bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the human and mouse genes encoding the tuberoinfundibular peptide of 39 residues, a ligand of the parathyroid hormone receptor family
Authors: Hansen IA , Jakob O , Wortmann S , Arzberger T , Allolio B , Blind E
Published: December 2004

The polypeptide TIP39 (tuberoinfundibular peptide of 39 residues) is a potent activator of the parathyroid hormone (PTH)-2 receptor (P2R) and an antagonist of the PTH-1 receptor (P1R). To clarify its possible physiological function(s), we studied its interaction with the human P1R and P2R and examined the expression of TIP39 in man and mouse. To find out possible sites of this ligand interaction in the organism, we identified the genes encoding the TIP39 protein precursors of Homo sapiens and Mus musculus in the databases of the human and mouse genome projects respectively. We then obtained the full-length cDNAs of both species by RACE-PCR. The deduced TIP39 preprohormones consist of an N-terminal 30 amino acid (aa) signal peptide followed by a 29 aa TIP39 precursor-related peptide, an Arg-Arg processing site, and the actual 39 aa TIP39 sequence. The first 23 aa of the actual TIP39 sequence, thought to contain the P2R receptor activation site, are identical in man and mouse and thus phylogenetically conserved. By contrast, the 16 aa C-terminal portion showed a higher degree of diversity (75% aa identity). By using RT-PCR, TIP39 was found to be highly expressed in human central nervous system tissues, trachea, fetal liver, and, to a lesser degree, in human heart and kidney. Using in situ hybridization, TIP39 mRNA expression was revealed in various areas of the mouse brain. In a homologous human cell model using human embryonic kidney 293 cells stably transfected with human P1R and P2R, human TIP39 did bind to P1R with moderate affinity (IC(50) approximately 10(-7)-10(-6 )M), but showed higher affinity binding to P2R (IC(50) approximately 10(-8)M), comparable to the affinity of human N-terminal PTH (hPTH(1-34)) to this receptor. In P2R-transfected cells, the cAMP pathway was activated more efficiently ( approximately 10-fold) by TIP39 as a ligand compared to hPTH(1-34). In P1R-transfected cells, only hPTH(1-34) but not TIP39 was able to elicit a cAMP response, but TIP39 was able to directly antagonize the cAMP-stimulating effect of hPTH(1-34) on this receptor. In conclusion, we could show a possible function of TIP39 for the human organism as a potent activator of P2R (e.g. in brain) as well as an antagonist of the action of PTH and/or PTH-related protein on P1R (e.g. in bone and kidney). The physiological role of TIP39 in calcium metabolism with regard to these actions remains to be determined. The tools developed in this work will allow us to investigate the possible role of TIP39 as a locally or systemically secreted ligand modulating the function of the PTH receptor family.
Regulation of lysophosphatidic acid receptor-stimulated response by G-protein-coupled receptor kinase-2 and beta-arrestin1 in FRTL-5 rat thyroid cells
Authors: Iacovelli L , Capobianco L , D'Ancona GM , Picascia A , De Blasi A
Published: December 2004

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that activates a variety of biological activities including cell proliferation. Three mammalian LPA receptor (LPAr) subtypes have been identified by molecular cloning, named lp(A1), lp(A2) and lp(A3), that are coupled to heterotrimeric G-proteins for signal transduction. The LPAr are endogenously expressed in the rat thyroid cell line FRTL-5 and we used the FRTL-5 cells permanently transfected to obtain moderate overexpression of G-protein-coupled receptor kinase-2 (GRK2) or beta-arrestin1 to study whether GRK2 and beta-arrestin1 desensitise LPAr-mediated signalling and regulate LPA-stimulated functional effects. Using RT-PCR we documented that lp(A1), lp(A2) and lp(A3) receptors are all expressed in FRTL-5 cells. We then analysed the signal transduction of the LPAr in FRTL-5 cells. Exposure to LPA did not stimulate inositol phosphate formation nor cAMP accumulation but reduced forskolin-stimulated cAMP. LPA was also able to stimulate MAP kinase activation and this effect was abolished by pertussis toxin pretreatment. These results suggest that LPAr are mainly coupled to a pertussis toxin-sensitive G-protein in FRTL-5 cells. In order to investigate whether GRKs and arrestins are involved in the regulation of LPAr-mediated signalling, we used the FRTL-5 cell line permanently transfected to overexpress GRK2 (named L5GRK2 cells) or beta-arrestin1 (L5betaarr1 cells). The ability of LPA to inhibit forskolin-stimulated cAMP accumulation was blunted in L5GRK2 and more markedly in L5betaarr1. The MAP kinase activation was also blunted in L5GRK2 and in L5betaarr1B cells. Exposure to 20 microM LPA increased the phosphorylation of extracellular signal-regulated kinases ERK1/2 by approximately 3-fold in L5pBJI cells (FRTL-5 cells transfected with the empty vector pBJI) while it induced a modest increase in L5betaarr1 and was ineffective in L5GRK2. We measured [3H]thymidine uptake in L5betaarr1B and in L5 GRK2 cells to test whether GRK2 and beta-arrestin1 could have a role in the regulation of LPAr-mediated cell proliferation. The mitogenic response induced by 35 microM LPA was substantially blunted in L5betaarr1 (-69+/-6%) and in L5GRK2 (-69.8+/-4.5%) cells as compared with L5pBJI. Our findings document that the receptor-mediated responses elicited by LPA are regulated by GRK2 and beta-arrestin1 in FRTL-5 cells and indicate that this mechanism is potentially important for the control of the LPA-stimulated proliferative response.
Physiological implications of pituitary trophic activity
Authors: Levy A
Published: December 2004

A complete inventory of pituitary trophic responses depends on precise estimates of mitotic activity and apoptotic events, and accurate characterization and quantification of pituitary cell subtypes irrespective of previous and current physiological demand. For a discrete structure that has been so extensively studied, it is disappointing but perhaps not surprising that none of these measures is available and therefore that the relative contributions to changes in the proportions of pituitary cellular subpopulations of trophic activity, differentiation of pluripotent cells and variations in the secretory profiles of apparently committed cells remain almost impossible to determine. To fully appreciate the extent of this dilemma, it should be remembered that conservative estimates of the proportion of corticotrophs in the rat anterior pituitary under basal conditions vary over twofold and that it is still not clear whether the apparent threefold increase in mammotrophs during pregnancy is the result of maturation of uncommitted cells, transdifferentiation of other cells such as somatotrophs, cell division, or a mixture of all three. Equally, while it has been known for some time that adrenalectomy results in a transient increase in anterior pituitary mitotic activity and appropriately timed supraphysiological glucocorticoid replacement with a wave of apoptosis, the precise identity of the cells involved in both of these responses is open to question. Thus, although many of the physiological stimuli associated with apparent changes in the proportions of pituitary cellular subpopulations are known, the precise mechanism of the changes and the consequences of the same remain obscure. This review summarizes the limited literature on pituitary trophic activity and asks what, if anything, analysis of pituitary trophic activity using current technology can tell us.
Co-localisation of gonadotrophins and granins in gonadotrophs at different stages of the oestrous cycle in sheep
Authors: Crawford JL , McNeilly AS
Published: December 2004

Associations between granins (secretogranin II (SgII) and chromogranin A and B (CgA and CgB)) and gonadotrophins (LH and FSH) have been reported in rodents and they may interact to facilitate differential storage and secretion of LH and FSH. This study investigated the relationship between granins and gonadotrophins in sheep at different stages of the oestrous cycle. Thirty-four cycling ewes had their oestrous cycles synchronised, and were divided into late luteal (LL; n=5) and early (EF; n=4), mid (n=3) and late (LF; n=11) follicular stages, and 24-53 h (n=5), 80-100 h (n=3) and 120-144 h (n=3) after the preovulatory LH surge (PS). LHbeta mRNA levels were low in LF ewes (when plasma levels and pulse frequency of LH were high) but had increased by 80-100 h PS. In contrast, FSHbeta mRNA levels decreased during the follicular phase and plasma FSH concentrations followed a similar pattern, to peak at 24-53 h PS due to low plasma oestradiol levels. While alpha-gonadotrophin subunit (alpha-GSU), SgII and CgA mRNA levels did not change, CgB mRNA levels were elevated in EF ewes and had declined in ewes around the surge. Four distinctly sized mRNA transcripts ( approximately 1.3, 2.0, 2.8 and 3.2 kb) were observed for CgA mRNA, while a double band was observed for LHbeta mRNA that was subsequently reduced to a single band after 3'-poly(A) tail truncation. The long and short LHbeta transcripts were prevalent in follicular and luteal ewes respectively. Numbers of LH(+ve)/FSH(-ve) granules stored within gonadotrophs were not different in LL and LF ewes (even though proportions of LH(+ve) granules were higher in LF ewes), but were reduced at 24-53 h PS. The majority of LH(+ve) granules also contained SgII, although few CgA(+ve) granules were found. Granule partitioning was evident whereby FSH and CgA were located near the periphery, and LH and SgII throughout the matrix. In conclusion, increases in both storage of LH(+ve) granules and secretion of LH in LF ewes despite constant LHbeta mRNA levels was facilitated, at least in part, by improved LHbeta mRNA transcript stability. Fewer LH(+ve)/FSH(-ve) granules were in storage after the PS, which was mirrored by a reduction in LH pulsatile release. Surprisingly, in view of results in rodents indicating significant changes, SgII and CgA mRNA levels did not change over the oestrous cycle in sheep. Conversely, CgB mRNA levels decreased around the time of PS. These novel results illustrate major differences in granin-gonadotrophin interactions between sheep and rodents.
Activation of angiotensin II type I receptor promotes protein kinase C translocation and cell proliferation in human cultured breast epithelial cells
Authors: Greco S , Muscella A , Elia MG , Salvatore P , Storelli C , Marsigliante S
Published: December 2004

The effect of angiotensin II (Ang II) on Ca(2+) signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca(2+) probe. Ang II (0.1-1000 nM) induced an intracellular free calcium ([Ca(2+)](i)) transient peak which was unchanged by external Ca(2+ )removal. In Ca(2+)-free medium pretreatment with thapsigargin abolished Ang II-induced Ca(2+ )release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca(2+) response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca(2+)](i) increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca(2+) response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation. Epithelial breast cells expressed PKC-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by Go 6976, a specific inhibitor of PKC-alpha and -beta1, the Ca(2+)-dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca(2+)](i )released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.
Decreased beta-cell proliferation impairs the adaptation to pregnancy in rats malnourished during perinatal life
Authors: Avril I , Blondeau B , Duchene B , Czernichow P , Breant B
Published: December 2004

We investigated the cellular mechanisms responsible for the inability of 8-month-old previously malnourished (PM) females to adapt their beta-cell mass during pregnancy. The evolution during pregnancy of beta-cell fraction, size and proliferation was studied. At day 21 of pregnancy beta-cell fraction increased less in PM than in control females, compared with their non-pregnant values. A slight beta-cell hypertrophy was observed during pregnancy in both groups. In control females, beta-cell 5-bromo-2'-deoxyuridine (BrdU) labelling index (LI) increased from 0.07+/-0.04% before pregnancy to 1.13+/-0.20% at day 12 and decreased thereafter to reach again basal levels at day 21. In PM females, beta-cell proliferation rate was decreased at day 12 (0.74+/-0.15%, P<0.05) but similar to controls at all other stages studied. Separate analysis of the head and tail parts of the pancreas in control animals revealed that the beta-cell fraction during pregnancy increased more in the head than in the tail; similarly, BrdU LI increased 20-fold in the head and 10-fold in the tail, compared with non-pregnant values. In PM females, no adaptation of beta-cell fraction could be observed in the head, where BrdU LI was decreased by half at day 12 of pregnancy. In PM females the lactogenic activity was twice that of controls at day 12 whereas all beta-cells expressed the prolactin receptor. In conclusion, perinatal malnutrition impairs subsequent adaptation to pregnancy by decreasing beta-cell proliferation in the head of the pancreas at a critical time during pregnancy.
Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice
Authors: Zawalich WS , Tesz GJ , Zawalich KC
Published: December 2004

We examined the effects of phosphatidylinositol 3-kinase (PI3K) inhibition by wortmannin or LY294002 on glucose-induced secretion from mouse islets. Islets were collagenase isolated and perifused or subjected to Western blot analyses and probed for insulin receptor-signaling components. In agreement with previous studies, mouse islets, when compared with rat islets, were minimally responsive to 10 mM glucose stimulation. The inclusion of 50 nM wortmannin or 10 microM LY294002 significantly amplified 10 mM glucose-induced release from mouse islets. The effect of wortmannin was abolished by the calcium channel antagonist nitrendipine or by lowering the glucose level to 3 mM. Wortmannin had no effect on 10 mM alpha-ketoisocaproate-induced secretion. In contrast to its potentiating effect on islets from CD-1 mice, wortmannin had no effect on 10 mM glucose-induced release from ob/ob mouse islets. Western blot analyses revealed the presence of the insulin receptor, insulin receptor substrate proteins 1 and 2 and PI3K in CD-1 islets. These results support the concept that a PI3K-dependent signaling pathway exists in beta-cells and that it may function to restrain glucose-induced insulin secretion from beta-cells. They also suggest that, as insulin resistance develops in peripheral tissues, a potential result of impaired PI3K activation, the same biochemical anomaly in beta-cells promotes a linked increase in insulin secretion to maintain glucose homeostasis.
Acipimox stimulates leptin production from isolated rat adipocytes
Authors: Wang-Fisher YL , Han J , Guo W
Published: December 2004

Acipimox is a nicotinic acid-derived antilipolytic drug devoid of major side effects, and has been used in a number of human trials. This work reports the effects of Acipimox on leptin production from isolated rat adipocytes, in comparison with nicotinic acid and insulin. For cells isolated from normal animals, all these three reagents stimulated leptin release to a similar extent. Acipimox and nicotinic acid were more potent than insulin in stimulating leptin release from cells isolated from diabetic animals, probably because of impaired insulin sensitivity in cells from these diseased animals. Co-incubation of Acipimox with norepinephrine or dibutyryl cAMP diminished its stimulatory effects on leptin release, in parallel with increased lipolysis, suggesting that intracellular free fatty acids play an important role in mediating leptin production in adipocytes.
Disruptions in feeding and body weight control in gastrin-releasing peptide receptor deficient mice
Authors: Ladenheim EE , Hampton LL , Whitney AC , White WO , Battey JF , Moran TH
Published: December 2004

Bombesin (BN) interacts with two mammalian receptor subtypes termed gastrin-releasing peptide (GRP)-preferring (GRP-R) and neuromedin B (NMB)-preferring (NMB-R) that may mediate the satiety action of BN. We examined the feeding behavior of mice that were deficient in the GRP-R (GRP-R KO) to assess the overall contribution of this receptor subtype in the feeding actions of BN-related peptides. GRP-R KO mice failed to suppress glucose intake in response to systemically administered BN and GRP(18-27), whereas both peptides elicited a potent reduction of intake in wild-type (WT) mice. Neither GRP-R KO nor WT mice suppressed glucose intake following NMB administration. Unlike the impaired responses to BN-like peptides, the feeding inhibitory action of cholecystokinin was enhanced in GRP-R KO mice. Consistent with behavioral results, GRP-R KO mice also exhibited a reduction in c-Fos immunoreactivity in the nucleus of the solitary tract (NTS) and paraventricular nucleus (PVN) following peripheral administration of BN. An evaluation of meal patterns showed that GRP-R KO mice ate significantly more at each meal than WT mice, although total 24 h food consumption was equivalent. A long-term analysis of body weight revealed a significant elevation in GRP-R KO mice compared with WT littermates beginning at 45 weeks of age. These data suggest that the GRP-R mediates the feeding effects of BN-like peptides and participates in the termination of meals in mice.
IGF-binding protein-1 inhibits IGF effects on adipocyte function: implications for insulin-like actions at the adipocyte
Authors: Siddals KW , Westwood M , Gibson JM , White A
Published: December 2004

IGF action in vivo is acutely regulated by IGF-binding protein-1 (IGFBP-1) and its phosphorylation state is implicated in modulating these effects. Since IGFs have an important regulatory role in adipocyte function, we investigated the effects of phosphorylated IGFBP-1 (pIGFBP-1) and non-phosphorylated IGFBP-1 (npIGF BP-1) on 3T3-L1 preadipocyte proliferation and adipocyte metabolism. IGFs stimulated clonal expansion of 3T3-L1 cells (IGF-I more potently than IGF-II (EC(50): 30 nM and 50 nM)). npIGFBP-1 inhibited IGF-I (50 nM) clonal expansion at a 5:1 molar ratio (P<0.01), whereas pIGFBP-1 (purified from HepG2 cell medium) abolished clonal expansion at a 1:1 molar ratio (P<0.005). In contrast, IGF-II-induced clonal expansion was inhibited 100% at a 1:1 molar ratio of npIGFBP-1. In mature adipocytes, IGF-I was equipotent with insulin in stimulating glucose uptake (EC(50): 10 nM) and inhibiting isoproterenol-induced lipolysis (EC(50): 15 nM). npIGFBP-1 completely reversed IGF-I effects at a 1:1 molar ratio (P<0.01). In summary, IGFs rather than insulin are potent regulators of clonal expansion in 3T3-L1 preadipocytes. Importantly, IGFs are equipotent with insulin in regulating adipocyte metabolism. IGFBP-1 inhibits IGF effects on preadipocyte proliferation and adipocyte metabolism, with pIGFBP-1 being more potent than npIGFBP-1 at inhibiting mitogenic actions. Since IGFBP-1 is acutely regulated by insulin, this could have important consequences in hyperinsulinaemic and insulin-resistant states.
Metalloproteinases and the modulation of GH signaling
Authors: Baumann G , Frank SJ
Published: December 2004

The metzincin metalloproteinase, tumor necrosis factor-alpha-converting enzyme (TACE), also known as ADAM (a disintegrin and metalloproteinase) 17, has recently been identified as an important enzyme for cleavage of the GH receptor (GHR) and shedding of GH-binding protein (GHBP). Proteolysis can be induced by phorbol esters, platelet-derived growth factor and serum; it is dependent on protein kinase C and partially on MAP kinase pathways. Proteolysis occurs at the cell surface, leading to extracellular release of GHBP and intracellular GHR remnant accumulation. The GHR remnant is further processed by gamma-secretase activity, possibly leading to biologically active products. TACE-dependent GHR proteolysis can be inhibited by GH as the dimerized GHR is resistant to cleavage. The cleavage site lies within a short juxtamembranous stem region that extends between the transmembrane helix and the globular dimerization domain of the GHR. GHR proteolysis leads to downregulation of functional GHRs at the cell surface, and has complex secondary effects on GH action via GHBP and GHR remnant generation.
Oestrogen-induced apoptosis in colonocytes expressing oestrogen receptor beta
Authors: Qiu Y , Waters CE , Lewis AE , Langman MJ , Eggo MC
Published: December 2004

Epidemiological studies of postmenopausal hormone replacement therapy show a reduction in the risk of developing colon cancer, and animal studies using 17beta-oestradiol (E(2)) demonstrate a decreased incidence of chemically-induced colon cancer. Using the colon cancer cell line, COLO205, we found that E(2) induced a dose-dependent increase in DNA fragmentation and nuclear condensation, significant effects being seen at 10(-12 )mol/l. BSA-conjugated E(2), which cannot enter cells, was ineffective at inducing apoptosis in COLO205 cells, indicating that E(2) was not acting through a cell-membrane receptor. E(2) did not induce the morphological changes characteristic of differentiation. Using RT-PCR we found that the oestrogen receptor alpha (ERalpha) isoform was absent in the COLO205 cell line in contrast to CACO-2, LoVo and SW620 cells, but mRNAs for ERbeta1, -beta2, -beta5 and -beta6 isoforms were detected. Western immunoblotting results showed full-length ERbeta protein but no detectable ERalpha in COLO205 cells. In normal human colon tissue samples immunoreactive ERbeta was found but ERalpha was barely detectable. Expression of ERbeta was lost in some colon cancer specimens and reduced in others. We conclude that E(2), through ERbeta, at concentrations found during replacement therapy, may inhibit the development of colon cancer by inducing apoptosis.
Proteasome inhibitors induce apoptosis in growth hormone- and prolactin-secreting rat pituitary tumor cells
Authors: Yu R , Ren SG , Melmed S
Published: December 2004

Proteasome inhibitors induce apoptosis in some malignant cells, and we show here that these inhibitors induce apoptosis in rat pituitary MMQ and GH3 tumor cells but not in normal pituitary cells. Three proteasome inhibitors, PSI, MG-132, and lactacystin, but not the calpain inhibitor, ALLM, dose- and time-dependently caused apoptosis in these cells, and 10 microM PSI caused apoptosis in 70% of MMQ cells and in 25% of GH3 cells within 24 h. A lower PSI dose (10 nM) inhibited GH3 cell growth without causing significant apoptosis or affecting prolactin secretion. Primary rat pituitary cells were resistant to both PSI and MG-132 and did not undergo apoptosis. In MMQ cells, DNA synthesis was slowed (approximately 30%) after 6 h of 10 microM PSI treatment and a partial cell cycle block at G2/M was evident after 8 h. Colorimetric caspase substrate assay and Western blotting of caspase substrates showed that caspases 2 and 3 are activated by PSI while caspases 6 and 8 remained inactive. A broad-range caspase inhibitor, caspase inhibitor III, prevented apoptosis induced by PSI. The results show that proteasome inhibitors induce apoptosis in rat pituitary tumor cells by specific caspase activation. This novel group of drugs may potentially be used in treatment of aggressive pituitary tumors, especially as their action appears relative for tumor cells.
Peripheral but not central leptin prevents the immunosuppression associated with hypoleptinemia in rats
Authors: Zhang Y , Wilsey JT , Frase CD , Matheny MM , Bender BS , Zolotukhin S , Scarpace PJ
Published: December 2004

Leptin is a peripheral immunoenhancing reagent that directly activates splenic lymphocytes in mice. We found that a 48 h fast in rats resulted in a decrease in serum leptin that was accompanied by a lower delayed-type hypersensitivity (DTH) response. Peripheral leptin replacement completely restored this response in fasted animals. We employed a recombinant adeno-associated virus (rAAV) system to deliver leptin gene directly into rat brain to assess the effect of sustained long-term central expression of leptin on immune responses. The rAAV-leptin rats had elevated central leptin over the 60 day duration of the experiment, whereas body fat and circulating leptin fell to near zero levels. The DTH response was significantly reduced by 10-20% in rats receiving rAAV-leptin compared with the control rats, and the difference was maintained for over 50 h. When the rats undergoing rAAV-leptin gene therapy were given either murine recombinant leptin or PBS s.c., rats receiving leptin had a 17% higher DTH response than rats receiving PBS. The isolated splenocytes from the former group also proliferated 34% more in vitro in response to the mitogen concanavalin A as compared with the latter group. These results suggest that peripheral leptin has a dominant role in maintaining T-cell-mediated immune responses in rats, and central leptin is unable to compensate for the immunosuppression associated with peripheral hypoleptinemia. Furthermore, preservation of normal cell-mediated immune responses does not require fat tissue as along as serum leptin levels are maintained.
Postnatal changes in ghrelin mRNA expression and in ghrelin-producing cells in the rat stomach
Authors: Sakata I , Tanaka T , Matsubara M , Yamazaki M , Tani S , Hayashi Y , Kangawa K , Sakai T
Published: December 2004

Ghrelin was recently isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. Although it is well known that a large amount of ghrelin is produced in the gastrointestinal tract, developmental changes in ghrelin mRNA expression and differentiation of ghrelin-immunopositive (ghrelin-ip) and mRNA-expressing (ghrelin-ex) cells in the stomach have not been elucidated. In this study, we therefore investigated the changes in ghrelin mRNA expression levels and in the numbers of ghrelin-ip and -ex cells in the stomachs of 1- to 8-week-old male and female rats by Northern blot analysis, immunohistochemistry and in situ hybridization. Northern blot analysis showed that the level of weak ghrelin mRNA expression was low in the postnatal period but then increased in a dimorphic pattern, i.e. transient stagnation at 4 weeks in the male rats and at 5 weeks in the female rats. The number of ghrelin-ip and ghrelin-ex cells also increased after birth, and more numerous ghrelin cells were found in female rats than in male rats, and this finding was confirmed by Northern blot analysis. Ghrelin-ip and -ex cells first appeared in the glandular base of the fundic gland and then they were found in the glandular base and the glandular neck at 3 weeks of age, suggesting that the distribution of ghrelin cells is extended from the glandular base to the glandular neck during the postneonatal development period. This is the first report on detailed changes in postneonatal ghrelin expression level and in the number of ghrelin cells in the rat stomach. The sexual dimorphism of ghrelin expression and ghrelin cell differentiation suggest that ghrelin plays an important physiological role in the stomach.
Gonadotropin-induced expression of pancreatitis-associated protein-III mRNA in the rat ovary at the time of ovulation
Authors: Yoshioka S , Fujii S , Richards JS , Espey LL
Published: December 2004

The ovulatory process in mammals involves gross physiological events in the ovary that cause transient deterioration of the ovarian connective tissue and rupture of the apical walls of mature follicles. This gonadotropin-induced process has features similar to an acute inflammatory reaction that affects most of the ovary. The present study reveals that the ovulatory events include induction of mRNA for pancreatitis-associated protein-III (PAP-III). Immature Wistar rats were primed with 10 IU equine chorionic gonadotropin s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU human chorionic gonadotropin (hCG) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12 and 24 h after the animals were injected with hCG. The RNA extracts were used for RT-PCR differential display to detect PAP-III gene expression in the stimulated ovarian tissue. Northern blotting showed that transcription was significantly greater at 4-12 h after the ovaries had been stimulated by hCG. In situ hybridization indicated that PAP-III mRNA expression was limited mainly to the hilar region of the ovarian stroma, with most of the signal emanating from endothelial cells that lined the inner walls of blood vessels, and from small secondary follicles. Treatment of the animals with ovulation-blocking doses of indomethacin (an inhibitor of prostanoid synthesis) or epostane (an inhibitor of progesterone synthesis) revealed that ovarian transcription of PAP-III mRNA was moderately dependent on ovarian progesterone synthesis. In conclusion, the present evidence of an increase in PAP-III gene expression in gonadotropin-stimulated ovaries provides further evidence that the ovulatory process is comparable to an inflammatory reaction.
Expression and localization of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP) in the human growth plate during pubertal development
Authors: Kindblom JM , Nilsson O , Hurme T , Ohlsson C , Savendahl L
Published: December 2004

Indian Hedgehog (Ihh) has been reported to control the rate of cartilage differentiation during skeletal morphogenesis in rodents through a negative feedback loop involving parathyroid hormone related protein (PTHrP). The role of Ihh and PTHrP in the regulation of human epiphyseal chondrocytes is unknown. The aim of the current study was to examine the expression and localization of Ihh and PTHrP in the human growth plate at various pubertal stages. Growth plate biopsies were obtained from patients subjected to epiphyseal surgery and the expression of Ihh and PTHrP was detected by immunohistochemistry. We show that Ihh and PTHrP are expressed mainly in early hypertrophic chondrocytes in the human growth plate. The levels of expression of Ihh and PTHrP are higher in early stages of puberty than later. Our results suggest that Ihh and PTHrP are present in the human growth plate and that Ihh and PTHrP may be involved in the regulation of pubertal growth in humans.
Comparative endocrinology of the insulin-like growth factor-binding protein
Authors: Kelley KM , Schmidt KE , Berg L , Sak K , Galima MM , Gillespie C , Balogh L , Hawayek A , Reyes JA , Jamison M
Published: December 2004

Emerging early in chordate evolution, the IGF-regulatory axis diverged from an insulin-like predecessor into a vertebrate regulatory system specializing in cell growth activation and allied anabolic functions. Essential to the divergence of the IGF and insulin systems was an early presence of soluble IGF-binding proteins (IGFBPs), which bind IGF peptides at much higher affinity than that of the insulin receptor but at comparable affinities to that of the IGF receptor. IGFBPs have no homology with IGF receptors. Instead, IGFBPs are a derived group of proteins within a superfamily of cysteine-rich growth factors, whose members are found throughout the animal taxa. While blocking IGF actions through the insulin receptor is a fundamental role, IGFBPs evolved within the vertebrate line into centralized, 'integrators' of the endocrine growth-regulatory apparatus. IGFBPs have substantial influences on the distribution and bioavailability of IGF peptides in the cellular and physiological environments, but they have a variety of other properties. The six principal mammalian IGFBPs exhibit an array of specialized properties that appear to be derived from a complex evolutionary history (including cell membrane association, interaction with proteins that post-translationally modify them, direct IGF-independent effects on cells, and others) and they are regulated by a diversity of 'outside' factors (e.g. other hormones, metabolic status, stress). Thus, IGFBPs are multifunctional integrators having diverse physiological 'agendas'. Much less is known about IGFBPs and their properties in the other vertebrate taxa. Increasingly, however, it is being recognized that they play equally important endocrine roles, in both conserved and non-conserved ways, when compared with those currently defined in mammals. This review highlights selected 'comparative aspects' in current IGFBP research, in an attempt to view this essential group of endocrine regulators from a wider, biological perspective.
Plasma binding proteins as mediators of corticosteroid action in vertebrates
Authors: Breuner CW , Orchinik M
Published: December 2004

Stressors elicit a complex but variable suite of endocrine events. Comparative studies of the stress response have focused primarily on the adrenocortical response to stress, in particular the measurement of plasma levels of glucocorticoids. However, a number of other factors contribute to and modify cellular and organismal responses to glucocorticoids. Notably, plasma corticosteroid binding globulins (CBGs) can regulate the general availability of steroid to tissues, and/or direct the delivery of hormones to specific sites. In this paper, we discuss possible functions of CBG and mechanisms of CBG action, review CBG characteristics among vertebrates, and discuss our recent studies indicating that CBG may indeed modulate responses to stressors. For example, in house sparrows, we found that basal and stress-induced concentrations of total corticosteroid (cortisol or corticosterone) (CORT) vary seasonally, but CBG concentrations change proportionally, so that free CORT concentrations appear static year-round. In contrast, in white-crowned sparrows and tree lizards, CBG concentrations change under conditions when total CORT levels do not, resulting in significant changes in circulating free CORT. These differences in free CORT are masked if CBG is not accounted for. We have also found that the binding properties of CBG vary considerably between species and need to be determined empirically. Such studies led to the observation that CBG in several species may also serve as a functional androgen binding protein; this is especially important for birds, because previous studies had concluded that birds lack androgen binding globulins. We propose that consideration of CBG is paramount to understanding the role of glucocorticoids in mediating behavioral and physiological responses to stress.
Sex hormone-binding globulin is synthesized in target cells
Authors: Kahn SM , Hryb DJ , Nakhla AM , Romas NA , Rosner W
Published: December 2004

Sex hormone-binding globulin (SHBG) is a multifunctional protein that acts in humans to regulate the response to steroids at several junctures. It was originally described as a hepatically secreted protein that is the major binding protein for sex steroids in plasma, thereby regulating the availability of free steroids to hormone-responsive tissues. SHBG also functions as part of a novel steroid-signaling system that is independent of the classical intracellular steroid receptors. Unlike the intracellular steroid receptors that are ligand-activated transcription factors, SHBG mediates androgen and estrogen signaling at the cell membrane by way of cAMP. We have reviewed the current state of knowledge on the SHBG gene and the role of SHBG in steroid signaling (we shall not address its function as a plasma-binding protein).
Albumin, steroid hormones and the origin of vertebrates
Authors: Baker ME
Published: December 2004

Albumin, the major serum protein, binds a wide variety of lipophilic compounds including steroids, other lipophilic hormones and various phytochemicals and xenobiotics that bind to receptors for steroids and other lipophilic hormones. Despite albumin's low affinity (K(d) approximately 10(-4) M to 10(-6) M) for these lipophilic compounds, the high concentration of albumin in serum makes this protein a major carrier of steroids and lipophilic hormones and a regulator of their access to receptors. Albumin also functions as a sink for xenobiotics, diminishing the binding of xenobiotics to hormone receptors and other cellular proteins. This protects animals from endocrine disruption by xenobiotics. We propose that these properties of albumin were important in protochordates and primitive vertebrates, such as jawless fish, about 600 to 530 million years ago, just before and during the Cambrian period. It is at that time that the ancestral receptors of adrenal and sex steroids - androgens, estrogens, glucocorticoids, mineralocorticoids, and progestins - arose in multicellular animals. Albumin regulated access of steroids to their receptors, as well as protecting animals from endocrine disruptors, such as phytochemicals, fungal chemicals and phenolics, and other chemicals formed at hydrothermal vents by geochemical processes. Thus, animals in which albumin expression was high had a selective advantage in regulating the steroid response and avoiding endocrine disruption by xenobiotics.
Herpes simplex virus type 1 infection and glucocorticoid treatment regulate viral yield, glucocorticoid receptor and NF-kappaB levels
Authors: Erlandsson AC , Bladh LG , Stierna P , Yucel-Lindberg T , Hammarsten O , Modeer T , Harmenberg J , Wikstrom AC
Published: December 2004

The interplay between the endocrine and immune systems has come into focus in recent years with the insight that endocrine parameters may affect susceptibility to both auto-immune and infectious diseases. Our interest in immunoendocrine regulation led us to investigate the effects of glucocorticoids on Herpes simplex virus type 1 (HSV-1) infections. Glucocorticoids used to treat inflammatory conditions are not yet recommended for HSV-1 therapy, since they have been reported to prolong viral shedding both in vivo and in vitro. Here we report that glucocorticoids did not alter the viral yield in human gingival fibroblast (HGF) cell culture when glucocorticoid treatment and viral infection occured simultaneously, but the viral yield increased when cells were treated with the glucocorticoid dexamethasone (dex) prior to viral infection. We found that viral infection in our primary cell system increased NF-kappaB levels and DNA binding. In addition, the amount of glucocorticoid receptor (GR) increased following viral infection, and HSV-1 infection as such could induce glucocorticoid-driven transcription of a reporter gene in human embryo kidney (HEK) 293 cells stably transfected with GR. Dex treatment did not affect HSV-1-induced binding of p65 to an NF-kappaB element in an electrophoretic mobility shift assay, and acyclovir was still efficient as an anti-viral drug in the presence of dex. Further studies of the observed effects of HSV-1 infection and glucocorticoid treatment on GR and NF-kappaB regulation could give insights into the immunoendocrine mechanisms important for defence and therapy against viral infections.
Effect of alloxan-induced diabetes on serum and cardiac butyrylcholinesterases in the rat
Authors: Dave KR , Katyare SS
Published: December 2004

Elevated serum butyrylcholinesterase (BChE) activity in the diabetic rat, mouse and human is very evident. The source of the increased level of BChE in the diabetic condition is not known. The effect of diabetes on cardiac BChE has not been studied so far, in spite of high BChE levels in the heart. In the present study, we investigated the effect of alloxan-induced diabetes on serum and on the soluble as well as the membrane-bound form of cardiac BChE activity and their substrate kinetics. We included rats of both sexes in the study. Serum BChE activity increased only in male diabetic rats (2.3-fold), while the activities of the soluble as well as the membrane-bound form of cardiac BChE activity increased 2.2- to 2.8-fold in male diabetic rats. A smaller increase (30%) was observed in the activity of the membrane-bound form of cardiac BChE in female diabetic rats. A slight reduction in BChE activity was observed in male and female rats after insulin treatment. The activity ratio of the soluble to the membrane-bound form of cardiac BChE was higher in diabetic and insulin-treated diabetic rats as compared with controls. The K(m) values of component II of the serum and soluble forms of cardiac BChE were comparable. In conclusion, the diabetes-induced increase in serum and cardiac BChE activity was sex dependent. Insulin was not able to rectify the diabetes-induced abnormalities in serum and cardiac BChE activity. The heart could be one of the possible sources of the increased level of serum BChE.
Recombinant interleukin-1 beta activates the hypothalamic-pituitary-interrenal axis in rainbow trout, Oncorhynchus mykiss
Authors: Holland JW , Pottinger TG , Secombes CJ
Published: December 2004

The present study provides the first direct evidence that implicates fish cytokines as the effector molecules by which the immune system signals the neuroendocrine system and activates the hypothalamic-pituitary-interrenal stress axis. I.p. injections of trout recombinant interleukin-1 beta (rIL-1 beta) or E. coli lipopolysaccharide (LPS), at concentrations known to induce immune/inflammatory responses in vivo (0.1-0.6 nmol/kg and 1.3 mg/kg respectively), significantly elevated plasma cortisol levels in a dose- and/or time-dependent manner. However, in contrast to general stress responses in fish, under the conditions employed in this study, no specific treatment effects on plasma glucose levels could be demonstrated. The trout IL-1 beta peptides (P1 and P3), which are homologous to receptor-binding sequences of human IL-1 beta, failed to influence the prevailing cortisol concentration even though an equivalent dose has been found to have immunostimulatory properties in vivo. Blockade of endogenous ACTH release by administration of the synthetic glucocorticoid dexamethasone prevented the rIL-1 beta/LPS-mediated elevation of plasma cortisol, suggesting that IL-1 beta and LPS modulate cortisol secretion via effects at the level of the hypothalamic-pituitary axis. These data indicate that, with respect to IL-1 beta, cytokine signalling between the immune and neuroendocrine systems in mammals appears to be conserved in lower vertebrates.
Prevention of solely estrogen-induced mammary tumors in female aci rats by tamoxifen: evidence for estrogen receptor mediation
Authors: Li SA , Weroha SJ , Tawfik O , Li JJ
Published: December 2004

There is increasing evidence that both endogenous and exogenously ingested estrogens play a primary role in sporadic breast cancer causation. To establish further that solely estrogen-induced mammary oncogenesis in female ACI rats is an estrogen receptor (ERalpha)-driven process, we show for the first time that concomitant treatment with the antiestrogen, tamoxifen citrate (TAMc), completely prevents the induction of 17beta-estradiol (E(2))-induced mammary gland tumors (MGTs). This finding is also supported by the reduced mammary gland (MG) lobulo-alveolar development and proliferative activity observed in TAMc+E(2)-treated animals compared with MGs from animals treated with E(2) alone. These data also correlated with a marked decrease in the number of MG cells expressing ERalpha and progesterone receptor (PR) in immunostained MG tissue sections from TAMc+E(2)-treated animals. Additionally, a marked decline in the level of expression of ERalpha 47, 56 and 66 kDa forms, and PR-A and PR-B was seen in TAMc+E(2)-treated MGs, compared with MGs treated solely with E(2). Thus, both ERalpha and PR MG profiles in TAMc+E(2)-treated rats essentially revert to their respective receptor profiles seen in untreated control and TAMc-alone-treated rats. The presence of 56 and 54 kDa isoforms in chronically E(2)-treated MGs and in MGTs respectively may contribute to fostering the enhanced E(2)-dependent growth response of both precursor and frank MGT epithelial cells. These findings are consistent with an ERalpha/PR-mediated mg cell proliferation, a prerequisite for generating the high frequency of chromosomal instability seen in E(2)-induced ductal carcinomas in situ and primary MGTs in female ACI rats reported by us previously.
Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor
Authors: Kovacs M , Schally AV , Lee EJ , Busto R , Armatis P , Groot K , Varga JL
Published: December 2004

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.
Production of cartilage link protein by human granulosa-lutein cells
Authors: Sun GW , Kobayashi H , Suzuki M , Kanayama N , Terao T
Published: December 2004

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.