Pro-TRH-connecting peptides in the rat pancreas during ontogenesis

Rat thyrotropin-releasing hormone prohormone (pro-TRH) is a protein containing five copies of TRH, separated by connecting peptides. We have recently developed radioimmunoassays to synthetic peptides corresponding to prepro-TRH(160-169) and prepro-TRH(178-199). In the present study we have used these assays to investigate the ontogenesis of pro-TRH-derived peptides in the rat pancreas. Reverse-phase HPLC analysis of pancreatic extracts from 2-day-old rats showed the presence of two major immunoreactive peptides exhibiting the same retention time as synthetic prepro-TRH(160-169) and prepro-TRH(178-199), respectively. The concentrations of TRH and pro-TRH cryptic peptides in the rat pancreas rose rapidly after birth, reached a maximum at day 2-4 and decreased gradually afterwards. Streptozotocin treatment of newborn rats induced a marked decrease of TRH (96%), prepro-TRH(160-169) (97%) and prepro-TRH(178-199) content (94%) in pancreatic extracts. These results indicate that the evolution of TRH and pro-TRH-derived peptides follows the same pattern during the postnatal period. Our results also suggest that beta-cells are the only source of pro-TRH-derived peptides in the rat pancreas.     

Specific processing of the thyrotropin-releasing prohormone in rat brain and spinal cord

Thyrotropin-releasing hormone (TRH) and TRH extended peptides were extracted from rat hypothalamus and spinal cord and resolved by gel exclusion chromatography under dissociating conditions. Peptides related to TRH were detected by trypsin digestion and radioimmunoassay with an antibody to TRH or an antibody raised against the pentapeptide Glp-His-Pro-Gly-Lys. In addition to the tripeptide hormone a series of C-terminally extended forms of TRH was shown to occur in both tissues; no N-terminally extended peptides were detected. The structure of the TRH-related peptides was confirmed by chromatographic identification of the N-terminal pentapeptide sequence released by trypsin. The TRH extended peptides, which accounted for 15-20% of the total TRH, were present in three groups of different molecular size corresponding to predicted fragments of the TRH prohormone. One of the peptides in the spinal cord was identified by chromatographic comparison with a synthetic 16-residue peptide representing residues 154-169 of the prohormone. In the spinal cord the TRH extended peptides differed in their relative concentrations from the corresponding peptides in the hypothalamus, possibly reflecting differences in processing. The finding of extended forms of TRH in which the extension occurs only on the C-terminal side of the hormone sequence shows that the prohormone undergoes highly specific processing.     

Processing of the thyrotropin releasing hormone (TRH) precursor in Xenopus skin and bovine hypothalamus: evidence for the existence of extended forms of TRH

Acid extracts of Xenopus laevis skin were fractionated by gel filtration on Sephadex G50 ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC). Peptides related to thyrotropin releasing hormone (TRH) were identified in the eluted fractions by trypsin digestion and radioimmunoassay (RIA) using antibodies to the TRH tripeptide pGlu-His-Pro amide or to a TRH-related pentapeptide pGlu-His-Pro-Gly-Lys. In addition to the tripeptide hormone, evidence was obtained for the presence of peptides containing 10-20 amino acid residues which were extended on the NH2-terminal or COOH-terminal side of TRH. The peptides extending on the NH2-terminal side predominated and were shown to comprise 5 components present in differing concentrations, indicating that the processing sites in the TRH prohormone vary in their susceptibility to proteolysis. Evidence was also obtained for the presence of small amounts of the TRH-related pentapeptide pGlu-His-Pro-Gly-Lys. Using similar procedures it was demonstrated that TRH extended peptides were present in bovine hypothalamus. In this species the peptides extended at the NH2-terminus of TRH occurred in similar concentrations to the peptides extended at the COOH-terminus. The results show that processing of the TRH prohormone in Xenopus and ox leads to the formation of peptides intermediate in size between the prohormone and the tripeptide amide; the TRH extended peptides occur in significant quantity and in Xenopus are formed with a high degree of specificity.     

Characterization of receptors for thyrotropin-releasing hormone-potentiating peptide on rat anterior pituitary membranes.

Previous studies (Bulant, M., Delfour, A., Vaudry, H., and Nicolas, P. (1988) J. Biol. Chem. 263, 17189-17196; Bulant, M., Roussel, J. P., Astier, H., Nicolas, P., and Vaudry, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4439-4443) have shown that post-translational processing of rat thyrotropin-releasing hormone prohormone (pro-TRH) generates, besides thyrotropin-releasing hormone (TRH), a connecting decapeptide corresponding to prepro-TRH-(160-169), i.e. Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr. This peptide, which is named TRH-potentiating peptide (Ps4), is co-localized with TRH in the median eminence nerve endings and is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo. To characterize the receptor(s) for TRH-potentiating peptide in the pituitary, a highly potent and metabolically stable derivative of Ps4, [I-Tyr0]Ps4, was radioiodinated. Binding of [125I-Tyr-0]Ps4 to rat pituitary membrane homogenates was specific, saturable, reversible, and linear with membrane protein concentration. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high affinity binding sites (Kd = 0.22 nM; Bmax = 517 fmol/mg of membrane proteins). Several naturally occurring neuropeptides and hormones, including TRH, did not compete with [125I-Tyr0]Ps4 in the binding, which suggests the binding sites are specific to Ps4. Using C-terminal deletion analogs of [Tyr0]Ps4, we further showed the critical role the C-terminal residues Thr10, Val9, and Asp8 play in conferring high binding affinity and selectivity. Binding site tissue distribution and cross-reactivity binding studies suggest that the action of TRH-potentiating peptide is mediated through interaction with a specific pituitary cell-surface receptor which differ from those for TRH. [I-Tyr0]Ps4 reported in this paper, through its high binding affinity and specificity, its very low nonspecific binding, its high resistance to enzymatic degradation, and its high potentiating action in vitro should allow further progress in understanding the in vivo physiological function of Ps4.     

Effect of precipitated morphine withdrawal on post-translational processing of prothyrotropin releasing hormone (proTRH) in the ventrolateral column of the midbrain periaqueductal gray

We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH-derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH-containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine-treated rats 24 h following naltrexone-precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post-translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH(83-106) peptide from the N-terminal side of the prohormone, while the levels of the C-terminal preproTRH(208-285) peptide were reduced, with no change in preproTRH(25-50) or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH(83-106) peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region-specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH-derived peptides in the manifestations of opiate withdrawal.     

Cellular colocalization and coregulation between hypothalamic pro-TRH and prohormone convertases in hypothyroidism

The prohormone convertases (PCs), PC1/3 and PC2, are involved in the tissue-specific endoproteolytic posttranslational processing of many hormonal precursors within the secretory pathway. One important prohormone, pro-thyrotropin-releasing hormone (TRH), is expressed in both hypophysiotropic (where it regulates the secretion of thyroid-stimulating hormone) and nonhypophysiotropic regions of the brain. Pro-TRH is processed at specific sites in the secretory pathway, primarily by PC1/3 followed by PC2. We hypothesized that thyroid hormone status in specific nuclei of the brain would alter pro-TRH processing by inducing changes in PC1/3 and PC2 expression. Therefore, we examined pro-TRH, PC1/3, and PC2 coexpression and coregulation in the paraventricular nucleus (PVN), lateral hypothalamus (LH), and ventromedial nucleus (VMN) of hypothyroid and euthyroid rats. Our results show that 6-n-propyl-2-thiouracil (PTU) treatment producing hypothyroidism induced a significant increase in the expression of PC1/3, PC2, and pro-TRH in the PVN and LH, but not VMN. When confocal studies were performed, an increase in colocalization of PC1/3 or PC2 in pro-TRH was observed only in PVN, a response that was especially prominent in the ventral and medial areas of the PVN. PTU did not regulate colocalization in the VMH or LH. Regulation of colocalization of processing enzyme and prohormone expression is a novel mechanism to alter hormonal biosynthesis.     

Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice

Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.     

Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the trans-Golgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.     

Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited <0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.     

Neuroanatomical connections between corticotropin-releasing factor (CRF) and somatostatin (SRIF) nerve endings and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus.

In order to investigate the possible involvement of corticotropin-releasing factor (CRF) and somatostatin (SRIF) on thyrotropin-releasing hormone (TRH) neuronal cell activity in the rat hypothalamic paraventricular nucleus, we have proceeded to the simultaneous localization of CRF or SRIF and TRH. For this purpose, we used a dual immunostaining procedure that employed antibodies to CRF and SRIF and peroxidase-labeled goat anti-rabbit IgG as a first sequence, and antibodies to a cryptic fragment (Phe178-Glu199) of pro-TRH (to label TRH neurons) and alkaline phosphatase-labeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive CRF and SRIF fibers was observed. A large number of CRF and SRIF nerve endings were seen intimate anatomic proximity and often appeared to surround TRH-containing cell bodies. These results strongly suggest that TRH neurons might be regulated by both CRF and SRIF. These interactions might be the neuroanatomical basis for the already observed inhibitory effects of CRF and SRIF on TRH release.     

Regional distribution of in vitro release of thyrotropin releasing hormone in rat brain

To increase our knowledge of the TRH functions in brain and the processes of TRH compartmentalization and release, we studied the in vitro release of endogenous TRH in different brain areas. We also determined the correlation between TRH levels and release under both basal and stimulated conditions. TRH concentration was measured in tissues and media by specific radioimmunoassay. TRH-like material detected in olfactory bulb and hypothalamic incubates (basal or K+ stimulated) were shown to be chromatographically identical to synthetic TRH. Different brain regions showed high variability in the basal release of TRH (1-20% of tissue content). This suggests the existence of different pools. The response to depolarizing stimulus (56 mM K+) was significant only in the following regions: median eminence, total hypothalamus, preoptic area, nucleus accumbens-lateral septum, amygdala, mesencephalon, medulla oblongata and the cervical region of the spinal cord. These regions have been shown to contain a high number of receptors, a high concentration of TRH nerve endings and are susceptible to TRH effects. These results support the hypothesis that TRH functions as neuromodulator in these areas.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Biosynthesis of proTRH-derived peptides in prohormone convertase 1 and 2 knockout mice

Prohormone convertases (PCs) 1 and 2 are the primary endoproteases involved in the post-translational processing of proThyrotropin Releasing Hormone (proTRH) to give rise to TRH and other proposed biologically active non-TRH peptides. Previous evidence suggests that PC1 is responsible for most proTRH cleavage events. Here, we used the PC1 and PC2 knockout (KO) mouse models to examine the effects of PC1 or PC2 loss on proTRH processing. The PC1KO mouse presented a decrease in five proTRH-derived peptides, whereas the PC2KO mouse showed only lesser reduction in three TRH (Gln-His-Pro), TRH-Gly (Gln-His-Pro-Gly), and the short forms preproTRH(178-184) (pFQ(7)) and preproTRH(186-199) (pSE(14)) of pFE(22) (preproTRH(178-199)). Also, PC1KO and not PC2KO showed a decrease in pEH(24) indicating that PC1 is more important in generating this peptide in the mouse, which differs from previous studies using rat proTRH. Furthermore, downstream effects on thyroid hormone levels were evident in PC1KO mice, but not PC2KO mice suggesting that PC1 plays the more critical role in producing bioactive hypophysiotropic TRH. Yet loss of PC1 did not abolish TRH entirely indicating a complementary action for both enzymes in the normal processing of proTRH. We also show that PC2 alone is responsible for catalyzing the conversion of pFE(22) to pFQ(7) and pSE(14), all peptides implicated in regulation of suckling-induced prolactin release. Collectively, results characterize the specific roles of PC1 and PC2 in proTRH processing in vivo.     

Identification, characterization and localization of thyrotropin-releasing hormone precursor peptides in perinatal rat pancreas

The sequence of rat hypothalamic prepro TRH, deduced from its complementary DNA, contains five TRH progenitor sequences and six cryptic sequences separated by paired basic amino acid residues. We have utilised antisera against two synthetic peptides corresponding to sequences within proTRH, [Tyr53] preproTRH (53-74), part of the amino terminal leader sequence of proTRH and [Cys 74,83] preproTRH-(75-82), representing a TRH progenitor sequence flanked by cysteine residues (pCC10) in radioimmunoassays (RIA) to identify and chromatographically characterize proTRH derived peptides in extracts of rat perinatal pancreas and to localize these peptides immunohistochemically. Two forms of immunoreactive pYT22 (ipYT22) were observed, similar in size to ipYT22 seen in extracts of adult rat brain. By RIA immunoreactive pCC10 was detectable in neonatal but not fetal pancreas. However, immunohistochemical double staining of both fetal and neonatal rat pancreas colocalized both ipYT22 and ipCC10 with immunoreactive insulin in the B-cell of the developing Islets of Langerhans. These findings indicate that the B-cell of the perinatal pancreas synthesizes TRH from a prohormone encoded by a mRNA similar to that present in adult rat hypothalamus.     

Differential response of TRHergic neurons of the hypothalamic paraventricular nucleus (PVN) in female animals submitted to food-restriction or dehydration-induced anorexia and cold exposure

TRH neurons of the hypothalamic paraventricular nucleus (PVN), regulate pituitary-thyroid axis (HPT). Fasting activates expression of orexigenic peptides from the arcuate nucleus, increases corticosterone while reduces leptin, and pro-TRH mRNA levels despite low serum thyroid hormone concentration (tertiary hypothyroidism). TRH synthesis is positively regulated by anorexigenic peptides whose expression is reduced in fasting. The model of dehydration-induced anorexia (DIA) leads to decreased voluntary food intake but peptide expression in the arcuate is similar to forced-food restriction (FFR), where animals remain hungered. We compared the response of HPT axis of female Wistar rats submitted to DIA (2.5% saline solution, food ad libitum, 7 days) with FFR (provided with the amount of food ingested by DIA) and na?ve (N) group fed ad libitum, as well as their response to acute cold exposure. Pro-TRH and pro-CRH mRNA levels in the PVN were measured by RT-PCR, TRH content, serum concentration of TSH and thyroid hormones by radioimmunoassay. DIA rats reduced 80% their food consumption compared to N, decreased PVN pro-CRH expression, serum estradiol and leptin levels, increased corticosterone similar to FFR. HPT axis of DIA animals failed to adapt: FFR presented tertiary hypothyroidism and DIA, primary. Response to cold stimulation leading to increased pro-TRH mRNA levels and TRH release was preserved under reduced energy availability in FFR rats but not in DIA, although the dynamics of hormonal release differed: TSH release augmented only in na?ve; thyroxine in all but highest in DIA, and triiodothyronine in FFR and DIA suggesting a differential regulation of deiodinases.     

Prothyrotropin-releasing hormone targets its processing products to different vesicles of the secretory pathway

Prothyrotropin-releasing hormone (pro-TRH) is initially cleaved by the prohormone convertase-1/3 (PC1/3) in the trans-Golgi network generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product.     

Leptin regulates prothyrotropin-releasing hormone biosynthesis. Evidence for direct and indirect pathways

The hypothalamic-pituitary-thyroid axis is down-regulated during starvation, and falling levels of leptin are a critical signal for this adaptation, acting to suppress preprothyrotropin-releasing hormone (prepro-TRH) mRNA expression in the paraventricular nucleus of the hypothalamus. This study addresses the mechanism for this regulation, using primary cultures of fetal rat hypothalamic neurons as a model system. Leptin dose-dependently stimulated a 10-fold increase in pro-TRH biosynthesis, with a maximum response at 10 nm. TRH release was quantified using immunoprecipitation, followed by isoelectric focusing gel electrophoresis and specific TRH radioimmunoassay. Leptin stimulated TRH release by 7-fold. Immunocytochemistry revealed that a substantial population of cells expressed TRH or leptin receptors and that 8-13% of those expressing leptin receptors coexpressed TRH. Leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter and the long form of the leptin receptor cDNA. Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, addition of alpha-melanocyte-stimulating hormone produced a 3.5-fold increase in TRH biosynthesis and release, whereas neuropeptide Y treatment suppressed pro-TRH biosynthesis approximately 3-fold. Furthermore, the melanocortin-4 receptor antagonist SHU9119 partially inhibited leptin-stimulated TRH release from the neuronal culture. Consequently, our data suggest that leptin regulates the TRH neurons through both direct and indirect pathways.     

Thyrotropin releasing hormine (TRH): distribution in the brain, blood and urine of the rat

Measurement of thyrotropin releasing hormone (TRH) in the rat by a radioimmunoassay capable of detecting 6 pg is described. TRH was found in high concentration in the hypothalamus, especially in the stalk median eminence (SME). Small but significant concentrations were also detected hroughout the extrahypothalmic brain. Quantitatively, these levels are substantial, and suggest that this tripeptide may have an extrathyroidal brain function. TRH was measurable in the blood only in low concentrations, but large amounts were excreted in the urine (18.4ng/day).     

Effect of immobilization stress on neuropeptides and their receptors in rat central nervous system

The effect of immobilization stress (IM-stress) on the concentration and the receptor binding of substance P (SP), methionine-enkephalin (ME) and thyrotropin-releasing hormone (TRH) was determined in eight brain regions and the spinal cord. The concentration of SP was decreased in the septum, striatum and hippocampus, and SP receptor binding was decreased in the septum, amygdala + pyriform cortex and hypothalamus. Scatchard analysis indicated that the decrease in the SP binding is mainly due to the decrease in the number of receptors. The concentration of ME was not changed, but ME receptor binding was decreased in the septum. The concentration of TRH was decreased in the frontal cortex, septum, amygdala + pyriform cortex and pons + medulla oblongata, but increased in the spinal cord. TRH receptor binding was decreased in the septum, amygdala + pyriform cortex and hypothalamus. Scatchard analysis indicated that the decrease in TRH binding is due to the decrease in the number of receptors. These results show that IM-stress affects the neuropeptide receptor as well as neuropeptide concentration, and that the septum is a very important region under IM-stress.