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.     

Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates pro-TRH-connecting peptides. Identification and characterization of prepro-TRH-(160-169) and prepro-TRH-(178-199) in the rat nervous system

Rat thyrotropin-releasing hormone prohormone (pro-TRH) contains five separate copies of the TRH progenitor sequence: Gln-His-Pro-Gly. Each of the five sequences is flanked by pairs of basic residues and linked together by one of several predicted connecting sequences. Two of the pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199), were detected in extracts of rat neural tissues by radioimmunoassay using antibodies directed against the corresponding synthetic probes. Endogenous prepro-TRH-(160-169) and prepro-TRH-(178-199) were purified by gel exclusion chromatography, reverse-phase high pressure liquid chromatography, and ion-exchange chromatography. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards, immunological analysis, and tryptic mapping. Equimolar amounts of these connecting fragments were observed in hypothalamus and spinal cord. Quantification of TRH in spinal cord and hypothalamus extracts revealed the presence of 4.9-6.3 mol of TRH/mol of prepro-TRH-(178-199) and 4.4-6 mol of TRH/mol of prepro-TRH-(160-169), respectively. By using the indirect immunofluorescence technique, prepro-TRH-(178-199) immunoreactive cell bodies were found in the paraventricular nucleus of the hypothalamus, and a dense plexus of immunopositive nerve terminals was observed in the external zone of the median eminence, in a distribution similar to that described for TRH. These studies demonstrate that prepro-TRH-(160-169) and prepro-TRH-(178-199) are, together with TRH, predominant storage forms of the TRH precursor in hypothalamus and spinal cord, being present in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. The presence of pro-TRH-connecting peptides in various brain regions, including the median eminence, suggests that these peptides might act as neuromodulators in the central nervous system and/or neuroendocrine signals at the pituitary level. In the olfactory lobes, prepro-TRH is processed differently since a C-terminally extended form of TRH, prepro-TRH-(172-199), is found as a major end product along with lower but significant amounts of prepro-TRH-(178-199) and prepro-TRH-(160-169). The striking difference in pro-TRH processing patterns among the various tissues examined suggests differential regulating mechanisms for TRH and/or TRH-related activities.     

Ergot alkaloids

The developmental transition of ergot from the sphacelial to the sclerotial phase is accompanied by marked changes in morphology, growth and metabolism. The production of alkaloids is paralleled by a concomitant protein synthesis, a decline in the contents of both total N and P, and an attendant rise in the value of the N/P ratio. The total level of free amino acids drops with proceeding development of the sclerotium. The most oonspicuous drop, observed at the beginning of the production phase, is due to a sharp decrease in the level of free lysine. This drop attests to profound changes in the metabolism of the parasite in the period of transition from nonproducing sphacelium to producing sclerotium. The gradually decreasing level of free proline reflects the accumulation of the alkaloids. In a parasitic culture, proline is probably supplied in sufficient amounts by the host plant and the main difference between the metabolism of fungal sphacelium and sclerotium is a different utilisation of acetyl-CoA.     

Feasibility of coronary blood flow simulations using mid-fidelity numeric and geometric models

Biomechanics and Modeling in Mechanobiology - The fractional flow reserve index (FFR) is currently used as a gold standard to quantify coronary stenosis’s functional relevance. Due to its...     

Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis.

Thyrotropin-releasing hormone receptor (TRHR) has already been cloned in mammals wherethyrotropin-releasing hormone (TRH) is known to act as a powerful stimulator of thyroid-stimulating hormone (TSH) secretion. The TRH receptor of amphibians has not yet been characterized, although TRH is specifically important in the adaptation of skin color to environmental changes via the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH). Using a dege-nerate PCR strategy, we report on the isolation of three distinct cDNA species encoding TRHR from the brain of Xenopus laevis. We have designated these as xTRHR1, xTRHR2 and xTRHR3. Analysis of the predicted amino acid sequences revealed that the three Xenopus TRHRs are only 54-62% identical and contain all the highly conserved residues constituting the TRH binding pocket. Amino acid sequences and phylogenetic analysis revealed that xTRHR1 is a member of TRHR subfamily 1 and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype awaiting discovery in other animal species. The three Xeno-pus TRHRs have distinct patterns of expression. xTRHR3 was abundant in the brain and much scarcer in the peripheral tissues, whereas xTRHR1 was found mainly in the stomach and xTRHR2 in the heart. The Xenopus TRHR subtype 1 was found specifically in the intestine, lung and urinary bladder. These observations suggest that the three xTRHRs each have specific functions that remain to be elucidated. Expression in Xenopus oocytes and HEK-293 cells indicates that the three Xenopus TRHRs are fully functional and are coupled to the inositol phosphate/calcium pathway. Interestingly, activation of xTRHR3 required larger concentrations of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation.     

Isolation and amino acid sequence of the TRH-potentiating peptide from bovine hypothalamus.

A neuropeptide termed TRH-potentiating peptide, which potentiates TRH-evoked thyrotropin secretion by antehypophysis in vitro, was isolated from an acetonic powder of bovine hypothalamus. The peptide was purified to homogeneity by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography and reverse phase high performance liquid chromatography. The complete amino acid sequence of the decapeptide was determined as Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr by automated Edman degradation with a solid-phase sequencer. Bovine TRH-potentiating peptide is structurally identical to Ps4, a decapeptide which was deduced from the cDNA encoding the rat TRH precursor. This study provides for the first time a direct chemical evidence for the existence of non-TRH peptides originating from posttranslational processing of the TRH precursor in vivo.     

A simple coronary blood flow model to study the collateral flow index

Biomechanics and Modeling in Mechanobiology - In this work, we present a novel modeling framework to investigate the effects of collateral circulation into the coronary blood flow physiology. A...     

A cDNA from brain of Xenopus laevis coding for a new precursor of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is found in large amounts in the skin of Xenopus laevis. In this tissue, 3 TRH precursor mRNAs can be detected of which the 2 more expressed encode almost identical proteins. However, Northern blot analysis of TRH precursor mRNAs in the brain of X. laevis revealed the existence of a new mRNA of about 1200 nucleotides which was present along with the larger TRH precursor mRNA identified in the skin. A cloned cDNA of a TRH precursor, corresponding in size to this new mRNA, was isolated and sequenced from a Xenopus brain lambda gt11 library. It encodes a precursor polypeptide which also contains 7 copies of TRH. However, at the amino acid level it differs by about 16% from the corresponding prepro-TRHs from skin. We have also attempted to characterize the gene encoding this prepro-TRH from Xenopus brain. Only the first and part of the second exon could be detected which are separated by an intron containing more than 8000 base pairs. Interestingly, the 5'-flanking region of this gene does not contain the characteristic promoter elements of the mammalian TRH genes suggesting marked differences in the regulation of their expression.     

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.