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.

The physiological role of thyrotropin-releasing hormone in the regulation of thyroid-stimulating hormone and prolactin secretion in the rat.

The physiological role of thyrotropin-releasing hormone (TRH) in the regulation of thyrotropin (thyroid-stimulating hormone, TSH) and prolactin (Prl) secretion has been assumed but not proven. Stimulation of their release requires pharmacologic doses of TRH. Lesions of the hypothalamus usually induce an inhibition of TSH secretion and an increase in Prl. To determine whether TRH is essential for TSH and Prl secretion in the rat, 0.1 ml of TRH antiserum (TRH-Ab) or normal rabbit serum was administered to normal, thyroidectomized, cold-exposed, and proestrus rats through indwelling atrial catheter. Serum samples were obtained before and at frequent intervals thereafter. Serum TSH concentrations in normal, thyroidectomized, cold-exposed, and proestrus rats were not depressed in specimens obtained up to 24 h after injection of normal rabbit serum. In contrast, serum TSH was significantly decreased after the administration of TRH-Ab in all normal (basal, 41+/-8 muU/ml [mean+/-SE]; 30 min, 6+/-2; 45 min, 8+/-3; 75 min, 4+/-2); thyroidectomized (basal, 642+/-32 muU/ml; 30 min, 418+/-32; 60 min, 426+/-36; 120 min, 516+/-146); coldstressed (basal, 68+/-19 muU/ml; 30 min, 4+/-3; 180 min, 16+/-8); and proestrus (basal, 11 a.m., 57+/-10 muU/ml; 1 p.m., 20+/-3; 3 p.m., 13+/-4; 5 p.m., 19+/-3) rats. However, 0.1 ml of TRH-Ab had no effect on basal Prl concentrations in normal or thyroidectomized rats and did not prevent the Prl rise in rats exposed to cold (basal, 68+/-7 ng/ml; 15 min, 387+/-121; 30 min, 212+/-132; 60 min, 154+/-114), or the Prl surge observed on the afternoon of proestrus (basal 11 a.m., 23+/-2 ng/ml; 1 p.m., 189+/-55; 3 p.m., 1,490+/-260; 5 p.m., 1,570+/-286). These studies demonstrate that TRH is required for TSH secretion in the normal, cold-exposed and proestrus rat and contributes, at least in part, to TSH secretion in the hypothyroid rat, but is not required for Prl secretion in these states.

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.

Peptides related to thyrotrophin-releasing hormone (TRH) in human prostate and semen.

The TRH-related peptide, pGlu-Glu-ProNH2, which was first identified in rabbit prostate has recently been named fertilization-promoting peptide (FPP) because of its ability to enhance the in vitro fertilizing potential of mouse epididymal spermatozoa. This study set out to examine the nature of the TRH-related peptides in human prostate and semen but, first, the optimal conditions for collection of semen samples were investigated. FPP was degraded slowly (t1/2 = 163 min, S.E. +/- 51.3, n = 6) in seminal plasma which has allowed us to measure accurately the concentrations of FPP, after extraction of the peptide in acidified acetone precisely 5 min after ejaculation. In this way, high levels of FPP (mean: 49.5 nmol/l) were detected in normal human semen, from young men, although other TRH-related peptides did not appear to be present. We have also examined the TRH-related peptides present in prostate samples from clinical patients both with and without evidence of benign prostatic hyperplasia (BPH), by ion-exchange chromatography followed by radioimmunoassay. Substantial concentrations of FPP were observed in normal (4.10 pmol/g tissue, S.E. +/- 1.46) and BPH prostate (6.27 pmol/g tissue, S.E. +/- 1.65). In addition, a second, neutral TRH-immunoreactive peptide was always detected in BPH tissue (7.40 pmol/g tissue, S.E. +/- 1.98) with only low levels generally present in normal prostate. The possibility that the presence of high levels of the neutral peptide in prostate may be used as an indicator of the onset of BPH deserves further scrutiny.

Properties of monoclonal antibodies to thyroliberin (TRH) induced by different immunogens: comparison with pituitary TRH receptor.

Thyroliberin E-H-P-NH2 (TRH) is a small neuropeptide pGlu-His-Pro-NH2 widely distributed in neural sites. The aim of this work was to obtain an antibody molecule with the nearest properties to that of TRH-receptor in GH3 cells. Different TRH-protein conjugates were prepared and utilized to induce monoclonal antibodies in mice. Several monoclonal antibodies were obtained using E-H-P-NH2 (TRH) coupled either to the histidyl residue (immunogen I) or to the prolyl residue (immunogen II). Antibodies generated using immunogen I and immunogen II were characterized in a radioimmunoassay system and an enzyme immunoassay system respectively. Their selectivities regarding a series of TRH related peptides were compared to those of rabbit polyclonal antibodies using three differently labelled TRH (tritiated-TRH, mono-iodinated-TRH and TRH-OH-acetyl-cholinesterase) as tracers and to prolactin secreting cells TRH receptors using 3H-TRH. Whatever the immunogen, the stereospecificity of monoclonal antibodies tested were found more different from TRH receptor characteristics than rabbit polyclonal antibodies.

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.

Abundance of immunoreactive thyrotropin-releasing hormone-like material in the alfalfa plant.

Immunoreactive TRH (IR-TRH) is found in high concentrations (157 +/- 6 ng/g) in extracts of a plant, alfalfa. On high performance liquid chromatography (HPLC), most alfalfa TRH showed an increased retention time compared to synthetic TRH. Since the plant extract eluted after synthetic TRH on both Sephadex G-10 and Biogel P-2 chromatography, it does not appear to be an aggregate or a "big" TRH. Incubation with rat serum caused degradation of alfalfa TRH at a rate similar to synthetic TRH. Since the antibody against TRH is highly specific for the N and C terminal ends of the molecule, a point change at the His could explain the nature of alfalfa TRH. The biologic function of material resembling a mammalian hypothalamic releasing factor in this location is unknown, but its presence in the plant kingdom may have evolutionary significance.

Suppression of prolactin and thyrotropin secretion in the rat by antiserum to thyrotropin-releasing hormone.

Administration of antiserum to synthetic thyrotropin-releasing hormone (TRH) to male and female rats cause a 50% and a 70% suppression in serum levels of prolactin and thyrotropin, respectively, as compared with controls injected with normal rabbit serum. The degree of suppression was similar in diestrous and proestrous female rats and in male rats. These findings support the view that, in addition to its original designation, TRH also has a physiological role in regulating release of pituitary prolactin.

Studies on the mechanism of growth hormone and thyrotropin responses to somatostatin antiserum in anesthetized rats.

An iv administration of 1 ml sheep antiserum to somatostatin (anti-SS) resulted in marked increases of both serum GH and TSH, with a peak 10--20 min after administration in male rats anesthetized with urethane or pentobarbital. Administration of anti-SS had no effect on serum PRL. Ablation of the basal medial hypothalamus abolished the rises of both serum GH and TSH after anti-SS administration. Intravenous injection of 1 ml rabbit antiserum to TRH (anti-TRH) decreased serum TSH levels 15 min after injection, whereas injection of normal rabbit serum did not affect TSH levels. Serum TSH levels did not rise after injection of anti-SS in rats pretreated with anti-TRH. On the other hand, pretreatment with anti-TRH did not affect the basal serum GH levels nor the anti-SS-induced GH release. The enhanced secretion of GH and TSH after anti-SS injections was not blocked by pretreatment with indomethacin, an inhibitor of prostaglandin synthesis. The following conclusions were made: 1) both GH and TSH responses to anti-SS require an intact basal medial hypothalamus; (2) TSH response to anti-SS is mediated by hypothalamic TRH; and 3) the GH response may be mediated by hypothalamic GH-releasing hormone which is not TRH or prostaglandins.

The peak phase of the proestrous prolactin surge is blocked by either posterior pituitary lobectomy or antisera to vasoactive intestinal peptide.

UNLABELLED: The proestrous surge of PRL could result from a decrease in dopamine, an increase in PRL-releasing factor (PRF) or both. The objectives were to determine whether PRF from the posterior pituitary regulates the proestrous PRL surge, and to examine if there are interactions between PRF and vasoactive intestinal peptide (VIP). Posterior pituitary lobectomy (LOBEX) and passive immunization against VIP were employed. Adult cycling rats were subjected at 0900 h on proestrus to LOBEX or sham surgery (SHAM) under short term anesthesia, and were injected iv at 1330 h with 0.75 ml anti-VIP serum or normal rabbit serum. Jugular blood was collected hourly from 1400-2300 h and analyzed for PRL and LH by RIA. Oviductal ova were examined on estrus. The rise in plasma PRL in normal rabbit serum-treated SHAM rats was biphasic, with an early peak between 1500-1700 h and a lower plateau between 1900-2100 h. This rise was similar in profile and magnitude to that seen in intact rats. In contrast, LOBEX significantly attenuated the early peak, but did not alter the plateau. Passive immunization against VIP of either SHAM or LOBEX rats mimicked the effect of LOBEX alone on PRL release. Neither surgery nor anti-VIP serum affected the profile of the LH surge which was sharp and symmetrical, and all rats ovulated with 15-16 ova per rat. To determine whether VIP is the posterior pituitary PRF, selected tissues removed on proestrus or diestrus-1 were analyzed for VIP by RIA. VIP was undetectable (less than 20 pg/organ) in the posterior pituitary on either day examined. The contents of VIP in the anterior pituitary, medial basal hypothalamus, and paraventricular nuclei were unchanged between diestrus-1 and proestrus. CONCLUSIONS: The proestrous surge of PRL consists of two components: an early peak and a late plateau. The peak phase appears to be dependent on PRF input from the posterior pituitary. This input might be regulated by VIP, and interactions between the two could occur at the level of the hypothalamus, anterior pituitary, or both. The plateau phase of the PRL surge is independent of the posterior pituitary and VIP, and might involve hypothalamic dopamine.

Control of prolactin release induced by suckling.

In the present study, the role of dopamine and TRH in suckling-induced PRL release was investigated. Bupropion, a dopamine reuptake blocker, increased hypophysial stalk dopamine levels and inhibited suckling-induced PRL release. A short period of suckling, thought to induce a transient decrease in hypothalamic dopamine release, led to higher PRL levels following an iv injection of TRH than in rats which had not nursed their young for a short period after 4- to 6-h separation. These results, in combination with previous data, suggest that a decrease in hypothalamic dopamine release is important for suckling-induced PRL release. Increased PRL release may be in part due to an augmented hypothalamic release of TRH. Since serotonergic mechanisms seem involved in TRH release, lactating rats were treated with drugs acting on serotonergic pathways. Parachlorophenylalanine and pizotifen did not alter suckling-induced PRL release. Methysergide, a serotonin receptor blocker, prevented this PRL release when administered ip but not when injected into the lateral brain ventricle. Since methysergide is converted peripherally into metabolite(s) with dopamine agonistic activity, its effect on suckling-induced PRL release may be due to this action, rather than to its action on serotonin receptors. Thus, these data do not indicate that serotonergic mechanisms are important for suckling-induced PRL release. Passive immunization against TRH inhibited suckling-induced PRL release, indicating that TRH is a hypophysiotropic mediator of this PRL release.

Immunocytochemical distribution in rat brain of putative peptides derived from thyrotropin-releasing hormone prohormone.

Processing of the TRH prohormone (Pro-TRH), a protein of approximately 26,000 mol wt, could yield 5 copies of TRH, as well as extended forms of TRH and several other non-TRH peptides. To determine whether some of these peptides are formed and transported by axons in the rat brain, we used antiserum to synthetic peptides corresponding to portions of pro-TRH. These included the N-tyrosyl analogs [Tyr0]prepro-TRH-(25-50) (pYE27) and [Tyr1]prepro-TRH-(53-74) (pYT22) contained within the N-terminal flanking region of the prohormone, the N-tyrosyl analog [Tyr0]prepro-TRH-(165-186) (pYS23), expanding the fourth progenitor sequence of TRH in the midportion of the prohormone, and the synthetic peptide pAC12 corresponding to the first 12 amino acids of the C-terminal flanking region or prepro-TRH-(208-219). All antisera showed staining in neuronal perikarya and processes in all regions of the brain previously demonstrated to immunostain for TRH, including dense innervation of the external zone of the median eminence. In addition, these antisera immunostained regions of the brain not previously immunopositive for TRH. Not all regions reactive with antiserum to [Tyr0]prepro-TRH-(25-50) were also recognized by anti-pYT, -pYS, and -pAC. These studies confirm the presence of the deduced non-TRH sequences within the TRH precursor and their formation and transport in vivo in the central nervous system. The presence of immunoreactivity in regions of the brain that do not contain TRH and the variability of immunostaining of the different antisera in some of these regions suggest regional preferential processing of pro-TRH to other peptides that may be biologically active.

Antithyrotropin-releasing hormone serum inhibits secretion of glucagon from isolated perfused rat pancreas: an experimental model for positive feedback regulation of glucagon secretion.

TRH is synthesized in the islets of Langerhans and was found in the perfusate of isolated rat pancreas. In the present study, designed to determine the role of endogenous TRH, we first characterized chromatographically the identity of immunoreactive TRH with synthetic pGlu-His-Pro-NH2. Since endogenous TRH secretion may mask the effects of exogenous TRH, we performed, in parallel to dose-response studies, immunoneutralization experiments using anti-TRH serum to neutralize the endogenous TRH secretion from isolated perfused rat pancreas. The data indicate that exogenous TRH enhances basal glucagon secretion; inversely, anti-TRH serum inhibits glucose plus arginine-induced glucagon secretion and produces a concomitant slight inhibition of somatostatin secretion. The present study shows a physiological contribution for endogenous TRH as a local modulator of intraislet hormone regulation; from these observations, we postulate a direct effect of pancreatic TRH on glucagon-containing (alpha) cell secretion, which, in turn, may produce the fluctuation in somatostatin secretion. Local TRH secretion provides a model for positive feedback regulation of glucagon secretion, frequently associated with diabetes.

Role of the hypothalamic paraventricular nucleus in the secretion of thyrotropin under adrenergic and cold-stimulated conditions in the rat.

To investigate the role of central noradrenergic systems in the regulation of TSH secretion, clonidine (an alpha 2-adrenergic agonist) and an electrolytic procedure were used. The administration of a small dose (50 micrograms/kg, iv) of clonidine induced a significant rise in immunoreactive TSH levels in the plasma with a short time lag in unanesthetized, unrestrained rats. The TSH-stimulating effect of clonidine was significantly reduced by passive immunization with rabbit antiserum to TRH. In rats with midbrain transection, the plasma TSH elevation in response to clonidine did not differ from that of sham-operated controls, whereas bilateral lesions in the paraventricular nucleus (PVN) markedly diminished the TSH secretory response. In contrast, the placement of electrolytic lesions in the dorsal raphe nucleus caused a significant increase in the basal TSH concentrations within 3 days, and magnified the TSH response to alpha 2-adrenergic stimulation. Acute exposure to cold (2-3 C) induced a prompt increase in the plasma TSH concentrations in normal rats. Bilateral lesions in the medial preoptic/anterior hypothalamus had no effect on the TSH response to cold exposure. In rats with the dorsal raphe nucleus lesions no significant effect was observed on the responsiveness to cold. In rats bearing PVN lesions, however, plasma TSH levels decreased significantly compared with sham-operated controls within 4 days, and also remained at basal levels during the whole period of cold exposure. The results of the present study provide evidence supporting the hypothesis that the PVN is essential for the process of TRH-TSH secretion, which is accelerated under cold-stimulated conditions, presumably via the central noradrenergic system(s).

Evidence for alpha 1-adrenergic stimulatory control of in vitro release of immunoreactive thyrotropin-releasing hormone from rat median eminence: in vivo corroboration.

The aim of this study was to investigate whether the alpha-adrenergic stimulation of TSH secretion may occur directly at the median eminence (ME) level by modulating the release of TRH. The effects of pharmacological manipulations of the two subtypes of central alpha-adrenergic receptors, alpha 1 and alpha 2, were tested on in vitro TRH release from medial basal hypothalami containing mainly the ME. Hypothalamic fragments were superfused with a modified Locke medium, and TRH was measured by RIA in samples collected every 10 min. After a preliminary period of 40 min to test TRH release during basal conditions, drug effects were checked for 20 min. Superfusion with norepinephrine (NE) (10(-10), 10(-8), 10(-6) M) induced a rapid and dose-dependent rise of TRH release; epinephrine (10(-8) M) induced an effect similar to that of NE 10(-8) M. Phentolamine (10(-7) M), an alpha-adrenergic antagonist, completely blocked the NE (10(-8) M)-induced release of TRH, which was not modified by the beta-adrenergic antagonist propranolol (10(-7) M). Neither antagonist had an effect on basal TRH release when added alone to the medium. The NE-induced release of TRH was completely suppressed by prazosin (10(-7) M), whereas yohimbine had no effect. Superfusion with clonidine (10(-9), 10(-8), 10(-7), 10(-6) M), an alpha 2-receptor agonist, did not alter basal TRH release. In contrast, phenylephrine (10(-8) and 10(-6) M), an alpha 1-receptor agonist, induced a significant (P less than 0.01) rise in TRH release. These results were corroborated in vivo in several unanesthetized rats bearing a push-pull cannula previously and stereotaxically implanted into the ME. Perfusion with artificial cerebrospinal fluid containing NE (10(-7), 10(-6) M) or phenylephrine (10(-7) M) elicited a rapid rise in TRH release, within 15 min after the onset of drug perfusion. Clonidine (10(-5) M), similarly perfused for 15 min, had no effect. Our data suggest a direct stimulatory influence of catecholamines on TRH release at the ME level that is mediated through alpha 1-adrenergic receptors.

Inhibition of degradation and measurement of immunoreactive thyrotropin-releasing hormone in rat blood and plasma.

A radioimmunoassay (RIA) for thyrotropin-releasing hormone (TRH) is described. The cross-reactivity of the antiserum was tested with 26 analogs of TRH, 5 amino acids, and LH-releasing hormone. Use of this RIA revealed that inactivation of TRH by rat blood was prevented if the blood was frozen and thawed prior to its incubation with TRH. This procedure did not interfere with the binding of TRH to its antibody. The degradation of TRH by blood or plasma was inhibited by 2,3-dimercaptopropanol (BAL) or benzamidine, but these compounds nonspecifically inhibited the binding of [125I]TRH to anti-TRH. At 37 C, 50% of the synthetic TRH added to rat blood was degraded within seconds, whereas at 1C, 60-65% was recovered after 90 min. When blood was frozen and thawed prior to its incubation with TRH at 1C, essentially all of the hormone was recovered after a 90-min incubation period. In contrast, incubation of TRH with frozen and thawed blood at 37 C resulted in a rapid loss of TRH. BAL (10 mM) or bensamidine (100 mM) afforded complete protection for TRH for at least 30 min at 1 C. At 37 C, protection was incomplete. Exposure of rats to cold (2C) resulted in a significant increase in serum TSH levels, but TRH was undetectable (less than3 pg) in 100 mul of blood regardless of whether the blood contained BAL (10 mM) or benzamidine (100 mM), or was frozen quickly and thawed before RIA. However, when 5-8 ml of trunk blood was extracted with methanol, 8-11 pg/ml of TRH was found, and the TRH levels were slightly but significantly elevated after cold exposure.

Gonadal steroids regulate rat anterior pituitary levels of TSH-releasing hormone- and pyroglutamyl-glutamyl-proline amide-like immunoreactivity.

The novel peptide, pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2, EEP), which has structural and immunological similarities to TRH (pGlu-His-ProNH2) has recently been shown to contribute to total TRH-like immunoreactivity (t-TRH-LI) detected in the rabbit prostate and rat and porcine anterior pituitary. This study was undertaken to determine the effects of gonadal steroids on t-TRH-LI and its components in the rat hypothalamus and pituitary. EEP-like immunoreactivity (EEP-LI) was separated from TRH-LI by ion exchange chromatography and detected by TRH RIA. Although male and female posterior pituitary and hypothalamic t-TRH-LI levels were similar, the mean t-TRH-LI in female anterior pituitaries was significantly lower than that in males, 10.3 +/- 2.9 pmol/g vs. 24.4 +/- 2.5 pmol/g (P < 0.01). Anion exchange analysis of control anterior pituitary samples distinguished two peaks of t-TRH-LI, corresponding to [125I]-TRH marker and [3H]-EEP markers. In control female anterior pituitaries EEP-LI accounted for 26.0 +/- 2.4% of t-TRH-LI, whereas in males it accounted for 43.3 +/- 5.3% of the total. Hypothalamic and posterior pituitary samples only contained t-TRH-LI corresponding to [125I]-TRH markers. There was no significant change in hypothalamic and posterior pituitary levels of t-TRH-LI after ovariectomy or orchidectomy. Anterior pituitary levels of t-TRH-LI, however, were increased by an estimated 6-fold after ovariectomy and 2-fold after orchidectomy. After ovariectomy, the proportions of t-TRH-LI accounted for by TRH-LI and EEP-LI were reversed in the female. EEP-LI now accounted for the majority of t-TRH-LI, constituting an increase of approximately 21-fold in pituitary EEP-LI levels. The changes in the levels of pituitary TRH-LI and EEP-LI induced by ovariectomy were reversed by 17-beta-estradiol. As in the ovariectomized samples EEP-LI was increased (2-fold) by orchidectomy. Both TRH-LI, which increased 1.6-fold, and EEP-LI were restored to control values after testosterone replacement. These findings confirm the hypothesis that pituitary TRH-LI and EEP-LI are regulated by gonadal status. The fact that these changes were not observed in the hypothalamus and posterior pituitary suggests that TRH-LI and EEP-LI have specific functional significance in the pituitary gland.

Identification and immunocytochemical localization of a new thyrotropin-releasing hormone precursor in rat brain.

Antisera were raised to a tridecapeptide, Ser-Asp-Val-Thr-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Phe, that was synthesized based on the sequence (residues 166-178) of a proposed cDNA for pro-TRH reported by Lechan et al. With this antiserum, immunostaining of Western blots of rat brain extracts revealed two major proteins with mol wt (Mr = 39,000 and 52,000) considerably larger than that of the largest protein (Mr = 29,000) that could be encoded by the cDNA of Lechan et al. Because these observations suggested the possibility of novel TRH precursors, we studied the immunocytochemical distribution of pro-TRH (39-52K) in rat brain. Our anatomical findings were 4-fold. 1) The distributions of 29K pro-TRH and 39-52K pro-TRH are not identical. 2) TRH is found only in regions containing 29K pro-TRH, 39-52K pro-TRH, or both. 3) There are regions that contain both 29K pro-TRH and 39-52K pro-TRH, but no TRH. 4) Regions containing only 39-52K pro-TRH do not contain 29K pro-TRH mRNA as mapped by Segerson et al. From these electrophoretic and anatomical observations, we postulate the existence of at least one and possibly two additional precursors that can be processed to TRH in rat brain.

Evidence of thyrotropin-releasing hormone activity in autopsy pancreata from newborns.

Pancreata, obtained from infants who had died during the neonatal period, were pooled and extracted with methanol. The extracts were submitted to cation exchange and high pressure liquid chromatography. The fractions were assayed for TRH by RIA and a mouse bioassay. The results indicate that the pancreata of newborns contain TRH.