bluuebunny/crossref_metadata_2025 huggingface dataset:
Sorting of PC2 to the regulated secretory pathway in AtT20 cells
Authors: Taylor NA , Jan G , Scougall KT , Docherty K , Shennan KI
Published: December 2004

PC2 and PC3 are neuroendocrine specific members of the eukaryotic subtilisin-like proprotein convertase (PC) family. Both are sorted via the regulated secretory pathway into secretory granules. In order to identify sequences in PC2 which are involved in targeting to the regulated secretory pathway we expressed a series of PC2 cDNAs containing mutations in the C terminal or propeptide domains in the mouse corticotrophic AtT20 cell line. Sorting of endogenous PC3 was used as a control. PC2 and PC3 were secreted with similar kinetics and sorted to secretory granules with similar efficiencies. Deletions of up to 50 amino acids from the C-terminus of proPC2 had no effect on secretion or sorting, but larger deletions completely prevented maturation or secretion. Two large deletions within the propeptide also prevented secretion. Smaller deletions between the primary and secondary cleavage sites, or of the primary cleavage site, reduced the amount of protein secreted but did not affect sorting to secretory granules. Replacement of the propeptide of PC2 with that of the endogenous PC3 also had no effect on secretion or sorting. The results indicate that targeting of proPC2 to the regulated secretory pathway is dependent on more than one region within the proPC2 molecule.
The upregulation of messenger ribonucleic acids during 17alpha, 20beta-dihydroxy-4-pregnen-3-one-induced ovulation in the perch ovary
Authors: Langenau DM , Goetz FW , Roberts SB
Published: December 2004

While progestins appear to be involved in the local ovarian regulation of vertebrate ovulation, their specific role is unclear. In yellow perch (Perca flavescens) the progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20-P), stimulates ovulation in vitro and this induction requires gene activation. Therefore, the perch model was used to isolate progestin-upregulated mRNAs. Perch ovaries were incubated for 32 h with or without 17,20-P (0.1 microg/ml). Messenger ribonucleic acids were isolated from the tissue and used for differential display PCR (DDPCR). From DDPCR, 5 bands were eventually obtained that were verified by Northern analysis to be consistently upregulated by 17,20-P at 32 h. Using these bands, full-length cDNAs were obtained by library screening and completely sequenced. Based on similarity to known sequences, four of the cDNAs presumably encode for perch forms of (1) neprilysin (PNEP-1; 63% identical); (2) a lysyl oxidase-type protein (PLO-2; 43.2% identical); (3) calmodulin (PCAL-1; 100% identical); and (4) a microtubule aggregate-like protein (PMAP-1; 29.6% identical). The fifth cDNA obtained from DDPCR most likely encodes for an egg protein and will be reported separately. Each of the cDNAs was used to probe Northern blots of ovarian mRNA taken at 0, 12, 24, 32 and 42 h of incubation with 17,20-P. This temporal Northern analysis verified that all four were upregulated by 32 h. In addition, PNEP and PMAP transcripts began to increase by 12 h, while PCAL and PLO transcripts remained elevated through 42 h. On Northern blots of RNA from other perch tissues, calmodulin was found in all tissues, PLO mRNA was ovarian specific, and PMAP mRNA was also present in the gills and liver. Multiple transcripts were observed for PNEP, but the ovarian form induced by 17,20-P was only found in high abundance in the heart. To our knowledge, this is the first report that specifically characterizes progestin upregulated mRNAs in the vertebrate ovary at ovulation.
Identification of Sp1 as the transcription factor for the alternative promoter P2 of the bovine growth hormone receptor gene
Authors: Jiang H , Okamura CS , Boyd CK , Lucy MC
Published: December 2004

Growth hormone receptor (GHR) mRNA variants that differ in the 5'-untranslated regions (5'-UTR) have been isolated in various species. These 5'-UTR variants are generated from the use of alternative promoters and/or alternative splicing. The 5'-UTR 1B is one of the GHR 5'-UTR variants isolated in the bovine but its homologues are also present in other species. The 5'-UTR 1B is a predominant GHR 5'-UTR expressed in many tissues. In the present study, we screened a bovine genomic library and isolated a 1.7 kb bovine GHR genomic sequence including exon 1B and its 5' flanking region from which the GHR 5'-UTR 1B is generated. Using primer extension, two major transcription start sites were mapped in the bovine exon 1B. Transient transfection analysis of the 5' flanking region of exon 1B confirmed its promoter activity (termed P2) in both Hep G2 and BHK-21 cells. Furthermore, analysis of deletion promoter-reporter constructs found that the basal activity of P2 resided in the proximal region of P2. DNase I footprinting analysis and electromobility shift assay (EMSA) identified the ubiquitous transcription factor Sp1 as the binding protein to a GC box-containing DNA element within the proximal P2. Deletion of the GC box greatly reduced the activity of P2 in cell lines. The GC box-containing site also appeared to bind Sp1 in the nuclear extracts from diverse bovine tissues. This suggests that interactions of Sp1 with the GC box-containing element in the proximal region of P2 may be part of the mechanism for the expression of the bovine GHR 5'-UTR 1B in diverse tissues.
The transcription factors SP1 and MAZ regulate expression of the parathyroid hormone/parathyroid hormone-related peptide receptor gene
Authors: Williams LJ , Abou-Samra AB
Published: December 2004

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor regulates extracellular calcium concentrations and is therefore important for mineral homeostasis. ROS 17/2.8 cells, a rat osteoblast-like osteosarcoma cell line, express the PTH/PTHrP receptor and provide a good model for examining the transcriptional regulation of its gene. The rat PTH/PTHrP receptor gene has two promoters, U1 and U3, which were shown to be important for its expression. Using extracts from ROS 17/2.8 cells, we have demonstrated two regions (termed FP1 and FP2) of nuclear protein/DNA interaction within promoter sequences previously shown to be important for the activity of the U3 promoter. Nuclear extracts from rat 2 fibroblasts, which do not express the PTH/PTHrP receptor, produced one site of protein/DNA interaction which was found at a position on the promoter identical to the position of FP1 produced by a ROS 17/2.8 nuclear extract. Mutation of these two sites of protein/DNA interaction resulted in reduced U3 promoter activity. Furthermore, we have demonstrated that the transcription factors SP1 and MAZ regulate U3 promoter expression and have shown their functional significance using mutational analysis. These data demonstrate that SP1 and MAZ bind to the PTH/PTHrP receptor promoter and that they are involved in cell-specific expression of its gene product.
Differential expression of secreted frizzled-related protein 4 in decidual cells during pregnancy
Authors: Fujita M , Ogawa S , Fukuoka H , Tsukui T , Nemoto N , Tsutsumi O , Ouchi Y , Inoue S
Published: December 2004

During pregnancy, the uterus shows marked morphological and physiological changes under the regulation of ovarian steroid. To elucidate the molecular cues of these changes, we tried to identify the transcripts differentially expressed in the pregnant rat uterus by using the suppression subtractive hybridization method. Seven independent clones were isolated and one of the up-regulated genes was secreted frizzled-related protein 4 (sFRP4). sFRP4 contains a Wnt-binding domain and belongs to the secreted frizzled protein family whose members are assumed to function as modulators of the Wnt signal. The expression level of sFRP4 mRNA reached a peak in the pregnant uterus on day 12, when uterine decidualization was almost complete in the rat. In situ hybridization histochemistry revealed that sFRP4 transcripts were observed in the decidual cells. In addition, proliferating cell nuclear antigen (PCNA)-positive cells were shown to be overlapped in decidua, suggesting that sFRP4 mRNA expression was accompanied by the late phase of decidual cell proliferation. Moreover, sFRP4 and estrogen receptor-alpha transcripts were co-localized. Furthermore, we analyzed the regulation of sFRP4 by estrogen using 17 beta-estradiol-treated ovariectomized rats. sFRP4 mRNA was detected in the uterus at 48 h after estrogen treatment, especially in endometrial stroma where PCNA-positive cells were also observed. The results in this study led us to the notion that sFRP4 mRNA may be up-regulated after estrogen treatment in the late phase of uterine cell proliferation.
Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene
Authors: Nandy A , Jenatschke S , Hartung B , Milde-Langosch K , Bamberger AM , Gellersen B
Published: December 2004

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.
The different effects of endocrine-disrupting chemicals on estrogen receptor-mediated transcription through interaction with coactivator TRAP220 in uterine tissue
Authors: Inoshita H , Masuyama H , Hiramatsu Y
Published: December 2004

An endocrine-disrupting chemical (EDC) can alter endocrine functions through a variety of mechanisms, including nuclear receptor-mediated changes in protein synthesis, interference with membrane receptor binding, steroidogenesis or synthesis of other hormones. Although major chemicals have been shown to disrupt estrogenic actions mainly through their binding to estrogen receptor (ER) or androgen receptor, it is not clear how EDCs affect endocrine functions in vivo. We present evidence that the EDCs bisphenol A and phthalate activate ER-mediated transcription through interaction with TRAP220. Moreover, bisphenol A had positive effects on the interaction between ER-beta and TRAP220 and on the expression of ER-beta and TRAP220 compared with phthalate and estradiol in uterine tIssue. These data suggested that some EDCs might alter endocrine function through the change of the receptor and coactivator levels in uterine tIssue and through the different effect on the interaction between ERs and coactivator TRAP220.
mRNA expression profiles for corticotrophin-releasing hormone, urocortin, CRH-binding protein and CRH receptors in human term gestational tissues determined by real-time quantitative RT-PCR
Authors: Sehringer B , Zahradnik HP , Simon M , Ziegler R , Noethling C , Schaefer WR
Published: December 2004

Increasing maternal plasma levels of corticotrophin-releasing hormone (CRH) during the last weeks of pregnancy suggest that this stress hormone plays an important role in the control of human parturition. Little is known about the quantitative contribution of gestational tissues (other than placenta) to intrauterine formation of CRH, urocortin and CRH-binding protein (CRH-BP), or about the distribution of CRH receptors within the uterus. We have investigated the mRNA expression of CRH, urocortin, CRH-BP and CRH receptors 1 and 2 (CRH-R1 and -R2) in gestational tissues by real-time RT-PCR. Placenta, myometrium and choriodecidua were collected after uncomplicated pregnancies at term, before the onset of labour. Distribution of CRH-R1 and CRH-R2 protein was also investigated by immunostaining with receptor subtype-specific antibodies. The placenta was identified as the main site of CRH and CRH-BP mRNA expression, displaying mRNA levels >1000 and >20 times higher than those found in the myometrium and choriodecidua respectively (P<0.05 in each case). mRNA expression of urocortin was low in all tissues investigated. Myometrium and choriodecidua expressed relevant amounts of both receptor subtypes, whereas the CRH receptor population in placenta consisted mainly of CRH-R2. The high expression of CRH in placenta and the substantial expression of CRH receptors in choriodecidua and myometrium suggested that CRH derived from placenta exerts direct or indirect actions on these tissues. Neither CRH produced by myometrium or choriodecidua nor urocortin from other intrauterine sources seem to play a major role in the control of labour.
Four natriuretic peptides (ANP, BNP, VNP and CNP) coexist in the sturgeon: identification of BNP in fish lineage
Authors: Kawakoshi A , Hyodo S , Inoue K , Kobayashi Y , Takei Y
Published: December 2004

The natriuretic peptide (NP) family is composed of three members: atrial, brain/ventricular and C-type NPs (ANP, BNP/VNP and CNP respectively) in tetrapods and teleostean fish, but only CNP in elasmobranch fish. In order to trace the process of divergence of the NP family in early vertebrate evolution, we attempted to detect NPs in the primitive ray-finned fish, the sturgeon (Acipenser transmontanus). Unexpectedly, we isolated four distinct NP cDNAs from the heart and brain of this chondrostean fish. The single NP from the brain was CNP, as judged from the lack of C-terminal 'tail' sequence extending from the intramolecular ring. Two of the three cardiac NPs were ANP and VNP, as judged by the presence of an amidation signal at its C-terminus (ANP) and a long and conserved C-terminal tail sequence (VNP) respectively. The third cardiac NP was most probably BNP because it possessed all the features characteristic of BNP including: (1) the presence of dibasic amino acids within the intramolecular ring; (2) the presence of AUUUA repeats in the 3'-untranslated region of its mRNA; (3) equivalent expression of its mRNA in the atrium and ventricle and appreciable expression in the brain. Based on the sturgeon BNP sequence, we further isolated BNP cDNA from the heart of tilapia and pufferfish for the first time in teleostean fish. Phylogenetic analysis of the precursors showed that newly identified NPs belong to each group of the four NPs. The current identification of both VNP and BNP in the sturgeon clearly showed that BNP and VNP are coded by distinct genes, and that the NP family consists of at least four members in the ray-finned fish. VNP has not been molecularly identified in mammals but its presence is suggested from physiological studies; heterologous fish VNP exhibited more potent vasorelaxant activity than homologous mammalian ANP in the isolated coronary artery of dogs.
Requirement for G proteins in insulin-like growth factor-I-induced growth of prostate cells
Authors: Lyons-Darden T , Daaka Y
Published: December 2004

Elevated levels of IGF-I in the circulation are associated with increased risk for the development of prostate cancer in men, and transgenic expression of human IGF-I in mouse epithelial prostate cells results in spontaneous prostate tumorigenesis. Little, however, is known about the mechanisms involved in the IGF-I-regulated growth of prostate cells. Here, we have demonstrated that treatment with IGF-I induces the activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and the growth of human prostate cells. Stimulation with IGF-I also promoted the tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Signal relay from IGF-I to ERK requires heterotrimeric G proteins and EGFR; inhibition of Gi/o protein activation by pertussis toxin, or EGFR by tyrphostin AG1478 obliterated the ability of IGF-I to promote ERK activation. Further, treatment with pertussis toxin inhibited the IGF-I-mediated prostate cell growth. These data demonstrated the requirement of heterotrimeric G proteins in IGF-I-regulated prostate cell growth and suggest the potential utility of the G proteins as effective drug targets to combat this common cancer.
Localization of 17β-hydroxysteroid dehydrogenase type 1 mRNA in mouse tissues
Authors: Pelletier G , Luu-The V , Li S , Ren L , Labrie F
Published: December 2004

The enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 catalyzes the conversion of estrone (E1) into 17β estradiol (E2). To gain information about the cellular localization of 17β-HSD mRNA type 1 expression, we performed in situ hybridization using a 35S-labeled cRNA probe in several tissues of adult mice of both sexes. In the ovary, high expression was found in granulosa cells of growing follicles. No specific labeling could be observed in corpora lutea or interstitial cells. In the pituitary gland of animals of both sexes, 17β-HSD type 1 mRNA was expressed in the intermediate lobe melanotrophs while no specific signal could be detected in the anterior or posterior lobes of the pituitary. In the prostate, 17β-HSD type 1 mRNA was exclusively found in the epithelial cells. In both male and female mouse dorsal skin, a specific hybridization signal was seen in the sebaceous glands while the epidermis, stroma, hair follicles and sweat glands were unlabeled. In the testis, a hybridization signal was detected in germ cells of the seminiferous tubules, Leydig cells being unlabeled. The present data indicate that E2 can be formed through the action of 17β-HSD type 1 in specific cells of the gonads and peripheral tissues. In the testes and peripheral tissues, the action of E2 is probably limited to the cells involved in its formation in an intracrine fashion.
Arachidonate 12-lipoxygenase in porcine anterior pituitary cells: its localization and possible function in gonadotrophs
Authors: Ikawa H , Yamamoto K , Takahashi Y , Ueda N , Hayashi Y , Yamamoto S , Ishimura K , Irahara M , Aono T
Published: December 2004

Arachidonate 12-lipoxygenase, which oxygenates positions 12 and 13 of arachidonic and linoleic acids, is present in porcine anterior pituitary cells. Colocalization of the 12-lipoxygenase with various pituitary hormones was examined by immunohistochemical double-staining using antibodies against 12-lipoxygenase and various anterior pituitary hormones. Under light microscopy, approximately 7% of the cells producing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were positive for 12-lipoxygenase, whereas the enzyme was detected in less than 2% of the cells producing thyrotrophin, prolactin, growth hormone (GH), and adrenocorticotrophin. In an attempt to examine the participation of 12-lipoxygenase metabolites in pituitary hormone release, we incubated the primary culture of porcine anterior pituitary cells with 12-hydroperoxy-arachidonic acid or 13-hydroperoxy-linoleic acid. Significant stimulation of LH and FSH release by these hydroperoxides was observed at 10 μm in a time-dependent manner. At doses around 10 μm these compounds produced responses of similar magnitude to 1 nm gonadotrophin-releasing hormone (GnRH), but higher concentrations (30 μm) of the compounds were required for GH release. In contrast, 12-hydroxy-arachidonic and 13-hydroxy-linoleic acids were almost ineffective. Furthermore, the gonadotrophin release by 1 nm GnRH was inhibited by nordihydroguaiaretic acid (a lipoxygenase inhibitor) with an IC50 of about 5 μm. Thus, the hydroperoxy (but not hydroxy) products of 12-lipoxygenase may be involved in the release of pituitary hormones especially LH and FSH.

Journal of Endocrinology (1996) 148, 33–41
Pig uterine luminal fluid contains the developmentally regulated neurotrophic factor, pleiotrophin
Authors: Brigstock D R , Kim G Y , Steffen C L
Published: December 2004

The pig conceptus produces a non-invasive placenta which is probably dependent on uterine secretory products during both the pre- and post-attachment periods. Since some of these factors are also produced during the oestrous cycle, uterine luminal flushings (ULF) from adult non-pregnant pigs were analysed to identify potentially important maternally-secreted growth factors or cytokines. ULF contained several proteins that were detected on Western blots by an antibody raised against the N-terminal 14 amino acids of rat pleiotrophin (PTN). One of the proteins co-migrated on SDS-PAGE gels and co-eluted from heparin-affinity columns with 18 kDa recombinant human PTN, suggesting its identity as the intact native porcine PTN (pPTN) molecule. Additional immunoreactive forms of pPTN were identified that were of lower molecular mass (14–16 kDa), had lower heparin-affinities, were more hydrophobic, and were apparently C-terminally truncated. Native pPTN was isolated from ULF using cation-exchange chromatography, heparin-affinity fast protein liquid chromatography and C4 reverse-phase high performance liquid chromatography. Structural analysis of the purified protein resulted in definitive identification of 15 out of 17 N-terminal amino acids; these were 100% conserved with the corresponding residues of human, bovine and rat PTN. These results demonstrate various biochemical properties of pPTN and suggest that, in addition to the apparent involvement of PTN in differentiation during early neonatal life, it may be delivered in uterine secretory fluids to, as yet, undefined target cells in the reproductive tract of the adult female.

Journal of Endocrinology (1996) 148, 103–111
A quantitative analysis of testosterone action on FSH secretion from individual pituitary cells using the cell immunoblot assay
Authors: Noguchi K , Arita J , Nagamoto A , Hosaka M , Kimura F
Published: December 2004

We investigated the effects of testosterone on FSH secretion from male rat anterior pituitary cells in culture at the single cell level. Anterior pituitary cells cultured with or without 10 ng/ml testosterone for 72 h were mono-dispersed and subjected to cell immunoblot assays for FSH. Cell blots specific for FSH were quantified by means of a microscopic image analyzer. The number of FSH-secreting cells detected as immunoreactive cell blots on the transfer membrane represented 4·1% of total pituitary cells applied on the membrane. The amount of FSH secreted by single cells varied from <20 to >8 000 fg/cell/h. The number of FSH-secreting cells was not changed by the addition of 10 ng/ml testosterone into the culture medium. Testosterone administration increased the mean FSH secretion by 64% after 3 h incubation, resulting in a shift to the right in the frequency distribution of FSH secretion from single cells. The total amount of FSH, namely the sum of FSH secreted by each FSH-secreting cell, was increased by 92% by the addition of testosterone. However, mean amounts of FSH secretion by the top ten cells of the largest secretor subgroup (>5 pg/cell/3 h) were not different between control and testosterone-treated groups.

The present study analyzed, for the first time, FSH secretion from rat anterior pituitary cells at the single cell level. The results suggest that stimulation by testosterone of FSH secretion in vitro is not due to an increase in the number of FSH-secreting cells but to an increase in FSH secretion from each cell.

Journal of Endocrinology (1996) 148, 427–433
Sodium channel heterogeneity in the apical membrane of porcine thyroid epithelial cells
Authors: Bourke J R , Abel K C , Huxham G J , Sand O , Manley S W
Published: December 2004

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl−. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g=4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g=10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na+-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na+-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.

Journal of Endocrinology (1996) 149, 101–108
Pituitary adenylate cyclase-activating peptide stimulates cyclic AMP accumulation in UMR 106 osteoblast-like cells
Authors: Kovacs C S , Chik C L , Li B , Karpinski E , Ho A K
Published: December 2004

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) share 68% homology and function as neurotransmitters or neuroendocrine factors. Although VIP immunoreactivity has been detected in bone cells, the presence of PACAP or PACAP receptors in bone has not been determined. In this study, we investigated the role of PACAP and VIP in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line.

PACAP 27 (10−9 to 3 × 10−7 m), PACAP 38 (10−9 to 3 × 10−7 m) and VIP (10−8 to 10−6 m) stimulated cAMP accumulation up to eightfold. PACAP 27 was slightly more potent than PACAP 38, and both were tenfold more potent than VIP. Both PACAP- and VIP-stimulated cAMP accumulation were potentiated by 4β-phorbol 12-myristate 13-acetate, an activator of protein kinase C. Two PACAP antagonists, PACAP 6–27 (3 × 10−6 m) and PACAP 6–38 (3 × 10−6 m), blocked PACAP- and VIP-stimulated cAMP accumulation. Two VIP antagonists ([Lys1,Pro2,5,Arg3,4,Tyr6]-VIP, and 4 Cl-d-Phe6,Leu17]-VIP) did not reduce the PACAP-or VIP-stimulated cAMP accumulation. Pretreatment with PACAP 27, PACAP 38 or VIP equally blocked PACAP- and VIP-stimulated cAMP accumulation.

These results suggest that PACAP is a more potent stimulator of cAMP accumulation than VIP in UMR 106 cells. PACAP and VIP may share a role in the paracrine or neuroendocrine regulation of bone metabolism.

Journal of Endocrinology (1996) 149, 287–295
Monoclonal antibodies specific for the binding site on the LH receptor alter progesterone production in cultured granulosa cells
Authors: Nau E A , Epsaro E M , Scherer M S , Abel J H
Published: December 2004

Monoclonal antibodies (mAbs) specific for the LH receptor (LHR) were generated through a modified auto-anti-idiotypic approach in which human chorionic gonadotropin (hCG) was used as the immunogen followed by cyclophosphamide to induce anti-idiotypic antibodies. The purpose of this study was to investigate the effectiveness of these antibodies to alter progesterone production in porcine granulosa cells in vitro. Anti-LHR mAbs were incubated with granulosa cells in the presence or absence of a stimulatory dose of hCG. Progesterone output by treated cells was measured using a RIA procedure. Most of the mAb could inhibit stimulated progesterone production by cultured granulosa cells. It was speculated that two possible mechanisms may cause the inhibition effect observed. Several of the antibodies appeared to block hCG binding thus removing the stimulatory effects of hCG. However, the most potent inhibiting mAbs for progesterone production had little or no effect on hCG binding, suggesting that some other mechanism was responsible for the observed inhibition. In addition, several of the antibodies were found to have a stimulatory effect on progesterone production by granulosa cells even in the absence of a stimulating dose of hCG. It is proposed that these antibodies were able to mimic hCG.

Journal of Endocrinology (1996) 150, 9–16
Dihydropyridine-sensitive and -insensitive voltage-operated calcium channels participate in the control of glucose-induced insulin release from human pancreatic β cells
Authors: Davalli A M , Biancardi E , Pollo A , Socci C , Pontiroli A E , Pozza G , Clementi F , Sher E , Carbone E
Published: December 2004

Calcium ion entry through voltage-operated calcium channels is a crucial step in the coupling of β cell depolarization with insulin secretion. Various calcium channel subtypes have been shown to be coexpressed in single neurons and endocrine cells. Using the patch-clamp technique, we investigated the biophysical and pharmacological properties of calcium channels in freshly dispersed human pancreatic β cells.

Both low and high voltage activated currents were expressed, the two current types being easily distinguishable on the basis of biophysical criteria. The high voltage activated currents were not homogeneous: one component was affected by the dihydropyridine antagonist nitrendipine and the agonist Bay-K-8644; the other was insensitive to both dihydropyridines and ω-conotoxin GVIA. In line with this pharmacology, nitrendipine reduced and Bay-K-8644 increased glucose-induced insulin secretion from perifused human islets, whereas ω-conotoxin GVIA had no effect. However, about 20% of the glucose-induced insulin release was found to be resistant to high nitrendipine concentrations.

These data show that human pancreatic β cells express heterogeneous voltage-operated calcium channels, only one of which is dihydropyridine-sensitive (L type). The L type channels are clearly involved in the control of insulin secretion, but our data suggest that dihydropyridine- and ω-conotoxin GVIA-insensitive channels may also play a role in the stimulus-secretion coupling of human β cells.

Journal of Endocrinology (1996) 150, 195–203
Role of phosphoprotein phosphatases in the corpus luteum: I Identification and characterisation of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells
Authors: Ford S L , Abayasekara D R E , Persaud S J , Jones P M
Published: December 2004

Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca2+-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions.

Journal of Endocrinology (1996) 150, 205–211
Mediation of the hormone- and serum-dependent regulation of thyroglobulin gene expression by thyroid-transcription factors in rat thyroid FRTL-5 cells
Authors: Kambe F , Seo H
Published: December 2004

The molecular mechanism for hormone- and serum-dependent regulation of thyroglobulin (TG) gene expression was studied. A construct of rat TG promoter (−178 to −3) linked to a luciferase gene was transfected into TSH-, insulin- and serum-deprived FRTL-5 cells. Addition of TSH, insulin or serum augmented the luciferase activity. The endogenous TG mRNA level was also increased, indicating that the promoter used confers responsiveness of TG gene to these additives. The possible involvement of thyroid-transcription factors, TTF-1, TTF-2 and Pax-8, in the induction of TG gene transcription was studied using an electrophoretic mobility shift assay. Since the protein/DNA ratio in FRTL-5 cell extracts was significantly increased by these additives, binding activities of these factors per unit of DNA were examined. It was demonstrated that TSH, insulin or serum increased not only TTF-2 binding activity but also the binding activities of TTF-1 and Pax-8. However, the magnitude of the increase in TTF-1 and Pax-8 mRNA levels per unit of DNA was less than that of the binding activity. Taken together, our results suggest that TSH, insulin and serum increase the binding activities of TTF-1 and Pax-8 to the TG promoter presumably through the posttranslational modification of the factors, thereby enhancing TG gene transcription.

Journal of Endocrinology (1996) 150, 287–298
Lectin-binding assays for the isoforms of human erythropoietin: comparison of urinary and four recombinant erythropoietins
Authors: Storring P L , Tiplady R J , Gaines Das R E , Rafferty B , Mistry Y G
Published: December 2004

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis.

Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Galβ1–4GlcNAc (RCA and ECA), NeuAcα2-3Galβ1–4GlcNAc (MAL), NeuAcα2–6Gal (SNA), or repeating Galβ1–4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAcα2–6GalNAc (SNA) and Galβ1–3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding.

Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA-and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Galβ1–4GlcNAc sequences and of those with O-glycans containing Galβ1–3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Galβ1–3GalNAc-containing O-glycans but not NeuAcα2–6GalNAc-containing O-glycans or NeuAcα2–6Gal-containing N-glycans.

Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

Journal of Endocrinology (1996) 150, 401–412
Evidence of thyrotropin-releasing hormone (TRH) gene expression in rat anterior pituitaries and modulation by estrogens of TRH-like immunoreactivity and TRH-elongated peptide contents
Authors: Croissandeau G , Schussler N , Grouselle D , Pagesy P , Rauch C , Bayet M C , Peillon F , Le Dafniet M
Published: December 2004

TRH gene expression in the anterior pituitary has previously been reported in the human in vivo and in the rat in vitro. Until now, modulation of this synthesis with glucocorticoids and thyroid hormones has been observed in rats. The present study demonstrates for the first time that the TRH gene is also expressed, in vivo, in the rat anterior pituitary and that anterior pituitary TRH-like immunoreactivity (TRH-LI) and elongated forms of the immediate TRH progenitor sequence (TRH-elongated peptide) contents are also modulated by estrogens (E2). To investigate the presence of proTRH mRNA in the rat anterior pituitary, total RNA was reverse transcribed (RT) and the RT products were then amplified by PCR. Treatments with E2 were performed on intact and ovariectomized (OVX) rats for 2 months. TRH-LI was measured by RIA with an antibody which did not recognize the TRH-like peptide, pGlu-Glu-Pro-NH2 (<EEP-NH2) (cross-reactivity <0·1%) and was characterized further as TRH-LI by HPLC. TRH-elongated peptides were measured by EIA and characterized by Sephadex G-50 chromatography and immunoblotting (molecular mass 25–35 kDa). The plasma prolactin levels and the pituitary sizes were increased by E2 treatment in both intact and OVX rats. Anterior pituitary TRH-LI increased in intact E2-treated rats compared with intact rats (82·7 ± 19·0 versus 39·6 ± 3·6 fmol/mg protein; means ± s.e.m.; P<0·001). This increase was greater when E2 was administered to OVX rats (599·0 ± 98·4 after E2 treatment versus 58·6 ± 3·6 fmol/mg protein; P<0·001). In intact rats, anterior pituitary TRH-elongated peptide contents were not modified by E2 treatment while they were significantly decreased in OVX E2-treated rats (144·6 ±8·8 versus 223·7 ± 9·5 fmol/mg protein; P<0·001). These results demonstrate TRH gene expression in the rat anterior pituitary in vivo and suggest that E2 treatment is responsible for an increase in anterior pituitary TRH-LI, together with a decrease in TRH-elongated peptide contents.

Journal of Endocrinology (1996) 151, 87–96
Effect of oxytocin receptor antagonists on the renal actions of oxytocin and vasopressin in the rat
Authors: Windle R J , Judah J M , Forsling M L
Published: December 2004

The effect of three oxytocin receptor antagonists on the renal actions of oxytocin and vasopressin was investigated in conscious male rats infused with hypotonic saline. Infusion of oxytocin at 100 pg/min produced plasma concentrations of 12·7 ± 3·3 pmol/l and led to significant increases in sodium excretion, urine flow and glomerular filtration rate (GFR). The increase in sodium excretion of 42 ± 9% during oxytocin infusion was significantly decreased by all three antagonists to 15 ± 5% (10 ng [mercapto-proprionic acid1,d-Tyr(Et)2, Thr4,Orn8]-oxytocin/min), 13 ± 5% (5 ng desGly9[d-Trp2,Thr4,Orn8]-dC6oxytocin/min) and 4 ± 5% (1 ng d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]-vasotocin/min). Similarly, the increase in urine production of 22 ± 5% associated with oxytocin infusion was significantly decreased to 4 ± 3% (5 ng desGly9[d-Trp2,d-Thr4,Orn8]-dC6oxytocin/min) and 1 ± 4% (1 ng d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]-vasotocin/min). All three antagonists blocked the oxytocin-induced increase in GFR when infused at 10 ng/min. Infusion of vasopressin at 160 pg/min produced plasma concentrations of 10·1 ± 2·1 pmol/l and this led to a significant increase in sodium excretion and a significant decrease in urine flow rate. None of the antagonists had any effect on the natriuretic or antidiuretic actions of vasopressin suggesting that different receptors are involved in these renal actions of the two peptides.

Journal of Endocrinology (1997) 152, 257–264
Tri-iodothyronine prevents the amiodarone-induced decrease in the expression of the liver low-density lipoprotein receptor gene
Authors: Hudig F , Bakker O , Wiersinga W M
Published: December 2004

Treatment with amiodarone, a potent antiarrhythmic drug, is associated with a dose-dependent increase in plasma cholesterol resulting from a decreased number of liver low-density lipoprotein (LDL) receptors. Similar changes occur in hypothyroidism, and it has been suggested that amiodarone acts via induction of a local 'hypothyroid-like' state in extrathyroidal tissues. The present study was designed to evaluate whether exogenous tri-iodothyronine (T3) could prevent the effects of amiodarone on LDL cholesterol.

Rats were treated for 14 days with water, amiodarone 10 mg/100 g body weight (BW), or amiodarone and 2·5, 5 or 10 μg T3/100 g BW respectively. Relative to controls, amiodarone increased plasma LDL cholesterol by 31% and decreased liver LDL receptor mRNA by 56% and protein by 45%; liver T3 content was reduced by 21%. Addition of T3 to the treatment with amiodarone dose-dependently reversed all these changes, with a return to control values of plasma cholesterol and the number of liver LDL receptors, although LDL receptor mRNA remained slightly lower. Treatment of rats for 14 days with T3 alone (5 μg/100 g BW) decreased plasma LDL cholesterol by 19% and increased liver LDL receptor mRNA by 41%.

In conclusion, T3 prevents the amiodarone-induced changes in plasma LDL cholesterol and liver LDL receptor gene expression. These findings suggest that the inhibitory effect of amiodarone on LDL receptor gene expression is mediated by T3-dependent pathways.

Journal of Endocrinology (1997) 152, 413–421
Gonadotrophs but not somatotrophs carry gonadotrophin-releasing hormone receptors: receptor localisation, intracellular calcium, and gonadotrophin and GH release
Authors: Bosma P T , Kolk S M , Rebers F E M , Lescroart O , Roelants I , Willems P H G M , Schulz R W
Published: December 2004

Gonadotrophs are the primary target cells for GnRH in the pituitary. However, during a limited period of neonatal life in the rat, lactotrophs and somatotrophs respond to GnRH as well. Also, in the adults of a number of teleost fishes (e.g. carp, goldfish, and tilapia but not trout), GnRH is a potent GH secretagogue. In studying hypophysiotrophic actions of the two forms of GnRH present in the African catfish (Clarias gariepinus), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH; cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH; cfGnRH), we have investigated the effects of GnRH on catfish gonadotrophs and somatotrophs. GnRH binding was examined by incubating dispersed pituitary cells attached to coverslips with 125I-labelled [d-Arg6,Trp7,Leu8,Pro9-Net]GnRH (sGnRHa), a salmon GnRH analogue with high affinity for the GnRH receptor. Following fixation and immunohistochemistry using antisera against catfish LH and GH, 125I-labelled sGnRHa was localised autoradiographically and silver grains were quantified on gonadotrophs and somatotrophs. Specific binding of 125I-labelled sGnRHa was restricted to gonadotrophs. Both cfGnRH and cGnRH-II dose-dependently inhibited 125I-labelled sGnRHa binding to gonadotrophs. To substantiate the localisation of functional GnRH receptors, the effects of cfGnRH and cGnRH-II on the cytosolic free calcium concentration ([Ca2+]i) were examined in Fura-2-loaded somatotrophs and gonadotrophs. GnRH-induced increases in [Ca2+]i appeared to be confined to gonadotrophs, in which both endogenous GnRHs caused a single and transient increase in [Ca2+]i. The amplitude of this [Ca2+]i transient depended on the GnRH dose and correlated well with the GnRHs' effect on LH release. In vivo experiments demonstrated that GnRH treatments which markedly elevated plasma LH levels had no effect on plasma GH levels, while a dopamine agonist (apomorphine) significantly elevated plasma GH levels. We conclude that the two endogenous forms of GnRH in the African catfish are not directly involved in the regulation of the release of GH, suggesting that GnRHs cannot be considered as GH secretagogues in teleosts in general.

Journal of Endocrinology (1997) 152, 437–446
Effect of pregnancy on rat myometrial β2-adrenoceptor mRNA and isoproterenol-induced relaxation of isolated uterine strips
Authors: Engstrøm T , Bratholm P , Vilhardt H , Christensen N J
Published: December 2004

The altered myometrial contractile activity near term of pregnancy is partly due to changes in the responsiveness to catecholamines. Previous experiments have basically been concerned with uterine adrenoceptor binding characteristics. In the present study we have evaluated total myometrial DNA, β2-adrenoceptor mRNA and isoproterenol-induced relaxation of rat isolated uterine strips pre-contracted with potassium on days 0, 7, 14 and 21 of pregnancy and on day 5 post-partum. Total myometrial DNA expressed per milligram wet tissue peaked at day 14 of pregnancy followed by a decrease at the end of gestation. This suggests that hyperplasia predominates in the growth of the uterus in early gestation, whereas hypertrophy may be more marked in late pregnancy. The concentration of β2-adrenoceptor mRNA decreased linearly throughout the gestational period (0·73 ± 0·20 amol/mg wet tissue on day 0 vs 0·34 ± 0·09 amol/mg wet tissue on day 21, P<0·05). Five days after parturition, at which time the uterus had returned to its pre-pregnant weight, β2-adrenoceptor mRNA was found to have increased 8-fold (2·79 ± 0·14 amol/mg wet tissue, P<0·05) as compared with day 21. The maximal effect of isoproterenol on pre-contracted uterine strips in which α-receptors were blocked by phentolamine showed a similar decrease which on day 21 reached 67% of the day 0 level (P<0·001). EC50 values were unchanged in all groups except day 21 pregnant rats in which an increase was observed. One-way ANOVA with Bonferroni's t-test showed statistically significant differences only between the day 21 group and either the day 5 post-partum group or the day 14 pregnant group (P<0·05).

The observed alteration in EC50 prior to the end of gestation indicates that the system becomes less sensitive to β2-adrenergic stimulation at this time. We conclude that a reduction of de novo synthesis of β2-adrenoceptors may play a role in contributing to the increased myometrial activity at term. We further suggest that the dramatic up-regulation of β2-adrenoceptor mRNA postpartum may protect the fully involuted uterus against excessive contractions induced by oxytocin secreted during lactation.

Journal of Endocrinology (1997) 153, 393–399