Plasma clearance, tissue uptake and expression of pituitary peptide 23/pancreatitis-associated protein in the rat.

The secretion of peptide 23 by rat pituitary cells is stimulated by growth hormone-releasing hormone and inhibited by somatostatin. Recent cloning of the cognate cDNA for peptide 23 revealed that it is identical to pancreatitis-associated protein (PAP). In the present study, the clearance and tissue uptake of recombinant peptide 23/PAP in normal adult male rats was assessed. The plasma half-life of recombinant peptide 23/PAP was 4.8 +/- 1.4 (S.D.) min. Maximal accumulation of radio-labelled peptide 23/PAP was observed in the kidney, stomach, small intestine and pancreas whereas negligible uptake was seen in the liver, lung or heart. Peptide 23/PAP was detected in a variety of tissue extracts using a radioimmunoassay. Extracts of ileum contained the highest concentrations of peptide 23/PAP. In situ hybridization analysis showed that peptide 23/PAP mRNA was highly expressed in the columnar epithelial cells of ileum, jejunum and duodenum. These observations demonstrate that peptide 23/PAP, a protein previously thought to be of pituitary origin, is widely expressed in the gastrointestinal tract and that it is rapidly removed from the circulation by the kidney and by tissues which express peptide 23/PAP.

Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone.

Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.

Molecular cloning and expression of peptide 23, a growth hormone-releasing hormone-inducible pituitary protein.

Peptide 23 is a newly identified protein secreted by rat pituitary cells in primary culture. Although the secretion of this protein is stimulated by GH-releasing hormone and inhibited by somatostatin, the N-terminal amino acid sequence of peptide 23 shows no homology to rat GH. Using the polymerase chain reaction technique, we cloned and sequenced the peptide 23 complementary DNA (cDNA). By means of the mixed oligonucleotide-primed amplification of cDNA technique, primers corresponding to the NH2-amino acid sequence of peptide 23 were used to amplify, clone, and sequence a 74-basepair cDNA of peptide 23. This polymerase chain reaction product was then used as a primer to amplify the complete peptide 23 cDNA by means of the rapid amplification of cDNA ends procedure. The cDNA of peptide 23 obtained by the rapid amplification of cDNA ends procedure contained 777 nucleotides and encoded a 175-amino acid protein with a 26-amino acid putative signal peptide. The calculated mol wt of the mature protein (16,613 daltons) was in good agreement with that estimated by polyacrylamide gel electrophoresis (16 kilodaltons). Northern blot analysis revealed a major messenger RNA species of about 0.9 kilobase and a minor species of about 1.7 kilobases in cultured rat anterior pituitary cells. In rats, peptide 23 was most abundant in the pancreas and gastrointestinal tract. A GenBank sequence search revealed complete sequence identity between peptide 23 cDNA and pancreatitis-associated protein cDNA, an approximately 73% homology with human hepatocellular carcinoma cDNA from human hepatocellular carcinoma, 64% homology with bovine pancreatic thread protein cDNA, and 55% homology with rat and human reg cDNAs, which have been reported to be expressed in regenerating pancreatic islets. Therefore, peptide 23 is identical to pancreatitis-associated protein and a member of the C-type lectin supergene family.

Expression of pituitary peptide 23 in the rat uterus: regulation by estradiol.

Peptide 23 was first identified in pituitary cell conditioned medium as a secreted protein which was regulated in a similar fashion to growth hormone. It was subsequently found to be a member of the C-type lectin gene superfamily and identical to pancreatis associated protein (PAP). It is widely expressed in the gastrointestinal tract. Our present study demonstrates that peptide 23 gene is also expressed in the uterus. Peptide 23/PAP mRNA was at highest levels during estrus and was not detectable in the immature rat uterus. A single injection of 17 beta-estradiol resulted in a transient induction of peptide 23/PAP mRNA in ovariectomized rats whereas a sustained induction was seen with diethylstilbestrol implants. In situ hybridization localized peptide 23/PAP mRNA to the luminal epithelial cells. During gestation, peptide 23/PAP mRNA was detected only in the uterine samples from day 12 to 18 of pregnancy with maximal expression on day 12. Peptide 23 expression was confined to the uterus itself and not expressed in either the decidua or the fetal tissues. PSP/reg, another closely related member of the C-lectin gene family was not expressed in any of these uterine tissues. These results indicate that estrogen may act as a physiological regulator of peptide 23 in the uterus and suggests that this protein may have some role in estrogen action.

Rat prolactin-like protein A partial gene and promoter structure: promoter activity in placental and pituitary cells.

Rat prolactin-like protein A (rPLP-A) is a member of a rapidly expanding family of prolactin-related proteins that are expressed during pregnancy by the rat placenta according to specific developmental patterns. Although the factors involved in the pituitary-specific expression of the prolactin and growth hormone genes themselves have been extensively studied, essentially nothing is known of the factors responsible for the placental expression of these new family members. In this paper we describe the isolation of rPLP-A genomic clones, analyze a portion of the 5' flanking sequence of this gene and use the recently described rat choriocarcinoma cell line, Rcho, in transient transfection studies to show that a 975 base-pair (bp) fragment of 5' flanking sequence is sufficient to specify placental expression of the rPLP-A gene.

In situ detection and distribution of stanniocalcin mRNA in the corpuscles of Stannius of sockeye salmon, Oncorhynchus nerka.

Stanniocalcin (STC) gene expression was examined in the corpuscles of Stannius of sockeye salmon by in situ hybridization. Generally, the STC gene was not expressed uniformly in all cells of the corpuscles of Stannius. However, in cases where gene expression was uniform throughout a gland, it appeared that virtually all cells contained STC mRNA, thereby supporting the 'one cell type' hypothesis for the corpuscles of Stannius. Correlative immunocytochemistry on adjacent tissue sections indicated that cellular levels of stored hormone paralleled the distribution of STC mRNA. Stanniocalcin mRNA was undetectable in numerous other salmon tissues, thereby further supporting the notion that the STC gene is expressed exclusively in the corpuscles of Stannius.

Steroids and tissue-specific modulation of galanin gene expression in the male rat reproductive system.

Galanin is a neuropeptide widely distributed throughout the vertebrate neural and endocrine system. Galanin can influence pituitary hormone secretion, intestinal motility, and other biological activities. The precise physiological role of galanin is unknown. We studied the control of galanin gene expression in peripheral organs in the male rat using Northern blot and in situ hybridization techniques. In the adrenals and prostate, galanin mRNA was undetectable in the controls and did not change after the administration of dexamethasone (0.0001-10.0 mg/kg, ip) and diethylstilbestrol (0.1 mg/kg, ip). In the testis, thymus, seminal vesicles, medial basal hypothalamus, and colon, galanin message was detectable, but was not influenced by steroids. On the other hand, dexamethasone (0.5-10.0 mg/kg) was very effective in enhancing galanin expression in the vas deferens and epididymis (4- to 7-fold in the vas deferens), with a peak 6-9 h after the treatment. Diethylstilbestrol (0.1 mg/kg) stimulated galanin mRNA transcription only in the vas deferens (2- to 3-fold), with a peak 1-3 h after the treatment. Dihydrotestosterone treatment (0.2-0.4 mg/kg) was ineffective in all tissues examined. In the vas deferens and seminal vesicles, galanin mRNA has been localized at a cellular level by in situ hybridization. In these tissues only fibroblast-like cells contained the message. These data demonstrate that galanin is expressed in the male rat reproductive system and that steroid hormones participate in the control of galanin gene expression in a tissue- and hormone-specific fashion.

Expression and secretion of galanin during pregnancy in the rat.

The expression of galanin messenger RNA (mRNA) in the pituitary and conceptus of pregnant rats has been studied at various stages of gestation. Using Northern blot analysis and in situ hybridization we have found that high levels of mRNA coding for galanin were detected in the conceptus during early pregnancy. The level of expression in conceptuses increased until day 11-12 after which the levels decreased rapidly. In contrast, in the pituitary galanin mRNA continued to increase throughout pregnancy, especially in the latter half of pregnancy as serum estradiol levels has been reported to be increased. The size of the galanin transcript was the same in the conceptus and pituitary (0.9 kilobase). Expression of this mRNA was confined to the decidua and was first seen at day 5 of pregnancy at a time when implantation swellings were first observed. Galanin antisense probe hybridized strongly to the decidual cells that surround the implantation site. At day 11 of pregnancy the galanin mRNA was found in both the antimesometrial and the mesometrial tissue with the highest concentration in the region lateral to the antimesometrial cells and continued toward the mesometrial cells. Serum galanin levels measured by an RIA using synthetic rat galanin as standard and antisera raised against porcine galanin, exhibited a temporal pattern similar to the pattern of mRNA expression in decidua, with a 7.1-fold increase at day 12 of pregnancy followed by a decline. In summary, decidual cells may differentiate into an endocrine cell during pregnancy. The galanin secreted by these cells, in addition to acting locally in a paracrine/autocrine fashion, may function as a placental hormone with systemic effects on distal target tissues.

Molecular cloning and expression of rat placental lactogen-Iv, a variant of rPL-I present in late pregnant rat placenta.

Late pregnant rat placenta was found to contain a messenger RNA (mRNA) that encodes an additional member of the prolactin-growth hormone family. This polypeptide was detected by hybridization of complementary DNA (cDNA) for rPL-I to a 1 kilobase mRNA transcript in late pregnant rat placenta. A cDNA clone for the new polypeptide was isolated from a phage lambda-gt10 library containing cDNA synthesized from day 18 rat placental mRNA. Sequencing of the day 18 cDNA clone revealed that it was most closely related structurally to rPL-I and at the amino acid terminal was identical to the rPL-I variant (rPL-Iv). The sequence of rPL-Iv (223 amino acids) is 85% homologous to rPL-I (230 amino acid). There are 71 base pair changes (85% nucleotide homology) between rPL-Iv and rPL-I which are not confined to any specific region of the molecule and which result in 36 amino acid changes. In vitro translation of rPL-Iv mRNA produced by transcription of the cDNA template yielded a 26 kilodalton polypeptide, the size of the expected precursor protein. In situ hybridization studies indicated that mRNA for rPL-Iv was present primarily in cytotrophoblasts of the basal zone as early as day 15 of gestation with some hybridization in a few giant cells as well.

Expression of rat prolactin-like protein B in deciduoma of pseudopregnant rat and in decidua during early pregnancy.

The rat PRL-like protein-B (rPLP-B) complementary DNA (cDNA), originally cloned from a late term placental library, hybridizes to transcripts in the deciduoma tissue artificially produced in pseudopregnant rats. The expression of rPLP-B in deciduoma is first observed 48 h (pseudopregnancy day 7) after the deciduogenic stimulus and increases to a maximum by 96-120 h (pseudopregnancy day 9-10). In situ hybridization studies show that rPLP-B hybridizes specifically to the antimesometrial cells of deciduoma tissue in day 9 pseudopregnant rats and to the homologous cells of the decidua that surround the embryo-trophoblastic structure at day 9 of pregnancy. A temporal study on the expression of rPLP-B during pregnancy shows that rPLP-B is concomitantly expressed by the decidual cells of maternal origin and the cytotrophoblast of fetal origin around day 13 of pregnancy.

Expression of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in the rat uterus during decidualization.

Decidualization of the uterus involves proliferation and differentiation of uterine cells. The effects of decidualization on uterine expression of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) have been examined in the hypophysectomized-ovariectomized (hypox-ovx) rat and the pituitary-intact (ovx) rat. Decidualization was induced by uterine stimulation of animals treated with a combination of 17 beta-estradiol and progesterone. The patterns of change in uterine IGF-I mRNA and IGFBP-1 mRNA abundance were similar to hypox-ovx rats, hypox-ovx rats replaced with GH and T4, and ovx rats. The changes in IGF-I mRNA abundance were temporally related to 17 beta-estradiol injections. IGFBP-1 mRNA was undetectable early in the decidualization process and reached maximal levels on day 6. Mechanical separation of the deciduoma tissue from the underlying myometrium revealed that the deciduoma tissue was depleted in IGF-I mRNA, while the majority of the IGFBP-1 was located in the deciduoma tissue. The in situ hybridization technique was used to localize IGF-I and IGFBP-1 mRNA in the decidualized uterus. The majority of the IGF-I expression was localized to the outer stroma and smooth muscle cell layer, whereas IGFBP-1 mRNA was detected in uterine epithelial cells and stromal glands. These experiments demonstrated that uterine IGF-I and IGFBP-1 expression during the process of decidualization are pituitary independent. Furthermore, our observations support the hypothesis that the expression of IGFBP-1, a protein capable of inhibiting the mitogenic activity of IGF-I, in deciduoma tissue may inhibit paracrine IGF-1 actin and allow for the differentiation of stromal tissue.

Molecular cloning and expression of rat placental lactogen-I complementary deoxyribonucleic acid.

A full-length cDNA clone for rat placental lactogen I (rPL-I) has been isolated from a phage lambda gt11 library containing cDNA synthesized from day 11 rat placental mRNA. By Northern blot analysis the rPL-I cDNA clone hybridizes to a 1.0-kilobase placental mRNA and appears as early as day 10 of gestation. Maximal expression of this mRNA was observed in day 11 and 12 placenta, and faint hybridization of the rPL-I cDNA was also detected in day 18 to term placenta. In contrast, the mouse clone hybridized to mRNA for mouse PL-I (mPL-I) only in day 10 mouse placenta (9). In vitro translation of rPL-I mRNA produced by transcription of the cDNA template yielded a 27-kDa polypeptide the size of the expected precursor protein which was immunoprecipitated by a monoclonal antibody to rPL-I. The rPL-I cDNA nucleotide sequence has been determined. The sequence is very similar to that for mPL-I and contains an open reading frame encoding a polypeptide of 230 amino acids compared to 224 for mPL-I. Comparison of the predicted primary translation product of rPL-I mRNA with that of mPL-I mRNA revealed that rPL-I shares 73% identity to mPL-I at the amino acid level. The predicted rPL-I protein shares 41% amino acid identity with rPL-II precursor, 24% with rat prolactin-like protein A, 26% with rat prolactin-like protein B, and 31% with rat PRL. In situ hybridization studies indicated that mRNA for rPL-I was present in a few rapidly dividing cells as early as day 8 of gestation, and by day 9 could be localized to giant cells which surround the conceptus.

Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization.

In situ hybridizations using 35S-labelled antisense and sense RNA probes of rPLII, rPLP-A and rPLP-B were carried out on developing rat placenta to determine which cell types synthesized each specific mRNA. Cellular localization of the sites of synthesis of these placental RNAs would help to decide whether these proteins were functioning in the mother of the fetus. The cells of the basal zone are known to have access only to the maternal blood supply, while the labyrinth region is supplied by both maternal and fetal blood vessels. The data in this paper show that at day 12 of pregnancy the rPLII mRNA is synthesized in the primary and secondary giant cells. At later days, hybridization is seen in both the giant cells of the basal zone, and cells in the labyrinth, suggesting that rPLII has a function not only in the mother, but also in the fetus. The rPLP-A mRNA is synthesized in both the giant cells and the cytotrophoblasts of the basal zone. No hybridization is seen to any cells in the labyrinth, even at the later days when it appears that all cytotrophoblasts synthesize rPLP-A mRNA. The rPLP-B mRNA is synthesized exclusively by the cytophoblasts of the fetal placenta. Like rPLP-A, all these cells synthesize this mRNA in the late term placenta. The synthesis of the rPLP-A and rPLP-B mRNAs in cells which have access only to the maternal circulation suggest that they have a role in the mother.

Estrogen induction of galanin synthesis in the rat anterior pituitary gland demonstrated by in situ hybridization and immunohistochemistry.

The distribution of galanin messenger (mRNA) and galanin-like immunoreactivity (Gal-LI) in the anterior and posterior pituitaries of control and estrogen-implanted female rats was determined by in situ hybridization and immunohistochemical methods. In control ovariectomized animals galanin mRNA was undetectable in the posterior, intermediate and anterior pituitary. However, 4 days after implantation with 10 mg of diethylstilbestrol, galanin mRNA was clearly present in the anterior pituitary, but not in the posterior or intermediate lobe. By immunohistochemistry Gal-LI was readily visualized and detected in the posterior lobe, but clearly undetectable in cells of the intermediate and anterior pituitary lobes of control animals. After estrogen administration numerous cells exhibiting intense Gal-LI were evident in the anterior lobe, while Gal-LI remained unchanged in the intermediate and posterior lobes. These results indicate that in control animals galanin is stored, but not synthesized, in the posterior pituitary and that after estrogen administration galanin production is substantially increased in the anterior pituitary. We conclude that the expression of galanin in the anterior pituitary is regulated by estrogen and suggest that galanin may be a pituitary hormone.