The post-translational processing of prothyrotropinreleasing hormone (pro-TRH 25-255 ) has been extensively studied in our laboratory, and the processing pathway to mature TRH has been elucidated. We have also demonstrated that recombinant PC1 and PC2 process partially purified pro-TRH to cryptic peptides in vitro and that pro-TRH and PC1 mRNAs are coexpressed in primary cultures of hypothalamic neurons. To further define the role of each convertase, and particularly PC1 and PC2, in pro-TRH processing, recombinant vaccinia viruses were used to coexpress the prohormone convertases PC1, PC2, PACE4, PC5-B, furin, or control dynorphin together with rat prepro-TRH in constitutively secreting LoVo cells or in the regulated endocrine GH4C1 cell line.Radioimmunoassays from LoVo-derived secreted products indicated that furin cleaves the precursor to generate both N-and C-terminal intermediates. PC1, PC2, and PACE4 only produced N-terminal intermediates, but less efficiently than furin. In GH4C1 cells, PC1, PC2, furin, PC5-B, and PACE4 produced both N-terminal and C-terminal forms. Significantly, TRH-Gly and TRH were mostly produced by PC1, PC2, and furin. Utilizing gel electrophoresis to further analyze the cleavage specificities of PC1 and PC2, we found that PC1 seems primarily responsible for cleavage to both intermediates and mature TRH, since it generated all products at significantly higher levels than PC2. The addition of 7B2 to the coinfection did not augment the ability of PC2 to cleave pro-TRH to either N-or C-terminal forms.Many neuropeptides are first synthesized as large prohormones, precursors that must be post-translationally proteolyzed to elaborate smaller bioactive peptides. This processing occurs through limited endoproteolytic cleavage at paired basic residues, either Lys-Arg or Arg-Arg, with cleavage at monobasic sites occurring less frequently (1, 2). Through such posttranslational processing, prothyrotropin-releasing hormone (pro-TRH) 1 is cleaved to mature TRH. Mature TRH is a modified 3-amino acid neuropeptide (pyroglutamate-histidine-proline-amide) that stimulates the synthesis and release of thyrotropin, prolactin, and growth hormone from the mammalian pituitary (3-6). In the rat, mature TRH is derived from a 231-amino acid prohormone, which contains five TRH progenitor sequences (Gln-His-Pro-Gly) (7,8). Each one of these sequences is flanked by dibasic residues, and another dibasic residue exists that does not flank a TRH progenitor sequence (7). Cleavage at all dibasic residues yields five copies of the TRH progenitor and seven cryptic peptides (7, 9). Mature TRH is achieved by exoproteolytic excision of the basic residues flanking the TRH progenitor by carboxypeptidase E, amidation of the carboxyl-terminal proline by peptidyl glycine ␣-amidating monooxygenase, and cyclization of the glutamine (10 -12).Much of the characterization of pro-TRH and its post-translational processing and sorting have been done using transfected AtT-20 cells, which yield high levels of prohormone expression (7). U...

Immunohistochemistry employing immunoglobulin G fractions raised against aromatase cytochrome P-450 and antiserum against 17 beta-estradiol dehydrogenase was used to localize these two steroid-converting enzymes in the human placenta. Immunostaining for both enzymes was found exclusively in the syncytiotrophoblast, while the underlying cytotrophoblast and the villus core did not stain. Ultrastructural examination of aromatase cytochrome P-450- and 17 beta-estradiol dehydrogenase-labeled sections disclosed immunoreactive product in the membranes of the endoplasmic reticulum; the nucleus, mitochondria, Golgi apparatus, and secretory granules were free of staining. These findings suggest that the syncytiotrophoblast is actively involved in the synthesis and metabolism of estrogens and in their role in placental endocrine function.

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 pre-proTRH 25 -15a) and a 10-kDa C-terminal peptide . Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa Cterminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (prepro-TRH25-,6 or preproTRH25-82) and a 3.8-kDa peptide (pre-proTRH 83 -108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide . The optimal pH for these cleavages was 5.5 . ZnCl 2 , EDTA, EGTA, and the omission of Ca t ' inhibited the formation of pYE2, (preproTRH25-50), 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.

The 24-h patterns of circulating cortisol and corticosterone were determined in male hamsters housed under a 14:10 light-dark cycle. Corticoid levels varied significantly over the 24-h sampling period with peak levels of both hormones occurring near the onset of the daily dark phase. The ratio of cortisol to corticosterone changed dramatically during the day. Corticosterone levels were significantly higher than cortisol during the early part of the light phase; however, cortisol levels became significantly higher than corticosterone when both hormones began their daily rise. To examine whether the circadian rhythm of cortisol secretion could be involved in the physiological control of hamster circadian organization, cortisol was infused at approximately physiological levels into adrenalectomized hamsters either continuously or in a 24-h rhythm. No significant differences were observed in the timing of circadian wheel-running rhythms in hamsters housed in LD 16:8, LD 14:10, or LL when cortisol was infused continuously, in a 24-h rhythm that mimicked the cortisol rhythm of intact hamsters, or in a 24-h rhythm several hours out of phase with the rhythm of intact hamsters. Provision of cortisol in a 24-h rhythm appeared to promote the survival of adrenalectomized hamsters since hamsters receiving a 24-h pattern of cortisol survived the experimental protocol significantly longer than those receiving the same dose of cortisol continuously.