FSH response was not affected (P > 0.1); the prolactin response was greater (P = 0.014) and the TSH response was smaller (P = 0.0005) in mares that had previously received TRH. Surprisingly, an immediate growth hormone response to EP51389 was absent in all mares. In conclusion, daily GnRH-A treatment stimulated plasma LH and FSH concentrations immediately after injection; although no long-term elevation in preinjection concentrations was achieved, the responses gradually increased over time, indicating a stimulation of gonadotropin production and storage. Daily treatment with TRH stimulated plasma TSH and prolactin concentrations, but the response diminished rapidly and was minimal within a few days, indicating a depletion of pituitary stores and little or no stimulation of production. There was no beneficial effect of adding TRH treatment to the daily GnRH-A regimen.

Production and Characterization of an Antiserum Which Recognizes the Native Receptor for Thyrotropin-Releasing Hormone

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50–60 kDa and a minor broad band at 100–120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.

Thyrotropin-releasing hormone stimulates phosphorylation of the epidermal growth factor receptor in GH3 pituitary cells.

TRH has been found to stimulate tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. A specific EGF receptor kinase inhibitor, tyrphostin AG1478, substantially reduced TRH-stimulated tyrosine phosphorylation of the EGF receptor. TRH-induced EGF receptor phosphorylation was found to lead to the recruitment of the adapter proteins Grb2 and Shc. TRH treatment also led to phosphorylation of the related receptor tyrosine kinase, HER2. HER2 activation likely contributes to downstream signaling events and enhances EGF receptor action. TRH-induced tyrosine phosphorylation of the EGF receptor was reduced by incubation with a protein kinase C (PKC) kinase inhibitor, GF109203X. EGF receptor phosphorylation was required for full TRH-induced activation of mitogen-activated protein kinase (MAPK) and stimulation of specific transcriptional responses.

THYROID HORMONES AND PROLACTIN IN PREMATURE INFANTS WITH AND WITHOUT RDS AFTER PREANTAL TRH TREATMENT. † 979

Since 1989 TRH plus glucocorticoids combination has been studying in humans. Aim: to evaluate the incidence and severity of RDS and chronic lung disease (CLD) after prenatal TRH plus betamethasone treatment. Hormonal studies included free T3 (FT3) [nd/dl], free T4 (FT4) [ng/dl], TSH[μU/ml] and prolactin (PRL) [ng/ml]. The correlation between RDS and thyroid function and PRL was analysed. Methods: study group (TRH+B) 58 newborns with mean 31.9 wga and mean birth weight 1795g. Control group(B) 68 neonates of 31.5 mean wga and 1714g mean birthweight. Hormonal analysis was performed on cord blood, and day 1 and 7 of life. For statistical analysis, Statistica 4.5 for Windows was used. Results: 1) no statistical differences in mean TSH level was found in any sample between study and control group (10.70 vs 10.62 in cord; 7.34 vs 5.48 after day 1; 3.33 vs 4.38 after day 7). 2) FT3 concentration did not differ in particular samples for TRH +B and B group (0.22 vs 0.25 in cord; 0.33 vs 0.31 after day 1 and 0.34 vs 0.33 after day 7). 3) There was statistically significant difference in FT4 level, in cord blood (1.26 in TRH +B vs 1.18 in B group; p< 0.05), but not in other samples (1.64 v. 1.63 after day 1 and 1.42 vs 1.28 after day 7). 4) PRL level was similar in study and control goup in each succeeding samples (97.07 vs 112.59; 108.88 vs 93.27; 73.64 vs 72.58). RDS and thyroid function. 5) Mean TSH level in cord blood was higher in group with RDS (12.90 vs 9.28). On day 1 it was lower in RDS group (5.21 vs 7.27; p < 0.01). On day 7 there was no statistical difference between both groups (4.28 in RDS and 2.85 in control group). 6) FT3 level was lower in RDS than in control group in each sample (0.23 vs 0.26; 0.24 * vs 0.39*; 0.31 vs 0.37; *p <0.001). 7) FT4 concentration was slightly higher in RDS group in cord blood (1.24 vs 1.19). On day 1 and 7 FT4 level was higher in control group (1.68 vs 1.59 and 1.44 vs 1.26). 8) Prolactin level was lower in RDS group in each following samples: cord blood; 99.82 vs 113.62; day 1: 79.55 vs 116.60, p<0.01; and day 7: 66.43 vs 81.02. Conclusions: fetal pituitary-thyroid axis is sensitive to exogenous TRH. Low thyroid hormones concentration ininfants with RDS may have an important role in protectig sick neonate from higher oxygen consumption in situations when it's delivery to organs and tissues is imparied. The rate of T4 to T3 conversion is low in these situations, with overproduction of rT3. We suggest that hormonal disturbances in infants with RDS are transient and contribute to a low T3 syndrome.

A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin α-subunit gene

Our previous studies demonstrated that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (-151 to -135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (-223 to -190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA-protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.

Basic Fibroblast Growth Factor Expression by Two Prolactin and Thyrotropin-Producing Pituitary Adenomas.

We investigated the expression of basic fibroblast growth factor (bFGF) in an aggressive type of PRL and TSH-producing pituitary adenoma. Immunocytochemistry and electron microscopy were used to characterize the tumors removed from two patients. lmmunoassays were used to measure hormone and bFGF levels in vitro and in vivo. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect bFGF mRNA expression by these tumors. Morphologically, these tumors were characterized by an unusual plurihormonal pattern with expression of PRL and TSH and the ultrastructural characteristics of the silent subtype 3 adenoma; in addition, both adenomas displayed marked interstitial fibrosis. bFGF was measurable in the circulation of these patients ranging from 7.5-20.5 pg/mL (normal < 1 pg/mL). bFGF concentrations were reduced following surgical adenomectomy. bFCF in culture media was present in concentrations of 197-387 pg/24h/10(5) cells. bFGF mRNA expression was identified in both adenomas examined. bFGF levels were unaltered in the culture media and in the serum of patients following GnRH and TRH treatment. In conclusion, the expression of bFGF by these plurihormonal pituitary adenomas suggests the possibility that it may play a role in the development of fibrosis and tumor cell proliferation of this unusual type of pituitary neoplasm.

Prolactin-synthesizing and prolactin-releasing activity of fetal and early postnatal rat pituitaries: in vivo and in vitro studies using RIA, reverse hemolytic plaque assay and immunocytochemistry.

In vivo and in vitro prolactin (PRL)-synthesizing and PRL-releasing activity of fetal (days 12-22) and early postnatal (days 1-10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18, 12-15% of monodispersed pituitary cells displayed PRL immunopositivity, but only 3-5% of PRL-positive cells were plaque-forming, i.e. released PRL. By the end of gestation 19-25% and on postnatal day 10 42-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL-positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathke's pouch cells have substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions.(ABSTRACT TRUNCATED AT 250 WORDS)

In Situ Hybridization Detection of TSH? Subunit Gene Expression in the Serum-Free Primary Culture of the Adult Rat Pituitary

This study was designed to construct the primary culture system to detect the change in TSH beta subunit (TSH beta) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3, or 6 days with or without 1 nM triiodothyronine (T3) under the serum-free condition. In some cultures, TRH was added to a final concentration of 1 microM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35S-labeled RNA probe complementary to TSH beta mRNA. Emulsion autoradiography was subsequently performed. T3 treatment markedly suppressed relative cellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSH beta mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T3 treatment suppressed basal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T3 treatment. These results show that the thyroid hormone and TRH regulate TSH beta gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.

Prenatal thyroid releasing hormone and thyroid releasing hormone plus dexamethasone lessen the survival of newborn rats during prolonged high O2 exposure.

Newborn rats prenatally treated with TRH or the combination of TRH + DEX have lower lung antioxidant enzyme activities at birth than control newborns but are able to induce an adaptive antioxidant enzyme response to hyperoxic exposure of similar or even greater magnitude compared to O2 control offspring. Because of this greater antioxidant enzyme response, we hypothesized that the hormonally pretreated newborns might demonstrate superior tolerance to prolonged high O2 exposure. However, when placed in greater than 95% O2 at birth, the survival rates were consistently lower in the TRH- and TRH + DEX-treated pups at all time periods in hyperoxia from 9 d [control = 74 of 92 (80%); TRH + DEX = 32 of 47 (68%); TRH = 29 of 48 (60%); p less than 0.05] to 14 d [control = 43 of 92 (47%); TRH + DEX = 11 of 47 (23%); TRH = nine of 48 (19%); (p less than 0.05)]. Other evidence of poorer O2 tolerance in the prenatal hormone-treated pups included a greater incidence of intraalveolar edema and elevated lung conjugated dienes, an index of lipid peroxidation, at 3, 5, and 7 d of O2 exposure. There was also a persistent elevation in 3,5,3'-triiodo-L-thyronine and thyroxine serum levels in the 10-d-old TRH-treated offspring. We conclude that prenatal TRH treatment, possibly working through the secretion of 3,5,3'-triiodo-L-thyronine and thyroxine, has some important lasting postnatal effect (not completely reversed by dexamethasone) that predisposes newborn rats to greater O2 radical-induced lung sequelae of prolonged hyperoxic exposure.

Lactotrophs secreting small amounts of prolactin reveal great responsiveness to thyrotropin-releasing hormone: analysis by the sequential cell immunoblot assay

The effects of TRH on PRL secretion from individual lactotrophs of female rats were investigated by using a sequential cell immunoblot assay. The same pituitary cells cultured on coverslips were first incubated to determine basal secretion of PRL and subsequently challenged by one of various concentrations of TRH. The PRL secreted from the single lactotrophs was absorbed on protein-blotting transfer membranes, immunostained, and quantified by microscopic image analysis. When no TRH was added to the medium used in the second incubation (controls), the amount of PRL secreted from individual lactotrophs was 93% of that secreted in the first incubation. Treatment with 3 x 10(-10)-10(-7) M TRH in the second incubation increased in a dose-dependent manner the proportion of lactotrophs whose PRL secretion was significantly greater than confidence limits for PRL secretion in the controls. However, the percentage of TRH-responsive lactotrophs remained less than 50% even at a maximally effective concentration of TRH. Proportions of the TRH-responsive lactotrophs were significantly greater in cells that secreted small amounts of PRL under basal conditions than in those that secreted large amounts. Furthermore, the small, but not the large, secretors showed a significant increase in mean absolute amounts of PRL secreted by 10(-7) M TRH, which represented no less than 45% of all PRL secreted from the lactotroph population by the TRH treatment. These results indicate that 1) there is a heterogeneity with respect to lactotroph responsiveness to TRH and that 2) a population of lactotrophs that secrete small amounts of PRL under basal conditions contains a much larger proportion of TRH-responsive lactotrophs than does a population of lactotrophs that secrete large amounts of PRL.

Lactotrophs secreting small amounts of prolactin reveal great responsiveness to thyrotropin-releasing hormone: analysis by the sequential cell immunoblot assay.

The effects of TRH on PRL secretion from individual lactotrophs of female rats were investigated by using a sequential cell immunoblot assay. The same pituitary cells cultured on coverslips were first incubated to determine basal secretion of PRL and subsequently challenged by one of various concentrations of TRH. The PRL secreted from the single lactotrophs was absorbed on protein-blotting transfer membranes, immunostained, and quantified by microscopic image analysis. When no TRH was added to the medium used in the second incubation (controls), the amount of PRL secreted from individual lactotrophs was 93% of that secreted in the first incubation. Treatment with 3 x 10(-10)-10(-7) M TRH in the second incubation increased in a dose-dependent manner the proportion of lactotrophs whose PRL secretion was significantly greater than confidence limits for PRL secretion in the controls. However, the percentage of TRH-responsive lactotrophs remained less than 50% even at a maximally effective concentration of TRH. Proportions of the TRH-responsive lactotrophs were significantly greater in cells that secreted small amounts of PRL under basal conditions than in those that secreted large amounts. Furthermore, the small, but not the large, secretors showed a significant increase in mean absolute amounts of PRL secreted by 10(-7) M TRH, which represented no less than 45% of all PRL secreted from the lactotroph population by the TRH treatment. These results indicate that 1) there is a heterogeneity with respect to lactotroph responsiveness to TRH and that 2) a population of lactotrophs that secrete small amounts of PRL under basal conditions contains a much larger proportion of TRH-responsive lactotrophs than does a population of lactotrophs that secrete large amounts of PRL.

Thyroid Vascular Conductance: Differential Effects of Elevated Plasma Thyrotropin (TSH) Induced by Treatment with Thioamides or TSH-Releasing Hormone*

We have reported previously that thyroid gland blood flow, expressed as vascular conductance (C) per mass, is decreased at very low and increased at very high chronic plasma TSH concentrations, but is apparently unchanged over a broad range of plasma TSH concentrations encompassing normal levels. The aim of the present study was to examine the apparently very steep dose-response relationship between elevated plasma TSH and thyroid vascular C/mass. In the first series of experiments, endogenous plasma TSH concentrations were manipulated by treating male Sprague-Dawley rats (250-280 g) for 6 days as follows: 1) controls (0.5 ml saline/day, ip), 2) propylthiouracil injections (2.0 mg PTU/day, ip), 3) PTU plus partial thyroid hormone replacement (2.0 mg PTU/day and 0.3-0.9 microgram T4 plus 0.075-0.225 microgram T3/100 g.day via continuous sc infusion), or 4) TRH (9-1200 micrograms TRH/100 g.day via continuous iv infusion). The vascular C values of the thyroid gland, salivary gland, kidney, and pancreas were determined using the reference sample version of the radioactive microsphere technique. PTU treatment led to the expected hypothyroidism, increased plasma TSH concentrations (959 +/- 66 vs. 154 +/- 22 ng/dl), increased thyroid weight (9.19 +/- 0.36 vs. 4.60 +/- 0.15 mg/100 g), and increased thyroid vascular C/mass (495 +/- 51 vs. 127 +/- 20 microliters/mm Hg.g/min). PTU-treated rats receiving partial thyroid hormone replacement demonstrated a dose-related suppression of plasma TSH, thyroid weight, and thyroid vascular C. Although, TRH treatments resulted in increased plasma TSH concentrations (e.g. 1200 micrograms TRH, 706 +/- 46 ng/dl) and thyroid weight (e.g. 1200 micrograms TRH, 7.45 +/- 0.41 mg/100 g), thyroid vascular C per tissue mass was not significantly increased after any TRH treatment (e.g. 1200 micrograms TRH, 166 +/- 19 microliters/mm Hg.g/min). Thus, at similarly elevated plasma TSH concentrations, the thyroid vascular C/mass of PTU- and TRH-treated rats constituted separate populations. Both PTU- and TRH-induced thyroid growth were accompanied by similar alterations in thyroid gland morphology (i.e. increased cellular mass with little change in the total amount of colloid). To investigate the mechanisms involved, groups of rats were treated for 6 days as follows: 1) control, 2) PTU or methimazole (25 mg MMI/day, ip), 3) PTU or MMI plus thyroid hormone replacement (1.2 micrograms T4 plus 0.3 microgram T3/d.100 g), 4) TRH (12 micrograms/100 g.day), and 5) PTU or MMI, thyroid hormones, and TRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe

Previous studies have demonstrated that TRH is a potent stimulator of alpha-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked alpha-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol. TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of alpha-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/l respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of alpha-MSH release from perifused neurointermediate lobes but did not impair TRH-induced alpha-MSH secretion. In the presence of LiCl, TRH (1 mumol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (-70%). These results indicate that, in frog pars intermedia, TRH-evoked alpha-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.

Alterations in cerebral glucose utilization in acute ischemic brain with and without TRH treatment

Alterations in cerebral glucose utilization in acute ischemic brain with and without TRH treatment

Placental and Pituitary Growth Hormone Secretion during Pregnancy in Acromegalic Women

It is now well established that during the second half of normal pregnancy, the human placenta secretes its specific GH variant (placental GH) in increasing amounts up to delivery. During the same period, pituitary GH secretion is progressively suppressed. The present study was aimed at clarifying the physiology of GH secretion in pregnant acromegalic women. Two young women remained acromegalic despite transphenoidal removal of their pituitary adenoma. Increased basal levels of GH and insulin-like growth factor-I (IGF-I) as well as paradoxical GH release after TRH injection were noted. Both women became pregnant and delivered term babies without any complication. In both patients, pituitary GH remained elevated during the entire pregnancy, contrary to the situation in normal women. Paradoxical GH release after TRH treatment was also present, whereas no response was observed in five normal control subjects. GH pulsatility studies revealed a highly pulsatile secretory pattern of pituitary GH, in contrast to that in normal woman, whose placental GH is secreted tonically. Tissue placental GH concentrations were within the range of levels in normal placentas. An increase in serum IGF-I in late pregnancy was also similar to that observed in normal pregnancy. These findings confirm that increased IGF-I levels are not pituitary GH dependent in late pregnancy. They add new evidence that adenomatous somatotrophs lack an IGF-I-dependent feedback regulation present in normal somatotrophs.

Interactions of Thyrotropin-Releasing Hormone, Phorbol Ester, and Forskolin-Sensitive Regions of the Rat Thyrotropin-β Gene

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the Circulating Renin-Angiotensin System on Prolactin Release in Humans*

We recently reported that renin, angiotensinogen, and angiotensin-converting enzyme were present in normal human pituitary lactotroph cells and PRL-secreting adenomas. Angiotensin-II and -III have also been shown to modulate PRL release in vitro. The present study was designed to determine whether angiotensin modulates PRL secretion in vivo. In 36 hypertensive patients with widely varying renin levels, active renin and basal PRL levels did not correlate. In 10 normal volunteers, both a sustained infusion of angiotensin-II and a graded infusion of angiotensin-III induced a 2- to 3-fold increase in aldosterone levels, but had no effect on PRL secretion. Administration of the angiotensin-converting enzyme inhibitor captopril had no effect on PRL circadian rhythm in 10 normal subjects or on PRL concentrations in 11 patients with PRL-secreting adenomas. Cross-over administration of placebo and captopril did not affect the peak PRL level measured after TRH treatment in 10 hypertensive men (placebo, 43.1 +/- 5.4; captopril, 40.0 +/- 6.2 micrograms/L; P = NS) or the rise in PRL induced by doperidone in 6 normal women (placebo, 129.5 +/- 16.2; captopril, 150.0 +/- 35.7 micrograms/L; P = NS). Further, administration of enalapril for 30 days to 6 hypertensive patients did not alter basal PRL concentrations or the peak concentrations induced by TRH. These data indicate that in humans the circulating renin-angiotensin system does not interact with diurnal PRL release or with the response to TRH or domperidone.

TRH treatment in cerebellar ataxia following a lightning stroke

TRH treatment in cerebellar ataxia following a lightning stroke