Thyrotropin-releasing Hormone Metabolism Research Articles

Marginal involvement of pyroglutamyl aminopeptidase I in metabolism of thyrotropin-releasing hormone in rat brain.

On thyrotropin-releasing hormone (TRH) metabolism, pyroglutamyl aminopeptidase II (PAP-II), a zinc-dependent ectoenzyme primarily located in the central nervous system, is believed to play a predominant role. Recently we cloned pyroglutamyl aminopeptidase I (PAP-I) which is known for specifically removing a L-pyroglutamate (L-pGlu) residue from the amino terminus of proteins and peptides including TRH. To investigate possible contribution of PAP-I toward TRH metabolism, we conducted biochemical and immunohistochemical characterization using recombinant rat, mouse and human PAP-Is and an antibody raised against rat PAP-I. The Km values toward TRH by the recombinant PAP-Is were about 0.05 mM, being similar value to the reported value of recombinant PAP-II. The L-pGlu-cleaving activities toward TRH in rat brain homogenate were inhibited by a PAP-II specific inhibitor 1,10-phenanthroline, but not inhibited by the antibody against rat PAP-I. Immunohistochemical study in rats revealed heterogeneous distribution of PAP-I in the pituitary, the target tissue of TRH, but the distribution was cytosolic. Taken together, these results suggested that PAP-I might not be dominantly involved in the degradation of TRH in rats. Additionally, we found that PAP-I was localized in the renal proximal tubules. Further investigations are needed for elucidating the function of PAP-I in these restricted sites.

Chronic ingestion of ethanol or glucose solutions affects hypothalamic and limbic TRH metabolism in dams and their pups

The effect of chronic ethanol consumption during pregnancy and lactation on thyrotropin releasing hormone (TRH) metabolism was investigated in the hypothalamus and limbic areas of female rats and their weaned pups. Pregnant female rats received ethanol or isocaloric glucose solution during pregnancy either alone, or also during the 3 weeks of lactation. Thyrotropin (TSH) and corticosterone levels were measured in serum; TRH and TRH-gly concentrations were determined in hypothalamus, hippocampus, n.accumbens, frontal cortex and amygdala of dams and pups at 21 days after parturition. Ethanol or glucose consumption during pregnancy and lactation produced a decrease in TSH levels compared with control animals fed at libitum; water replacement during lactation normalized TSH levels only in glucose-fed dams. Pups from ethanol or pair-fed dams showed low weight and increased TSH levels compared with normal rats. Variations in TRH metabolism were detected in limbic areas. Chronic ethanol caused a decrease in the levels of TRH in the hippocampus and frontal cortex of dams. In contrast, glucose chronic ingestion increased TRH content specifically in n.accumbens and amygdala of dams. Most of the variations in TRH content of limbic areas of pups were not specific for glucose or ethanol treatment and correlated with the deleterious effect of the mother’s thyroid condition, although some differences were observed depending on pup’s gender. These results support the involvement of TRHergic neurons in the limbic system of the female rat exposed to alcohol or glucose during pregnancy and lactation.

El colesterol de la dieta modifica la actividad piroglutamil aminopeptidasa de la corteza frontal del ratón. Diferencias sexuales

Pyroglutamyl aminopeptidase (pGluAP) is an omega peptidase widely distributed in fluid and tissues, which hydrolyses biological active peptides including thyrotropin-releasing-hormone (TRH). In the last years, different endocrine and extraendocrine functions have been attributed to TRH. The aim of present work is to study the influence of high dietary cholesterol on soluble and membrane bound pGluAP activity in frontal cortex of male and female mice. Material and methods. Soluble and membrane bound pGluAP activities of frontal cortex of mice feeding during 15 days, a standard diet enriched with cholesterol (1%) and cholic acid (0.5%) were measured using fluorimetric assays. Significant increases were observed in membrane bound pGluAP activity in males and females. No changes were detected in soluble pGluAP activity in frontal cortex of females but a significant increase was observed in this enzymatic activity in males. High dietary cholesterol induces a significant increase in tissue pGluAP activity. These increases may produce changes in TRH metabolism which may be related with alterations in its neuromodulatory functions and with the possible relationship between TRH and other neurotransmitter systems.

Orexis, anorexia, and thyrotropin-releasing hormone.

The hypothalamus, long known to play a determinant role in food intake and satiety, has recently been shown to exert this homeostatic function via peptidergic neuronal circuits. The major peptide that has been identified as orexigenic, namely neuropeptide Y (NPY), is suppressed by leptin, an adipocyte-derived hormone, in a potential circuit that seems to function as an adipostat. Information regarding energy balance is fed back to the paraventricular nucleus of the hypothalamus where a complex interplay between thyrotropin-releasing hormone (TRH) and corticotrophin-releasing hormone (CRH) determines consequent effects in thermogenesis and stress reactions. Inflammatory mediators that have been implicated in anorexia simultaneously suppress TRH in a dominant way that overcomes the feedback effects of the thyroid hormones. Moreover, endogenous opioids and melanotropic peptides modulate orexigenic and thermogenic effects in a complex, yet poorly understood, way. However, TRH metabolism, which is affected by dietary modifications, seems to be involved in the orexigenic events that take place in the hypothalamus. It is, therefore, evident that TRH is directly involved in the complex hypothalamic networks that establish energy balance by modulation of food intake, satiety, thermogenesis, and other autonomic responses.

Limited proteolysis and physiological regulation: an example from thyrotropin-releasing hormone metabolism.

Proteases like trypsin, elastase, and many others play important regulatory functions by generating new biologically active molecules through limited proteolysis of larger proteins and peptides. The limited proteolysis of thyrotropin-releasing hormone (TRH) by Pyroglutamate aminopeptidase yields cyclo(His-Pro) or CHP, a new biopeptide associated with a variety of pharmacological activities, including regulation of body temperature, inhibition of prolactin secretion, and modulation of motor functions. Although the mechanism by which CHP elicits these biological activities is not well understood, it appears that the cyclic peptide may function at least in part by modulating central amine transport mechanisms.

Multifactorial modulation of TRH metabolism.

1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through median eminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary. 2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell. 3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure. 4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times. TRH mRNA are thus controlled by transsynaptic and hormonal influences. 5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglutamyl peptidase II (PPII). 6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity. 7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.

Transfer and metabolism of thyrotropin releasing hormone across the perfused human term placenta.

The transport and uptake of TRH was investigated in the maternal-fetal-placental unit of perfused human term placenta. The degradation of TRH in biological fluid was first determined by incubating [125I]TRH with 100 microL 50% maternal or cord sera with or without pretreatment with 200 microM of p-hydroxymercuriphenyl sulfonic acid (p-HMSA), a proline dipeptidase inhibitor. Transplacental transfer of TRH was then studied by adding 10 microCi of [125I]- or [3H]TRH to the maternal circulation of dually perfused isolated lobule of human term placenta with or without 200 microM p-HMSA. Creatinine was used as an internal marker. The rate of degradation of TRH (P < 0.001) and inhibition by p-HMSA were significantly higher in maternal than cord sera (P < 0.05). In the maternal circulation, TRH concentration declined rapidly from 100% at time 0 to 33.5 +/- 1.2% at 120 min. The fetal concentration increased from undetectable levels to a maximum of 1.8 +/- 0.3% at 120 min with a low feto-maternal ratio (0.08 +/- 0.02). Perfusion in the presence of p-HMSA, however, did not significantly change fetal concentration, or the maternal and fetal concentration-time integral levels of TRH. Chromatography of maternal, fetal, and placental homogenates showed that TRH was metabolized by the placenta into small molecular weight fragments predominantly released in the maternal circulation. These results suggest that human placenta acts as an enzymatic barrier to the free passage of TRH.

Transfer and metabolism of thyrotropin releasing hormone across the perfused human term placenta

Transfer and metabolism of thyrotropin releasing hormone across the perfused human term placenta

Alterations in Local Cerebral Glucose Metabolism and Endogenous Thyrotropin-Releasing Hormone Levels in Rolling Mouse Nagoya and Effect of Thyrotropin-Releasing Hormone Tartrate

To identify the brain region(s) responsible for the expression of ataxic gaits in an ataxic mutant mouse model, Rolling mouse Nagoya (RMN), changes in local cerebral glucose metabolism in various brain regions and the effect of thyrotropin-releasing hormone tartrate (TRH-T), together with alterations in endogenous thyrotropin-releasing hormone (TRH) levels in the brains of RMN, were investi gated. Ataxic mice [RMN (rol/rol)] showed significant decreases in glucose metabolism in regions of the diencephalon: thalamic dorsomedial nucleus, lateral geniculate body and superior colliculus; brain stem: substantia nigra, raphe nucleus and vestibular nucleus; and cerebellar nucleus as compared with normal controls [RMN (+/+)]. When RMN (rol/rol) was treated with TRH-T (10 mg/kg, equivalent to 7 mg/kg free TRH), glucose metabolism was significantly increased in these regions. These results suggest that these regions may be responsible for ataxia. We also found that TRH levels in the cerebellum and brain stem of RMN (rol/rol) were significantly higher than those of RMN (+/+). These results suggest that ataxic symptoms in RMN (rol/rol) may relate to the abnormal metabolism of TRH and energy metabolism in the cerebellum and/or brain stem and that exogenously given TRH normalizes them.

Influence of thyroid status on TRH metabolism in rat olfactory bulb

The effect of thyroid hormones (TH) on the metabolism of thyrotropin-releasing hormone (TRH) in the olfactory bulb (OB) was compared with the hypothalamic response to TRH. Two methods were used to induce hypothyroidism: propylthiouracyl-methimazole (PTU-M) or 131I treatment. Hyperthyroidism was produced by 3,3′,5-triiodo- l-thyronine (T 3) injections to the hypothyroid animals. With PTU-M treatment, paraventricular TRH mRNA levels increased 57% and returned to the euthyroid level with T 3 treatment. In OB, TRH mRNA was not altered. The TRH content was unaffected in the mediobasal hypothalamus of PTU-M-treated animals whereas it was reduced in OB (31%) with no further response upon T 3 treatment. 131I-induced hypothyroidism did not modify the OB TRH content but it was decreased (31%) in hyperthyroids. In the median eminence, TRH increased 26% in hypothyroids, and the response was reversed with T 3. Our results demonstrate that treatments that change thyroid status can alter TRH levels in the OB, probably at a translational or postranslational level, though the effects may be pharmacological.

A fast protein liquid chromatography (FPLC) method for study of thyrotropin-releasing hormone (TRH) and its metabolite histidyl-proline diketopiperazine (CHP) in human blood: Degradation in liver and pancreatic diseases

We have developed a convenient method combining fast protein liquid chromatography (FPLC) with sensitive radioimmunoassay (RIA) for thyrotropin-releasing hormone (TRH) to separate and identify TRH and its metabolite histidyl-proline diketopiperazine (CHP) and applied this to study inactivation of TRH by blood extracts from patients with liver cirrhosis (LC) and acute edematous pancreatitis (AP). Blood samples spiked with TRH and CHP were extracted by cold methanol and injected on a reverse-phase FPLC column. A linear gradient was applied for separation. Subsequent analyses of fractions by RIA for TRH revealed that only fractions 9–10 contained TRH. Separation by retention time (9.9 ± 0.8 min for TRH, 10.5 ± 0.6 min for CHP, mean ± SEM) was highly reproducible. For degradation studies, pooled sera from patients with LC and AP were incubated with TRH and CHP for 60 min. Inactivation of TRH was less rapid in the presence of blood extract from LC patients than that from normal subjects or AP patients. CHP was more stable than TRH. These data suggest that activity of TRH-degrading enzymes is reduced in liver disease, whereas it does not appear to be altered in AP. Degradation of CHP does not closely reflect metabolic processing of its major precursor. This rapid and sensitive method may be applicable for further investigations on the metabolism of TRH in organic fluids.

Transport of thyrotropin releasing hormone in rabbit buccal mucosa in vitro.

The transport of thyrotropin releasing hormone (TRH) in rabbit buccal mucosa in vitro has been investigated with respect to (a) rate and type of metabolism of TRH on mucosal and serosal sides of buccal mucosa, (b) mechanism of TRH transport including charge effect on its permeability, and (c) pathway and rate-limiting regions of TRH movement. In addition, the integrity of excised buccal mucosa has been evaluated for purposes of in vitro solute diffusion experiments using tissue ATP level data, transmission electron microscopy, and TRH transport kinetic data. The results indicate that excised rabbit buccal mucosa can be used for TRH diffusion studies for approximately 6 hr. In addition, TRH apparently traverses buccal mucosa by simple diffusion with a steady-state permeability of about 10(-7) cm/sec, and this permeability is independent of pH. Moreover, the primary pathway appears to be via the intercellular space in the rate-limiting barrier, i.e., the upper 50 microns of the epithelium. Finally, TRH is degraded predominantly by deamidase activity, which is followed by, to a lesser degree, carboxypeptidase metabolism.

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

TRH-Induced Tachyphylaxis: Present in the Smooth Muscle but Not in the Cardiorespiratory Effects

The development of tachyphylaxis to the pressor, tachycardiac, ventilatory and smooth muscle effects of thyrotropin-releasing hormone (TRH) was studied. Subsequent administrations of increasing doses of TRH (0.1-1000 nmol/kg intracerebroventricularly), at 20 min. intervals in urethane-anesthetized rats dose-dependently increased the mean arterial blood pressure, heart rate and minute ventilation volume. At all doses the effect on mean arterial blood pressure was apparent at least 20 min. after each injection whereas at low doses (0.1-1 nmol/kg) the duration of the effects on heart rate or minute ventilation volume were shorter. The cardiorespiratory effects of TRH 10 nmol/kg intracerebroventricularly did not subside completely within an observation period of 1 hr. However, a repeated administration of TRH at 1 hr resulted in the levels of mean arterial blood pressure, heart rate and minute ventilation volume not statistically different from the peak levels of the first injection. While the cardiorespiratory effects of TRH were long-lasting, the TRH induced inhibition of the contractions of the rat duodenal smooth muscle lasted only 12-18 sec. The TRH-treated duodenal preparation responded normally to noradrenaline but remained unresponsive to further administrations of TRH until washed extensively. When the medium after cessation of the TRH response was transferred into another chamber with an untreated preparation, a full response was resulted. Inhibitors of TRH metabolism, bacitracin, EDTA and iodoacetamide, did not prolong nor enhance the response. Likewise, a stable TRH-analogue MK-771 did not produce any longer response than TRH. It is concluded that no tachyphylaxis was produced to the neurally mediated pressor, tachycardiac or ventilatory effects of TRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Some Events of Thyrotropin-Releasing Hormone Metabolism Are Regulated in Lactating and Cycling Rats

Levels of thyrotropin-releasing hormone (TRH), TRH mRNA and pyroglutamyl peptidase II were analyzed in the hypothalamus-adenohypophyseal axis during lactation and estrous cycle. Mediobasal hypothalamic levels of TRH dropped 41% (p less than 0.01) from pregnancy levels (taken as 100%) on the first day of lactation, recovering until day 15 to the values observed at pregnancy. A sharp decrease was also observed during weaning (36%, p less than 0.01 compared to last day of lactation). TRH levels in the neurohypophysis increased during lactation and dropped at weaning. Highest TRH mRNA levels in the paraventricular nucleus were found at the end of pregnancy and beginning of lactation; they decreased 37% (p less than 0.05) at day 5 of lactation and stayed constant thereafter. Pyroglutamyl peptidase II adenohypophyseal activity was not modified during lactation but changed during estrous cycle. Relative to estrous values, activity diminished 58% (p less than 0.05) at 10.00 h (57% at 14.00 h) during diestrus 2 and 27% at 10.00 h (37% at 14.00 h) during proestrus. Hypothalamic TRH mRNA levels fluctuated in an opposite manner to adenohypophyseal pyroglutamyl peptidase II during the estrous cycle with a peak at diestrus 2: 183% of the estrous value (p less than 0.05). These data point to a regulation of TRH metabolism in conditions where prolactin (PRL) secretion fluctuates. They also suggest a sharp release of TRH between the end of pregnancy and the first day of lactation and that translational efficiency or post-translational processing of TRH precursor in the paraventricular neurons (projecting to the median eminence) increases during lactation and drops at weaning, concomitantly with PRL secretion.

Exogenous Triiodothyronine Lowers Thyrotropin-Releasing Hormone Concentrations in the Specific Hypothalamic Nucleus (Paraventricular) Involved in Thyrotropin Regulation and also in Posterior Nucleus

Recent evidence indicates that thyroid hormones can regulate thyrotropin secretion in vivo in part by inhibiting thyrotropin-releasing hormone (TRH) secretion itself. Therefore, to explore whether triiodothyronine (T3) interacts with the specific hypothalamic area involved in thyrotropin (TSH) secretory regulation, the paraventricular nucleus (PVN), Palkovitz micropunches from eight nuclear regions were obtained from 1,000-microns frozen coronal brain slices for immunoassay determinations of TRH. Rats were treated either with parenteral L-T3 for 6 days to induce experimental thyrotoxicosis, or 0.15 M saline. The induction of thyrotoxicosis was confirmed by demonstrating that mean plasma TSH concentrations fell from 108 to less than 10 microU/ml (p less than 0.01). TRH concentrations in the PVN were reduced concomitantly after L-T3 from 1.9 to 1.1 ng/mg protein (p less than 0.05). No reductions in TRH concentrations during T3 treatment occurred in other nuclear groupings except in the posterior hypothalamic nucleus. Total TRH content in the median eminence declined also in T3-treated animals from 1.77 to 1.29 ng, representing a 32% reduction (p less than 0.01). No significant change was seen in the median eminence content of the TRH structurally related dipeptide, cyclo(His-Pro). The data herein indicate that experimental thyrotoxicosis in the rat is associated with a selective reduction in TRH concentrations in the PVN, documenting T3 effects upon hypothalamic TRH metabolism per se.

Pyroglutamate aminopeptidase activity in human cerebrospinal fluid decreases with age

The activity of pyroglutamate aminopeptidase, the major enzyme catalyzing thyrotropin-releasing hormone (TRH) metabolism in human CSF, decreased with age. This decrement is not due to age-dependent appearance of any enzyme inhibitor in CSF. The results of these studies underline the importance of using age-matched controls in assessing abnormalities of TRH metabolism in CSF during disease states.

Thyrotropin-releasing hormone metabolism is attenuated in the cerebrospinal fluid of the human neonate

The present study was designed to investigate the ontogeny of thyrotropin-releasing hormone (TRH) metabolism in human cerebrospinal fluid (CFS). The activity of pyroglutamate aminopeptidase (EC 3.4.11.8), the major enzyme catalyzing TRH metabolism in human CSF, was measured in CSF of 11 premature infants (gestational age, 29–39 weeks; birth weight, 1774 ± 274 g), 8 newborn infants (term delivery; birth weight, 3648 ± 240 g), and 11 adults (mean age, 29.6 ± 1.5 years). Pyroglutamate aminopeptidase activity in CSF of premature and newborn infants was significantly lower (p < 0.05) than that of adult CSF. These observed differences in the enzymatic activities were not due to changes in the affinity of the enzyme for its substrate TRH or the presence of enzyme inhibitor(s)/stimulator(s).

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Thyrotropin-Releasing Hormone Metabolism and Extraction by the Perfused Guinea Pig Placenta*

This report describes the extraction of synthetic TRH and its metabolic conversion in the perfused guinea pig placenta. These studies were performed to obtain an estimate of fractional fetal TRH losses through the placenta and to determine if some of these losses are due to TRH metabolism. The in situ guinea pig placenta was perfused through an umbilical artery for 90 min, and placental effluent fractions were collected at timed intervals from the umbilical vein. Experiments were performed in which the perfusion buffer contained 0.01, 1, and 10 micrograms/ml or no synthetic TRH. Synthetic TRH was always perfused in the presence of 3H2O. In experiments in which TRH was perfused, the perfusion reservoir contents and placental effluent fractions were counted for 3H, and TRH and deamido-TRH were determined by RIA. Similarly, cyclo(His-Pro) was measured when 10 micrograms/ml TRH were perfused. When no TRH was perfused, the perfusion reservoir and placental effluent contents were processed to determine their content of TRH immunoreactivity. When synthetic TRH was perfused, steady state TRH concentrations were achieved in placental effluent fractions by 20-30 min. The single pass extraction of TRH by the placenta was 11.4 +/- 2.6% (mean +/- SE) compared to 56.9 +/- 7.0% for 3H2O (P less than 0.001). No significant difference was detected regardless of whether 10, 1, or 0.01 micrograms/ml TRH were perfused. A portion of the TRH that perfused the placenta was converted to deamido-TRH at all concentrations of perfused TRH. No conversion of TRH to cyclo(His-Pro) was noted when the highest concentration (10 micrograms/ml) of TRH was perfused. The conversion of TRH to TRH-OH was 4.2 +/- 0.7% in a single pass. When the perfusion buffer was devoid of synthetic TRH, a small but significant increase in the content of TRH immunoreactivity was noted in the placental effluent compared to that in the perfusion reservoir. This was not large enough to affect calculations of the placental extraction of TRH. These studies, in addition to demonstrating that the placenta contains TRH deamidase activity, suggest that losses of fetal TRH through the placenta are not large. They do not support the current impression, based on the fetal TSH response to maternal TSH administration, that the placenta is freely permeable to TRH.