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.     

Pro-Thyrotropin-Releasing Hormone Processing by Recombinant PC1

Abstract: Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH₂), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [³H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH_25–152 or preproTRH_25–158) and a 10-kDa C-terminal peptide (preproTRH_154–255 or preproTRH_160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH_25–76 or preproTRH_25–82) and a 3.8-kDa peptide (preproTRH_83–108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH_206–255). The optimal pH for these cleavages was 5.5. ZnCl₂, EDTA, EGTA, and the omission of Ca²⁺ inhibited the formation of pYE₂₇ (preproTRH_25–50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.     

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.     

Neuropeptide processing profile in mice lacking prohormone convertase-1

Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of Met-Enkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.     

Role of a pro-sequence in the secretory pathway of prothyrotropin-releasing hormone

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31-52 sequence. The decreased steady state level of the mutant DeltaPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of DeltaPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.     

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.     

Role of PC2 in Proenkephalin Processing: Antisense and Overexpression Studies

Abstract: The contribution of the prohormone-processing enzyme PC2 to the proteolytic maturation of proenkephalin was examined in three sets of studies. In the first, the processing of this precursor was compared in PC2-rich (Rin5f) and PC2-lacking (AtT-20) cell lines expressing proenkephalin by virtue of stable transfection. These studies showed that the time frame for processing of this precursor is cell line specific, with AtT-20 cells processing proenkephalin to peptide B much more rapidly than Rin cells. However, the latter cell line processed proenkephalin much more extensively, i.e., produced a greater proportion of the penta- to octapeptide enkephalins. The involvement of PC2 in these later processing events was analyzed by examining the processing of proenkephalin in PC2-overexpressing AtT-20 cell lines. These experiments yielded a processing profile similar to that observed for Rin cells, although the time frame of initial processing was similar to that found in AtT-20 cells. To confirm the physiological involvement of proenkephalin in the production of the small opioid peptides, we generated a Rin cell line in which the production of PC2 was impaired due to stable expression of antisense mRNA to this enzyme. These experiments provided conclusive evidence that the generation of Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu, but not the larger enkephalin-containing peptides, is mediated by PC2. Taken together, our data support the idea that PC2 is physiologically capable of mediating only the later processing steps of neuropeptide precursors. PC2 thus appears to be the primary enzyme responsible for the generation of bioactive opioid peptide species from proenkephalin.     

Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides

Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.     

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.     

Tissue distribution and processing of proSAAS by proprotein convertases

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.     

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.     

Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide

Purified recombinant prohormone convertase 1 and 2 (PC1 and PC2) cleave a peptide containing cholecystokinin (CCK) 8 Gly Arg Arg and the carboxyl-terminal peptide liberating CCK 8 Gly Arg Arg. PC1 and PC2 also cleave purified pro CCK, liberating the amino terminal pro-peptide while no carboxyl-terminal cleavage was detected. Under the conditions of the in vitro cleavage assay, it appears that the carboxyl-terminal cleavage site of pro CCK is not accessible to the enzymes while this site is readily cleaved in a synthetic peptide. Additional cellular proteins that unfold the prohormone may be required to expose the carboxyl-terminal site for cleavage.     

Disruption of proprotein convertase 1/3 (PC1/3) expression in mice causes innate immune defects and uncontrolled cytokine secretion

The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.     

Thyrotropin-releasing hormone gene expression in normal thyroid parafollicular cells

The rat TRH gene encodes a 255-amino-acid precursor polypeptide, preproTRH, containing five copies of TRH and seven non-TRH peptides. Expression of this gene is well documented in the central nervous system, particularly in the hypothalamus. Thyroids also contain TRH immunoreactivity, but it is unknown whether this immunoreactivity results from expression of the TRH gene or from other genes encoding TRH-like products. Since the CA77 neoplastic parafollicular cell line expresses the TRH gene, we investigated whether TRH gene expression also occurs in normal thyroid parafollicular cells. Northern analysis of total thyroid RNA with a preproTRH-specific RNA probe identified a single hybridizing band the same size as authentic TRH mRNA found in hypothalamus and CA77 cells. Gel filtration analysis of thyroid extracts identified the same 7-kilodalton and 3-kilodalton species of immunoreactive preproTRH53-74 previously identified in hypothalamus and CA77 cells. Immunoreactive preproTRH115-151, not previously identified, was found in all three tissues. Part of this immunoreactivity comigrated with the synthetic preproTRH115-151 standard on gel filtration and reversed-phase HPLC. PreproTRH53-74 was localized to thyroid parafollicular cells by immunostaining. These findings demonstrate authentic TRH gene expression by normal rat thyroid parafollicular cells and establish the CA77 cell line as the only model system of a normal TRH-producing tissue. In addition to expanding the range of neuroendocrine peptides known to be produced by parafollicular cells, these results also suggest a potential paracrine regulatory role for TRH gene products within the thyroid.     

Defective prodynorphin processing in mice lacking prohormone convertase PC2

Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.     

Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain

Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1–24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.     

proTRH gene expression by fetal pancreatic islets in culture

The hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), has been detected in neonatal pancreatic tissue and localized by immunocytochemistry in the islets of Langerhans. To determine whether the TRH gene is expressed in islets, we have extracted RNA from cultured rat islets and probed for proTRH mRNA using a [32P]-labeled antisense RNA. Islet proTRH mRNA comigrated with the 1.6 kilobase proTRH mRNA present in the rat hypothalamus. Normalized to total RNA, islets cultured for 7 days contained at least 10 times more proTRH mRNA than day 1 whole pancreas. We conclude that pancreatic TRH is synthesized in situ in the islets of Langerhans. This is the first attempt to characterize and quantify proTRH mRNA using neoformed foetal islets. We propose that quantitative analysis of proTRH mRNA concentrations in this culture system will enable study of the direct regulation of TRH biosynthesis in the pancreas.     

Molecular Characterization and Differential Gene Induction of the Neuroendocrine-Specific Genes Neurotensin, Neurotensin Receptor, PC1, PC2, and 7B2 in the Human Ocular Ciliary Epithelium

Abstract: The ocular ciliary epithelium is a bilayer of neuroepithelial cells specialized in the secretion of aqueous humor fluid and the regulation of intraocular pressure. In this study, we report on the expression of the regulatory peptide neurotensin (NT) and a set of differentiated neuroendocrine markers including neurotensin receptors (NTrs), the prohormone convertases furin, PC1, and PC2, and the neuroendocrine polypeptide 7B2 in the ciliary epithelium. Using a human cell line, ODM-2, derived from the nonpigmented ciliary epithelium, we demonstrate that (1) NT expression is highly activated by nerve growth factor, glucocorticoid, and activators of adenylate cyclase; (2) NTr expression is up-regulated by selective ligand-activated β₂-adrenergic receptor; and (3) PC1 and PC2 expression are up-regulated via distinct signaling transduction pathways. PC1 gene expression is activated by phorbol ester, and PC2 by the same inducers as those of NT expression. A radioimmunoassay for NT detected an NT-like immunoreactivity in human ciliary epithelium and ODM-2 cell extracts, in aqueous humor, and in conditioned culture medium. The results support the view that the entire ciliary epithelium functions as a neuroendocrine tissue, synthesizing, processing, and releasing NT into the aqueous humor where it may exert important physiological functions through autocrine and/or paracrine mechanisms.     

Corticotrophs and peptides

Corticotrophs were long thought to be a static, homogeneous population of cells that respond positively to hypothalamic stimulation, are inhibited by glucocorticoid feedback and secrete a single biologically active peptide, ACTH(1-39). Our current understanding is that this is an oversimplification and corticotrophs are a dynamic and more complex group of cells. The biosynthetic precursors of ACTH and other cleavage products of proopiomelanocortin (POMC) have been found to be secreted by anterior pituitary cells, to circulate and to have biological activity. POMC and the biosynthetic intermediate, pro-ACTH, exert activity antagonistic to ACTH(1-39) on glucocorticoid secretion by adrenal cells, and other derivatives of POMC are mitogenic to adrenocortical cells. In terms of responses to hypothalamic and peripheral factors, corticotrophs are functionally heterogeneous. This is reflected in the sensitivity of individual subtypes of corticotrophs to CRH, vasopressin and glucocorticoids. There is a functional plasticity amongst the various types of corticotrophs. During gestation, in fetal sheep, changes occur in the overall ACTH-secretory responses to CRH relative to vasopressin, the proportions of total corticotrophs that respond to the respective peptides and the average secretory response of individual cells. Corticotrophs also respond to locally produced pituitary factors. Local actions of leukaemia inhibitory factor are demonstrated by the effects of immunoneutralization of the peptide in pituitary cells. Urocortin and preproTRH(178-199) are locally produced peptides with potent stimulatory and inhibitory actions on corticotrophs, respectively. The specific roles of these peptides are under investigation.