Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni): functional expression, gene structure, and evolution.

A PCR approach was used to clone thyrotropin-releasing hormone receptors (TRH-R) from the brain and anterior pituitary of the teleost Catostomus commersoni (cc), the white sucker. Two distinct TRH-R, designated ccTRH-R1 and ccTRH-R2, were identified. ccTRH-R1 was similar to mammalian TRH-R of the subtype 1, whereas ccTRH-R2 exhibited the highest identity (61% at the amino acid level) with the recently discovered rat TRH-R2. It is postulated that ccTRH-R2 and rat TRH-R2 are members of the same TRH-R subfamily 2. Functional expression of ccTRH receptors in human embryonic kidney cells and in Xenopus laevis oocytes demonstrated that both ccTRH receptors were fully functional in both systems. Oocytes expressing either receptor responded to the application of TRH by an induction of membrane chloride currents, indicating that ccTRH-R of both subtypes are coupled to the inositol phosphate/calcium pathway. The analysis of genomic clones revealed, for the first time, both similarities and differences in the structure of TRH-R subtype genes. Both ccTRH-R genes contained an intron within the coding region at the beginning of transmembrane domain (TM) 6. The position of this intron is highly conserved, as it was found at an identical position in the human TRH-R1 gene. The ccTRH-R2 gene contained an additional intron at the end of TM 3 that was not found in any of the TRH-R1 genes identified so far. The analysis of the gene structure of ccTRH-R and the amino acid sequence comparisons of mammalian and teleost TRH-R of both subtypes suggest that TRH receptors have been highly conserved during the course of vertebrate evolution. A common ancestral TRH receptor gene that could be found much earlier in evolution, possibly in invertebrates, might be the origin of ccTRH-R genes.

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.

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.

Glucocorticoids modulate the biosynthesis and processing of prothyrotropin releasing-hormone (proTRH).

The thyrotropin- (TRH) releasing hormone precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating N-terminal 15 kDa/9.5 kDa or C-terminal 16.5/10 kDa intermediate forms that are processed further to yield five copies of TRH-Gly and seven non-TRH peptides. Glucocorticoids (Gcc) have been shown to enhance TRH gene expression in three different cell systems in vitro, an effect that occurs, at least in part, through transcriptional activation. Although this implies that an increase of TRH prohormone biosynthesis would take place, this had not been demonstrated as yet. We report here that the synthetic glucocorticoid dexamethasone (Dex) substantially elevated the de novo biosynthesis of the intact 26-kDa TRH prohormone and its intermediate products of processing in cultured anterior pituitary cells, an observation that is consistent with an overall upregulation of both the biosynthesis and degradation of the TRH precursor. We reasoned that Gcc may act not only at the transcriptional, but also at the translational/posttranslational level. To address this question we chose a different cell system, AtT20 cells transfected with a cDNA encoding preproTRH. Since TRH gene expression in these cells is driven by the CMV-IE promoter and not by an endogenous "physiological" promoter, these cells provide an ideal model to study selectively the effects of Gcc on the translation and posttranslational processing of proTRH without interference from a direct transcriptional activation of the TRH gene. Dex caused a significant 75.7% increase in newly synthesized 26-kDa TRH prohormone, suggesting that the glucocorticoid raised the translation rate. We then demonstrated that Dex treatment accelerated TRH precursor processing. Of interest, processing of the N- vs the C-terminal intermediate was influenced differentially by the glucocorticoid. Although the N-terminal intermediate product of processing accumulated, the C-terminal intermediate was degraded more rapidly. Consistent with these observations was the finding that the intracellular accumulation of the N-terminally derived peptide preproTRH 25-50 was enhanced, but levels of the C-terminally derived peptide preproTRH208-255 were reduced. Accumulation of TRH itself, whose five copies are N- and C-terminally derived, was also enhanced. We conclude that Gcc induce changes in the biosynthesis and processing of proTRH by increasing the translation rate and by differentially influencing the processing of N- vs C-terminal intermediates of the precursor molecule. These effects of Gcc at the translational and posttranslational levels result in an increase in TRH production accompanied by differential effects on the accumulation of N- and C-terminal non-TRH peptides.

Evidence for diverse pathways of hypophysiotropic hormone transport in mammals.

Comparative studies of mammalian hypothalamic-pituitary relationships have revealed striking variations in hypophysiotropic systems and in portal vascular architecture. Immunocytochemical studies indicate that mammalian GnRH, GHRH and somatostatin systems can project to all portions of the neurohypophysis (median eminence, infundibular stem and pituitary neural lobe). In rats, primary secretion sites are located within the median eminence and upper infundibular stem, whereas in bats, most projections extend into the lower infundibular stem and pituitary neural lobe. In ferrets and monkeys, sites of secretion appear to extend throughout the neurohypophysis, from median eminence to proximal neural lobe. In this review, these interspecific differences are examined in light of observed structural variations in portal vascular systems. Correlations suggest that hypophysiotropic hormones can be delivered to target cells in the pars distalis by diverse routes, with some species relying more heavily on long and others on short portal transport. These patterns may have important functional implications with respect to regulatory mechanisms operating within the hypothalamic-pituitary complex.

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.

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.

Thyrotropin-releasing hormone gene expression in cultured anterior pituitary cells: role of gender.

The present studies were undertaken to investigate the effect of gender on thyrotropin-releasing hormone (TRH) gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male, female, or female pups that had been neonatally treated with testosterone propionate (TP), were cultured for up to 18 days in a modified DMEM/L-15 medium containing 10% fetal calf serum. TRH and AP hormones including GH, prolactin (PRL), luteinizing hormone (LH) and thyrotropin (TSH) were measured by RIA, proTRH mRNA was determined by in situ hybridization using a full-length riboprobe followed by quantification with a computer-assisted image analysis system. Cultures derived from female rats contained significantly (p < 0.01) higher amounts of TRH and secreted approximately twice (p < 0.01) as much TRH under basal conditions and in response to activators of the protein kinase A and C pathways, respectively. In situ hybridization studies revealed that 'female' cultures contained significantly higher amounts of proTRH mRNA compared to 'male' cultures. Computer-assisted image analysis demonstrated that proTRH mRNA levels were 3.5 times higher in 'female' compared to 'male' cultures (p < 0.01), an effect that was the result of a significantly higher number (3 times; p < 0.01) of cells expressing proTRH mRNA in 'female' cultures. Neonatal TP treatment did not affect either proTRH mRNA or TRH peptide levels. In vitro testosterone treatment resulted in a moderate rise (p < 0.05) of intracellular TRH accumulation in cultures from both sexes, however, proTRH mRNA levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Onset of pro-thyrotropin-releasing hormone gene expression in cultured rat anterior pituitary cells is expedited by dexamethasone.

The early onset of proTRH gene expression in anterior pituitary (AP) cells in culture and its regulation by dexamethasone (DEX) were investigated. AP cells derived from 15-day-old rats were cultured for up to 4 days in the presence or absence of 10(-7) M DEX. TRH peptide levels, which could be detected only after 3 days of culture in control cells, were detectable after 1 day in DEX-treated cells. Levels rose from undetectable (< 35 fmol/well/0.2 x 10(6) cells) to 121 +/- 11 fmol/well in control cells and from 59 +/- 3 to 2978 +/- 88 fmol/well in DEX-treated cells (Day 1 to Day 4; means +/- SEM, n = 6). ProTRH mRNA levels as analyzed by in situ hybridization showed an excellent correlation with TRH peptide levels: mRNA was already detectable on Day 1 in DEX-treated cells and on Days 2-3 in control cells. DEX stimulated proTRH mRNA levels as determined by Northern blot analysis within 4 h. The half-life of proTRH mRNA was calculated based on a first-order decay model by measuring mRNA levels after addition of 5 micrograms/ml actinomycin D with or without DEX. The t1/2 of proTRH mRNA in control cells was 13.1 +/- 2.8 h and was not influenced by DEX treatment (12.5 +/- 2.8 h). Since DEX stimulated proTRH mRNA levels acutely without any increase in mRNA stability, we propose that DEX expedites proTRH gene expression in our AP cell culture system by acting at the transcriptional level.

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.

Postsomatostatin hypersecretion of growth hormone from perifused rat anterior pituitary cells is dependent on calcium influx.

The role of ionic calcium (Ca2+) in the rebound secretion of growth hormone (GH) following termination of somatostatin (SRIF) administration was investigated in vitro by perifusion of acutely dispersed rat anterior pituitary cells. Treatment with 10 nM SRIF for 40 min significantly reduced the mean GH secretory rate by 3.3 +/- 0.2 ng min-1 representing a 58% decrease from baseline (p < 0.01). Following the withdrawal of SRIF treatment, GH levels surged 3- to 5-fold relative to baseline with the mean secretion rate increasing by 4.5 +/- 0.99 ng min-1 (p < 0.05). GH rebound secretion following SRIF removal from the perifusion medium was completely abolished (p < 0.01) when zero calcium medium (0 Ca2+) or medium containing 2 mM cobalt chloride (Co2+) were administered after SRIF termination. Perifusion with 0 Ca2+ caused the GH release rate to return to above baseline levels. In contrast, Co2+ perifusion caused the GH secretion rate to remain at the level observed during SRIF treatment (-4.52 +/- 0.38 ng min-1 relative to baseline; p < 0.01). Similarly, when cells were exposed to Co2+ alone, a reduction in the rate of GH secretion (-3.96 +/- 0.56 ng min-1; p < 0.01) was evident. After termination of Co2+ treatment, either by itself or following SRIF pretreatment, and upon changing from 0 Ca2+ to normal calcium-containing medium following SRIF pretreatment, a significant overshoot in GH release similar to SRIF withdrawal-induced GH release was observed (p < 0.05 and 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Abnormalities of the thyroid hormone negative feedback regulation of TSH secretion in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are characterized by several neuroendocrine abnormalities including a chronic hypersecretion of thyrotropin (TSH) of unknown etiology. We hypothesized that the inappropriately high TSH secretion in SHR may be the result of an impaired thyroid hormone negative feedback regulation of hypothalamic thyrotropin-releasing hormone (TRH) and/or pituitary TSH production. To test this hypothesis, SHR or their normotensive Wistar-Kyoto (WKY) controls were treated with either methimazole (0.02% in drinking water) to induce hypothyroidism or administered L-thyroxine (T4) at a dose of 0.8 or 2.0 micrograms/100 g body weight/day to induce hyperthyroidism. All treatments were continued for 14 days after which animals were killed under low stress conditions. TSH concentrations in plasma and anterior pituitary tissue were 2-fold higher (P less than 0.01) in euthyroid SHR compared to WKY control rats while thyroid hormone (T3 and T4) levels were in the normal range. Hypothyroidism induced by either methimazole or thyroidectomy caused a significant (P less than 0.01) rise of plasma TSH levels in both WKY and SHR rats. However, relative to the TSH concentrations in control animals, the increase of plasma TSH in SHR was significantly blunted (P less than 0.01) in comparison to the WKY group. Hypothyroidism caused a significant depletion of TRH in stalk-median eminence (SME) tissue in both groups of rats. However, no differences between SHR and WKY rats were observed. The administration of thyroid hormone caused a dose dependent suppression of plasma TSH levels in both strains of rats. However, at both doses tested plasma TSH concentrations in SHR rats were significantly less suppressed (P less than 0.05) than those in WKY animals. Under in vitro conditions basal and potassium induced TRH release from SMEs derived from SHR was significantly (P less than 0.05) higher than that from WKY rats, whether expressed in absolute terms or as percent of content. These findings suggest that the thyroid hormone negative feedback regulation of TSH secretion may be impaired in SHR rats. Our data do not allow conclusions as to whether defects in the regulation of TSH production are located exclusively at the hypothalamic level. Since the overproduction of hypothalamic TRH and hypophysial TSH should lead to an increased thyroid hormone biosynthesis other defects in the hypothalamus-pituitary-thyroid-axis may contribute to the abnormal regulation of TSH secretion in SHR rats.

Transplantation of fetal growth hormone-releasing factor-immunoreactive neurons into the ventricular system of adult MSG-treated rats.

Fetal (17-18 days of gestation) mediobasal hypothalamic tissue (MBH) was transplanted into the third ventricle of adult, male rats which had been treated neonatally with monosodium glutamate (MSG). MSG treatment caused a marked reduction of growth hormone-releasing factor-like-immunoreactive (GRF-i) perikarya in the arcuate nucleus and GRF-i fibers in the median eminence (ME), as compared to littermate controls. When normal fetal MBH was transplanted into the third ventricle of MSG recipients, numerous GRF-i perikarya were located within the graft four weeks following surgery. GRF-i fibers in the ME of MSG-treated rats were enhanced when MBH grafts were in close contact with the ME, but not when transplants were located dorsally or rostrally in the third ventricle without making contact with the recipient's ME. Fetal cerebral cortex, which was grafted as a control tissue, did not contain GRF-i neurons. These immunohistochemical results suggest that grafted fetal GRF-i perikarya may contact the recipient's ME to increase the content of GRF previously depleted by exposure to MSG.

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.

Hypothyroidism reduces content and increases in vitro release of pro-thyrotropin-releasing hormone peptides from the median eminence.

To determine the effect of thyroid status on proTRH-derived peptide processing and secretion, the content and release of TRH and prepro-TRH25-50 (PYE27), as well as somatostatin (SRIF) from median eminence (ME) or olfactory lobe (OL) tissue was studied in the rat. In hypothyroid animals treated by thyroidectomy (Tx), the ME content of TRH and PYE27 was reduced by more than 50%; further, when compared with euthyroid controls there was a significant 2-fold enhancement of the in vitro release of these peptides from ME fragments in response to depolarizing concentrations (60 mM) of potassium. Hyperthyroidism (T4 treatment) caused either no change or an increase in the ME content of these peptides and their response to K+ in vitro did not differ from control animals. The OL content of TRH and PYE27 was unaffected by thyroid status. SRIF levels in both ME and OL as well as in vitro secretion from the ME did not change with either Tx or T4 treatment. The ratio of TRH/PYE27 secretion throughout release and content studies remained stable at 3:1 to 4:1. These findings support the view that TRH in the hypothalamus but not OL is regulated by thyroid hormone. In this location hypothyroidism enhances not only pro TRH synthesis but also release of TRH and another proTRH-derived peptide. The consistent ratio of TRH/PYE27 suggests that regulation of TRH production by thyroid hormone occurs predominantly at the transcriptional level and not through posttranslation processing.

Immunocytochemical localization of growth hormone and growth hormone-releasing hormone immunoreactivity in the brain and pituitary of the little brown bat.

Anterior pituitary cells exhibiting growth hormone (GH) immunoreactivity and forebrain neurons containing growth hormone-releasing hormone (GHRH) immunoreactivity were identified in little brown bats (Myotis lucifugus) using light microscopic immunocytochemistry. Pituitary somatotropes appeared as ovoid or polyhedral cells that were distributed throughout most of the pars distalis, with the exception of its most rostral region where this cell type was scarce. GH-immunoreactive cells occupied approximately one-third of the total volume of the pars distalis; this proportion did not differ significantly between males and females or in bats collected at different times of year. Neuronal perikarya containing immunoreactive GHRH were observed in the hypothalamic arcuate and suprachiasmatic nuclei, as well as in the cortical and subcortical telencephalon. Fibers were most evident in the median eminence, paraventricular and periventricular nuclei, and molecular layer of the cerebral cortex. Fine fibers were also accumulated in the bed nucleus of the stria terminalis and in the amygdala.