A novel mba-based Real time PCR approach for genotyping of Ureaplasma parvum validated in a cohort of Mongolian mothers and offspring.

The role of Ureaplasma parvum in abnormal outcomes of human pregnancy has been discussed controversially in the past. Of the 14 known ureaplasma serovars, the Ureaplasma parvum serovars 1, 3, 6 and 14, have been found to derive from smaller genomes. Serovars 3 and 6 have been described more often to cause complications in pregnancy. To elucidate the serovar distribution in U. parvum positive specimens of 200 Mongolian mothers and their offspring, a new set of mba-targeting PCRs was developed enabling a fast and reliable serovar differentiation by melting peak analysis in a Real time PCR approach or by conventional agarose gel electrophoresis. 92% maternal and 55% neonatal samples were retrospectively genotyped and a dominance of serovars 3 and 6 was detected while serovar 14 was almost absent. Transmission from mothers to newborns was detected in 83% of U. parvum positive neonates exhibiting serovar patterns identical to their mothers. No statistically significant correlation between a distinct serovar and pregnancy outcome could be detected. However, neonatal colonization with serovar 1 declined with progressing pregnancy suggesting that a higher ureaplasma load shortened pregnancy and thereby had a potential negative effect on offspring health. Our novel mba-based Real time PCR approach, which can also be used in conventional PCR and gel electrophoretic analysis, provides the proof of principle that the four U. parvum serovars 1, 3, 6 and 14 can be differentially detected and quantified. A larger scale study outside the scope of this work should be conducted to clarify the impact of serovar 1 on pregnancy outcome.

Ontogeny of hypothalamus-pituitary function in the fetal pig: gonadotropin release in response to electrical and electrochemical stimulation of the hypothalamus.

The ontogeny of hypothalamic control of anterior pituitary gonadotropin secretion was studied in anesthetized fetal pigs at different gestational ages (60, 80, and 105 days gestation; term, 114 days). Three or four fetuses from one mother simultaneously received either no treatment (control), sham operation, electrical stimulation (EL), or electrochemical stimulation (EC). Electrodes were implanted unilaterally into the hypothalamus. Fetuses remained in utero during surgery. A distinct difference in the development of basal LH and FSH secretion was observed. Basal plasma LH concentrations almost doubled (P less than or equal to 0.001) between days 60 and 80, with no further significant increase between 80 and 105. Plasma FSH concentrations did not change significantly between days 60 and 80, but increased more than 5-fold (P less than or equal to 0.001) between days 80 and 105. EL or EC did not affect LH secretion at 60 days. At 80 days, EL and EC significantly (P less than or equal to 0.05) elevated plasma LH concentrations 30 and 50 min after the onset of stimulation. At 105 days, EL and EC caused a rise in plasma LH levels within 10 min; the maximum level, reached 30 min after the onset of stimulation, was double that in 80-day-old fetuses. Plasma FSH values were not significantly affected by EL or EC in any age group. The results indicate that the fetal pig hypothalamus is able to influence pituitary LH secretion by day 80 (70% of gestation). Further, maturation of hypothalamic control of LH secretion becomes demonstrable between days 80 and 105. The development of hypothalamic control of FSH secretion is delayed relative to LH.

Glucocorticoids stimulate thyrotropin-releasing hormone gene expression in cultured hypothalamic neurons.

Although there is much evidence indicating that glucocorticoids (GC) inhibit the hypothalamic-pituitary-thyroid axis in both rat and man in vivo, there have been no previous studies on the direct effect of GC on hypothalamic TRH neurons in vitro. In this laboratory, we developed fetal rat (day 17) diencephalic neuronal cultures in the presence of 5'-bromo-2-deoxyuridine, a cell-differentiating agent that stimulates TRH gene expression. In 12 separate experiments, dexamethasone (Dex) induced a 2.2-fold increase in TRH content vs. the control value (P < 0.01). Dex (10(-8)M) enhanced TRH messenger RNA (mRNA) 1.6-fold (n = 75 wells; P < 0.01) by nonisotopic in situ hybridization. On Northern blot analysis using a 32P-labeled complementary RNA probe, TRH mRNA was enhanced 3-fold (n = 4; P < 0.01). Nuclear run-on analysis revealed that Dex enhanced transcription 7.7 fold (n = 3; P < 0.01). We conclude that 1) Dex stimulates the expression of TRH peptide and TRH mRNA in cultured hypothalamic neurons; 2) the increase in TRH mRNA results (at least in part) from enhanced transcription; and 3) the reported in vivo depression of TRH in the paraventricular nucleus after GC stimulation suggests that this effect must be mediated indirectly on the TRH neuron.

Hypophysiotropic regulation of adrenocorticotropin secretion in response to insulin-induced hypoglycemia.

The hypophysiotropic coding of ACTH secretion resulting from insulin-induced hypoglycemia was investigated in urethane-anesthetized fasted rats. The participation of corticotropin-releasing factor (CRF), arginine vasopressin (AVP), and catecholamines in the ACTH response was first investigated by systemic administration of CRF antiserum, an AVP pressor antagonist, or a ganglionic blocking agent. These treatments were without effect on the hypoglycemic response, which was characterized by a 67% fall in systemic glucose levels within 30 min of insulin administration. ACTH secretion in response to insulin-induced hypoglycemia was differentially affected by these pharmacological treatments. Administration of antiserum to CRF abolished the ACTH response, whereas ganglionic blockade was without significant effect. However, administration of a vasopressinergic pressor antagonist significantly attenuated ACTH secretion after insulin treatment. These observations suggested the participation of both CRF and AVP in mediation of the ACTH secretory response to hypoglycemia. Infusion of glucose to counter the hypoglycemia action of insulin injection prevented the ACTH secretory response. Measurement of immunoreactive (ir) CRF, irAVP, and ir-oxytocin in sequential collections of hypophysial portal plasma revealed a significant elevation of irAVP concentration without concomitant elevation of irCRF or ir-oxytocin levels. We propose that CRF functions in a permissive role, maintaining a relatively constant portal concentration and thereby allowing expression of the weaker ACTH-releasing activity of AVP and other secretagogues. Thus, AVP, not CRF, appears to represent the dynamic mediator of ACTH secretion accompanying insulin-induced hypoglycemia. These observations provide additional support for the hypothesis of multifactor stimulus-specific hypophysiotropic coding of ACTH secretion.

Evidence for multifactor regulation of the adrenocorticotropin secretory response to hemodynamic stimuli.

We have examined the contributions of corticotropin-releasing factor (CRF), arginine vasopressin (AVP), epinephrine, and oxytocin to the ACTH secretory responses to hemorrhage. The relative significance of each of these putative ACTH regulatory factors is undefined with respect to net ACTH secretion. Initially, the effects of selective systemic pharmacological blockade of individual factors on the ACTH response were examined. Immunoneutralization of CRF reduced resting ACTH levels below the detection limits of our RIA and abolished the secretory response to hemorrhage. Ganglionic blockade or treatment with a potent AVP antagonist reduced the ACTH secretory response by 55% and 38%, respectively. Further evidence for multifactor regulation of hemodynamically evoked alterations in ACTH secretion was obtained by measurement of the concentrations of these factors in the hypophysial portal circulation during hemorrhage. Immunoreactive CRF, AVP, oxytocin and epinephrine were present in the portal plasma at concentrations within a range shown to evoke ACTH secretion from cultured pituitary cells when presented alone or in combination. The concentrations of all of these were significantly elevated during hemorrhage. During atrial pulsation, a stimulus mimicking volume loading and associated with a reduction of systemic ACTH levels, we observed a significant decline in portal concentrations of immunoreactive AVP coupled with a nonsignificant trend toward reduced portal immunoreactive CRF levels. These observations are highly suggestive of multifactor regulatory control of ACTH secretion in response to hemodynamic stimuli.

Central modulation of immunoreactive corticotropin-releasing factor secretion by arginine vasopressin.

Arginine vasopressin (AVP) is regarded as facilitatory to adenohypophysial ACTH secretion at the level of the corticotropic cell. A central facilitatory action of AVP on hypothalamic corticotropin-releasing factor (CRF) has also been postulated, although conclusive evidence on this point is lacking. We directly tested this hypothesis and have found that intracerebroventricular administration of AVP attenuates secretion of immunoreactive CRF (irCRF) into the hypophysial portal circulation in urethane-anesthetized rats. This suppression occurred in a dose-dependent fashion. Conversely, immunoneutralization of AVP or treatment with an AVP antagonist increased portal concentrations of irCRF by 53% and 30%, respectively. These unexpected observations provide evidence for a tonic inhibitory role of central AVP in regulation of irCRF and thus ACTH secretion.

Central modulation of immunoreactive arginine vasopressin and oxytocin secretion into the hypophysial-portal circulation by corticotropin-releasing factor.

Corticotropin-releasing factor (CRF), a potent ACTH secretagogue, has been found to exhibit many characteristics of central neurotransmitter/neuromodulatory substances. In this capacity, hypothalamic CRF might participate in postulated autoregulatory processes which regulate net secretion of adenohypophysical ACTH. We have examined the actions of centrally injected ovine CRF on the secretion of immunoreactive CRF, arginine vasopressin (AVP) and oxytocin (OT) into the hypophysial-portal circulation of urethane-anesthetized rats. Our observations do not support a short-term autoregulatory role for CRF. However, central administration of CRF was associated with a dose-dependent inhibition of hypophysial-portal concentrations of immunoreactive AVP and OT, suggesting potentially important central interactions among putative ACTH-regulatory factors.

Effect of paraventricular lesions on corticotropin-releasing factor (CRF)-like immunoreactivity in the stalk-median eminence: studies on the adrenocorticotropin response to ether stress and exogenous CRF.

Corticotropin-releasing factor-like immunoreactivity (CRF) and plasma ACTH were measured in rats bearing bilateral lesions of the paraventricular nucleus (PVN). Four to 6 days after stereotaxic surgery, CRF-like immunoreactivity content of the stalk-median eminence was reduced by 87-90% and the ACTH response to a 3-min ether stress was greatly attenuated (70-85% reduction). In order to differentiate between possible effects of CRF, catecholamines, and arginine vasopressin (AVP), the following treatments were used: group I, sham/vehicle; group II, sham/AVP-antagonist; group III, sham/ganglionic blocker chorisondamine; group IV, PVN-lesions/vehicle; group V, PVN-lesion/AVP antagonist; group VI, PVN-lesion/chlorisondamine. Blood was sampled at 0, 5, and 15 min after a 3-min exposure to ether vapor. PVN lesions alone greatly attenuated the ACTH response to ether stress by 70-85% (group IV), whereas basal ACTH levels were not affected. Chlorisondamine alone (group III) was as effective as PVN lesions in reducing the secretion of ACTH due to stress. When PVN-lesioned animals were treated with the ganglionic blocker, the residual ACTH response was completely abolished (group VI). The AVP-antagonist alone reduced the response at +15 min by 45%, whereas the antagonist given to PVN-lesioned animals completely abolished the response at 15 min. Injection of 0.15 nmol ovine CRF into PVN-lesioned rats resulted in a dramatically increased ACTH response (328% at 15 min) in comparison to the response of sham-operated rats. We conclude that: 1) CRF originating from neurons within the PVN is the predominant regulator of stress-induced ACTH secretion; 2) catecholamines and AVP are involved in mediating stress-induced ACTH secretion, most probably as CRF-potentiating agents; and 3) pituitary hyperresponsiveness to exogenous CRF results from removal of endogenous CRF.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. IV. Evidence for paracrine and autocrine regulation.

Disulfiram (Dis), an inhibitor of peptidyl-glycine alpha-amidating monooxygenase, the enzyme responsible for the production of alpha-amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated. Dis (8 microM) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nM), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (-73%) and TSH (-40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion. Dis significantly elevated prepro-TRH (25-50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.

Presence of growth hormone-releasing factor-like immunoreactivity in rat duodenum.

Growth hormone-releasing factor-like immunoreactivity (GRF-LI) was detected in partially purified extracts of rat hypothalamus and duodenum with a highly specific RIA for rat GRF (rGRF). The concentration of rGRF-LI in the rat hypothalamus was 32.5 fmol/mg dry weight (455 fmol or 3.18 ng/region), while the rat duodenum contained 2.15 fmol/mg dry weight (111 fmol or 0.78 ng/region). Partially purified hypothalamic and duodenal extracts exhibited displacement of the tracer parallel to rGRF. GRF-LI levels in all other brain regions (brainstem, cortex) or gastrointestinal tract tissues (forestomach, antrum, ileum, colon, pancreas) measured were close to the detection level of the RIA. rGRF-LI in hypothalamic and duodenal extracts behaved like synthetic rGRF in both gel filtration and reverse-phase HPLC systems. Coelution of rGRF-LI contained in both these tissue extracts with synthetic rGRF suggests that these molecules are similar, if not identical.

Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents.

The present studies were undertaken to investigate the effect of 5-bromo-2'-deoxyuridine (BrdU; 50 microM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 microM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.

Protrh peptides are synthesized and secreted by anterior pituitary cells in long-term culture.

The expression of two ProTRH derived peptides, thyrotropin--releasing hormone (TRH) and PrePro-TRH25-50 (PYE27) was studied in anterior pituitary (AP) cells cultured in monolayer for up to 21 days. TRH levels in extracted cells rose from undetectable at 3 days to 267 +/- 22.5 fmol/well (p less than 0.01) at 21 days in culture. When AP tissue was extracted without dissociation or culture TRH was undetectable. The molar ratio of TRH/PYE27 was approximately 5:1 as predicted by the structure of PreProTRH. Extracts of cultured AP cells coeluted with TRH and PYE27 standards when subjected to HPLC analysis. Basal TRH secretion was 13.2 +/- 1.8 fmol/well/30 min at 18 days in culture; depolarizing concentrations of K+ (55 mM) caused a 2.2 fold (p less than 0.01) Ca++ dependent increase in TRH release. Immunostaining for PYE27 was found in approximately 10% of the cell population. Our results suggest that authentic ProTRH peptides are synthesized by AP cells in long term culture but not in situ. While the mechanism of activation of the PreProTRH gene needs to be elucidated we propose that TRH and/or other ProTRH derived peptides may exert paracrine effects on AP function.

Corticotropin-releasing factor in the dog adrenal medulla is secreted in response to hemorrhage.

To characterize the nature of CRF-like immunoreactivity (CRF-LI) in the dog adrenal, adrenal medullary, adrenal cortical, or hypothalamic tissue was extracted and subjected to RIA after partial purification on C-18 cartridges or Sephadex G-50. Using N- and C-terminal-directed antisera against rat/human (r/h) CRF, significant levels of CRF-LI were found in the adrenal medulla and hypothalamus, but not in the adrenal cortex. Immunocytochemical analysis revealed that CRF-immunoreactive cells were located in the adrenal medulla, many of them concentrated in the vicinity of blood vessels and at the border between adrenal medulla and cortex. However, the cortex was devoid of any CRF-positive cells. On reverse phase HPLC, CRF-LI in the adrenal medulla coeluted with synthetic r/hCRF. In a bioassay system, using dispersed rat anterior pituitary cells, purified adrenal CRF caused a dose-dependent increase in ACTH secretion parallel to the r/hCRF standard, indicating that dog adrenal medulla contains authentic r/hCRF. Evidence of CRF-LI secretion from the adrenal was supported by its presence in adrenal venous, but not in peripheral arterial, plasma. Adrenal venous plasma CRF-LI coeluted with r/hCRF on reverse phase HPLC after affinity chromatographic purification. The CRF-LI secretory rate in conscious trained dogs was 68 +/- 19 pg/min (concentration, 27 +/- 5 pg/ml). In response to 20% hemorrhage, the CRF-LI secretion rate rose 3-fold within 15 min and was associated with increased catecholamine secretion. The existence of a biologically active CRF-like substance in the dog adrenal medulla and its secretion in conjunction with catecholamines after a hemorrhage suggest a physiological role for this peptide other than pituitary or central nervous system regulation.

Analysis of pulsatile secretion of thyrotropin and growth hormone in the hypothyroid rat.

To characterize the role of TRH in the generation of TSH pulsatility as well as the effect of hypothyroidism on episodic GH secretion, blood was constantly withdrawn (30-60 microliters/min) from rats treated with 0.02% methimazole in the drinking water for 8-10 days. This treatment significantly reduced circulating levels of both T3 and T4 and elevated plasma TSH; however, since thyroid hormone titers were still detectable (T3, 39.6 +/- 5.3 vs. 89.8 +/- 5.3 ng/dl in euthyroid animals), methimazole-treated rats were referred to as being mildly hypothyroid. TSH was found to be secreted in secretory bursts, consisting of one to several peaks in these rats. Pulsar analysis of TSH secretory profiles revealed a mean pulse frequency of 2.8 pulses/h, a mean pulse amplitude of 10 ng/pulse, and a mean pulse duration of 0.2 h. Euthyroid rats exhibited similar fluctuations of circulating TSH levels; however, due to the variability of the TSH RIA in the range of euthyroid TSH titers, no significant pulsatility was detected by Pulsar. Mean plasma TSH levels in eu- and hypothyroid rats were 2.3 +/- 0.3 and 14.6 +/- 1.8 ng/ml, respectively. To confirm that the TRH antiserum (TRH-AS) used in the present study for passive immunization had sufficient binding capacity to absorb endogenous TRH release, euthyroid rats were pretreated with either normal rabbit serum or TRH-AS, followed by the injection of clonidine (100 micrograms/kg BW, iv). This alpha 2-adrenergic agonist caused a significant (P < 0.01) 12.7-fold rise in plasma TSH levels in normal rabbit serum-treated animals, which was completely abolished by TRH-AS pretreatment, indicating that clonidine stimulates TSH secretion via activation of hypothalamic TRH release. When TRH-AS was slowly infused into hypothyroid rats that were sampled frequently for the detection of TSH pulsatility, it caused a significant (60.3%; P < 0.01) decrease in mean TSH levels, with TSH titers approaching euthyroid concentrations 1 h after the infusion of TRH-AS. The antiserum treatment also caused the disappearance of statistically significant (Pulsar) TSH secretory pulses. Mild hypothyroidism shifted the GH secretory profiles from a low frequency, high amplitude in euthyroid animals to a high frequency, low amplitude pattern in hypothyroid rats. Mean GH levels in hypothyroid rats were 76% lower than those in euthyroid controls. These findings show that TSH is secreted in a pulsatile fashion in the hypothyroid rat and that TRH is predominantly responsible for the generation of TSH pulsatility.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the role of angiotensin II in mediation of adrenocorticotropin secretion.

It has been suggested that ACTH secretion in response to selected stimuli may be modulated by angiotensin II (AII) via direct action at the level of the corticotrope or through central actions to facilitate CRF secretion into the hypophysial-portal circulation. These hypotheses were evaluated in the present series of experiments. Our failure to observe a significant portal to peripheral plasma immunoreactive (ir) AII gradient suggested that AII does not act as a physiologically significant ACTH secretagogue at the level of the corticotrope. Central administration of synthetic AII evoked a modest dose-related stimulation of hypothalamic irCRF secretion into the portal circulation, which was reversed by the AII receptor antagonist saralasin. Activation of a known AII-positive neuronal pathway projecting from the subfornical organ (SFO) to the hypothalamic paraventricular nuclei resulted in an elevation of hypophysial-portal plasma irCRF levels and increased circulating ACTH. Pretreatment with saralasin prevented SFO stimulation-induced irCRF secretion. These observations suggest that central AII-containing pathways may participate in mediation of ACTH secretion in response to hypovolemia or hyperosmolality by facilitating irCRF secretion. Involvement of the SFO, a circumventricular organ, in this circuit is intriguing as it provides a means of monitoring peripheral irAII concentration and then converting this humoral signal into a neural signal distributed to regions regulating drinking behavior, neurohypophysial AVP secretion, and activation of the hypothalamic-pituitary-adrenal axis.

Juxtamembrane regions in the third intracellular loop of the thyrotropin-releasing hormone receptor type 1 are important for coupling to Gq.

Juxtamembrane residues in the putative third intracellular (I3) loops of a number of G protein-coupled receptors (GPCRs) have been shown to be important for coupling to G proteins. According to standard hydropathy analysis, the I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 amino acids from position-213 to position-263. We constructed deletion and site-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulated signaling activities. As expected, the effects of these mutations on TRH binding were small (less than 5-fold decreases in affinity). No effect on TRH-stimulated signaling activity was found in a mutant receptor in which the I3 loop was shortened to 16 amino acids by deleting residues from Asp-226 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 or from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In the region near transmembrane helix 6, single site-specific substitution of either Arg-261 or Lys-262 by neutral glutamine had little effect on signaling, but mutant TRH-Rs that were substituted by glutamine at both basic residues exhibited reduced TRH-stimulated activity. The reduced signaling activity of this doubly substituted mutant was reversed by over expressing the a subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.

Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2).

We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical with rat TRH-R2 and 50% identical with mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9, and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of G protein-coupled receptor activity under normal physiologic circumstances.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. II. Stimulation by glucocorticoids.

The present studies were undertaken to determine whether glucocorticoids (GC) regulate TRH gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male rats were cultured for up to 18 days in Dulbecco's Modified Eagle's Medium-L-15 medium supplemented with 1) fetal calf serum (FCS), 2) charcoal-treated FCS, 3) normal rat serum, or 4) serum from rats that were adrenalectomized, rendered hypothyroid, and gonadectomized (ATG rat serum). Dexamethasone (Dex) or corticosterone (Cort) was added to the culture medium at various concentrations with exposure times ranging from 4-18 days. TRH and prepro-TRH-(25-50) in cellular extracts and release media were measured by RIA, and pro-TRH mRNA was determined by Northern blot analysis and in situ hybridization. Dex substantially stimulated cellular TRH and prepro-TRH-(25-50) accumulation under all culture conditions investigated, i.e. in medium supplemented with any of the four sera. TRH gene expression did not occur in medium supplemented with charcoal-treated FCS or ATG rat serum. Pretreatment with 10(-8) M Dex caused a significant increase in basal as well as cAMP- or phorbol ester-stimulated release of the peptide. Steady state pro-TRH mRNA levels rose 6.8- and 4.2-fold (both P < 0.01) after treatment with 10(-8) M Dex for 4 and 12 days, respectively. In situ hybridization experiments revealed that this rise in pro-TRH mRNA levels was probably the result of an increase in the number of AP cells expressing pro-TRH. Both Dex and Cort caused a dose-dependent increase in TRH accumulation, but Cort was approximately 40 times less potent than Dex. These results indicate that GC stimulate TRH gene expression in cultured AP cells. The presence of GC in culture medium is a prerequisite for the occurrence of TRH gene expression in the AP. As GC have been reported to reduce pro-TRH mRNA levels in the hypothalamus in vivo, our results may provide an example of the tissue-specific effects of GC on TRH gene expression.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. III. Stimulation by thyroid hormone: potentiation by glucocorticoids.

The present study was designed to investigate the effect of thyroid hormone on TRH gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male rats were cultured for up to 14 days in Dulbecco's Modified Eagle's Medium-L-15 medium supplemented with either fetal calf serum (FCS) or FCS devoid of thyroid hormones. T4 or T3 were added at various concentrations to the medium for a duration of 2-14 days. TRH and GH were measured by RIA, and pro-TRH mRNA levels were determined by semiquantitative in situ hybridization. Addition of both T3 and T4, but not the biologically inactive diiodothyronine, significantly stimulated TRH accumulation in AP cells. T3 increased TRH content in a time- and dose-dependent fashion and was much more potent than T4. Dexamethasone (Dex) also raised the content of TRH significantly. The combination of 10(-9) M T3 and 10(-8) M Dex dramatically potentiated the effect of either treatment alone (T3, 8.9-fold rise; Dex, 37.2-fold rise) and increased TRH accumulation 251.2-fold (all P < 0.01). Levels of pro-TRH mRNA mirrored TRH content data. T3, Dex, or the combination of both raised pro-TRH mRNA levels 1.9-, 2.7 (both P < 0.05)-, and 11.1 (P < 0.01)-fold, respectively. The visualization of pro-TRH mRNA by in situ hybridization revealed that the combination of T3 and Dex treatment caused a substantial increase in the number of cells expressing pro-TRH. The results presented here demonstrate that T3 increases pro-TRH gene expression in cultured AP cells and that glucocorticoids markedly potentiate this effect. As pro-TRH is expressed in a subpopulation of somatotrophs, our data suggest that the TRH gene in this location may be coordinately regulated with the GH gene.

Thyrotropin-releasing hormone (TRH) gene expression in the anterior pituitary. I. Presence of pro-TRH messenger ribonucleic acid and pro-TRH-derived peptide in a subpopulation of somatotrophs.

We have previously reported the presence of authentic pro-TRH-derived peptides in cultured anterior pituitary (AP) cells. The present studies were undertaken to determine whether pro-TRH mRNA could be demonstrated in the AP and to elucidate the cell type expressing pro-TRH. AP cells were cultured for up to 18 days, during which time the content of both TRH and prepro-TRH-(25-50) rose significantly (P < 0.01). In contrast, the cellular contents of LH, FSH, TSH, and ACTH fell significantly (P < 0.01), whereas that of GH increased by 45.9% (P < 0.05). Northern blot analysis revealed that the levels of pro-TRH mRNA extracted from AP cells (18 days in culture) were similar to those in hypothalamic tissue from adult male rats, indicating a high relative abundance of this mRNA in the AP. In situ hybridization experiments showed a dense accumulation of silver grains over a subpopulation of cultured AP cells. A combination of in situ hybridization for pro-TRH mRNA and immunocytochemistry for pituitary hormones revealed colocalization of pro-TRH mRNA and GH in a subpopulation of somatotrophs. No colocalization with LH-, TSH-, PRL-, or beta-endorphin-containing cells was observed. Immunocytochemistry at the electron microscopic level demonstrated that prepro-TRH-(25-50) was contained in a subpopulation of secretory granules in AP cells expressing this pro-TRH-derived sequence. These studies demonstrate that pro-TRH mRNA is present in cultured AP cells in high concentration and that the pro-TRH gene is expressed within a subpopulation of somatotrophs.