Thyrotropin-Releasing Hormone (TRH) and Somatostatin (SST), but not Growth Hormone-Releasing Hormone (GHRH) nor Ghrelin (GHRL), Regulate Expression and Release of Immune Growth Hormone (GH) from Chicken Bursal B-Lymphocyte Cultures.

It is known that growth hormone (GH) is expressed in immune cells, where it exerts immunomodulatory effects. However, the mechanisms of expression and release of GH in the immune system remain unclear. We analyzed the effect of growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), ghrelin (GHRL), and somatostatin (SST) upon GH mRNA expression, intracellular and released GH, Ser133-phosphorylation of CREB (pCREBS133), intracellular Ca2+ levels, as well as B-cell activating factor (BAFF) mRNA expression in bursal B-lymphocytes (BBLs) cell cultures since several GH secretagogues, as well as their corresponding receptors (-R), are expressed in B-lymphocytes of several species. The expression of TRH/TRH-R, ghrelin/GHS-R1a, and SST/SST-Rs (Subtypes 1 to 5) was observed in BBLs by RT-PCR and immunocytochemistry (ICC), whereas GHRH/GHRH-R were absent in these cells. We found that TRH treatment significantly increased local GH mRNA expression and CREB phosphorylation. Conversely, SST decreased GH mRNA expression. Additionally, when added together, SST prevented TRH-induced GH mRNA expression, but no changes were observed in pCREBS133 levels. Furthermore, TRH stimulated GH release to the culture media, while SST increased the intracellular content of this hormone. Interestingly, SST inhibited TRH-induced GH release in a dose-dependent manner. The coaddition of TRH and SST decreased the intracellular content of GH. After 10 min. of incubation with either TRH or SST, the intracellular calcium levels significantly decreased, but they were increased at 60 min. However, the combined treatment with both peptides maintained the Ca2+ levels reduced up to 60-min. of incubation. On the other hand, BAFF cytokine mRNA expression was significantly increased by TRH administration. Altogether, our results suggest that TRH and SST are implicated in the regulation of GH expression and release in BBL cultures, which also involve changes in pCREBS133 and intracellular Ca2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.

Prolactin and natural killer cells: evaluating the neuroendocrine-immune axis in women with primary infertility and recurrent spontaneous abortion.

PROBLEM: An association between serum prolactin (PRL) and peripheral blood natural killer (NK) cells has been described in healthy women. We explored for the first time the PRL response to the thyrotrophin-releasing hormone (TRH) test and the association between PRL and NK cells in women with reproductive failure. METHODS: A total of 130 women [31 primary infertility, 69 recurrent spontaneous abortion (RSA), and 30 fertile women] were evaluated by a TRH test to analyze the following: basal PRL (bPRL), peak-time PRL, PRL absolute and relative increase, decline-time PRL. Hyperprolactinaemia (HPRL) was defined as bPRL ≥15 ng/mL. NK cells were characterized by immunophenotyping. RESULTS: Significantly higher bPRL levels were found in the infertile women than in controls. Both the infertile and the RSA women showed significantly elevated NK levels. bPRL levels correlated with NK cells in HPRL-infertile women. CONCLUSIONS: In patients with HPRL, an association between NK cell and bPRL results. The dynamic test in the infertile women would help in the management of the pregnancy impairment.

Brainstem thyrotropin-releasing hormone regulates food intake through vagal-dependent cholinergic stimulation of ghrelin secretion.

The brainstem is essential for mediating energetic response to starvation. Brain stem TRH is synthesized in caudal raphe nuclei innervating brainstem and spinal vagal and sympathetic motor neurons. Intracisternal injection (ic) of a stable TRH analog RX77368 (7.5-25 ng) dose-dependently stimulated solid food intake by 2.4- to 3-fold in freely fed rats, an effect that lasted for 3 h. By contrast, RX77368 at 25 ng injected into the lateral ventricle induced a delayed and insignificant orexigenic effect only in the first hour. In pentobarbital-anesthetized rats, RX77368 (50 ng) ic induced a significant bipeak increase in serum total ghrelin levels from the basal of 8.7+/-1.7 ng/ml to 13.4+/-2.4 ng/ml at 30 min and 14.5+/-2.0 ng/ml at 90 min, which was prevented by either bilateral vagotomy (-60 min) or atropine pretreatment (2 mg/kg, -30 min) but magnified by bilateral adrenalectomy (-60 min). TRH analog ic-induced food intake in freely fed rats was abolished by either peripheral atropine or ghrelin receptor antagonist (D-Lys-3)-GHRP-6 (10 micromol/kg) or ic Y1 receptor antagonist 122PU91 (10 nmol/5 microl). Brain stem TRH mRNA and TRH receptor 1 mRNA increased by 57-58 and 33-35% in 24- and 48-h fasted rats and returned to the fed levels after a 3-h refeeding. Natural food intake in overnight fasted rats was significantly reduced by ic TRH antibody, ic Y1 antagonist, and peripheral atropine. These data establish a physiological role of brainstem TRH in vagal-ghrelin-mediated stimulation of food intake, which involves interaction with brainstem Y1 receptors.

Prolactin-releasing peptide is essential to maintain the prolactin level and osmotic balance in freshwater teleost fish.

We administered prolactin-releasing peptide (PrRP) or anti-PrRP antiserum to goldfish in fresh water and analyzed their effects on prolactin and osmoregulatory mechanisms. The pituitary mRNA level of prolactin increased by PrRP but decreased by anti-PrRP. The rate of water inflow in the gills decreased by PrRP and increased by anti-PrRP, showing that PrRP restricts branchial water permeability, as also restricted by prolactin. PrRP also expanded the mucous cell layers on the scales, which may restrict efficiently water inflow by the mucous system. Eventually, the plasma osmotic pressure decreased by anti-PrRP. We conclude that PrRP is essential to maintain prolactin levels and osmotic balance in fresh water.

Evidence for the biosynthesis of a prolactin-releasing factor from the ovine pars tuberalis, which is distinct from thyrotropin-releasing hormone.

This study demonstrates the presence of two prolactin-releasing (PR) factors in media conditioned by primary pars tuberalis cells prepared from dispersed pars tuberalis tissue. One factor was identified as thyrotropin-releasing hormone (TRH) on the basis of immunoreactivity and following purification by high-performance liquid chromatography and mass spectrometry. The origin of TRH in the pars tuberalis conditioned media was investigated by measuring the expression of glutaminyl-cyclase (QC) by in situ hybridization. QC expression was not detected in pars tuberalis-specific cells, but was relatively abundant in cells in the pars distalis and hypothalamic paraventricular nucleus. These data suggest that TRH is not synthesized by the ovine pars tuberalis and more likely originated from the hypothalamic neuronal processes from the paraventricular nucleus that terminate in the median eminence. The second component of the conditioned media PR bioactivity was insensitive to the TRH-antiserum, less than 1 kDa and was not retained by the C18 reverse-phase column. The biosynthesis of the PR bioactivity by pars tuberalis cells was investigated using cycloheximide, forskolin and melatonin. Cycloheximide reduced the level of PR bioactivity produced by the pars tuberalis cells. Melatonin inhibited the increased level of PR bioactivity stimulated by forskolin. Collectively, these data demonstrate the synthesis of at least one regulator of prolactin secretion by ovine pars tuberalis-specific cells.

Activation of raphe pallidus neurons increases insulin through medullary thyrotropin-releasing hormone (TRH)-vagal pathways.

INTRODUCTION: Pancreatic insulin secretion is regulated by the vagus nerve. Medullary thyrotropin-releasing hormone (TRH) containing projections from the raphe pallidus (Rpa) neurons innervate vagal preganglionic motor neurons in the dorsal vagal complex (DVC) and are involved in vagal regulation of gastric functions. AIM: To investigate whether chemical stimulation of Rpa neurons influences circulating insulin levels through brain medullary TRH-vagal pathways. METHODOLOGY: In fasted, pentobarbital-anesthetized rats, kainic acid (10 ng/50 nL) was microinjected into the Rpa, and serum insulin levels were measured. Gastric acid secretion was monitored as a control of vagally mediated visceral response. RESULTS: Chemical stimulation of Rpa neuronal cell bodies significantly increased serum insulin levels. Values before and at 30, 60, and 90 minutes after the microinjection of kainic acid were 0.34 +/- 0.02, 0.54 +/- 0.06, 0.60 +/- 0.06, and 0.99 +/- 0.13 ng/mL, respectively. In the same rats, gastric acid secretion was stimulated (basal, 2.3 +/- 0.6, versus 26.1 +/- 8.6 micromol/15 min at 30 minutes). Microinjections outside of the Rpa had no effect. The Rpa stimulation-induced increase in serum insulin could be mimicked by DVC microinjection of TRH analog, completely prevented by bilateral cervical vagotomy, and significantly reduced by bilateral microinjection of TRH antibody into the DVC. CONCLUSION: Chemical activation of Rpa neurons increases pancreatic insulin release through medullary TRH and vagal-mediated pathways.

Thyrotropin-releasing hormone regulates the diurnal variation of pyroglutamyl aminopeptidase II activity in the male rat adenohypophysis.

OBJECTIVE: Thyrotropin-releasing hormone (TRH) is inactivated in the extracellular compartment by pyroglutamyl aminopeptidase II (PPII), a narrow specificity ectopeptidase present in the brain and in the lactotrophs of the adenohypophysis. TRH and various hypothalamic/paracrine agents regulate the activity of PPII on the surface of adenohypophyseal cells in primary culture. The activity of the hypothalamic-pituitary-thyroid axis presents circadian variations including an increase of serum thyrotropin levels in the early hours of the day. The purpose of this study was to determine whether adenohypophyseal PPII activity fluctuates during the daytime in the male rat and the role of TRH in these regulatory events in vivo. RESULTS: Adenohypophyseal PPII specific activity and mRNA levels presented diurnal variations. A decrease in specific activity occurred with a minimum between 0930 and 1130 h, associated with increased serum thyrotropin levels. PPII mRNA levels were lowest at 0800 h. Intraperitoneal injection at 0800 or 1000 h of [3-Me-His(2)]-TRH, a potent agonist of the TRH receptor, reduced PPII specific activity at 30 min post-injection which was followed by a return to basal levels at 2 h. A second phase of decrease occurred between 4 and 8 h post-injection. Intravenous injection of a TRH-immune serum induced, at 2 h post-injection, an increase in adenohypophyseal PPII specific activity, which lasted up to 6 h. CONCLUSIONS: Adenohypophyseal PPII activity and mRNA levels fluctuate during the day; TRH down-regulates PPII activity in vivo, contributing to some of these variations. These new findings, and previous data, suggest that adenohypophyseal PPII activity varies in distinct physiological events, in response to endocrine and hypothalamic/paracrine factors, potentially modulating responses to TRH.

Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians.

The presence of thyrotropin-releasing-hormone-immunoreactive (TRH-ir) amacrine cells in the retina of amphibians is reported for the first time. The anuran and urodele retinas studied exhibit major differences in the distribution of TRH-ir cells. In the two urodele species investigated, most TRH-ir amacrine cells were located in the ganglion cell layer (GCL). These pear-shaped cells originate a dense TRH-ir dendritic plexus in strata 4-5 of the inner plexiform layer (IPL). A small number of TRH-ir amacrine cells were observed in the inner nuclear layer (INL). Most of these INL TRH-ir cells were multipolar neurons with radiating dendrites that originate a loose plexus in the IPL stratum 1. In the three anuran species investigated, most TRH-ir amacrine cells were located in the INL. Distribution of TRH-ir processes in the IPL of anurans was not so clearly layered as in urodeles, dendrites being observed throughout strata 1-5. In the toad retina THR-ir material was also observed in the outer plexiform layer, which suggests that toads may have some TRH-ir interplexiform neurons. In the frog and toad, TRH-ir fibers were also observed in the optic nerve, although their origin could not be ascertained. The number of TRH-ir amacrine cells per whole retina was higher in anurans than in urodeles, though urodeles have higher cell densities. The marked differences in distribution of TRH-ir amacrine cells observed between anurans and urodeles, and among the three anuran species, suggest different functions of TRH in retinal processing, perhaps related to the different specializations of the visual systems of these species.

Prostate-thyroid axis: prostatic TRH is one of the stimulators of thyroid hormone.

Ventral prostatectomy decreased serum thyroid hormones and histology of the thyroid gland indicate that hypothyroid condition. Co-culture of thyroid gland and ventral prostate stimulates thyroid hormone secretion. In the present study we report prostatic thyrotropin releasing hormone (TRH) is the stimulating factor of thyroid hormone secretion. Mature rat (90 days old) ventral prostate, anterior pituitary and thyroid glands were co-cultured in vitro with or without TRH antibody to assess the direct influence ofprostatic TRH on thyroid hormone secretion. Total thyroxine (T4) and triiodothyronine (T3) were increased significantly in the culture media of ventral prostate, anterior pituitary and thyroid gland when compared with thyroid gland plus anterior pituitary culture media. However, media T4 and T3 concentration decreased significantly in thyroid gland alone; also in thyroid gland plus ventral prostate, thyroid gland plus anterior pituitary and thyroid gland plus anterior pituitary plus ventral prostate were co-cultured with TRH antibody (Ab) in a dose dependent manner. The results suggest that ventral prostatic TRH is one ofthe stimulating factors of thyroid hormone secretion under these in vitro conditions.

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

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

Thyrotropin-releasing hormone and its receptor in glia.

The presence of thyrotropin-releasing hormone (Thyroliberin, TRH) and its receptor (TRH-R) in frozen coronal sections of the adult rat spinal cord and neonatal rat astroglial cultures was investigated by means of immunocytochemistry and Western blot using polyclonal antibodies generated against the hormone and monoclonal antibodies originated against discrete sequences of the type 1 rat TRH receptor (TRH-R1). TRH-R1 and TRH are present both in astroglial cells from adult rats and in cultured cells from newborn animals. The localization of TRH and TRH-R1 in nonneuronal cells in the central nervous system may reflect that some of the neurotrophic actions of TRH upon the central nervous system are mediated by glial cells.

Effect of intratesticular administration of TRH or anti-TRH antiserum on function of rat testis.

The effect of intratesticular administration of thyrotropin-releasing hormone (TRH) and anti-TRH antiserum on steroidogenesis was studied in immature and adult rats. In 9-day-old animals local administration of the neuropeptide resulted in an increase in basal testosterone secretion in vitro. Similar treatment of 15-day-old rats suppressed hCG-stimulated testosterone secretion with no change in basal testosterone production. In both immature groups the treatment did not affect serum testosterone concentration. By contrast, in adults TRH decreased serum testosterone level, but did not influence basal and hCG-stimulated testosterone secretion. Both in immature and adult rats, the changes in steroidogenesis were evident 1 hour posttreatment. Five days after the administration of anti-TRH antiserum into the remaining testis of immature rats subjected to hemicastration just prior to the antiserum treatment, the alterations in steroidogenesis were opposite to those detected after treatment with TRH. In 9-day-old rats the antiserum suppressed steroidogenesis, while in 15-day-old animals it stimulated testosterone secretion. The results suggest that testicular TRH might exert a local action on testicular steroidogenesis, and the effect is age-dependent.

TRH-like peptides in prostate gland and other tissues.

This minireview is aimed to recapitulate the occurrence of TRH-like peptides in the prostate gland and other tissues and to discuss their known functions in the organism. The hypothalamic thyrotropin-releasing hormone (TRH) was the first chemically defined hypophyseotropic hormone with the primary structure pGLU-HIS-PRO.NH2. However, the presence of extrahypothalamic TRH-immunoreactive peptides was reported in peripheral tissues including the gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. It was supposed that this TRH immunoreactivity can partially originate from TRH-homologous peptides and that these peptides have significant cross-reactions with the antibody specific against authentic TRH. This assumption was confirmed by the identification of prostatic TRH immunoreactivity as pyroGLU-GLU-PRO.NH2 using fast atom bombardment mass spectrometry and gas phase sequence analysis. TRH-like peptides are characterized by substitution of the basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acids, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological role of TRH-like peptides in peripheral tissues is not precisely known, but they possess a C-terminal amide group which is characteristic for many biologically active peptides. The occurrence of these peptides in the male reproductive system can influence male fertility. They are also closely related to circulating thyroid and steroid hormones. There might be an important connection of TRH-like peptides to the prostatic local autocrine/paracrine network mediated by extrahypothalamic TRH immunoreactivity corresponding to TRH-like peptides and extrapituitary thyrotropin (TSH) immunoreactivity also found in the prostatic tissue. A similar system of intraepithelial lymphocyte hormonal regulation due to the local paracrine network of TRH/TSH has been described in the gastrointestinal tract. The local network of TRH-like peptides/TSH may be involved in possible regulation of prostatic growth.

Thyrotropin-releasing hormone immunoreactivity in the brain and the pituitary during Bufo arenarum development.

The ontogeny of the thyrotropin releasing hormone (TRH) neuronal system was evaluated by immunocytochemistry in Bufo arenarum. The first appearance of TRH immunoreactive fibers was at early premetamorphosis. These fibers were found in small numbers and weakly stained in the median eminence and pars nervosa. With the advance of larval development, TRH-like material stained intensely and tended to aggregate in the median eminence, pars nervosa and pars intermedia. At climax stages immunoreactive fibers and perikarya (weakly stained) were also identified in the preoptic area. In adult specimens TRH perikarya and neuronal fibers were found in the preoptic and infundibular nuclei of the hypothalamus and in the amygdala, septum and diagonal band of Broca of the telencephalon. In addition, TRH neuronal fibers and endings were found in the preoptic-hypophyseal tract, the external zone of the median eminence, the pars nervosa and pars intermedia. Fibers were absent in the pars distalis. This study represents the first immunocytochemical demonstration of TRH in Bufo species, and serves as a basis for clarification of the neuroendocrine regulation of metamorphosis.

Hypothyroidism induces Fos-like immunoreactivity in ventral medullary neurons that synthesize TRH.

Altered thyroid statuses are associated with autonomic disorders. Thyrotropin-releasing hormone (TRH) in medullary nuclei regulates vagal efferent activity. Induction of Fos-like immunoreactivity (IR) in medullary TRH-synthesizing neurons was investigated in 24-h fasted rats with different thyroid statuses. Hypo- and hyperthyroidism were induced by 6-N-propyl-2-thiouracil (PTU) in drinking water and a daily intraperitoneal injection of thyroxine (T(4); 10 microgram. 100 g(-1). day(-1)), respectively, for 1-4 wk. The numbers of Fos-like IR positive neurons in the raphe pallidus, raphe obscurus, and parapyramidal regions, which were low in euthyroid rats (0-2/section), increased remarkably as the hypothyroidism progressed and were negatively correlated with serum T(4) levels. At the 4th wk, Fos-like IR positive neurons were 10- to 70-fold higher compared with euthyroid controls. Simultaneous T(4) replacement (2 microgram. 100 g(-1). day(-1)) prevented the increases of Fos-like IR in PTU-treated rats. Hyperthyroidism did not change the number of Fos-like IR neurons in the raphe nuclei but reduced it in the parapyramidal regions. Double immunostaining revealed that most of the Fos-like IR induced by hypothyroidism was located in the prepro-TRH IR positive neurons. The selective and sustained induction of Fos-like IR in TRH-synthesizing neurons in ventral medullary nuclei by hypothyroidism indicates that these neurons play a role in the autonomic disorders observed in altered thyroid statuses.

Neuropeptide Y innervation and neuropeptide-Y-Y1-receptor-expressing neurons in the paraventricular hypothalamic nucleus of the mouse.

The paraventricular hypothalamic nucleus (PVH) serves as integrator and link between the neuroendocrine and autonomic nervous systems. Neuropeptide-Y (NPY)-producing neurons in the arcuate nucleus project to the PVH, where neurons expressing NPY Y1 receptor (Y1R) have been demonstrated. This projection has been suggested to be involved in the regulation of parameters related to energy metabolism, e.g. food intake and thermoregulation. The present study aimed at characterizing this pathway and chemically defining Y1R-expressing neurons by means of immunohistochemistry. The densely distributed NPY-immunoreactive (ir) terminals in the PVH co-stained for agouti gene-related protein (AGRP) mainly in the medial parvocellular regions, indicating an origin in the arcuate nucleus. This was in contrast to noradrenergic/adrenergic terminals in the PVH, which were less frequently seen to contain NPY-like immunoreactivity. Furthermore, AGRP-ir terminals were seen forming abundant close appositions on Y1R-ir cell bodies. Double staining revealed co-existence of Y1R-like immunoreactivity and immunoreactivities for thyrotropin-releasing hormone (TRH) and, to a minor extent, cocaine- and amphetamine-regulated transcript peptide in parvocellular neurons. No Y1R-like immunoreactivity was noted in parvocellular neurons expressing corticotropin-releasing hormone or in magnocellular neurons expressing vasopressin or oxytocin. The present results suggest that the arcuatoparaventricular NPY projection targets the TRH neurons preferentially via the Y1R, whereas the NPYergic regulation of corticotropinergic and magnocellular neurons may be relayed through other subtypes of NPY receptors. This study further defines the link between NPY-induced feeding and the hypothalamus-pituitary-thyroid axis.

Identification of the TRH-like peptides pGlu-Glu-Pro amide and pGlu-Phe-Pro amide in rat thyroid: regulation by thyroid status.

Rat thyroid contains thyrotropin-releasing hormone (TRH) and TRH-like peptides which react with TRH antisera. We have identified the TRH-like peptides in the thyroid and examined whether their levels are influenced by thyroid status. The peptides were extracted from the thyroid glands of five hyperthyroid rats and purified by ion-exchange chromatography on SP-Sephadex C25 and reversed-phase high performance liquid chromatography. The principal TRH-immunoreactive component exhibited the same retention on HPLC as synthetic pGlu-Glu-Pro amide and a secondary component corresponded to synthetic pGlu-Phe-Pro amide. In agreement with these assignments the main peptide was shown to be acidic when chromatographed on DEAE-Sephadex A25 and the second peptide neutral. The levels of TRH and TRH-like peptides in the thyroid were investigated in hyper-, hypo- and euthyroid rats. Hyperthyroidism was induced by chronic subcutaneous administration of triiodothyronine (T3) and hypothyroidism was produced by addition of propylthiouracil (PTU) to the drinking water. The amounts of the peptides were determined by radioimmunoassay with a TRH-antiserum, carried out after extraction from the tissues and purification by ion exchange chromatography. The mean concentration of TRH-like peptides in the thyroids of the hyperthyroid rats was 95.5+/-25.5 pmol/g, the mean concentration in the hypothyroid rats was 11.7+/-3.4 pmol/g, and in the euthyroid rats 17.6+/-3.2 pmol/g. The concentrations of TRH were less influenced by thyroid status: the values in hyper-, hypo- and euthyroid rats were 47.5+/-9.4, 42.1+/-6.3, and 17.2+/-1.6 pmol/g respectively. The results show that the levels of the TRH-like peptides in rat thyroid are highly sensitive to thyroid status, suggesting a possible involvement in thyroid regulation.

Enzyme immunometric assay of thyroliberin (TRH).

An enzyme immunometric assay of thyroliberin (TRH) using monoclonal antibodies and a derivatization procedure is described. This assay, named SPIE-IA, involves a four step procedure after chemical derivatization of TRH and biological samples by diazotized APEA. Step 1: derivatized TRH was immunocaptured by a monoclonal anti-TRH antibody coated on a 96-well microtiter plate. Step 2: after washing, derivatized TRH was cross-linked via its amino group to the wells using glutaraldehyde. Step 3: washing and treatment with NaOH. Step 4: measurement of bound TRH using a monoclonal anti-TRH antibody labeled with acetylcholinesterase. The minimal detectable concentration was 0.1 pmol/ml: with a coefficient of variation less than 10% in the 0.156-10 pmol/ml range. This assay is 26-fold more sensitive and more specific than the competitive enzyme immunoassay using the same monoclonal capture antibody, derivatized TRH and TRH-acetylcholinesterase conjugate as tracer. Good correlation was observed between SPIE-IA and a sensitive competitive enzyme immunoassay using polyclonal antibodies.

[Antigenicity study on montirelin hydrate (NS-3)].

An antigenicity study of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was conducted in Hartley guinea pigs. Animals were sensitized twice by subcutaneous injections of montirelin hydrate in combination with Freund's complete adjuvant and twice by intramuscular injections of montirelin hydrate alone. Montirelin hydrate was found to be negative in the homologous passive cutaneous anaphylaxis test carried out with sera from the sensitized guinea pigs. Negative results were also obtained in the active cutaneous anaphylaxis test and the active systemic anaphylaxis test in guinea pigs sensitized with montilerin hydrate. These results show that montirelin hydrate has no antigenicity under the present experimental conditions.

Thyrotropin-releasing hormone inputs are preferentially directed towards respiratory motoneurons in rat nucleus ambiguus.

In the present study, we assessed the extent of the thyrotropin-releasing hormone (TRH) input to motoneurons in the ambigual, facial, and hypoglossal nuclei of the rat using a combination of intracellular recording, dye filling, and immunohistochemistry. Twelve motoneurons in the rostral nucleus ambiguus were labelled by intracellular injection in vivo of Neurobiotin (Vector). Seven out of 12 ambigual motoneurons displayed rhythmic fluctuations of their membrane potential in phase with phrenic nerve discharge, whereas the other five had no modulations of any kind. Seven facial motoneurons and seven hypoglossal motoneurons were also filled with Neurobiotin. All three motor nuclei contained TRH-immunoreactive varicosities, with the largest numbers found in the nucleus ambiguus. Close appositions were seen between TRH-immunoreactive boutons and every labelled motoneuron. Respiratory-related motoneurons in the nucleus ambiguus received the largest number of TRH appositions with 74 +/- 38 appositions/neuron (mean +/- S.D.; n = 7). In contrast, nonrespiratory ambigual motoneurons received significantly fewer TRH appositions (11 +/- 5; n = 5; P < 0.05; Mann-Whitney U test). Facial motoneurons received about the same number of TRH appositions as nonrespiratory ambigual motoneurons, with 13 +/- 4 (n = 7). Hypoglossal motoneurons received the fewest appositions from TRH-containing boutons, with 8 +/- 2 (n = 7). There were no differences in the TRH inputs to respiratory and nonrespiratory motoneurons in the facial and hypoglossal nuclei. These results demonstrate that, among motoneurons in the medulla, respiratory motoneurons in the rostral nucleus ambiguus are preferentially innervated by the TRH-immunoreactive boutons.